Effects of renin-angiotensin system inhibitors on pancreatic

injury in cerulein-induced acute pancreatitis: potential role

of pancreatic renin-angiotensin system in exocrine pancreas

%. J 广-

TSANG，Siu Wai

A Thesis Submitted in Partial Fulfillment

Of the Requirements for the Degree of

Master of Philosophy

in

Physiology

The Chinese University of Hong Kong

June 2003

The Chinese University of Hong Kong holds the copyright of this thesis. Any

person(s) intending to use a part or whole of the materials in this thesis in a proposed

publication musk seek copyright release from the Dean of the Graduate School.

P ( 0 3 111 20M m

Abstract

Background — Acute pancreatitis is an inflammatory disease, which arises from the

premature activation of acinar digestive zymogens in the pancreas, thus causing an

autodigestion of the gland. The pathogenesis of acute pancreatitis is thought to be

multi-factorial. Apart from the activation of proteolytic enzymes, the activation of

kinins and vasoactive peptides such as angiotensin II (Ang II) would also be involved.

The renin-angiotensin system (RAS) in the pancreas has been shown to be

dramatically up-regulated by an experimental model of cerulein-induced acute

pancreatitis. Angiotensin converting enzyme (ACE) inhibitor has been demonstrated

to provide defensive effects against pancreatic inflammation and fibrosis. The

present study hypothesized that inhibition of the pancreatic RAS by specific RAS

inhibitors could have a role for changes in the severity of pancreatic injury and

oxidative stress induced by acute pancreatitis. In addition, the present study also

investigated the existence of RAS in the exocrine pancreas, and its involvement in

regulating the acinar digestive enzyme secretion and in acute pancreatitis.

Methodology — A rat model of cerulein-induced acute pancreatitis was utilized in

assessing the inhibition of the RAS on the changes in the severity of tissue injury and

oxidative stress in the pancreas. Biochemical and histopathological approaches were

exploited to evaluate the differential effects of RAS inhibitions. Furthermore, the

existence of RAS components and their regulation in isolated pancreatic acini were

examined by means of reverse transcription-polymerase chain reaction analysis. On

the other hand, the potential role of Ang II in acinar digestive secretion coupled with

specific antagonists for Ang II receptors was explored.

Results — Indicators for pancreatic injury, such as tissue edema, plasma

hyperamylasemia and hyperlipasemia, were significantly reduced in acute

i

pancreatitis-induced pancreas when the specific ATi receptor antagonist, losartan

and the non-specific Ang II receptor antagonist, saralasin were applied. Suppression

in oxidative stress was also observed as indicated by a marked decrease in

glutathione depletion, protein and lipid oxidative modifications. However, the ACE

inhibitor, ramiprilat and the specific AT2 receptor antagonist, PD123319 did not

provide any suppressive properties against the acute pancreatitis-induced injury and

oxidative stress. Histopathological evaluations were in close agreement with those

results from biochemical assays. On the other hand, exogenous Ang II was found to

stimulate a concentration-dependent release of a-amylase in isolated pancreatic acini.

Upon the application of ATi receptor antagonist, the Ang Il-evoked a-amylase

secretion was significantly inhibitable from acinar cells; however, the AT2 receptor

antagonist did not exhibit such an effect. In those acute pancreatitis-induced

pancreatic acini, the a-amylase secretion was subjected to a marked increase, which

was again inhibitable by the ATi receptor antagonist. On the other hand, preliminary

data showed that ATi receptor antagonist also provided defensive effects against

acute pancreatitis-induced systemic inflammation.

Conclusion — The experimental results suggest that the pancreatic RAS may play an

important role in the regulation of exocrine acinar functions such as digestive

enzyme secretion. The activation of such a pancreatic RAS by acute pancreatitis

may exert critical influences in the severity of tissue injury and oxidative stress. To

this end, the differential effects of RAS inhibitors on the changes in pancreatic injury

could provide an alternative strategy for the potential treatment for acute pancreatitis;

otherwise acute pancreatitis may lead to serious complications such as systemic

inflammatory response syndrome thus multi-organ dysfunction syndrome.

ii

摘要

背景-急性胰腺炎是一種炎症性疾病。它的成因主要是由於胰腺泡裡的消化

酶原過早活化,令胰腺體自身溶解而引起。其實急性胰腺炎的發病機理是由多

種因素所構成,除了因蛋白酶活化外,激肽和血管活性肽(如血管緊張素11)的活

化也可能是箇中的原因。從實驗模型裡發現,由eemlein誘發的急性胰腺炎能把

胰腺內在的腎素-血管緊張素系統大幅調升。有論證顯示，血管緊張素轉換酶抑

制劑能有效抑制胰腺炎症及纖維化的發生。當前的硏究假定了針對性的腎素-血

管緊張素系統抑制劑能在抑制胰腺內在的腎素-血管緊張素系統中,扮演一個關

鍵的角色,從而能夠調節由急性胰腺炎誘發胰腺損傷的嚴重性和氧化應激狀態。

此外,當前的硏究亦對胰臟外分泌腺是否附有腎素-血管緊張素系統,及此系統

在調節胰腺泡分泌消化酶時的參與作出調查。

方法一我們利用了一個由cemlein誘發急性胰腺炎的實驗模型來評估抑壓腎

素-血管緊張素系統能否改變胰臟腺體損傷的嚴重性和氧化應激狀態,同時也運

用了生物化學和組織病理學技術來評定不同性質的腎素-血管緊張素系統抑制

劑有何差別。除此以外,我們亦利用逆轉錄聚合酶連鎖反應檢測方法來測試胰

腺泡是否附有賢素-血管緊張素系統的成員及其在胰腺泡中的調節作用。另一方

面,從血管緊張素II受體對抗劑的反應中,探索到血管緊張素n在胰腺泡分泌消

化酶時潛在的影響力。

結果一沒有針對性的血管緊張素II受體對抗劑(saralasin)和血管緊張素II受

體一型對抗劑(losartan)能有效減輕由急性胰腺炎所誘發的胰腺損傷,例如減輕

了組織水腫,血清內高濃度的澱粉酶甲和脂肪酶。此外,它們對減輕氧化應激，

如穀胱甘肽的缺失,蛋白質和脂肪的氧化,亦有相當的抑壓效果。但血管緊張素

iii

轉換酶抑制劑(ramiprilat)和血管緊張素11受體二型對抗劑(PD123319)就沒有

在由急性胰腺炎所誘發的胰腺損傷及氧化應激這兩方面提供任何抑壓效果。對

那些賢素-血管緊張素系統抑制劑作出的測試，從組織病理學評估和生物化學

測定兩方面得到的結果都十分配合。另一方面,對從胰腺分離出來的胰腺泡添

加血管緊張素II,結果發現胰腺泡能依血管緊張素n劑量增加的剌激而隨著增

力口澱粉酶的分泌。血管緊張素II受體一型對抗劑能對血管緊張素II所提供的剌

激作出有效抑制,但血管緊張素II受體二型對抗劑就不能提供這樣的反應。那

些已誘發了急性胰腺炎的胰腺泡在澱粉酶的分泌方面有了大幅調升,而這樣的

調升又能被血管緊張素II受體一型對抗劑所抑制。此外,初部的實驗數據顯示

出血管緊張素II受體一型對抗劑同時亦可對由急性胰腺炎所弓丨致的系統性炎症

提供防禦性的效果。

結論-從實驗數據中看到,胰腺內在的腎素-血管緊張素系統也許會在胰腺泡

的外分泌作用上(如消化酶的分泌)扮演一個重要的角色。它會在急性胰腺炎發

生時被活化,因而也許會影響到胰臟腺體損傷的嚴重性和氧化應激狀態。因不

同性質的腎素-血管緊張素系統抑制劑產生的效果能影響胰臟腺體的損傷,所以

抑制胰腺的腎素-血管緊張素系統或可能對治療急性胰腺炎是一個另類的方法；

否則,急性胰腺炎也許會弓丨致多種嚴重的倂發症,如系統性的炎症反應綜合症及

多器官機能異常綜合症。

iv

Acknowledgements

This thesis is the result of my two years of work in the field of physiological research.

I have received different kinds of support from numerous people and I am

particularly grateful to have the opportunity of working with them. It is a pleasant

occasion for me to express my gratitude for all of them.

I would like to give my deepest and sincere thankfulness to my supervisor

Prof. P.S. Leung who has provided me with invaluable guidance and advice in many

aspects throughout the completion of my two-year research project. He has always

kept an eye on my work progress, and his incomparable patience and encouragement

have always kept me in good spirits. I appreciate Prof. Leung for having shown me a

comprehensive view of research and I have learned an immense amount from him.

A heartfelt gratitude goes to Dr. S.P. Ip of the School of Chinese Medicine

for all his scientific advice, valuable comments and precious help in constructing

experimental methods and overcoming technical obstacles throughout my research

study.

I wish to convey special thanks to Mr. T.P. Wong who is a very smart

laboratory technician in our lab. His technical supports and the sharing of his

practical experiences have been an indispensable part of my study. His humorous

character provided the Lab321 with an enjoyable and comfortable working

atmosphere. A warmest thank goes to Miss S.Y. Lam, a wonderful pretty lady, for

technically helping me in experimental applications as well as acquainting me with

facts and duties for my study in school.

Last I wish to thank my parents, my brother and buddies for their never-

ending love and sweetest supports.

V

Table of Contents

Abstract i

摘要 iii

Acknowledgements v

Table of Contents vi

List of Abbreviations x

Chapter 1 Introduction

1.1 Renin-angiotensin system (RAS) 1

1.1.1 Circulating RAS 2

1.1.2 Tissue-specific RAS 5

1.2 RAS inhibitors 7

1.2.1 Angiotensin converting enzyme inhibitor 8

1.2.2 Non-specific angiotensin II receptor blocker 9

1.2.3 Specific AT 1 receptor antagonist 10

1.2.4 Specific AT2 receptor antagonist 11

1.3 Pancreas and functions of exocrine pancreas 14

1.3.1 Structure of pancreas 14

1.3.2 Exocrine secretions and pancreatic enzymes 16

1.3.3 Regulation of exocrine secretions 17

1.4 Pancreatic RAS 18

1 A. 1 Expression and localization 18

1.4.2 Regulation 19

1.4.3 Clinical relevance to the pancreas 20

1.5 Acute pancreatitis 21

1.5.1 Pathogenesis 21

1.5.2 Experimental models of acute pancreatitis 22

1.5.3 Criteria of acute pancreatitis 23

1.5.4 Oxidative stress in acute pancreatitis 24

1.6 RAS and acute pancreatitis in exocrine pancreas 26

1.6.1 RAS and acute pancreatitis 26

1.6.2 RAS and pancreatic microcirculation 26

vi

1.6.3 RAS and tissue injury 27

1.6.4 Exocrine pancreatic RAS and acute pancreatitis-induced injury 28

1.7 Aims of study 29

Chapter 2 Materials and Methods

2.1 Animal models and RAS inhibitors 30

2.1.1 Cerulein-induced acute pancreatitis 30

2.1.2 Prophylactic treatment with RAS inhibitors 31

2.1.3 Therapeutic treatment with RAS inhibitors 32

2.2 Evaluation of pancreatic injury 32

2.2.1 Assessment of pancreatic water content 33

2.2.2 Measurement of a-amylase activity in plasma 33

2.2.3 Measurement of lipase activity in plasma 34

2.3 Histopathological examinations 34

2.3.1 Preparation of paraffin blocks 3 5

2.3.2 Hematoxylin and eosin staining 35

2.4 Biochemical assay of pancreatic oxidative status 37

2.4.1 Sample preparation 37

2.4.2 Quantification of protein content 37

2.4.3 Measurement of glutathione levels 38

2.4.4 Assessment of protein oxidation 38

2.4.5 Assessment of lipid peroxidation 39

2.4.6 Measurement of NADPH oxidase activity 40

2.5 Studies of pancreatic digestive enzyme secretions from isolated acini 40

2.5.1 Dissociation of acini from pancreatic tissue 40

2.5.2 Treatment with peptides and RAS inhibitors 42

2.5.3 Quantification of protein and DNA contents 43

2.5.4 Measurement of a-amylase and lipase secretions 44

2.5.5 RT-PCR analysis of RAS components in acinar cells 44

2.6 Studies of RAS inhibitors on acute pancreatitis-induced systemic

inflammation 45

2.6.1 Systemic inflammation treatment 45

2.6.2 Measurement of myeloperoxidase activity in lung and liver 46

2.7 Statistical analysis 47

vii

Chapter 3 Results

3.1 Time-course experiment of acute pancreatitis model 48

3.1.1 Effect of acute pancreatitis on tissue injury 48

3.1.2 Effects of acute pancreatitis on oxidative status 48

3.2 Evaluation of ramiprilat and saralasin on changes of acute pancreatitis-

induced pancreatic injury 50

3.2.1 Changes in tissue injury and histopathology 50

3.2.2 Changes in oxidative status 57

3.3 Evaluation of losartan and PD123319 on changes of acute pancreatitis-

induced pancreatic injury 61

3.3.1 Changes in tissue injury and histopathology 61

3.3.2 Changes in oxidative status 68

3.4 Evaluation of acinar secretions of digestive enzymes 71

3.4.1 Cholecystokinin octapeptide-induced acinar secretions 71

3.4.2 Angiotensin Il-induced acinar secretions 71

3.4.3 Effects of losartan and PD123319 on a-amylase secretion 74

3.5 Existence and regulation of acinar RAS by acute pancreatitis 75

3.5.1 Expression of angiotensinogen and its regulation by acute

pancreatitis in acini 76

3.5.2 Expression of ATi receptor and its regulation by acute

pancreatitis in acini 76

3.5.3 Expression of AT2 receptor and its regulation by acute

pancreatitis in acini 76

3.5.4 Evaluation of RAS inhibitors in acute pancreatitis-induced

acinar cells 80

3.6 Preliminary data on acute pancreatitis-induced systemic inflammation 81

3.6.1 Time-course experiment on lung injury 81

3.6.2 Time-course experiment on liver injury 83

3.6.3 Evaluation of losartan on systemic inflammation 85

Chapter 4 Discussion

4.1 Actions of RAS inhibitors on the changes of tissue injury, oxidative status

and histopathology in acute pancreatitis-induced pancreas 87

4.1.1 Differential effects of ramiprilat and saralasin 88

viii

4.1.2 Differential effects of losartan and PD123319 92

4.2 Potential functions of RAS in pancreatic acinar secretions 95

4.2.1 Potential role of ATi receptor 96

4.2.2 Potential role of AT2 receptor 98

4.3 Regulation of RAS in acute pancreatitis-induced acini 98

4.3.1 Regulation of RAS components in acinar cells 99

4.3.2 Differential actions of losartan and PD123319 100

4.4 Potential role of RAS in acute pancreatitis 102

4.4.1 Regulation of RAS components by acute pancreatitis 102

4.4.2 Differential functions of ATi and A丁2 receptors in acute

pancreatitis 103

4.5 Conclusion 104

4.6 Further studies 105

Chapter 5 Bibliography 107

ix

List of Abbreviations

Angiotensin converting enzyme ACE

Angiotensin I Ang I

Angiotensin II Ang II

Angiotensin III Ang III

Angiotensin IV Ang IV

Angiotensin II receptor type 1 ATi receptor

Angiotensin II receptor type 2 AT2 receptor

Cholecystokinin CCK

Cholecystokinin octapeptide CCK-8

Glutathione GSH

Intraperitoneal i.p.

Intravenous i.v.

Malondialdehyde MDA

Myeloperoxidase MPO

Nicotinamide adenine dinucleotide phosphate NAD(P)H

Renin-angiotensin system RAS

Reverse transcription-polymerase chain reaction RT-PCR

X

Chapter 1 Introduction

1.1 Renin-angiotensin system (RAS)

Historically, the story and study of RAS had begun when two Swedish physiologists

Robert Tigerstedt and Per Bergmann [1896] at the Karolinska Institute proposed their

observation of a kidney-derived hormone [Lavoie and Sigmund 2003]. They named

the discovered hormone renin, which could produce an acute elevation of blood

pressure. Their discovery of the vasopressor property of renin was first published in

the Scandinavian Archives of Physiology [1898]. In the second place, the production

of experimental renal hypertension by Harry Goldbatt [1940] implicated the linkage

between the kidney-derived vasopressor hormone and the decrease in blood supply.

Through a lot of research studies, the interaction between renin and blood pressure

was eventually revealed until the circulating endocrine system RAS had been

delineated by the pioneering research of Irvine Page [1958]. Renin and angiotensin

converting enzyme (ACE) are the two main enzymatic characters of the complex

hormonal system RAS. The macroglobulin angiotensinogen is the precursor for the

generation of RAS products including angiotensin I (Ang I), angiotensin II (Ang II),

angiotensin 1-7, angiotensin III (Ang III) and angiotensin IV (Ang IV) [Semple et al.

1976; Ferrario et al. 1991; Swanson et al 1992]. Physiological actions of the RAS

are carried out when the angiotensins bind to their cognate receptors ATia, ATib, AT2,

AT3, AT4 and AT7 [de Gasparo et al 2000]. The RAS has been studied extensively

over the past three decades for its systemic functions as a circulating RAS.

Contemporary research studies have put great interest and emphasis on the specific

activities of intrinsic RAS in local tissues. Up to now, the importance of RAS is still

a hotbed of research.

I

1.1.1 Circulating RAS

The integral RAS plays an important homeostatic role in regulating blood volume,

arterial pressure, aldosterone secretion, sodium and fluid balance in the circulation

[Peach 1977; Ferrario 1990]. The circulating RAS cascade (Fig. 1-1 A), which has

been well documented, starts from the cleavage of angiotensinogen into a

decapeptide Ang I by the aspartyl protease renin. Angiotensinogen is originated

from the liver and is abundant in blood. Its synthesis is stimulated by a number of

hormones such as glucocorticoids, thyroid hormone as well as Ang II [Menard 1983;

Vallotton 1987]. Renin is released from the juxtaglomerular cells of kidney into the

bloodstream in response to hypovolaemia and hypotension. Neurogenic and

hormonal signals also participate in the regulation of renin secretion [Hackenthal et

al. 1990]. The ACE from the pulmonary vasculature immediately converts the

biological inactive product Ang I into an active octapeptide Ang II, which is the

principal effector peptide of the RAS [Menard et al. 1993]. Ang II targets, at least, at

two of its cognate receptor subtypes, designated ATi and AT2. It exhibits its

biological effects at various levels to increase the vascular resistance and blood

pressure in order to restore homeostasis of the circulation [Unger et al. 1996].

Therefore, Ang II is believed to be the principal vasoactive hormone of the RAS

responsible for vasoconstriction, aldosterone release as well as trophic effects. It is

generally accepted that the G-protein coupled ATi receptor mediates most of the

well-established physiological and pathophysiological actions of Ang II

[Timmermans et al 1993]. However, the role of AT2 receptor is still largely

unknown; it may play a counter-balancing role to the ATi receptor [Hein et al. 1995].

On the other hand, Ang II could be further converted into a number of

bioactive metabolites such as Ang III and Ang IV by various aminopeptidases.

2

Another angiotensin metabolite, Ang 1 -7 could also be generated upon the action of

prolyl-endopeptidase that exerts a cleavage of Pro-Phe bond from Ang I [Corvol et al

1995] (Fig. 1-1 A). In addition to the dominant role of Ang II, emerging evidence has

shown that Ang III, Ang IV and Ang 1 -7 also exhibit physiological RAS functions

via their respective receptors (Fig. 1-lB) [Ardaillou and Chansel 1997]. In fact, Ang

1-7 has been shown to act as a vasodilator as well as a cell-growth inhibitor [Ferrario

and Iyer 1998]. It is also reported to perform biological effects opposite to Ang II,

such as stimulating phosphatase activities and nitric oxide production through the

mediation of AT7 receptor [Leung and Chappell 2003]. Ang II acts on the ATi and

ATi receptors; however, the AT3 receptor for Ang III has been characterized to be

different from the ATi and AT2 receptors since neither AT 1-specific nor AT2-specific

agents could block its binding to Ang II [Chaki and Inagami 1992]. Ang III is

suggested to be involved in the production of chemokine and the regulation of cell

growth [Ruiz-Ortega et al. 2000]; it may also be involved in the evolution of renal

damage [Ruiz-Ortega et al. 2001]. The AT4 receptor was identified and appeared to

be responsible for the dilatation of arterioles in the brain [Haberl et al 1991]. Ang

IV seems to increase cell proliferation [Pawlikowski and Kunert-Radek 1997] and

may take part in proinflammatory processes [Ruiz-Ortega et al 2001] through the

mediation of AT4 receptor.

The RAS has been suggested to regulate the production of reactive oxygen

species and reactive nitrogen species in the circulation [Fernandez-Alfonso and

Gonzalez 1999]. The imbalance production of Ang II and reactive oxygen/nitrogen

species would probably lead to endothelial dysfunction, cardiovascular disease and

renal injury [de Gasparo et al 2002]. Over the past decades, a number of studies

3

suggested that the RAS may contribute to regulation of dysfunction and injury of the

organ systems.

A ‘ Angiotensinogen

丄 Renin

Angiotensin I

” A C E

Angiotensin II . � . . endopeptidase

Angiotensin III Angiotensin IV

Angiotensin 1-7 ^

B.

Angiotensin Amino acid sequences Respective peptides receptors

Ang I Aspi-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-LeU]o N/A Ang 11(1-8) AsprArg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8 ATia, ATib, AT: Ang m (2-8) Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8 — ATi, AT�,AT3 Ang IV (3-8) Val3-Tyr4-Ile5-His6-Pro7-Phe8 AT4

Ang 1-7 Aspi-Arg2-Val3-Tyr4-Ile5-His6-Pro7 ATy

Fig. 1-1 (A) The schematic presentation of the renin-angiotensin system pathway. (B) Amino acid sequences of angiotensin peptides and their respective receptors.

4

1.1.2 Tissue-specific RAS

A large body of investigations has ascertained the existence of the so-called tissue-

specific RAS. Such an intrinsic tissue RAS may produce angiotensinogen, renin and

ACE thus the locally formed Ang II within individual tissue [Campbell 1987a;

Campbell 1987b]. This tissue-specific RAS is found to have potential roles in

mediating autocrine/paracrine effects in order to regulate the end-organ functions

independently of or additionally to the systemic RAS [Ferrario 1990b, Vinson et al.

1995]. Key components of the intrinsic RAS have been successionally identified in a

wide range of tissues, including adrenals [Capponi and Catt 1980], brain

[Mendelsohn et al. 1984], kidney [Okura et al. 1992], pituitary [Trolliet and Phillips

1992), heart [Phillips et al. 1993], gonads [Vinson et al 1997], pancreas [Leung and

Chappell 2003] and lung [Cargill and Lipworth 1995].

The detection of renin and angiotensinogen mRNA in vascular tissue

implicated the local generation of Ang T as well as its consequent product Ang II

[Kato et al 1993; Campbell and Habener 1986]. The presence of RAS in the

vascular wall of normoreninemic renal hypertensive rats has been demonstrated to

play a significant role in maintaining the high blood pressure as implicated by the

hypotensive effect of intravenous administration of EXP 3174, a specific ATi

receptor antagonist [Basso et al 1995]. Besides, the cerebral RAS could play an

important role in the regulation of cerebral blood flow (CBF) during hypoxia in

rabbit as the increase in CBF was attenuated by RAS inhibitions [Mazen et al 1995].

Furthermore, the adrenal RAS has been reported to regulate the tissue growth and

functions of glomerulosa in rat [Vinson 1995]. The cardiac RAS has been shown to

play a regulatory role in cardiac hypertrophy and remodeling [Dostal 2000] as well

as in the pathogenesis of atherosclerosis [Schieffer et al 2000].

5

Activation of a local RAS is believed to result from the local elevation of

ACE activity and the generation of Ang II. An increase in cardiac ACE activity has

been observed after an experimental model of myocardial infarction [Johnston et al

1991]. This data suggested that harmful effects might be inducible from the

activation of tissue cardiac RAS, rather than just the circulating RAS. Therefore,

ACE inhibitor could probably be able to inhibit tissue RAS in order to manage not

only hypertension, but also cardiovascular diseases [Barsotti et al. 2001], renal

diseases [Gansevoort et al 1993] and tissue fibrosis [Border and Noble 2001]. A

large body of works has substantiated that the locally generated Ang II may carry

significant proinflammatory actions, such as the production of reactive oxygen

species, inflammatory cytokines, chemokines, and the modulating abilities in cell

growth/apoptosis and injury/repair of the end-tissue. In this respect, the liver-derived

Ang II was reported to be responsible for the hepatic fibrogenesis [Paizis et al 2001].

In addition, the pulmonary Ang II has been demonstrated to act as a potential

profibrotic mediator in the lung via the activation of ATi receptor [Marshall et al

2000]. Since Ang II is responsible for the most physiological and pharmacological

effects of the RAS, the inhibitions of this system by RAS blockers such as ACE

inhibitors and receptor antagonists for Ang II could produce beneficial effects from

blocking the synthesis of Ang II. A fully understood of tissue-specific RAS could

give rise to important clinical implications for a number of diseases.

6

1.2 RAS Inhibitors

As the RAS plays important physiological and pathophysiological conditions, its

inhibition could provide a therapeutic value for hypertension, myocardial infarction,

cardiac failure, renal diseases, and tissue inflammation as well as diabetes. ACE

inhibitor and Ang II receptor antagonists are the two most widely used RAS

inhibitors (Fig. 1-2). More complete inhibition of the RAS would be definitely

beneficial by virtue of using combination therapy of the two inhibitors.

Vasodilation .(^^^^giotensinT^ Pain

/ r Prostaglandin synthesis f \ Increased vascular permeability

(^^^giotens inog^ 1

ACE Inhibitor C S g i o t e n s i ^ j ^ Kininase I/II

Non-specific Angll / • receptor blocker / T \ ： ： ^ ^

C J n ^ t i v e f r a g m e n t ^

ATi receptor antagonist AT2 receptor antagonist

C ^ ^ T i r e c e p t o ^ - ^ - - ^ ^ r e c e p t o ^

t t Vasoconstriction Vasodilation Cell growth Anti-proliferation Superoxide generation Nitric oxide production Salt/water reabsorption P450 metabolites Aldosterone secretion Apoptosis

Fig. 1-2 Schematic presentation of the RAS cascade with its relationship to ACE inhibitor and Ang II receptor antagonists.

7

1.2.1 Angiotensin converting enzyme inhibitor

ACE is a bivalent dipeptidyl carboxyl metallopeptidase, which presents as a soluble

form in blood and as a membrane-bound form in endothelial and epithelial cells of

several organs. The ACE is a key enzyme in the RAS dedicated to convert the bio-

inactive Ang I into the effector peptide Ang II (Fig. 1-2). Cushman and Ondetti

found that modification of carboxypeptidase inhibitor could block the activity of

ACE and they made a revolutionary approach in 1975. They created the first orally

active ACE inhibitor Captopril (Fig. 1-3 A) with the application of structure-based

drug design technology. The FDA approved captopril in 1982 for the treatment of

hypertension. Besides, ACE inhibitors were reported to attenuate myocardial

infarction and cardiovascular diseases [Pfeffer et al 1988; Yusuf et al 2000].

The available ACE inhibitors can be divided into 3 groups: the sulphydryl-

containing group such as captopril and zofenopril, the carboxyl-containing group

such as enalapril, quinapril and ramipril, and the phosphorous-containing group such

as fosinopril and ceranapril [Wyvratt 1988; Voors et al 1995]. Generally, ACE

inhibitors belonging to the carboxyl group are more potent than captopril but

carrying lower bioavaliabilities while the phosphorous-containing group has longer

duration of effectiveness. Some studies suggested that the sulphydryl-containing

ACE inhibitors may act as free radical scavengers and provide beneficial effects on

reperfusion-induced depression of contractility and arrhythmia [Pi and Chen 1989].

Ramiprilat, being utilized in this study, belongs to the carboxyl group and is a white

crystalline drug modified with a hepatic cleavage of the ester group of ramipril (Fig.

1-3A). ACE inhibitors are generally well tolerated by most individuals; however,

some side effects such as persistent cough, diarrhea and rash may occur. In some

8

occasions, ACE inhibitors may cause angioedema, kidney and liver failure as side

effects [Voors et al 1995].

The blockade of ACE could reduce the formation of Ang II; however, the

ACE-independent mechanisms including chymase and cathepsin G systems may

alternatively supply Ang II for completing the flow of the RAS cascade [Urata et al

1996]. On the other hand, ACE is also known as kininase II, which participates in

metabolizing bradykinin to inactive kinins in the kallikrein-kinin system (Fig. 1-2).

The inhibition of ACE has been reported to produce elevation of bradykinin in

plasma level. Bradykinin is a well-known vasodilator, pain producer and stimulator

of prostaglandin synthesis [Bumier 2001]. The therapeutic effect of ACE inhibitors

on bradykinin accumulation is still uncertain.

1.2.2 Non-specific angiotensin II receptor blocker

Since Ang II is the key active peptide of the RAS, saralasin is thought to be more

effective than the actions of ACE inhibitor to block the RAS pathway more

specifically at the last step, without worrying about the accumulation of bradykinin

and the ACE-independent formation of Ang II (Fig. 1-2). Saralasin, which is a

peptide-based analog of Ang II, is the first Ang II receptor antagonist developed in

1971. The Asp-1 and Phe-8 residues of Ang II are replaced with Sar and lie

respectively in saralasin and the chemical formula of saralasin becomes

C42H65N13O10. Apart from saralasin, sarmesin and sarthran are also analogs of Ang

II containing similar chemical structures. Saralasin acts as an antagonist for Ang II

by competitively blocking both the ATi and AT2 receptors; however, it exhibits some

unwanted partial agonist activities at high dose usage. Saralasin was found effective

in treating hypertension in patients with high renin concentration, but not in patients

9

with low plasma level of renin [Satia et al. 1995]. In addition, saralasin has been

reported to improve systemic hemodynamics in patients with congestive heart failure

[Bumier 2001].

Since saralasin is a peptide-based antagonist, it has to be administrated

parenterally. This characteristic has limited the clinical usefulness of saralasin.

Other Ang II receptor blockers developed in the 90,s, such as EXP597 and XR510,

also have equal binding affinities for both ATi and AT2 receptors; indeed, they have

been improved with higher oral bioavailability and longer duration of action [Emm et

al 1996]. Given that ATi and AT2 receptors may counteract to each other, if so, the

blockade of RAS by saralasin could only represent the compensated results of the

two receptor subtypes. To this end, the utilization of ATi receptor antagonist became

more noteworthy.

1.2.3 Specific ATi receptor antagonist

The ATi receptor belongs to the seven transmembrane class of G-protein coupled

receptor and is encoded by a 359-amino acid protein. Two isoforms, ATia and ATib,

have been identified in rodents [de Gasparo et al. 2002]. The specific ATi receptor

antagonist interacts with amino acids in the transmembrane region of the ATi

receptor and occupies space among the seven helixes; thus prevents the binding of

Ang II. Losartan potassium (DuPont pharmaceuticals, Dup 753; Merck and Co., MK

954) is the first marketable non-peptide ATi receptor antagonist, which has been

developed based on the modification of chemical structures of imidazoles S-8307

and S-8308 [Timmermans et al 1993]. The “sartan’’ family including candesartan

cilexetil, irebesartan and valsartan are biphenyl analogs of losartan (Fig. 1-3B)

carrying different oral bioavailabilities, protein binding properties and acting

10

duration time. Losartan and candesartan cilextil are the only two compounds possess

metabolites, and they are rapidly and completely converted to EXP-3174 and

candesartan respectively [Goodfriend et al 1996].

Losartan overcomes the drawbacks of saralasin, that is to say improved with

higher bioavailability, selective antagonism to ATi receptor and without exhibiting

agonist effect in the course of RAS blockade. The ATi receptor antagonist was

approved by FDA in 1995 for the treatment of hypertension, either alone or in

combination with other antihypertensive agents. The application of ATi receptor

antagonist has been reported to have fewer side effects than ACE inhibitors such as

not causing persistent cough and angioedema, which are mediated by bradykinin

[Satia et al. 1995]. However, similar to ACE inhibitors, the ATi receptor antagonist

is also fetotoxic. In fact, most individuals generally tolerate the ATi receptor

antagonist treatment very well. Nevertheless, CNS effects, back pain and diarrhea

may still occur. Adverse effects such as hyperkalemia and upper respiratory tract

infection may happen in atypical cases.

1.2.4 Specific ATi receptor antagonist

The binding affinity of Ang II with AT! receptor is similar to that of ATi receptor

[de Gasparo et al 1995]. The AT! receptor also possesses a seven transmembrane

domain but is encoded by a 3 63-amino acid protein. Only one isoform of AT2

receptor has been discovered. In AT2 receptor-knockout mice, an increased

vasopressor response to Ang II injection and an impaired drinking response to water

deprivation were observed [Hein et al 1995]. In addition, the AT2 receptor-lacking

mice had higher sensitivity to pain [Sakagwa et al 2000]. The results suggested that

AT2 receptor might play a role in RAS-mediated cardiovascular and central nervous

11

system functions. In other studies, the AT2 receptor has been also suggested for

mediating anti-proliferation, cellular differentiation and apoptosis [Dinh et al 2001].

For a better understanding of the functions of AT2 receptor, the application the

specific AT2 receptor antagonist would be of assistance. A series of 1,2,3,4-

tetrahydrosioquinoline-3-carboxylic acids including EXP655, EXP801, PD121981,

PD123317 and PD123319 have been developed with high binding affinities

selectively to AT2 receptor for Ang II. The PD 123319-ditrifluoroacetate is a

commonly used non-peptide AT2 receptor antagonist, which is industrialized by the

Parke-Davis Pharmaceutical Research (Fig. 1-3C). PD121981 (WL-19), PD123177,

EXP801 and EXP655 are the former versions of PD 123319 containing similar base

chemical structures. The peptide ligands CGP42112A and /?-aminophenylalanine6-

Ang II are found to act as agonists for the AT2 receptor [Timmermans et al. 1992].

12

A.

cooc^h, CH, Q 口

5h3 V O “ ^ ° 7

COjH \

Captopril Ramipril

‘ ^ f c , c m 雄

Losartan Candesartan cilexetil

C.

CH, CH,

PD123317 PD123319

Fig. 1-3 Commonly used RAS inhibitors and their chemical structures. (A) ACE inhibitors: captopril and ramipril. (B) ATi receptor antagonists: losartan and candersartan cilexetil. (C) AT! receptor antagonists: PD123317 and PD123319.

13

1.3 Pancreas and functions of exocrine pancreas

1.3.1 Structure of pancreas

Pancreas is a flat and elongated gland associated with the upper duodenum (Fig. 1-

4A). It consists of a head, a body and a tail, and is grouped into lobules by reticular

septae. The main pancreatic duct, with connections to interlobular and intralobular

ducts, runs through the whole length of the gland and empties into the upper

duodenum at duodenal papilla. Indeed, the pancreas carries both exocrine and

endocrine functions. The exocrine functions are carried out by the pancreatic acini

and ductal epithelia. The pancreatic acini are composed of columnar to pyramidal

epithelial cells with minimal stroma and contain well-developed Golgi complex (Fig.

1-4B). The acinar cells form apically oriented complex with zymogen granules

containing digestive enzymes. Those zymogen granules migrate to the apical plasma

membrane in order to release its contents into the lumen in response to hormonal or

neuronal stimulation. Centroacinar cells are located at the junctions between

intercalated ducts and acini, and believed to serve as signal transducers for regulating

the degranulation of acinar cells. The exocrine acini are densely packed throughout

the pancreas and connected by intercalated ducts, interlobular ducts and the main

pancreatic duct to form an extensive network for the organ. The squamous ductual

epithelial cells are located at different types of ducts in the pancreas and participating

in bicarbonate and water secretions [Gray 1918].

The pancreatic endocrine action is performed by the islets of Langerhans,

which are round, compact and highly vascularized with scanty connective tissue (Fig.

1-4B). The endocrine subunit is composed of small spheroid cluster of cells, namely

a, p, 5 and PP cells. The a cells are responsible for producing glucagons, (3 cells for

insulin, 6 cells for somatostatin and PP cells for pancreatic polypeptides. The islets

14

are distributed throughout the pancreas, which consist of about 5 % of the total

pancreatic mass; whereas the acinar cells make up most the pancreas. The exocrine

secretions are released through the pancreatic duct into the duodenum for proper

digestions of food while the endocrine subunit discharges insulin and glucagon

directly into the bloodstream for blood glucose homeostasis.

A.

COMMON „ N P L PANCREATIC BILE DUCT I | N / D U C T

TAIL

、DUODB4UM

STEINS CMIEE IMAITHE

’ • � ; 蒙擔：海鍵 S 二 Lns

rg^&es , I I i n t e — d u c t I

Fig. 1-4 Structure of pancreas (A) Overview of the pancreas. (B) Histological examination (H&E staining) of the rat pancreatic tissue. (Magnification x 400)

15

1.3.2 Exocrine secretions and pancreatic enzymes

Most of the essential enzymes in the gastrointestinal tract required for digestion are

secreted from the exocrine pancreas. Besides, bicarbonate and water are also the

major secretory products of the exocrine gland. The bicarbonate and water secreted

by the pancreatic ductal cells neutralize the gastric acid before entering the

duodenum and provide an optimal alkaline condition for the activation of various

pancreatic digestive enzymes, including a-amylase, lipase, proteases, ribonuclease

and deoxyribonuclease, which are secreted by the acinar cells. Most of the

proteolytic enzymes are packaged as inactive precursor zymogens, such as

chymotrypsinogen and trypsinogen, in order to prevent the pancreatic self-

destruction. In addition, trypsin inhibitors are present in the ductal and acinar

secretions for stabilizing the proteolytic enzymes from mopping up any active trypsin.

The trypsinogens are activated into trypsin only in the duodenum by membrane-

bound enterokinases. Other zymogens are then activated in response to the intra-

pancreatic release of trypsin and become active endopeptidases to carry out the

breakdown of proteins into polypeptides or amino acids. The major dietary

carbohydrate, starch, is hydrolyzed by the actively secreted a-amylase, and is

converted into trisaccharide, maltose and dextrins. The pancreatic lipase breaks

down the major dietary fat, triglyceride, into glycerides and free fatty acids. The

ribonuclease and deoxyribonuclease are also secreted in active forms and they are

responsible for breaking down nucleic acids (RNA and DNA) into nucleotides.

Sufficiency of pancreatic exocrine secretion is essential for efficient digestion of

food, and the subsequent normal absorption [Singer 1993; Hoist 1993].

16

1.3.3 Regulation of exocrine secretions

Exocrine secretions of the pancreas are regulated in response to the food taken into

the gastrointestinal system. When all the food is emptied from the gastrointestinal

system, a certain level of exocrine secretion still occurs and that is considered as the

basal secretion. The basal pancreatic secretion could be mediated through the very

low circulating levels of cholecystokinin (CCK) and secretin, as well as the

metabolically dependent secretion of enzymes from the acinar cells. The food-

induced exocrine secretion is modulated by both neural stimulations and hormonal

controls. Neurotransmitters from pancreas, gut and vagus nerve contribute to the

neural control of the exocrine secretion regulation. In addition, number of studies

showed that acetylcholine released from the intrapancreatic postganglionic

cholinergic neurons acts directly on acinar and ductal cells in order to regulate the

exocrine secretions, so as the vasoactive intestine peptide from peptidergic nerves

[Solomon 1994; Chey and Chang 2001]. On the other hand, CCK, secretin and

gastrin are the three important gastrointestinal hormones responsible for controlling

the pancreatic exocrine secretions. In response to the small peptides and fatty acids

in small intestine, the synthesis of CCK by the endocrine I cells of duodenum is

highly stimulated and CCK is then released into the bloodstream. As CCK binds to

its receptors on pancreatic acini, large quantity of digestive enzymes is secreted from

the pancreas into the lumen of duodenum [Rosewicz et al. 1989]. As the release of

hormone secretin is rapidly increased in response to gastric acid in the duodenum,

the exocrine pancreas is modulated to secrete bicarbonate and water in order to be

cooperative with the elevated secretion of digestive enzymes. Gastrin is a hormone

released by stomach in response to food stimuli. Similar to CCK, it also induces

secretion of digestive enzymes from the pancreas [Grant 2001]. Besides the classical

17

neuronal and hormonal controls, many regulatory factors such as cytokines,

prostaglandin and vasoactive peptides are found to stimulate exocrine pancreatic

secretion of bicarbonate and digestive enzymes. The vasoactive peptide Ang II has

been demonstrated to play a role in stimulating the a-amylase secretion in the AR42J

acinar cell line [Cheung et al. 1999]. Indeed, the pancreatic endocrine hormones

such as glucagon, somatostatin and pancreatic polypeptide are factors for inhibiting

the exocrine secretion of pancreas. The vital role of exocrine pancreas is manifested

with the fact that the insufficiency in pancreatic exocrine secretion as occurred in

pancreatitis leads to maldigestion and malabsorption of our body.

1.4 Pancreatic RAS

1.4.1 Expression and localization

Most of the early studies of tissue RAS were focused on the vascular, cardiac and

renal levels. Recently, a wide range of tissues and organs has been investigating

steadily in the past two decades. Majority of tissues were found to express primarily

the Ang II type 1 receptor. However, the pancreas possesses a dominant amount of

the AT2 receptor subtype [Leung and Chappell 2003]. Expression of Ang II as well

as its metabolites Ang III and Ang 1 -7 were documented within the canine pancreas

[Chappell et al. 1991]. The characterization of key RAS components in pancreatic

tissue has been reported in a variety of species, including canine, [Chappell et al

1991], rat [Ghiani & Masini 1995; Leung et al. 1999], mouse [Leung et al. 1998] and

human [Tahmasebi et al 1999]. In rat pancreas, gene expressions of RAS

components including renin, angiotensinogen and Ang II receptors, and the

localization of angiotensinogen protein have been well demonstrated [Leung et al.

1997, 1999]. All these results could consolidate the existence of a pancreatic RAS.

18

By means of autoradiography, Ang II receptors were shown to be present in both

acinar and islet cells [Chappell et al. 1992, 1995]. By means of the in-situ

hybridization and immunohistochemistry techniques, angiotensinogen and Ang II

receptors (ATi and AT2) have been shown to localize primarily in the epithelia of

pancreatic ducts and the endothelia of vasculature [Leung et al 1997, 1999]. In rats,

two isoforms of ATi receptor, ATia and ATib, were detected in the RT-PCR analysis

and the ATia was found to be the predominant isotype present in pancreatic tissue

[Leung et al 1999]. Taken together, all these data from expression and localization

studies support the existence of an intrinsic RAS in the pancreas, which may be of

importance in the regulation of pancreatic exocrine as well as endocrine functions.

1.4.2 Regulation

It is known that the expression of major RAS components is regulated by multiple

factors of physiological and clinical aspects including hormones, electrolytes,

hypoxic stress and pathological conditions [Phillips et al 1993]. The same principle

should apply to the pancreatic RAS. Recent studies demonstrated that the pancreatic

RAS has been up-regulated in rat models under the treatments of experimental

pancreatitis [Leung et al. 2000; Ip et al. 2003] and chronic hypoxia [Chan et al 2000;

Ip et al 2003]. RAS components, notably angiotensinogen and Ang II receptors

(both ATi and AT2) in the mRNA and protein levels were elevated in a significant

amount in pancreatitis and chronic hypoxia models when compared to their

respective controls. In addition, the pancreatic RAS has been also subjected to

regulation by pancreatic endocrine tumors (PET). In that study, the gene expressions

of AT2 receptor and angiotensinogen were significantly increased whereas the

mRNA for ATi receptor was decreased in human PET samples [Lam and Leung

19

2002]. In view of these observations, the localization, expression and regulation of a

pancreatic RAS may have physiological and pathophysiological consequences in the

exocrine and endocrine pancreas.

1.4.3 Clinical Relevance to the pancreas

Emerging data on the expression and localization of key RAS components,

particularly angiotensinogen, ATi and AT2 receptors, have provided solid evidence

for the existence of an intrinsic, angiotensin-generating system in the pancreas. This

tissue RAS may play a potential role in regulating exocrine and endocrine functions

of the pancreas such as acinar secretion of digestive enzymes and islet hormonal

secretion. Interestingly, the pancreatic RAS is responsive to a number of

physiological and pathophysiological stimuli. These include chronically hypoxic

condition and during several other clinical conditions, e.g. acute pancreatitis and

endocrine tumor. The significance of tissue RAS in the pancreas and its regulation

by pancreatitis and hypoxia could be of potential importance in the physiology and

pathophysiology of the pancreas. The target for the inhibition of pancreatic RAS by

virtue of using specific RAS inhibitors could provide an insight into several exocrine

and endocrine pancreatic diseases such as acute pancreatitis, pancreatic cancer,

diabetes mellitus and cystic fibrosis. The pancreatic RAS could also have

implications for pancreatic islet transplantation [Leung and Carlsson 2001].

20

1.5 Acute pancreatitis

1.5.1 Pathogenesis

Acute pancreatitis is an inflammatory disease of the pancreas, which happens as a

sudden onset. It is believed to be a result of an inappropriate activation of proteolytic

zymogens occurred within the pancreatic acinar cells. The pre-mature activation of

trypsin, which is restored from trypsinogen through the proteolytic cleavage of the

N-terminal peptide trypsinogen-activating peptide (TAP), is thought to be the initial

event in the evolution of pancreatitis [Neoptolemos et al. 2000]. The lysosomal

hydrolase cathepsin B has been suggested to activate trypsinogen when trypsinogen

is erroneously co-localized with cathepsin B in the cytoplasmic vacuoles, thus

causing its premature activation [Steer 1998]. Other inactive zymogens, such as

procarboxypeptidase Al, procarboxypeptidase B and chymotrypsinogen 2 packaged

in the secretory granules, are impulsively activated by trypsin in a cascade reaction

within the pancreatic acini and restored into proteolytic enzymes. The abnormally

activated zymogens escape into the interstitium of the pancreas and lead to the

disruption of acinar cells, thus the autodigestion of the gland [Gorelick et al. 1992].

The proteolysis of zymogens within pancreatic acini has been demonstrated as an

association with acinar cell damage [Grady et al. 1998]. Besides, high doses of CCK

have been shown to stimulate intracellular activation of zymogens within isolated

pancreatic acini [Leach et al 1991]. Activation of the inflammatory cascade is also

considered to be involved in the pathogenesis of acute pancreatitis. Cytokines, free

radicals and nitric oxide are participating in the progression of the disease [Mitchell

et al 2003]. In fact, gallstone and alcoholism are the common leading causes of

acute pancreatitis. In addition, the cationic trypsinogen gene has been identified to

21

cause hereditary pancreatitis and the development of recurrent acute pancreatitis

[WhitcomWfl/. 1996].

1.5.2 Experimental models of acute pancreatitis

Intraperitoneal injection of CCK secretagogue, cerulein, is the most widely used

method for the production of an experimental acute pancreatitis in animal models by

means of hyperstimulating the pancreatic secretion of digestive enzymes [Lampel

and Kem 1977]. A supramaximally stimulating dose at 50 }ig/kg/hr is used for

inducing a mild edematous pancreatitis in an experimental period varying from one

to twelve hours [Kaiser et al 1995]. In addition to the well-established method of

cemlein-induced acute pancreatitis, other models such as pancreatic bile duct

occlusion, intra-ductal infusion with sodium taurocholate, glycodeoxycholic acid and

digestive enzymes are also commonly used for the induction of pancreatitis. With

regard to the induction of a severe form of acute pancreatitis with necrosis and

hemorrhage, the biliopancreatic duct could be retrogradely infused with bile salts or

acids including sodium taurocholate (5 %) and glycodeoxycholic acid (10 mmol/L)

for 10 to 20 minutes and generally followed by a continuous intravenous infusion of

cerulein (5 昭/kg/hr) for 6 hours [Nevalainen and Aho 1992]. Intra-ductal infusion

with digestive enzymes such as trypsin (3 |ig/kg/h) and enterokinase (30 U/kg/h),

partial occlusion of pancreatic bile duct and continuous regional arterial perfusion of

the pancreas with isotonic saline are also frequently utilized for inducing

experimental pancreatitis in animals [Kaiser et al. 1995]. In addition, pancreatitis

could also be initiated by ischemia, free fatty acid infusion and ethionine-

supplemented diet in order for the production of a moderate form of pancreatitis

[Sanfey et al 1985].

22

1.5.3 Criteria for acute pancreatitis

As the pancreas is damaged, amylase and lipase would escape from the tissue and

rush into the bloodstream, thus plasma hyperamylasemia and hyperlipasemia are two

primary biochemical indicators for the diagnosis of acute pancreatitis [Clavien et al

1989]. However, normal level of plasma a-amylase may occur occasionally in

pancreatitis. Furthermore, hyperamylasemia could be associated with other

extrapancreatic diseases, such as acute cholecystitis, small bowel obstruction and

peptic ulcers [Clavien et al 1989]. Similarly, hyperlipasemia may also be observed

in chronic or acute renal disease. Therefore, the simultaneous measurements of both

a-amylase and lipase are often performed in order to supplement the diagnosis of

acute pancreatitis. Since the serum lipase level is found to stay elevated longer than

the serum amylase, it is believed that serum lipase would be helpful in the late

detection of acute pancreatitis. In addition, plasma level of pancreatitis-associated

protein (PAP) has been also utilized clinically to diagnose and monitor the evolution

of acute pancreatitis. The mRNA for secretory protein PAP is almost absent in

normal pancreas but is found to be overexpressed in the acute pancreatitis-induced

pancreatic tissue [Su et al 1999]. Furthermore, the serum level of trypsinogen 2,

which is the anionic isoenzyme of trypsinogen, could represent a correlation to the

severity of the disease [Hedstrom et al. 1996]. On the other hand, morphological

features such as interstitial edema, acinar cell damage, fat necrosis, infiltration of

macrophage and hemorrhage are often characterized for the development of acute

pancreatitis [Spormann et al. 1989]. Histological tissue sections that show interstitial

edema and peripancreatic fat necrosis can be described as mild acute pancreatitis

whereas those display parenchymal necrosis and hemorrhage are regarded as severe

forms. Release of toxin factors into the systemic circulation is often associated with

23

severe acute pancreatitis. Severity of experimental acute pancreatitis could be

defined according to the morphological alterations or the grading of necrotic

parenchyma [Friess et al 1992].

1.5.4 Oxidative stress in acute pancreatitis

Oxidative radicals have been suggested to play a significant role in the evolution of

experimental acute pancreatitis, leading to oxidative stress, and consequently causing

inflammation and tissue injury [Sanfey et al. 1984]. Oxidative-derived free radicals

are the normal side-products of metabolism, and they are immediately removed by

the anti-oxidant system in normal condition. Majority of the radicals are originated

from oxygen and nitrogen, so they are termed reactive oxygen and reactive nitrogen

species respectively. However, oxidative stress develops when the production rate of

free radicals overwhelms the homeostasis. The intracellular formation of reactive

oxygen species such as superoxide anion, hydroxyl free radical and hydrogen

peroxide could cause inflammation and cellular injury in pancreatic acini during the

course of acute pancreatitis [Schonberg et al. 1994] (Fig. 1-5). Enzymatic radical

scavengers such as GSH peroxidase and superoxide dismutase are regarded as the

primary defense against the reactive oxygen species. The reduced-glutathione level

in pancreatic tissue was rapidly depleted by GSH peroxidase after the onset of acute

pancreatitis [Neuschwander-Tetri et al 1992]. In addition, the reactive oxygen

species have been reported to attack membrane lipids and proteins thus causing the

formations of thiobarbituric acid-reactive substance malondialdehyde (MDA) and

protein carbonyls, which are the direct markers respectively for lipid peroxidation

and protein oxidation [Reinheckel et al. 1999]. Moreover, the expression of

oxidative stress-response genes, including c-fos, heme oxygenase-1 and

24

metallothionein-I, was significantly enhanced in the cerulein-induced pancreatic

tissue [Sato et al 1997; Fu et al 1997]. Transgenic mouse overexpressing with the

Cu2+/Zn2+-superoxide dismutase gene was found to significantly reduce the elevated

levels of serum a-amylase and lipase in animals with acute pancreatitis [Kikuchi et

al 1997]. These results showed that the involvement of reactive oxygen species in

the pathogenesis of acute pancreatitis could be assessed in terms of the generation of

oxidative products. Furthermore, the activities of reactive oxygen species and

production of oxidative products are found directly proportional to the severity of

cerulein-induced acute pancreatitis [Dabrowski et al. 1999]. The conversion of

molecular oxygen to superoxide anion is believed to be the initial step for the

formation of reactive oxygen species, and the membrane-bound NADPH oxidase

system is the major potential source of the superoxide anion generation [Parks 1989].

In rat aortas, endogenous Ang II was demonstrated to activate NADPH oxidase as

implicated by the increase in reactive oxygen species formation [Heitzer et al. 1999].

Indeed, acute pancreatitis is a mild and self-limited disorder; however, it may also be

severe to an extent of mortal systemic inflammation thus causing multiple organ

dysfunction syndromes [Debeaux et al 1995; Tenner et al 1997].

f 一 一 s ) fe=s’) I 一 e s )

人 (Proteases) C ^ ^ ^ I n A a m m a t i o n )

C ^ ^ ^ e l l I n j u r T ] ^ > Tissue r e p a ^

Fig. 1-5 Schematic presentation of pathophysiological relevance of reactive oxygen species (ROS) and reactive nitrogen species (RNS).

25

1.6 RAS and acute pancreatitis in exocrine pancreas

1.6.1 RAS and acute pancreatitis

Although the cause of acute pancreatitis is multifactorial, some of the crucial factors

may include the activation of proteolytic enzymes, lipase, kinins and other vasoactive

peptides such as Ang II [Agarwal and Pitchumoni 1993; Lembeck and Griesbacher

1996]. In fact, it was previously reported that the plasma renin was significantly

activated in acute pancreatitis, suggesting a role of RAS in the evolution of acute

pancreatitis [Barrett et al 1982; Greenstein et al 1987; Pitchummoni et a/. 1988].

However, the association between acute pancreatitis and RAS, in particular emphasis

on a tissue RAS in the pancreas, has received scant attention in the literature.

Previous studies in our laboratory indicated that the pancreatic RAS was up-

regulated in the rat model of experimental acute pancreatitis, as implicated by the

activation of major RAS components in both mRNA and protein levels in pancreatic

tissue [Leung et al 2000]. In this regard, pancreatic ACE activity was extensively

increased approximately 6 folds in the course of acute pancreatitis [Ip et al. 2003].

These results gave an integral view of the up-regulation of pancreatic RAS in the

evolution of an experimental acute pancreatitis. To this end, blockade of the

pancreatic RAS with ACE inhibitors and Ang II receptor antagonists could confine a

deeper investigation and confirmation of the interaction between the tissue RAS and

acute pancreatitis.

1.6.2 RAS and pancreatic microcirculation

Vasoconstriction, leukocyte adherence, changes in capillary permeability and

hemorrhaging alterations are the major disturbances affecting the microcirculation of

the pancreas [Zhou and Chen 2002; Chen et al. 2001]. Besides, the intra-pancreatic

26

ductal pressure may be also influenced by the activation of the pancreatic RAS

during the course of acute pancreatitis. Number of works showed that RAS could

play a regulatory role in the generation of reactive oxygen species [Dabrowski et al.

1999]. The Ang Il-mediated generation of reactive oxygen species in the

microenvironment of pancreatic duct may lead to the changes in the permeability of

capillary. In addition, leukocyte adherence may be initiated by lipid peroxidation,

thus causing changes in the permeability and impairment of capillary [Menger et al

2001]. Therefore, a local RAS in the pancreas is believed to have modulating

capability in the generation of reactive oxygen species, thereby the pancreatic

microcirculation. On the other hand, activation of the pancreatic RAS occurs during

the exposure to some forms of stress, such as cardiogenic [Reilly et al 1997], septic

[Oldner et al. 1999] or trauma [Kincaid et al 1998] shocks. In cardiogenic shock, a

selective Ang Il-mediated pancreatic vasoconstriction is seen that causes severe

pancreatic ischemia/hypoxia [Reilly et al 1997]. In this regards, splanchnic blood

perfusion is markedly improved following an Ang II receptor antagonism [Kincaid et

al. 1998; Oldner et al 1999]. Recently, it has been shown that infusion of Ang II

induced a dose-dependent reduction in both whole pancreatic and islet

microcirculation [Carlsson et al 1998]. In that study, administration of ACE

inhibitor and Ang II receptor blocker could increase the pancreatic circulation.

1.6.3 RAS and tissue injury

Recent studies have demonstrated the effect of Ang II in stimulating the synthesis

and secretion of vascular permeability factor. This in turn leads to cell infiltration

and exudation plasma macromolecules [Pupilli et al. 1999; Williams et al 1995].

Ang II has also been shown to stimulate the synthesis of adhesion molecules and

27

chemokines [Scheiffer et al. 2000] as well as the migration and adhesion of

monocytes and leukocytes [Kintscher et al. 2001; Alvarez and Sanz 2001]. These

data indicate that RAS plays a critical role in the formation of tissue inflammation

and disease injury. A number of recent studies demonstrated that blockade of

intrinsic RAS could attenuate injury in local tissues and organs. Gene and protein

expressions of major RAS components are found to be up-regulated in diseased liver

parenchyma of the bile duct-ligated rats. The antifibrotic effects of RAS blockade

implicated the participation of the local RAS in fibrogenic responses in liver [Paizis

et al 2002]. In addition, the administration of Ang II receptor blocker has been

shown to produce beneficial effects on the postischemic endothelial dysfunction in

isolated working rat heart [Barsotti et al 2001]. Pulmonary RAS has been

demonstrated to play a role in the increasing of fibroblast activity and vascular

permeability. Both ACE inhibitor and Ang II receptor antagonist are reported to

attenuate experimental lung injury [Marshall 2003]. Since Ang II is found to induce

vascular damage and cardiovasvular diseases through mediating the production of

chemokines, cytokines and adhesion molecules [Ruiz-Ortega et al 2001], the

applications of RAS inhibitors could be of assistance in studying the role of local

RAS in tissue injury.

1.6.4 Exocrine pancreatic RAS and acute pancreatitis-induced injury

Expression of RAS components has been recently reported in the AR42J acinar cell

line [Chappell et al. 1995]. The mRNA and protein levels of major RAS

components in pancreatic tissue are increased significantly in the evolution of acute

pancreatitis [Leung et al 2000]. More interestingly, the pancreatic inflammation and

fibrosis caused by chronic pancreatitis could be attenuated by administration of ACE

28

inhibitor [Kuno et al 2003]. This result further supports the involvement of RAS in

pancreatits-induced injury. Emerging data from different aspects implicated the

existence of a local RAS in the exocrine acini and such a system would be expected

to be up-regulated by acute pancreatitis as seen in the pancreatic tissue. The

applications of RAS inhibitors should have some beneficial effects against acute

pancreatitis-induced tissue injury as well as the acute pancreatitis-associated

systemic inflammation.

1.7 Aims of study

Based on the available information, the present study is designed specifically to

investigate the effects of RAS inhibitors on acute pancreatitis-induced pancreatic

injury and the potential role of RAS in exocrine pancreas. Therefore, the aims of

present study are set as follows:

• Biochemical analysis of RAS inhibitors on the changes in acute pancreatitis-

induced pancreatic injury and oxidative stress

• Histopathological analysis of RAS inhibitors on the pancreatic histology in acute

pancreatitis

• Potential role of RAS in exocrine acinar secretion

• Existence of RAS and its regulation by acute pancreatitis in acinar cells

• Potential role of RAS in acute pancreatitis

• Preliminary study of RAS in acute pancreatitis-induced systemic inflammation

29

Chapter 2 Materials and Methods

2.1 Animal Models and RAS inhibitors

Male Sprague-Dawley (SD) rats aged 28 days weighing in the range of 75 to 100 grams

were used in all experiments. The animals were bred in the Laboratory Animal Services

Center of the Chinese University of Hong Kong, fed with standard rat chow, and

supplied with water ad libitum. The rats were housed under a 12-hour light/dark cycle

with constant ambient temperature of 2 2 � � ± 1 � � a n d relative humidity in the range of

60 to 80 %. Ethical approval for experimental procedures has been obtained from the

Animal Ethical Committee of the Chinese University of Hong Kong. The animals were

subjected to pentobarbital-anesthetic management during the whole period of

experiment and were killed by cardiac excision.

Four RAS inhibitors were used in the present study. The non-specific Ang II

receptor ligand saralasin and the specific ATi receptor antagonist PD123319 were

purchased from Sigma Co., USA. The specific ATi receptor antagonist losartan and the

ACE inhibitor ramiprilat were received as gifts from Merck, USA and Aventis,

Germany respectively.

2.1.1 Cerulein-induced acute pancreatitis

Prior to the experiments, the SD rats were starved overnight but allowed with free

access to water. Animals were randomly assigned to acute pancreatitis group and

control group. In the acute pancreatitis group, the rats were treated with CCK-

secretagogue, cerulein (Pharmacia GmbH, Germany), for the induction of disease

[Lampel and Kem 1977]. A time-course experiment was performed in order to figure

out the minimum time required for the onset of acute pancreatitis. A single intra-

peritoneal (i.p.) injection of cerulein was given at 50 ig per kilogram of body weight to

30

animals at minute 0 and if applicable one more at minute 60. The control did not

receive any cerulein injection. Two-hour was chosen as the intending experimental

period for development of acute pancreatitis. During the 2-hour experimental period,

two i.p. injections of cerulein were given hourly to animals in the acute pancreatitis

group. A supramaximal stimulating dose was given at 50 |ag/kg/hr in order to construct

a mild edematous pancreatitis. The second injection of cerulein was given to the animal

60 minutes after the first injection, and the animal was sacrificed 60 minutes thereafter.

Rats in the control group received 0.9 % NaCl saline solution injection instead of

cerulein in same volume and at same time intervals. The experimental protocols for the

induction of acute pancreatitis and the treatment with RAS inhibitors were summarized

in Table 2-1.

2.1.2 Prophylactic treatment with RAS inhibitors

Animals were randomly assigned to 4 groups, acute pancreatitis, acute pancreatitis +

prophylactic treatment (AP + PT), control and placebo (placebo PT). All four

experimental groups were handled the same during the 2-hour experimental period as

mentioned in section 2.1.1, except that rats in both AP + PT and placebo PT groups

were pre-treated with RAS inhibitors (ramiprilat, saralasin, losartan or PD123319) 30

minutes prior to the first i.p. injection of cerulein. RAS inhibitor was given as an

intravenous (i.v.) injection to the tail vein of the rat model. All animals were sacrificed

120 minutes after the first cerulein injection (Table 2-1). Saralasin and ramiprilat were

given to animals at doses in the range of 5 to 40 ^ig/kg whereas losartan and PD 123319

were given to animals at doses in the range of 50 to 800 |ag/kg.

31

2.1.3 Therapeutic treatment with RAS inhibitors

Animals were randomly assigned into 4 groups, acute pancreatitis, acute pancreatitis +

therapeutic treatment (AP + TT), control and placebo (placebo TT). All four

experimental groups were handled the same as mentioned in the previous section,

except that the AP + TT and placebo TT groups were intravenously injected with RAS

inhibitor 30 minutes after the second cerulein injection during the 2-hour experimental

period. All animals were sacrificed 120 minutes after the first injection of cerulein

(Table 2-1). Dosages of inhibitors were given as the same as in the prophylactic

treatment groups.

Prophylactic and therapeutic treatments with RAS inhibitors

^ ^ ^ ^ TimeO Time 30 Time 60 Time 90 Time 120 Time 150

“ 7 1 0.9%NaCl “ 0.9%NaCl ^ Control N/A (i.p.) N/A (i p ) N/A Killed

, Cerulein ../A Cerulein , . 丁 , AP N/A 二 1 N/A 二 N/A Killed

Placebo ||RAS(i.v.) 0.9%NaCl 0.9%NaCl ^ ^ Killed PT Inhibitor q^O N/A (j.p.) N/A Jellied

AP+PT n /A N/A Killed Inhibitor (i.p.) (i.P.) Placebo 0.9%NaCl ^ 0.9%NaCl RAS (i.v.) 丁,.” ^ ^

TT (Z.) N/A (i；.) Inhibitor Killed N/A Cerulein , Cerulein RAS (i.v.) 丁,.” j � t / a AP+TT M/A 广、 T 1 ‘ Killed N/A

八 丄 (i.p.) 丄�'八 （i.p.) Inhibitor

Table 2-1 Summary of the time schedule for prophylactic and therapeutic treatments; PT: prophylactic treatment, TT: therapeutic treatment, N/A: not applicable.

2.2 Evaluation of pancreatic injury

Tissue injury is one of the distinguished consequences of acute pancreatitis. Formation

of pancreatic edema, hyperamylasmia and hyperlipasmia are the primary indicators of

the acute pancreatitis-induced pancreatic tissue injuries [Clavien et al 1989; Nevelainen

and Aho 1992].

32

2.2.1 Assessment of pancreatic water content

Pancreatic edema results from the gain of water content in acute pancreatitis-induced

pancreatic tissue. This can be reflected by the gain of tissue weight. Pancreatic tissue

was removed, freed from fat and blotted dry. Weights of each animal and its pancreatic

tissue were measured. The obtained weights were expressed as a ratio of pancreatic

weight to body mass (g/kg) in order to assess the formation of pancreatic interstitial

edema.

2.2.2 Measurement of a-amylase activity in plasma

Traditionally, the diagnosis of acute pancreatitis is based on the elevation of serum

amylase activity [Clavien et al. 1989]. Heparinized blood sample was drawn from

anesthetized animal by cardiac puncture and plasma was obtained by centrifuging the

whole blood at 2,000 x g at 4 � C . The activity of a-amylase in plasma was measured

with an assay kit from Sigma Co., USA by following the manufacturer's instruction.

Briefly, the activity was measured based on the hydrolytic rate of a-amylase in

hydrolysis of the substrate 4,6-ethylidene-G7-p-nitrophenol to G2, G3 and G4 fragments

and further to j9-nitrophenol and glucose, using a kinetic spectrophotometric method.

The reaction started once 4 jil of plasma sample was added to 200 |LI1 of the substrate

reagent at 37 °C. The increase in light absorbance of p-nitrophenol at 405 nm in a 2-

minute period was taken with the micro-plate reader (Bio-tek Instrument Inc., USA).

The rate of increase was calculated and the enzyme activity was expressed as U/L.

33

2.2.3 Measurement of lipase activity in plasma

Another diagnostic parameter for acute pancreatitis is the elevation of serum lipase

activity, which is believed to remain elevated longer than serum amylase [Clavien et al.

1989]. Plasma samples were obtained as mentioned in the previous section. The

activity of lipase was measured based on a colorimetric method using the Lipase-PS

assay kit from Sigma Co., USA. By following the manufacturer'S instruction, 10 |LI1 of

plasma sample was incubated with 90 \i\ of reconstituted substrate solution at 37 °C for

4 minutes allowing the pancreatic lipase to hydrolyze the substrate 1,2-diglyceride to

monoglyceride and fatty acid. Upon the addition of 30 il of activator solution, the

mixture was incubated for another 3 minutes at 37 A quinone diimine dye was

formed, which absorbs light at 550 nm. Readings were taken with the micro-plate

reader and the rate of increase in absorbance at 550 nm was used in calculating the

pancreatic lipase activity, which was expressed as U/L.

2.3 Histopathological examinations

Acute pancreatitis is morphologically characterized by acinar cell damage, interstitial

edema, parenchymal necrosis and hemorrhages. All these morphological alterations

could be examined through histological slides under light microscopy. Acinar cell

damage can be defined as cytoplasmic vacuolization or shrinkage. The interstitial

edema could be determined by measuring the expansion area developed in interlobular

space of the pancreatic parenchyma. Loss of membrane integrity and disintegration of

organelles are morphological markers for parenchymal necrosis [Nevalainen and Aho

1992].

34

2.3.1 Preparation of paraffin blocks

Pancreatic tissues were harvested, trimmed from fat and fixed in 4% (w/v)

paraformaldehyde (PFA) at 4 overnight. The PFA-treated tissues were further

processed in a tissue-processing machine for 18 hours with repeated sequential ethanol

dehydrations and xylene clearing procedures. Processed tissue samples were embedded

in molten paraffin and cast into blocks after cooling.

2.3.2 Hematoxylin and eosin staining

Paraffin-embedded tissue blocks were sectioned into 5 [im slices using the biocut rotary

microtome machine (Reichert-Jung, Germany). The sliced pieces were transferred to a

4 0 � C water bath and allowed floating out. After trimming, the slices were fixed onto

0.5 % gelatin-coated slides and allowed to dry. The slides holding the sectioned tissues

were de-paraffinized with xylene 3 times, and hydrated with 100 %, 90 %, 80 % and 70

% ethanol in a series (Table 2-2A). After rinsing with water, the sections were stained

with hematoxylin and eosin (H&E) for 8 minutes and 2 minutes respectively. A brief

rinsing with 1 % acid alcohol and Scott's tap water was done in between the 2 staining

steps. The cytoplasm was stained red with eosin while the nucleus was stained blue

with hematoxylin (Table 2-2B). The stains were sequentially fixed with a series of

graded ethanol, 70 %, 80 %, 90 % and 100%. The fixing was further enhanced with

xylene. The H&E stains were mounted on the sections using the Clearmount solution

and covered up with transparent slips (Table 2-2C). The stained slides were examined

under light microscope (Leica, Germany) at magnification of 400 x. Images were

captured using the Leica DC200 software.

35

De-paraffinize the tissue sections: Reagents: Time: Xylene 5 minutes Xylene 5 minutes Xylene 5 minutes 100% ethanol 3 minutes 90% ethanol 3 minutes 80% ethanol 3 minutes 70% ethanol 3 minutes ELinse with water briefly

Table 2-2A Summary of the de-paraffmization procedures

Stain sections with H&E: Regents: Time: Hematoxylin ？里！"：进 fs 1 % acid alcohol 10 seconds Scott's tap water 15 seconds Eosin 2 minutes Rinse with water briefly

Table 2-2B Summary of the H&E staining procedures

Fix stained sections: Reagents: Time: 70% ethanol ...iminutss 80% ethanol 3 minutes 90% ethanol 3 minutes 100% ethanol 3 minutes 100% ethanol 3 minutes 100% ethanol 3 minutes Xylene 5 minutes Xylene 5 minutes Xylene 5 minutes Mount the stained slides

Table 2-2C Summary of fixing procedures

36

2.4 Biochemical assay of pancreatic oxidative status

Oxidative stress is implicated to be an important factor in the pathogenesis of acute

pancreatitis. Depletion of GSH, lipid peroxidation and protein oxidation are the most

common indicators for the assessment of oxidative stress development. Therefore, the

levels of GSH, MDA and protein carbonyls in pancreatic tissue were measured in the

present study. Moreover, the membrane-associated NADPH oxidase is believed to be

one of the potential sources of reactive oxygen species in tissue injury. The changes of

NAD(P)H oxidase activity in acute pancreatitis were also measured in the present study

[Parks 1989; Dabrowski et al 1999].

2.4.1 Sample preparation

Tissue samples were immediately removed, and frozen in - 7 0 � C of liquid nitrogen.

Upon biochemical assays, tissue samples were thawed. 10 % (w/v) tissue homogenates

were prepared in 100 mM ice-cold phosphorous buffer saline (PBS), pH 7.4, by

homogenization with a Telfon-Glass homogenizer.

2.4.2 Quantification of protein content

Protein content in each pancreatic sample was quantified using a colorimetric method

with a micro-plate reader. The 10 % (w/v) pancreatic homogenates were diluted 50

folds with 100 mM PBS (pH 7.4) and the final dilution volume was made to 1 ml.

Following the instructions provided by the Bio-Rad protein assay kit, 4 |LI1 of the diluted

homogenate was mixed with 200 il of reagent dye diluent, which was made from a 5-

fold dilution of the original concentrated stock. Concentrations of bovine serum

albumin (BSA) at 0.125, 0.25, 0.5 and 1.0 mg/ml were used for the construction of the

protein-assay standard curve. The absorbance at 595 nm was taken with the micro-plate

37

reader. Protein concentration of each pancreatic sample was calibrated with the protein

standard curve and expressed as mg per ml. Triplicate sets of measurement were

performed in order to average out the reading or pipetting errors.

2.4.3 Measurement of glutathione levels

Depletion of the antioxidant glutathione in pancreatic tissue was measured

spectrophotometrically in order to assess the cellular antioxidant capacity against the

oxidative stress [Godin and Gamett 1992]. 400 [il of 10 % pancreatic homogenate was

treated with 100 |al of 25 % (w/v) trichloroacetic acid (TCA) for precipitation of the

cellular protein. The mixture was centrifuged at 13,000 rpm for 2 minutes in the

Eppendorf micro-centrifuge. 200 jil of protein-free supernatant was added into 1000 |al

of 0.1 M phosphorous buffer. Upon the addition of 50 of freshly prepared 3 mM 5'5-

dithiobis-2-nitrobenzoic acid (DTNB) (Sigma, USA) in 0.1 M phosphate buffer, the

reaction started and was observed by the color change. The mixture was allowed 5

minutes for reaction to complete. Optical density (OD) of each pancreatic sample was

taken at 412 nm and corrected with its protein content. Known concentrations of DTNB

were used in preparing the standard curve and pancreatic glutathione levels were

expressed as nmol per mg protein.

2.4.4 Assessment of protein oxidation

Spectrophotometric quantification of protein-bound carbonyls was used to assess the

degree of oxidative protein modification in tissue injury. The assessment was employed

according to Reznick and Packer [1994]. Briefly, 500 jul of 10 % pancreatic

homogenate was incubated with 500 )al of 2 % (w/v) streptomycin solution for 15

minutes at room temperature in order to remove the nucleic acids in samples. The

38

mixture was centrifuged at 6,000 x g for 15 minutes. 400 |il of the nucleic acid-free

supernatant was treated with 400 [d of 20 mM 2,4-dinitrophenylhydrazine (DNPH)

(Fluka, Switzerland) in 2 M hydrochloric acid (HCl) for 1 hour at room temperature.

Another 400 il of supernatant was treated with 2 M HCl only to serve as a blank, which

was used to determine the protein recovery. The reaction was terminated by the adding

of 400 111 of 20 o/o (w/v) TCA. The mixture was subjected to a centrifUgation at 6,000 x

g for 2 minutes. The pellets were then rinsed with 800 il of ethanol — ethylacetate (1:1)

for 3 times and once with 800 of 10 % (w/v) TCA. The protein pellets were

dissolved in 750 of 6 M guanidine hydrochloride and the absorbance was read at 370

nm with a micro-plate reader. The absorbance of the blank was subtracted from the

obtained DNPH-treated reading in order for calculating the contents of protein carbonyl.

2.4.5 Assessment of lipid peroxidation

Lipid peroxidation in pancreatic tissue in terms of quantification of its degradation

product, MDA, was measured following the method described by Kruse et al [2001].

Briefly, 250 [i\ of 10 % (w/v) pancreatic homogenate was incubated with 250 ILII of 1.22

M phosphoric acid and 500 |LI1 of 0.5 % (w/v) thiobarbituric acid (TBA) (Sigma, USA)

solution in a 9 5 � C water bath for 30 minutes. 400 |il of the TBA-treated sample was

mixed with 720 of methanol and 80 of 1 M sodium hydroxide (NaOH) followed

by a centrifugation at 13,000 rpm for 2 minutes in a micro-centrifuge. 20 |li1 supernatant

was loaded into the jaBondapak C-18 column of the high-performance liquid

chromatography (HPLC) autosampler (Hewelett Packard, USA) and eluted in a mobile

phase, the MP solution, which was prepared with HPLC-grade methanol — 25 niM

phosphate buffer (1:1) and was adjusted to pH 6.5. The separation of the loaded sample

was monitored using the photodiode array detector and the eluted peak was observed at

39

532 nm. The integral area of the eluted peak calibrated with the protein concentration in

pancreatic tissue was used to calculate the amount of the produced MDA content. A

standard curve was prepared with known concentrations of MDA standards. The

amount of pancreatic MDA was expressed as pmol per mg protein.

2.4.6 Measurement of NADPH oxidase activity

Reactive oxygen species generation including the superoxide anion production was

measured in terms of NADPH oxidase activity with a chemiluminescent-based method

as demonstrated by Zhang et al. [1999]. The 10 % (w/v) pancreatic homogenate was

centrifuged at 1000 x g for 2 minutes at 4 to remove all cell debris and fat. 20 |LI1 of

supernatant was diluted with 800 of PBS. 100 of 1 mM reduced form NADPH

(Oriental Yeast Co. Ltd., Japan) was added as a substrate while 100 of 2.5 mM bis-TV-

methylacridinium nitrate (lucigenin) (Sigma, USA) was added as a chemiluminescent

agent. Upon the addition of substrate and lucigenin, the release of photons started. The

rate of light release was recorded and integrated with the TD-20/20 luminometer

(Promega, USA) over a period of 5 minutes. The activity of NADPH oxidase was

expressed as relative light unit (RLU) per second per mg protein.

2.5 Studies of pancreatic digestive enzyme secretions from isolated acini

2.5.1 Dissociation of acini from pancreatic tissue

The dissociation of functional intact acini from pancreatic tissue using collagenase and

hyaluronidase digestions with mild shearing forces has been demonstrated by

Amsterdam and Jamieson [1972] and Williams et al [1977]. Pancreatic tissue was

harvested, rinsed with PBS and trimmed from fat. The pancreas parenchyma was

inflated with 5 ml dissociation medium using a 27 G needle in order to distend the

40

tissue. The dissociation medium (Table 2-3) was made with Kerbs-Henselit

Bicarbonate (KHB)-Minimal Eagles Medium (MEM) (1:1), 60 U/ml purified

collagenase (Sigma, USA), 1.8 mg/ml hyaluronidase (Sigma, USA), 0.05 U/ml a -

chymotrypsin (Sigma, USA), 0.2 mg/ml soybean trypsin inhibitor (Sigma, USA), 100

U/ml penicillin and 0.1 mg/ml streptomycin (GIBCO, USA). The inflated tissue was

dissected and transferred into a vial containing another 5 ml fresh dissociation medium.

The vial was then placed in a 37 water bath with vigorous shaking for 20 minutes.

Pancreatic acini were dispersed to a greater extent by gently pipetting up and down, and

next filtered through a 150-|Lim mesh nylon screen (Sigma, USA). Acini were rinsed

with 10 ml KHB-MEM (1:1) buffer containing 1 % (w/v) fraction V BSA and purified

by centrifugation at 1000 rpm at 4 °C for 3 minutes. The acinar pellet was rinsed 3

more times, each with 10 ml KHB containing 4 % (w/v) BSA. After rinsing, the pellet

was re-suspended in 20 ml of incubation medium (Table 2-3). The incubation medium

is similar to KHB but containing 10 mM -hydroxyethyl) piperazine-A/"-(2-

ethanesulfonic acid) (HEPES) and without bicarbonate addition. Its pH was adjusted to

the value of 7.35. In the incubation medium, 0.3 mM phenylmethylsufonyl floride

(PMSF) and 2 mM ethylenediamine-tetraacetic acid (EDTA) were added for the

inhibition of pancreatic proteases. In the acute pancreatitis-induced model, edematous

pancreas was harvested. The acute pancreatitis-induced pancreas was also handled with

the same dissociation process except supplied with 4-fold enzymatic contents and

extensive shearing forces.

41

Dissociation medium (10ml) Reagents: Amount required KHB ^ MEM 5 ml Collagenase 600 U Hyaluronidase 18 mg a-chymotrysin 0.5 U Soyabean trypsin inhibitor 2 mg Penicillin 1000 U Streptomycin 1 mg

Incubation medium (10ml) Reagents: Amount required KHB (without HCO3) 20 ml HEPES 10 mM PMSF 0.3 mM EDTA 2 mM

Table 2-3 Summary of the reagents in dissociation and incubation media

2.5.2 Treatment with peptides and RAS inhibitors

The suspension of acini was randomly divided into 4 main groups, CCK-treated, Ang

Il-treated, Ang II +RAS inhibitor-treated and RAS inhibitor placebo groups. 3 ml of the

acini suspension was transferred to each incubation vial for enzymatic secretion analysis.

At the beginning of the 30-minute incubation period, 0.5 ml acinar suspension was

transferred out from each vial and subjected to a centrifugation at 5000 rpm at 4 °C for

2 minutes. In the CCK-treated and Ang Il-treated groups, 25 jul of the CCK or Ang II at

different final concentrations (0.1 nM to 100 nM) was added to the incubation vials

each containing 2.5 ml cell suspension. The suspension samples were incubated in a 37

� C water bath with vigorous shaking for 30 minutes. At the end of the incubation,

another 0.5 ml acinar suspension was taken from each vial, and was also subjected to

centrifugation. The supernatant was saved for analysis of a-amylase and lipase

secretion.

42

For the Ang II + RAS inhibitor-treated and RAS inhibitor placebo groups, the 3

ml acini suspension in each vial was pre-incubated with 30 |LI1 of 1 |iM RAS inhibitors

(losartan or PD 123319) for 15 minutes at room temperature. After the pre-incubation

period, the acini suspension was handled with same procedures as the Ang Il-treated

group received. However, the final concentration of Ang II was fixed at lOOnM in Ang

II + RAS inhibitor-treated group while same volume of PBS was given to the inhibitor

placebo group.

For the acini from acute pancreatitis-induced models, treatments with CCK,

Angll or RAS inhibitors were given exactly in same conditions as given to the non-

induced acini.

2.5.3 Quantification of protein and DNA contents

Prior to the centrifugation of the 0.5 ml acini suspension transferred out from the

incubation vial, 4 il of the cell suspension was mixed with 200 |al of protein reagent

dye in a micro-plate in order to determine the protein content in each suspension vial.

Absorbance at 595 nm from the micro-plate reader was calibrated with the BSA

standard curve in calculating the protein concentration. The protein quantification assay

was done with triplicate sets of measurements.

After the centrifugation of the acini suspension, pellets were saved for the acinar

DNA extraction while the supematants were removed for enzymatic assays. 1 ml of

DNAzol reagent (Invitrogen, USA) was added to each acinar pellet and the acini were

lysed by gentle pipetting. To each sample, 500 |al of 100 % ethanol was added to

precipitate the DNA. After centrifugation at 4000 x g for 2 minutes at 4 DNA

pellets were obtained. The pellets were washed twice with 800 il of 75 % ethanol and

were dissolved in 200 |LI1 of 8 mM sodium hydroxide. OD taken at 260 nm with the

43

micro-plate reader was used to calculate the DNA concentration. One A260 unit equals

50 i g of double-stranded DNA per ml.

2.5.4 Measurement of a-amylase and lipase secretions

The secretion of a-amylase or lipase in the incubated samples was expressed as the rate

of the enzymatic release during the incubation. The changes in absorbance throughout

the 30-minute incubation were used to calculate the rate of release, i.e. reading taken at

time 0 subtracted from the reading taken at time 30. The reading taken at time 0 was

considered as the basal value of enzyme secretion. The assay procedures were

mentioned in sections 2.2.2 and 2.2.3 for a-amylase and lipase respectively, except

plasma samples were replaced with acinar cell suspension supernatant.

2.5.5 RT-PCR analysis of RAS components in acinar cells

Experimental procedures with appropriate optimization for semi-quantitative RT-PCR

of pancreatic RAS components have been reported previously [Chan et al 2000]. The

mRNA expression of RAS components including angiotensinogen, ATi receptor and

AT2 receptor was assessed using RT-PCR standardized by co-amplification with the

house-keeping gene P-actin, which served as the internal control. Briefly, total RNA

was extracted from isolated pancreatic acinar cells, induced with or without AP, using

Trizol reagent (invitrogen, USA) by following the manufacturer's instruction.

Concentration and purity of RNA sample was determined from the OD measured with

micro-plate reader at 260 nm. 5 |Lig of the total RNA was reverse-transcribed into

CDNA according to the manufacturer's instruction (SuperScript II, GIBCO-BRL, USA)

using oligo dT as a primer. In the reverse transcription process, the mixture was

incubated at 42 °C for 50 minutes and the reaction was inactivated by heating at 72 °C

44

for 15 minutes. The cDNA was subjected to amplification by the polymerase-chain

reaction with rat-specific primers for angiotensinogen, ATia receptor, AT2 receptor and

p-actin. Each set of PGR mixture contained 0.8 |LI1 of cDNA template, 18 |LI1 of PGR

buffer, 1 [x\ of primer mix (sense and anti-sense) and 0.2 id of Taq polymerase, and the

reaction was carried out using a thermal cycler. The target sequences were amplified

with 28 repeated denaturation cycles at 94 for 1 minute, annealing at 62 for 90

seconds and elongating at 7 2 � C for 90 seconds. The PGR products were separated on 2

0/0 agarose gels containing 0.5 \ig/m\ ethidium bromide and visualized by UV

transillumination. The band sizes of the PGR products were determined using a size

ladder as reference. The intensities of the bands were measured using the molecular

imaging system (FluroChem, USA) and normalized to that of the P-actin internal

control gene.

2.6 Studies of RAS inhibitors on acute pancreatitis-induced systemic

inflammation

Measurement of myeloperoxidase (MPO) activity is a frequently used method for

assessing the neutrophil infiltration in tissues, such as the lung and liver [Haqqani et al

1999]. On the other hand, histopathology would be of assistance in the determination of

the tissue injury.

2.6.1 Systemic inflammation treatment

SD rats were randomly assigned into control, acute pancreatitis, RAS inhibitor placebo

(placebo) and acute pancreatitis + RAS inhibitor (AP+Inh) groups. Animals were

injected with cerulein for induction of acute pancreatitis-associated systemic

inflammation. A time course experiment was performed in addition to the designated

45

two-hour period. Cerulein, at 50 |ig/kg/hr, was given to animals in hourly intervals with

maximal number of doses up to four and the animals were sacrificed at hour 4, 6, 8 and

24. For the examination of the systemic inflammation induced by acute pancreatitis, the

8-hour experimental period was selected. In the 8-hour experimental period, animals in

acute pancreatitis and AP+Inh groups were given 4 i.p. cerulein injections in hourly

intervals for the first 4 hours and were sacrificed 4 hours thereafter. In control and

placebo groups, rats received 0.9 % NaCl saline instead of cerulein in same volume and

at same time intervals. For prophylactic treatment, a single dose of RAS inhibitor

losartan was intravenously given to placebo and AP+Inh groups 30 minutes prior to the

first cerulein. For continuous treatment, the placebo and AP+Inh groups received 2

hourly i.v. injections of losartan 30 minutes after the first injection of cerulein in

addition to the prophylactic dose. Losartan was given at dosage of 200 |Lig/kg. The

control and acute pancreatitis groups received isovolumetric i.v. injections of 0.9 %

NaCl saline instead of losartan.

2.6.2 Measurement of myeloperoxidase activity in lung and liver

Neutrophil sequestration in tissues was assessed by measuring the myeloperoxidase

(MPO) activity [Bhatia et al 2000]. Lung and liver tissues were removed, blotted dry

and stored in -70 °C. After thawing, 10 % (w/v) tissue homogenate was prepared in

ice-cold 20 mM phosphate buffer (pH 7.4) using a polytron homogenizer (Kinematica,

Switzerland). The pellet was obtained after centrifugation at 10,000 x g for 10 minutes

at 4 � C and was resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5 %

hexadecyltrimethylammonium bromide (Fluka, Switzerland). The suspension was

sonicated for 40 seconds with a sonicator probe, and centrifuged at 10,000 x g for

another 5 minutes at 4 °C. 20 |LI1 of supernatant was incubated with the freshly-prepared

46

reaction mixture for 110 seconds at 3 7 � C . The reaction mixture was prepared with

both 1.6 mM tetramethylbenzidine (TMB) (Farco Chemical Supply, USA) and 0.3 mM

hydrogen peroxide dissolved in 80 mM phosphate buffer (pH 5.4). The 110-second

reaction was terminated by the addition of 50 \i\ of 0.18 M sulphuric acid (H2SO4).

Absorbance at 450mn was taken for calculating the tissue MPO activity (Afmai — Ainitiai).

2.7 Statistical analysis

For the measurement of hyperamylasmia, hyperlipasmia and parameters of oxidative

stress, results were analyzed with one-way analysis of variance (ANOVA) and the

Tukey's post hoc test in order to establish whether the differences among the control,

acute pancreatitis group and inhibitor treatment groups were statistically significant

(n=5). All values were expressed as mean 士 SEM. For the measurements of acinar

secretions of pancreatic amylase and lipase, results were also analyzed with one-way

ANOVA and Tukey's post hoc test for the multiple determinations from the Ang II-

treated and RAS inhibitor-treated groups (n=4). For RT-PCR analysis, all gel images

were analyzed and quantified by an UV illuminator (FluroChem 8000 Advanced

Fluorescence, Chemiluminescence and Visible Light Imaging, Alpha Innotech

Corporation, CA, USA). The mRNA expression of RAS components was normalized

to the image intensity of the internal control P-actin. Results were also expressed as the

mean 士 SEM. Statistical comparisons and differences between mean values of control

and acute pancreatitis groups were compared using unpaired Student's t-test. In all

instances, p values lower than 0.05 were considered to be statistically significant.

47

Chapter 3 Results

3.1 Time-course experiment of acute pancreatitis model

The effects of cerulein-induced acute pancreatitis in terms of the changes in

pancreatic tissue injury and oxidative status were examined with time-course

experiments. The minimum time required for a distinguishable and detectable onset

the cerulein-induced disease was determined to be 120 minutes in the present study.

Two major parameters for the characterization of pancreatic tissue injury and

oxidative stress, the plasma a-amylase activity and pancreatic GSH depletion

respectively, were presented in the next sections.

3.1.1 Effect of acute pancreatitis on tissue injury

Hyperamylasemia in blood is one of the major markers for monitoring acute

pancreatitis and its severity. After the induction of cerulein, the plasma level of a -

amylase started to rise at the earliest time-point minute 15 and elevated to the highest

at the last time-point minute 240 as studied. However, the designated experiment

period for acute pancreatitis model in the present study was set to 120 minutes

because it was the shortest time period to statistically achieve the significant

difference from the non-cemlein-treated control in terms of a-amylase activity after

the onset of AP (Fig. 3-1 A). Pancreatic injury was also significantly detected in

other parameters, such as plasma lipase activity and weight ratio of pancreas to body,

at minute 120 (data not shown).

3.1.2 Effect of acute pancreatitis on oxidative status

With respect to the effect of cerulein on changes in oxidative status, the GSH

depletion assay was performed for further confirmation that the time period chosen

48

was correct. The pancreatic GSH levels dropped dramatically as soon as at minute

15. Again, minute 120 was the first time-point found to achieve the significant

difference from the control in GSH depletion (Fig. 3-lB). Other markers for

oxidative stress including protein oxidation and activation of NAD(P)H oxidase were

also detected with significant difference at minute 120 (data not shown).

A.

� 3000-1 >

CO —P-

S) 2 0 0 0 - I

H _ _ _ _ Control 15 30 60 120 240

Time after the first cerulein injection (minutes)

B. 20-|

志 I 15- _

" • l i l l i iB Control 15 30 60 120 240

Time after the first cerulein injection (minutes)

Fig. 3-1 (A) Time-dependent effect of cerulein on plasma a-amylase activity in rats. (B) Time-dependent effect of cerulein on pancreatic GSH depletion in rats. Acute pancreatitis was induced by i.p. injections of cerulein at 50 |ig/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the control.

49

3.2 Evaluation of ramiprilat and saralasin on changes of acute pancreatitis-

induced pancreatic injury

The ACE inhibitor, ramiprilat, is known to block the ACE from converting Ang I to

Ang II; whereas the non-specific Ang II receptor antagonist, saralasin, is

acknowledged to block Ang II from binding to its cognate receptors including both

ATi and ATi subtypes.

3.2.1 Changes in tissue injury and histopathology

Release of digestive enzymes from the pancreas into the circulation is one of the

most commonly used indictors for pancreatic injury as observed in cemlein-induced

acute pancreatitis. In the present study, the plasma a-amylase activity in the

cemlein-induced group was found to be 3 folds higher than the non-cemlein-treated

control after the 2-hour induction of cerulein at 50 |ig/kg/hr. Prophylactic treatment

with the non-specific Ang II receptor, saralasin, provided significant suppression in

the aspect of plasma a-amylase activity in the animals with acute pancreatitis. The

application of saralasin in the range of 5 to 40 )ag/kg was found effective. The result

indicated an U-shaped dose response with respect to the protective effect starting

from 5 |ag/kg and the less than maximal effect began at 40 ^ig/kg. As regards, the

optimal dose of saralasin appeared to be 20 |ig/kg (Fig. 3-2). The same dosage-

response experiment was also applied to the ACE inhibitor ramiprilat. However, no

protective action of ramiprilat against the a-amylase elevation was found within the

range of 5 to 40 [ig/kg. In contrast, adverse effects appeared to occur at the doses

higher than 40 |jg/kg as implicated by the higher level of plasma a-amylase (Fig. 3-

3).

50

• Norvoeruleirhtreated _ Cenjiein-treated

� 2 0 0 0 n

o 1500 ^ ^ A *

liJ _ l . i l HJJJJJ 0 5 10 20 40

Dose of saralasin (log/kg) Fig. 3-2 Prophylactic dosage effect of saralasin on plasma a-amylase activity in rats. Acute pancreatitis was induced by i.p. injections of cemlein at doses of 50 |ag/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cemlein-treated control. A Significantly different from the cemlein-treated control.

wmi Norvcerulein-treated ^ Cemleirvtreated

^ 2000-, *

H j u j j 0 5 10 20 40

Dosage of ramiprilat (lag/kg) Fig. 3-3 Prophylactic dosage effect of ramiprilat on plasma a-amylase activity in rats. Acute pancreatitis was induced by i.p. injections of cemlein at doses of 50 l^g/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cemlein-treated control. A Significantly different from the cemlein-treated control.

51

Apart from hyperamylasemia, hyperlipasemia in blood is also a common

consequence observed in cerulein-induced acute pancreatitis. After the induction of

cerulein at 50 |ig/kg for 2 hours, the plasma lipase activity in acute pancreatitis group

was found to be 4 folds higher than the non-cerulein-treated control in the current

study. Prophylactic treatment with saralasin at 20 [ig/kg significantly reduced the

plasma lipase activity in the acute pancreatitis-induced animal; however, the

prophylactic treatment with ramiprilat did not inhibit the pancreatic lipase from

releasing into the circulation in the course of acute pancreatitis. In addition, the

hyperlipasemia was even more severe in animals pre-treated with ramiprilat at doses

higher than 40 |ag/kg (Fig. 3-4).

On the other hand, pancreatic edema formation is another primary marker for

cerulein-induced acute pancreatitis, and is commonly evaluated by the gain of water

content in pancreatic tissue. After the inductions of cerulein, the weight ratio of

pancreas to body in the acute pancreatitis model was drastically increased more than

3 folds when compared with the non-cemlein induced control in the current study.

That was probably due to the formation of pancreatic interstitial edema in the course

of acute pancreatitis. The prophylactic administration of saralasin at 40 |ag/kg was

demonstrated to reduce the pancreatic edema formation as reflected by the decrease

in weight ratio of pancreas to body. However, the treatment with ramiprilat did not

provide any protective effect in this aspect (Fig. 3-5).

52

Norhcemlein-treated 國 Cemlein-treated

300n A

r ^ l i i j j J Saline Saralasin-20 Saralasin-40 Ramiprilat-20 Ramiprilat-40

Fig. 3-4 Effects of saralasin and ramiprilat on plasma lipase activity in rats. Acute pancreatitis was induced by i.p. injections of cerulein at 50 ^ig/kg/hr. Animals were given a single i.v. injection of saralasin or ramiprilat at 20 ^ig/kg or 40 ^ig/kg 30 minutes prior to the cerulein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated saline control. A Significantly different from the cemlein-treated saline control.

mm Non-cemlein-treated 20-, ^ Cerulein-treated

I : U J U J Saline Saralasin-20 Saralasin-40 Ramiprilat-20 Ramiprilat-40

Fig. 3-5 Effects of saralasin and ramiprilat on pancreatic weight ratio. Acute pancreatitis was induced by i.p. injections of cerulein at 50 jig/kg/hr. Animals were given a single i.v. injection of saralasin of ramiprilat at 20 )ig/kg or 40 |ag/kg 30 minutes prior to the cerulein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated saline control. A Significantly different from the cemlein-treated control.

53

Under the histological examinations, the control pancreas was

morphologically intact (Fig. 3-6A) whereas the acute pancreatitis-induced pancreas

showed severe interstitial edema, cytoplasmic shrinkage and acinar necrosis (Fig, 3-

6B). No histological alteration was noted in pancreas treated with saralasin placebos

(20 and 40 |ag/kg) when compared to the control pancreas (Fig. 3-6C, 3-6D). The

focal expansion of the interlobular septae in pancreatitic pancreas was greatly

improved by the prophylactic treatment with saralasin at both 20 lag/kg and 40 |ig/kg

(Fig. 3-6E, 3-6F). In addition, the acute pancreatitis-induced cytoplasmic shrinkage

and acinar necrosis were also reduced by the pre-treatment with saralasin. The

positive histological influences of prophylactic treatment with saralasin were in close

agreement with the experimental results obtained in previous analyses of tissue

injury, i.e. reductions in release of pancreatic digestive enzymes and pancreatic water

content.

On the other hand, prophylactic treatments with ramiprilat at both 20 and 40

|ig/kg did not provide any improvement in the aspects of acute pancreatitis-induced

pancreatic edema formation and acinar cell damage. The pancreatitic pancreas pre-

treated with ramiprilat was observed with cytoplasmic swelling and acinar necrosis

in a more severe form (Fig. 3-7E, 3-7F). Slight tissue edema formation was found in

the pancreas pre-treated with ramiprilat placebos (Fig. 3-7C, 3-7D).

54

•It (A) (B) mm (C) (D)

mm (E) (F)

Fig. 3-6 Histological examinations (H&E staining) of rat pancreas on induction of acute pancreatitis with/without prophylactic treatment with saralasin. (A) Control pancreas, non-cemlein-treated; (B) acute pancreatitis-induced pancreas, cerulein-treated; (C) prophylactic treatment with saralasin (placebo) at 20 jiig/kg; (D) prophylactic treatment with saralasin (placebo) at 40 |Lig/kg; (E) acute pancreatitis-induced pancreas with saralasin treatment at 20 jag/kg; (F) acute pancreatitis-induced pancreas with saralasin treatment at 40 [ig/kg. (Magnification x 400)

55

mm (A) (B)

(C) (D)

(E) (F)

Fig. 3-7 Histological examinations (H&E staining) of rat pancreas on induction of acute pancreatitis with/without prophylactic treatment with ramiprilat. (A) Control pancreas, non-cerulein-treated; (B) acute pancreatitis-induced pancreas, cerulein-treated; (C) prophylactic treatment with ramiprilat (placebo) at 20 |ag/kg; (D) prophylactic treatment with ramiprilat (placebo) at 40 |Lig/kg; (E) acute pancreatitis-induced pancreas with ramiprilat treatment at 20 jiig/kg; (F) acute pancreatitis-induced pancreas with ramiprilat treatment at 40 jig/kg. (Magnification, x 400)

56

3.2.2 Changes in oxidative status

The Ang II receptor antagonist saralasin has been demonstrated in previous sections

to possess protective properties against the cemlein-induced pancreatic injury. Its

positive role in regulating oxidative stress would be lead to a great attention.

Glutathione depletion, protein oxidation, lipid peroxidation and activation of

NAD(P)H oxidase are the common markers for the detection of oxidative stress. In

the current study, glutathione in acute pancreatitis-induced pancreatic tissue was

depleted drastically by more than 50% when compared to the non-cerulein-treated

control. The prophylactic treatment with saralasin at 10 )ag/kg and 20 |Lig/kg

significantly suppressed the glutathione depletion in the cemlein-induced pancreas.

The treatment at such doses restored the GSH levels by approximately 40% (Fig. 3-

8).

H NorvoerJein-treatecl 20-1 • CenJeirvtredted

' i L M i i 0 5 10 20 40

Dose of saralasin (ju^kg) Fig. 3-8 Dosage effects of saralasin on GSH depletion in rat pancreas. Acute pancreatitis was induced by i.p. injections of cerulein at 50 )ig/kg/hr. Animals were given a single i.v. injection of saralasin at doses from 5 |ag/kg to 40 [ig/kg 30 minutes prior to the cerulein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cerulein-treated control.

57

Induction of cerulein could result in significant oxidative modification of

proteins. An intensive increase in the content of DNPH-reactive protein carbonyls

was obtained by approximately 50 % in the acute pancreatitis-induced pancreatic

tissue when compared to the non-cerulein-treated control as a result of protein

oxidation. Pre-treatment with saralasin from 5 |ag/kg to 40 idg/kg greatly reduced the

level of the oxidatively modified proteins in the cemlein-treated pancreatic tissues.

The protein carbonyl formation was obviously reduced even lower than the control

level when saralasin was applied at the dose of 20 |ag/kg (Fig. 3-9).

I H Non-cerulein-treated ^ ^ Cemlein-treated

1 i " 它 w

i i l i i l i 0 5 10 20 40

Dose of saralasin (^ig/kg)

Fig. 3-9 Dosage effects of saralasin on protein oxidation in rat pancreas. Acute pancreatitis was induced by i.p. injections of cerulein at 50 |ag/kg/hr. Animals were given a single i.v. injection of saralasin at doses from 5 |ag/kg to 40 |ag/kg 30 minutes prior to the cerulein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cemlein-treated control.

58

Apart from the oxidative modification of proteins, the cerulein induction

could also cause lipid peroxidation as a consequence of acute pancreatitis. MDA, a

thiobarbituric acid reactive substance, is the major product of lipid peroxidation. The

pancreatic MDA level was markedly elevated by about 34 % in the cemlein-induced

animals when compared to the non-cemlein-treated control. Prophylactic treatment

with saralasin at doses from 5 to 20 |Lig/kg significantly attenuated the lipid

peroxidation in acute pancreatitis-induced pancreatic tissue as reflected by the

decrease in pancreatic level of MDA. However, the same protective effect was not

observed at the higher dose of 40 [xg/kg (Fig. 3-10). That might be due to the

toxicity of the high dose usage.

The generation of superoxide anion radical catalysed by NAD(P)H oxidase is

also one of the major consequences of oxidative stress. The NAD(P)H oxidase

system was activated in the acute pancreaetitis-induced pancreas as its activity was

found to be enhanced by virtue of using the cheminluminscent assay. Treatment of

saralasin declined, to appreciable extent, the NADPH oxidase activation in cemlein-

induced pancreatic tissue; however, the inhibitory effect was not considered

statistically significant (Fig. 3-11). In the four parameters mentioned, the saralasin

placebos did not show significant alterations from the control pancreas.

59

• NxKHiien-tre^ed 國 Oeoieirvtreded

<£ *

mnii 0 5 10 20 40

Dose of saralasin (jugfkg)

Fig. 3-10 Dosage effects of saralasin on lipid peroxidation in rat pancreas. Acute pancreatitis was induced by i.p. injections of cemlein at 50 |ig/kg/hr. Animals were given a single i.v. injection of saralasin at doses from 5 |ag/kg to 40 ^ig/kg 30 minutes prior to the cemlein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cemlein-treated control. A Significantly different from the cemlein-treated control.

mm Norvcerulein-treated ^ Cemlein-treated

B J j J j J 0 5 10 20 40

Dose of saralasin (i ig/kg)

Fig. 3-11 Effects of saralasin on NADPH oxidase activity in rat pancreas. Acute pancreatitis was induced by i.p. injections of cemlein at 50 }ig/kg/hr. Animals were given a single i.v. injection of saralasin at doses from 5 |ag/kg to 40 |ag/kg 30 minutes prior to the cemlein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cemlein-treated control. A Significantly different from the cemlein-treated control.

60

3.3 Evaluation of losartan and PD123319 on changes of acute pancreatitis-

induced pancreatic injury

The administration of non-specific Ang II receptor antagonist saralasin was

demonstrated to be protective against the acute pancreatitis-induced pancreatic injury

and oxidative stress. It is therefore intriguing to define the differential effects of the

specific Ang II receptor inhibitions of ATi and AT2 on the changes in acute

pancreatitis-induced pancreatic injury, not only prophylactically but also

therapeutically.

3.3.1 Changes in tissue injury and histopathology

As a major indicator of pancreatic injury, pancreatic edema was assessed in terms of

the gain of water content in the pancreatic tissue in order for scoring the severity of

acute pancreatitis. Prophylactic administration of the specific ATi receptor

antagonist, losartan, reduced the formation of pancreatic edema in the cerulein-

induced pancreas at doses from 100 to 800 i^g/kg. The reduction was reflected by

the decrease in the weight ratio of pancreas to body. Pre-treatment with losartan at

400 and 800 ^ig/kg showed significant reductions in the pancreas to body weight

ratio (Fig. 3-12A); however, pre-treatment with PD123319, the selective AT2

receptor antagonist, at the same range of doses studied did not provide such effects

(Fig. 3-12B). In the therapeutic treatment groups, losartan and PD123319 at 400

j^g/kg exhibited similar results to the cerulein-induced animals as in the prophylactic

treatment groups (Fig. 3-13).

61

A.

1 ^ Non-cerulein-treated 1 ^ ^ C e m l e i n - t r e a t e d

l i o - i 1 i i � ：

丨 J J J M 0 50 100 200 400 800

Dose of Losartan (jag/kg)

B.

• • • Non-cerulein-treated 15-| ^ ^ Cerulein-treated

0 50 100 200 400 800 Dose of PD123319 (|ig/kg)

Fig. 3-12 Dosage effects of prophylactic treatments with RAS inhibitors on the weight ratio of pancreas to body. (A) Prophylactic treatment with losartan; (B) prophylactic treatment with PD123319. Acute pancreatitis was induced by i.p. injections of cerulein at 50 jig/kg/hr. Animals were given a single i.v. injection of RAS inhibitor from 50 |Lig/kg to 800 [ig/kg 30 minutes prior to the cerulein induction. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cerulein-treated control.

62

H H Non-cemlein-treated 15 ^ ^ Cerulein-treated

Saline Losartan PD123319

Fig. 3-13 Effects of therapeutic treatment with losartan and PD123319 on the weight ratio of pancreas to body. Acute pancreatitis was induced by i.p. injections of cerulein at 50 |ig/kg/hr. Animals were given a single i.v. injection of RAS inhibitor at 800 jig/kg 30 minutes after the onset of acute pancreatitis. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cemlein-treated control. A Significantly different from the cerulein-treated control.

The assessments of plasma a-amylase and lipase activities are the widely

established indicators for cemlein-induced pancreatic tissue injury. In the present

study, the activities of a-amylase and lipase in plasma were dramatically elevated

more than 3 folds and 4 folds respectively in the cerulein-treated rat model. Both the

prophylactic and therapeutic administration of losartan at 200 |Lig/kg greatly

suppressed the cemlein-induced pancreatic injury, as evidenced by the decreases in

plasma levels of a-amylase (Fig. 3-14) and lipase (Fig. 3-15). On the other hand,

neither the prophylactic nor the therapeutic treatment of PD 123319 at 200 lug/kg

provided protective effect against the acute pancreatitis-induced injury, as indicated

by the high levels of plasma a-amylase (Fig. 3-14) and lipase (Fig. 3-15).

63

A.

• • • Non-cerulein treated 2000 " zzzzA Cerulein-treated

Hill Saline Losartan PD123319

B.

Non-cerulein treated 2QQQ ^ ^ Cerulein-treated

丨遍 Saline Losartan PD123319

Fig. 3-14 Differential effects of losartan and PD123319 on the plasmas a-amylase activity in rats. (A) Prophylactic treatment, animals were given a single i.v. injection of RAS inhibitor at 200 ^ig/kg 30 minutes prior to the cerulein induction; (B) therapeutic treatment, animals were given a single i.v. injection of RAS inhibitor at 200 )dg/kg 30 minutes after the onset of acute pancreatitis. Acute pancreatitis was induced by i.p. injections of cerulein at 50 |Lig/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cerulein-treated control.

64

A.

^ • i Non-cerulein treated 一 300 - j ^ ^ Cemlein-treated

丨IbJ Saline Losartan PD123319

B.

warn Non-cerulein treated � 3 0 0 - 1 ^ ^ C e m l e i n - t r e a t e d

i i J i J Saline Losartan PD123319

Fig. 3-15 Differential effects of losartan and PD 123319 on the plasma lipase activity in rats. (A) Prophylactic treatment, animals were given a single i.v. injection of RAS inhibitor at 200 jag/kg 30 minutes prior to the cerulein induction; (B) therapeutic treatment, animals were given a single i.v. injection of RAS inhibitor at 200 |Lig/kg 30 minutes after the onset of acute pancreatitis. Acute pancreatitis was induced by i.p. injections of cerulein at 50 |ag/kg/hr. Values were expressed as mean 士 SEM, with n=5. * Significantly different from the non-cerulein-treated control. A Significantly different from the cemlein-treated control.

65

In addition to the biochemical analyses, the effects of cerulein on histological

alterations in pancreatic tissues were also examined in the present study. The control

pancreas retained morphologically intact (Fig. 3-16A) whereas the cerulein-induced

pancreatitic pancreas was detected with substantial damages including interstitial

edema, cytoplasmic shrinkage and acinar cell necrosis (Fig. 3-16B). In those acute

pancreatitis-induced pancreatic tissues, prophylactic (Fig. 3-16C) and therapeutic

(Fig. 3-16D) treatments with losartan at 200 |Lig/kg ameliorated the severity of acute

pancreatitis, as observed with less interstitial edema and acinar necrosis. In the

losartan placebos, no notable change in intact integrity of pancreatic morphology was

observed (data not shown).

(A) (B)

mMm9Mmm. ( C ) 孤 （D)

Fig. 3-16 Histological examinations (H&E staining) of rat pancreas on induction of acute pancreatitis with losartan treatment. (A) Control pancreas, non-cemlein-treated; (B) Acute pancreatitis-induced pancreas, cerulein-treated; (C) cerulein-induced pancreatitic pancreas with prophylactic treatment with losartan; (D) pancreatic pancreas with therapeutic treatment with losartan. (Magnification x 400)

66

Under the histological examination, the cerulein-induced pancreatitic tissues

with prophylactic (Fig. 3-17C) and therapeutic (Fig. 3-17D) treatments with

PD123319 at 200 jag/kg were shown with no improvement in the severity of acute

pancreatitis when compared to the cemlein-treated pancreas (Fig. 3-17B). The

treatments with PD123319 did not reduce the formation of interstitial edema, acinar

necrosis and cytoplasmic shrinkage in the course of acute pancreatitis. However, no

significant histological alteration in the intact integrity of pancreatic morphology was

observed in the PD123319 placebos (data not shown).

mm (A) (B)

(C) (D)

Fig. 3-17 Histological examinations (H&E staining) of rat pancreas on induction of acute pancreatitis with PD123319 treatment. (A) Control pancreas, non-cemlein-treated; (B) acute pancreatitis-induced pancreas, cemlein-treated; (C) cerulein-induced pancreatitic pancreas with prophylactic treatment with PD123319; (D) cerulein-induced pancreatitic pancreas with therapeutic treatment with PD 123319. (Magnification x 400)

67

3.3.2 Changes in oxidative status

Saralasin treatment has been demonstrated in previous sections to ameliorate

cerulein-induced oxidative stress. Nevertheless, saralasin could not suppress the

activation of NADPH oxidase to a significant level. The NADPH oxidase activity

was enhanced by 40 % in the cerulein-treated pancreatic tissue when compared to the

non-cemlein-treated control. Prophylactic treatment with the specific ATi receptor

antagonist, losartan, at 200 ^ig/kg could reduce the enhanced activity by nearly 30 %;

however, such a reduction was not observed in the prophylactic treatment with the

selective A T � receptor antagonist, PD123319 (Fig. 3-18A). Similar suppressive

results on NADPH oxidase activity were obtained in the therapeutic treatments with

losartan and PD123319 (Fig. 3-18B). For a further confirmation of the protective

actions of the specific ATi receptor antagonism, protein oxidation was assessed as

another indicator for monitoring the changes in oxidative status. The pancreatic level

of protein carbonyls was greatly elevated by 60 % in the cerulein-induced pancreatic

tissue as proteins were oxidatively modified in the course of acute pancreatitis.

Animal model pre-treated with losartan at 200 [ig/kg was found with a significant

reduction in the formation of pancreatic protein carbonyls whereas no positive effect

was provided by the pre-treatment of PD123319 (Fig. 3-19A). In the therapeutic

treatment groups, losartan also exhibited similar effective reduction against the

protein carbonyl formation; however, PD 123319 again provided no beneficial effect

(Fig. 3-19B).

68

A.

• • • Non-cerulein treated 30"! ^ ^ Cerulein-treated

Saline Losartan PD123319

B.

H H Non-cerulein treated 1 ^ ^ Cerulein-treated

"S O 25- *

t - _ 工 _

mMt Saline Losartan PD123319

Fig. 3-18 Differential effects of losartan and PD123319 on the NAD(P)H oxidase activity. The activity of NAD(P)H oxidase was expressed in relative light unit (RLU)/second/mg protein. (A) Prophylactic treatment; (B) therapeutic treatment. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cemlein-treated control. A Significantly different from the cerulein-treated saline control.

69

A.

• • • Non-cerulein-treated 5 ^ ^ Cerulein-treated

I f 4- ^ ^

_ _ i Saline Losartan PD123319

B.

w^m Non-cerulein-treated 5n ^ ^ Cerulein-treated

4 - " T

" l i M Saline Losartan PD123319

Fig. 3-19 Differential effects of losartan and PD123319 on the oxidative modification of protein. The protein oxidation was expressed in terms of nmol protein carbonyl formed/mg protein. (A) Prophylactic treatment; (B) therapeutic treatment. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cerulein-treated saline control.

70

3.4 Evaluation of acinar secretions of digestive enzymes

Activities of a-amylase and lipase in the freshly isolated pancreatic acini were

measured in order to investigate the roles of CCK-8 and Ang II in the regulation of

acinar digestive enzyme secretions. In addition, the effects of RAS inhibition on

acinar a-amylase secretion were also studied.

3.4.1 Cholecystokinin octapeptide-induced acinar secretions

In the present study, the secretory function of the freshly dispersed pancreatic acini

was primarily examined in terms of their responsiveness to exogenous secretagogue

CCK-8, which is the well-established hormone for stimulating exocrine secretion.

The isolated pancreatic acini were found highly functional as implicated by the

extensive secretions of a-amylase and lipase after a 30-minute incubation with CCK-

8. The CCK-stimulated release of a-amylase (Fig. 3-20) and lipase (Fig. 3-21) from

pancreatic acini was exhibited in a concentration-dependent manner. The secreted

enzyme activities started to level off at 10 nM of CCK-8.

3.4.2 Angiotensin Il-induced acinar secretions

Similar to the CCK-8 stimulation, exogenous Ang II was also demonstrated to

stimulate acinar secretion of digestive enzymes in the current study. The acinar

secretion of a-amylase evoked by Ang II was also found to be dose-dependent, i.e.

from 0.1 to 100 nM (Fig. 3-22). However, the stimulatory potency of Ang II was not

as high as CCK-8. For example, at the half-maximal dose of Ang II (10 nM), the

acinar release of a-amylase was stimulated to only one forth of the effect of CCK-8.

Apart from a-amylase, the release of lipase from pancreatic acini was also stimulated

by all doses of Ang II studied. Nevertheless, the stimulation of lipase at low

71

concentrations of Ang II, 0.1 and 1 nM, were not statistically different from the basal

secretion (Fig. 3-23).

secreted a-amylase activity

i l l ! 0.0 0.1 1.0 10.0 100.0

[CCK-8] (nM)

Fig. 3-20 Dosage influence of CCK-8 on a-amylase secretion from isolated pancreatic acini. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the non-CCK-8-treated control (basal).

secreted lipase activity

2 0 0 0 - 1

丨 i l l 0.0 0.1 1.0 10.0 100.0

[CCK-8] (nM)

Fig. 3-21 Dosage influence of CCK-8 on lipase secretion from isolated pancreatic acini. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the non-CCK-8-treated control (basal).

72

secreted a-amylase activity

1500-1

*

• I • * 麵動

0.0 0.1 1.0 10.0 100.0

[Angll] (nM)

Fig. 3-22 Dosage influence of Ang II on a-amylase secretion from isolated pancreatic acini. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the non-Ang Il-treated control (basal).

secreted lipase activity

750-1

0.0 0.1 1.0 10.0 100.0

[Angll] (nM)

Fig. 3-23 Dosage influence of Ang II on lipase secretion from isolated pancreatic acini. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the non-CCK-8-treated control (basal).

73

3.4.3 Effects of losartan and PD123319 on a-amylase secretion

Exogenous dose of Ang II at 100 nM was demonstrated to significantly stimulate the

release of a-amylase from isolated pancreatic acini as mentioned in previous sections.

In the current study, pre-incubation with the specific ATi receptor antagonist,

losartan, (1 |LIM) diminished the Ang IL-evoked release of a-amylase from pancreatic

acini almost back to the basal level. On the other hand, the pre-incubation with

losartan alone at same dosage did not alter the basal release of a-amylase from

isolated acinar cells (Fig. 3-24).

secreted a-amylase activity

1500".

� J _ _ _ _ CON Angll Los Los+Angll

Fig. 3-24 Effect of losartan on Ang Il-stimulated a-amylase secretion from isolated pancreatic acini. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the control (basal). A Significantly different from the Ang Il-treated control.

74

Dissimilar to the ATi receptor antagonism, the pre-incubation with the

selective ATi receptor antagonist, PD 123319, (1 |aM) did not provide any inhibitory

effect against the Ang Il-evoked secretion of a-amylase from the isolated pancreatic

acinar cells. Nevertheless, the PD123319 placebo also did not exhibit any executive

effect on the basal acinar secretion of a-amylase (Fig. 3-25).

secreted a-amylase activity

1500-1

i L l CON Angll PD PD+Angll

Fig. 3-25 Effect of PD123319 on Ang Il-evoked a-amylase secretion from isolated pancreatic acini. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the control (basal).

3.5 Existence and regulation of acinar RAS by acute pancreatitis

The evidence for the presence of RAS components in pancreatic acini and their

regulations by acute pancreatitis were examined in this study. These were achieved

by means of semi-quantitative RT-PCR.

75

3.5.1 Expression of angiotensinogen and its regulation by acute pancreatitis in

acini

RT-PCR analysis showed that mRNA of angiotensinogen, the mandatory component

for a local RAS, was expressed in pancreatic acini. The amplified PGR product was

312 base pairs (bp) in size as expected (Fig. 3-26A). There was a significant effect

of acute pancreatitis on the expression of angiotensinogen mRNA. Acute

pancreatitis elicited a significant increase in the mRNA expression for

angiotensinogen when compared with its internal control, P-actin. The relative

change of expression was about 1.5-fold, as demonstrated by the RT-PCR and image

analyses (Fig. 3-26B).

3.5.2 Expression of ATi receptor and its regulation by acute pancreatitis in

acini

The mRNA expression of ATi receptor was noted in the freshly isolated pancreatic

acinar cells. The isoform ATia was the dominant type and its PGR product was 385

bp in size as expected (Fig. 3-27A). The mRNA expression of ATia receptor was

demonstrated with an up-regulation in acute pancreatitis when compared with its

internal control. The RT-PCR and image analyses showed that the relative

expression of mRNAs for ATi receptor to P-actin in acute pancreatitis was increased

by approximately 1.8-fold (Fig. 3-27B).

3.5.3 Expression of AT! receptor and its regulation by acute pancreatitis in

acini

The expression of AT! receptor mRNA was also detected in the freshly isolated

pancreatic acini by RT-PCR. The expected size of the amplified PCR product of

76

ATi receptor was 511 bp (Fig. 3-28A). The relative expression of mRNA for AT2

receptor was elevated by acute pancreatitis nearly 1.9-fold when compared with its

control P-actin, as demonstrated by the image analysis (Fig. 3-28B).

A.

312 bp ->

M 1 2 3 4

B. 0 . 2 0 - 1

I 丁

c C .2 •云 0.15-

s ？ 0) CO. X <D 0 5) > -i r ^ 1 I 0.10-O： O

O) c <

O-OS-I Control AP

Fig. 3-26 (A) RT-PCR analysis of mRNA expression for angiotensinogen in pancreatic acini. Lane M, DNA marker; lane 1, angiotensinogen expression in normal acinar cells; lane 2, angiotensinogen expression in acute pancreatitis-induced acini; lane 3, B-actin expression in control; lane 4, B-actin expression in acute pancreatitis-induced acini. The arrows indicate the expected size of the amplified products — angiotensinogen (312bp) and B-actin (240bp). (B) The relative expression was shown as angiotensinogen/B-actin mRNA in normal and acute pancreatitis-induced pancreatic acini. Data were expressed as mean 土 SEM, (n=3/group). * Significant different from control.

77

A.

385 bp—

240 b p —

M 1 2 3 4

B. *

0 . 8 -<

l l ^ O c o <p 0.6-& 营 (D O O笠 •| 8 芸 T Q^ ， 0 . 4 -

！5

0.2 J C o n t r o l A P

Fig. 3-27 (A) RT-PCR analysis of mRNA expression for ATia receptor in pancreatic acini. Lane M, DNA marker; lane 1, ATia expression in normal acinar cells; lane 2, ATia expression in acute pancreatitis-induced acinar cells; lane 3, B-actin expression in control; lane 4, B-actin expression in acute pancreatitis-induced acini. The arrows indicate the expected size of amplified products 一 ATia receptor (385bp) and B-actin (240bpX (B) The relative expression was shown as ATia/B-actin mRNA in normal and acute pancreatitis-induced pancreatic acini. Data were expressed as mean 土 SEM, (n=3/group). * Significant different from control.

78

A.

511 bp

240 bp ^ ^ ^ B I ^ H I ^ H ^ I ^ Z ^ S ^ Z Z H

M 1 2 3 4

B. -k

0 . 8 -

< Z 0 a： c £ .2 c (A •云 8 <p 0.6-

t t 1 I 1 O — r -<D L 1

CN 0 . 4 -<

C o n t r o l A P

Fig. 3-28 (A) RT-PCR analysis of mRNA expression for AT! receptor in pancreatic acini. Lane M, DNA marker; lane 1, AT2 expression in normal acinar cells; lane 2, AT2 expression in acute pancreatitis-induced acinar cells; lane 3, B-actin expression in control; lane 4, B-actin expression in acute pancreatitis-induced acini. The arrows indicate the expected size of amplified products — AT2 receptor (51 Ibp) and B-actin (240bp). (B) The relative expression was shown as AT2/B-actin mRNA in normal and acute pancreatitis-induced pancreatic acini. Data were expressed as mean 土 SEM, (n=3/group). * Significant different from control.

79

3.5.4 Evaluation of RAS inhibitors in acute pancreatitis-induced acinar cells

In the freshly isolated pancreati acini, the stimulatory role of Ang II has been shown

in the previous sections. In this study, exogenous Ang II was also demonstrated to

stimulate a-amylase secretion from the acute pancreatitis-induced pancreatic acini.

The stimulated release of a-amylase from acute pancreatitis-induced acini was more

than 2-fold higher than that from the normal acinar cells. The acinar release of a -

amylase was normalized with the protein content in the cells. Pre-incubation with

the specific ATi receptor antagonist, losartan, significantly reduced the Ang Il-

evoked release of a-amylase from acinar cells. However, the AT! receptor

antagonist, PD123319, did not exhibit any effect on the acinar release of a-amylase

(Fig. 3-29). Both losartan and PD123319 were applied at dose of 1 ^M as previously

tested in the normal acinar cells. In addition, the release of a-amylase was also

normalized with the DNA content in acinar cells and was expressed as units per [ig

DNA (Fig. 3-30).

3000-1

n^i l i i CON Angll AP+Angll A^+Aigll fiP+AngW

+Los +PD Fig. 3-29 Effects of losartan and PD 123319 on a-amylase secretion from acute pancreatitis-induced pancreatic acini. Los: losartan; PD: PD123319; AP: acute pancreatitis. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the control (basal). A Significantly different from the AP+Ang II group (non-RAS inhibitor-treated control).

80

7500-|

I

H - i l i i CON Angll AP+Angll PP+AngW AP+Aigll

+Los +PD

Fig. 3-30 Effects of losartan and PD123319 on a-amylase secretion from acute pancreatitis-induced pancreatic acini. The units of activity were expressed per jig DNA. Los: losartan; PD: PD123319; AP: acute pancreatitis. Values were expressed as mean 士 SEM, (n=4/group). * Significantly different from the control (basal). A Significantly different from the AP+Ang II group (non-RAS inhibitor-treated control).

3.6 Preliminary data on acute pancreatitis-induced systemic inflammation

3.6.1 Time-course experiment on lung injury

The plasma hyperamylasemia is a common indicator for acute pancreatitis. A time-

course experiment up to 24 hours was performed to investigate the AP-associated

injury in tissues other than the pancreas. Acute pancreatitis was induced by 4 i.p.

injections of cerulein at 50 )ag/kg/hr; the plasma a-amylase activity was elevated to

the highest level at hour 8 and almost returned to basal level at hour 24 (Fig. 3-31).

On the other hand, the MPO activity, a measure of neutrophil infiltration, in

pulmonary tissues was also increased time-dependently with the highest level found

at hour 8. The MPO activity at hour 8 was about 3 folds higher than the control (Fig.

3-32).

81

7500-1

Q) *

l i s o o o - * . 4

i i ^ o o . j i i

Control 2 4 6 8 24 Time after the first cerulein injection

(hours)

Fig. 3-31 Time-dependent effect of cerulein on a-amylase activity in rats. Acute pancreatitis was induced by i.p. injections of cerulein at 50 jag/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the control.

i i l l i l i Control 2 4 6 8 24

Time after the first cerulein injection (hours)

Fig. 3-32 Time-dependent effect of cerulein on lung MPO activity in rats. Acute pancreatitis was induced by i.p. injections of cerulein at 50 |ag/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the control.

82

Besides the measurements of MPO activity, the acute pancreatitis-associated

lung injury was further confirmed by the histological alterations in lung sections.

The control lung retained morphologically intact aveoli (Fig. 3-33A). However, the

acute pancreatitis-induced lung consisting of pulmonary inflammation, such as

edema, alveolar thickening and infiltration of inflammatory cells, was observed as

early as from hour 2 (Fig. 3-33B). The severity of pulmonary injury was time-

dependently increased up to hour 8 (Fig. 3-33C, D, E). The pulmonary injury was

histologically improved greatly at hour 24 (Fig. 3-33F).

3.6.2 Time-course experiment on liver injury

In addition to the injury in lung, acute pancreatitis could cause injury in liver as well.

A time-course experiment up to 24 hours was also performed to investigate the acute

pancreatitis-associated liver injury. The liver MPO activity was also found to elevate

significantly. However, the severity of acute pancreatitis-associated tissue injury in

liver was not as serious as found in the lung. The liver was only influenced shortly

after the onset of acute pancreatitis. The highest level of MPO activity in liver was

found at hour 2. The activity was almost back to the normal level from hour 4

onwards (Fig. 3-34).

83

U S (A) (B)

mm (C) (D)

mm (E) (F)

Fig. 3-33 Histological examinations (H&E staining) of rat lung on induction of AP. (A) Control lung, non-cemlein-treated; (B) lung harvested after 2 hours of acute pancreatitis; (C) lung harvested after 4 hours of acute pancreatitis; (D) lung harvested after 6 hours of acute pancreatitis; (E) lung harvested after 8 hours of acute pancreatitis; (F) lung harvested after 24 hours of acute pancreatitis. (Magnification x 400)

84

4- |

’H_i___« Control 2 4 6 8 24

Time after the first cerulein injection (hours)

Fig. 3-34 Time-dependent effect of cerulein on liver MPO activity in rats. Acute pancreatitis was induced by i.p. injections of cerulein at 50 j^g/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the control.

3.63 Evaluation of losartan on systemic inflammation

As lung injury was significantly detected at hour 8 in the time-course experiment, the

designated induction period for acute pancreatitis-associated injury in lung was set to

8 hours by giving 4 i.p. injections of cerulein. Protective effect against acute

pancreatitis-associated lung injury was found when 3 continuous hourly injections of

the selective ATi receptor antagonist, losartan, at 200 |ag/kg were given to the acute

pancreatitis-induced animals. The treatment with losartan significantly suppressed

the plasma a-amylase activity by approximately 30 % when compared to the

cemlein-treated control (Fig. 3-35). The decrease in pulmonary MPO activity by the

administration of losartan was in agreement with the measurement of

hyperamylasemia (Fig. 3-36).

85

I H Non-cerulein-treated 7 5 0 0 ， 國 Ceruleirhtreated

h J j Saline Losartan

Fig. 3-35 Effect of losartan on plasma a-amylase activity in rats. Acute pancreatitis was induced with 4 i.p. injections of cemlein at 50 )Lig/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cemlein-treated saline control.

• • Non-cerulein-treated L 4-| ^ Cemlein-treated

!i I . • I i IJL

Saline Losartan Fig. 3-36 Effect of losartan on pulmonary MPO activity in rats. Acute pancreatitis was induced with 4 i.p. injections of cemlein at 50 |Lig/kg/hr. Values were expressed as mean 士 SEM, (n=5/group). * Significantly different from the non-cerulein-treated control. A Significantly different from the cemlein-treated saline control.

86

Chapter 4 Discussion

4.1 Actions of RAS inhibitors on the changes of tissue injury, oxidative

status and histopathology in acute pancreatitis-induced pancreas

Acute pancreatitis is an inflammatory disease that arises from the premature

activation of acinar proteolytic zymogens in the pancreas. The inappropriate

activation of digestive enzymes consequently causes acinar cell damage and finally

leads to autodigestion of the gland. Since the pathogenesis of acute pancreatitis is

complicated and undefined, the involvement of pancreatic digestive enzymes is just

partly accountable to this disease. In fact, other important mechanisms such as the

release of various inflammatory mediators [Osman and Jensen 1999], activation of

phagocytes [Beger et al 2000] as well as the activation of the RAS and other

vasoactive peptide systems could be involved. The RAS in the pancreas has been

documented for its existence in a wide range of species including canine, rat, mouse

and human [Leung and Chappell 2003]. In rats, the pancreatic RAS has been

subjected to an up-regulation in an experimental model of cerulein-induced acute

pancreatitis [Leung et al. 2000; Ip et al 2003]. Such an activation of the pancreatic

RAS is believed to play an important role in the pathogenesis of acute pancreatitis.

A body of evidence has been substantiated that Ang II may mediate inflammation by

stimulating the synthesis of cytokines, chemokines and other inflammatory factors

such as phospholipase A � and TGF-pl [Brady et al 1999; Saluja and Steer 1999].

The chain of events would be initiated subsequently, via the participation of the RAS,

thereby resulting in the development of acute pancreatitis. To this end, the

application of RAS inhibitors would be beneficial to the changes in the severity of

pancreatic injury and the oxidative stress, as induced by acute pancreatitis.

87

4.1.1 Differential effects of ramiprilat and saralasin

ACE inhibitor and Ang II receptor antagonist are commonly used as anti-

hypertensive drugs; besides, they are also applied to treat acute myocardiac

infarction and diabetic nephropathy [Drexler et al 1987]. Most of their actions are

dedicated to the inhibition of the RAS. In this regards, the ACE inhibitor, ramiprilat,

exerts its effect at the mid-point of the RAS cascade by blocking the ACE from the

conversion of Ang I to Ang II; whereas the Ang II receptor antagonist, saralasin,

exhibits its effect at rather down-stream of the RAS pathway by blocking Ang II

from binding to its cognate receptors.

The pancreatic injury caused by acute pancreatitis is commonly characterized

by the release of digestive enzymes from the pancreas into the circulation, the gain of

pancreatic water content and the formation of tissue edema. In the present study,

prophylactic treatment with ramiprilat did not show any inhibition on the acute

pancreatitis-induced release of pancreatic a-amylase (Fig. 3-3) and lipase (Fig. 3-4)

into the circulation. In addition, the treatment also did not reduce the gain of water

content in pancreas (Fig. 3-5) and the formation of tissue edema (Fig. 3-7). When

the ramiprilat was applied at a higher dose, the severity of pancreatic injury was even

enhanced. On the contrary, prophylactic treatment with saralasin provided

significant suppressions in plasma levels of a-amylase (Fig. 3-2) and lipase (Fig. 3-4)

in animals with acute pancreatitis. The pancreatic water content (Fig. 3-5) and

interstitial edema (Fig. 3-6) were also greatly reduced when saralasin was

prophylactically administrated to the cerulein-induced animals.

The differential effects of the two RAS inhibitors may be due to the

involvement of bradykinin accumulation during the RAS blockades. ACE, is also

known kininase II, cleaves its C-terminal dipeptide, Phe-Arg from bradykinin, thus

88

regulating the balance between the RAS and kallikrein-kinin system [Deddish et al.

1996]. The application of ACE inhibitor not only inhibits the synthesis of Ang II,

but also blocks the kininase II from the breakdown of bradykinin. Bradykinin is

acknowledged to evoke vasodilation and increase vascular permeability [Voors et al.

1995]. In addition, the ACE inhibition may also elevate the endogenous level of

nitric oxide (NO) along with the level of bradykinin. It is known that NO is a potent

inhibitor of leukocyte and hence could attenuate inflammatory processes. However,

excess generation of NO under oxidative stress may lead to inflammatory responses

through its reaction with superoxide anion to form peroxynitrite (ONOO ), which is a

potent oxidant [Beckman et al 1990; Peng et al 1998]. In fact, ACE inhibitors have

been reported to induce localized angioedema, pancreatic duct obstruction and even

pancreatitis [Iliopoulou et al 2001; Muchnick and Mehta 1999]. On the other hand,

Ang II could be generated from the non-ACE pathways such as chymase and

cathepsin G systems [Dzau 1988], and thereby full suppression of the RAS cascade

could not be achieved by the ACE inhibition alone. In this context, the harmful

effects of ramiprilat in the acute pancreatitis-induced pancreas have been implicated

by both biochemical and histological evaluations in the present study.

Nevertheless, a recent study reported that chronic oral administration of ACE

inhibitor alleviated chronic inflammation and fibrosis in pancreas, thus might be

useful for treating chronic pancreatitis [Kuno et al 2003]. In that study, high doses

of lisinopril (20 — 200 mg/L) were utilized to suppress the overexpression of the

TGF-pi mRNA, which consequently prevented the activation of pancreatic stellate

cells and the synthesis of extracellular matrix protein. In the current study, i.v.

injections of ramiprilat at 40 |dg/kg enhanced the plasma levels of a-amylase and

lipase, thus causing a more severe acute pancreatitis. Although both ramiprilat and

89

lisinopril are carboxy-containing ACE inhibitors, lisinopril contains a dicarboxylate

in its chemical structure and possesses a higher potency relatively [Voors et al 1995].

The heterogeneity of different ACE inhibitors may lead to beneficial but also

harmful outcomes in the treatments whereas the involvement of bradykinin and NO

elevations would be more critical in the pathology [Hartman 1995]. The difference

in pathogenesis of acute and chronic pancreatitis may also be accountable to the

properties of ACE inhibitors in dissimilar manners.

In support for the role of RAS in the pathogenesis of cemlein-induced acute

pancreatitis, the prophylactic treatment with Ang II receptor antagonist, saralasin,

was shown to be effective against the acute pancreatitis-induced oxidative stress. If

the generation of reactive oxygen species overwhelms the capacity of the anti-

oxidant mechanisms in the cells, oxidative stress develops [Dabrowski et al. 1999].

The NAD(P)H oxidase system is a major source for the cellular formation of reactive

oxygen species. Superoxide anion is the primary product of the system [Parks 1989;

Babior 1999]. Increase in intracellular calcium and/or activation of protein kinase C

could play a role in the regulation of the superoxide anion generation [Matsubara and

Ziff 1986]. In this study, the activity of NAD(P)H oxidase was significantly

suppressed by the prophylactic treatment with saralasin (20 |ag/kg) in acute

pancreatitis-induced pancreas (Fig. 3-11). The result implicated that the

development of oxidative stress via the activation of NAD(P)H oxidase system,

together with the activation of RAS, was involved in the pathogenesis of acute

pancreatitis. In other studies, Ang II has been suggested to stimulate the production

of superoxide anion via the membrane-bound NAD(P)H oxidase system in

phagocytes, and such stimulation could be inhibited by Ang II receptor antagonists

[Dijkhorst-Oei et al 1999; Wolf 2000].

90

GSH depletion, lipid and protein oxidative modifications are common

markers for oxidative stress. GSH is generally regarded as the first line of defense

against oxidative stress [Sweiry and Mann 1996; Dabrowski et al 1999]. The

administration of glutathione precursor, GSH monoethyl ester, has been shown to

limit the severity of cerulein-induced pancreatitis [Neuschwander-Tetri et al 1992;

Demols et al 2000]. In the present study, the prophylactic treatment with saralasin

at doses of 5 and 10 jag/kg demonstrated a significant GSH restoration (Fig. 3-8).

Therefore, the RAS could have a role in regulation of GSH depletion and

consequently the oxidative stress caused by acute pancreatitis. On the other hand,

the increase in other markers for oxidative damage, such as protein oxidation and

lipid peroxidation, would proceed to the disruption of cell integrity. In the course of

acute pancreatitis, the degradation products of protein oxidation and lipid

peroxidation, i.e. protein carbonyls and MDA, respectively, were found significantly

elevated along with severe pancreatic tissue damage [Dabrowski et al. 1999]. The

levels of protein carbonyls and MDA were significantly reduced by the prophylactic

treatment with saralasin at doses from 5 to 20 ^ig/kg (Fig. 3-9, 3-10). Thus, the RAS

could also have a role in the regulation of protein oxidation and lipid peroxidation

caused by acute pancreatitis. However, the application of saralasin at dose of 40

|Lig/kg appeared to potentiate lipid peroxidation (Fig. 3-10). That might probably due

to the toxicity of the drug at high dose usage. Based on the experimental data

obtained in the study, it is believed that the activation of pancreatic RAS is

associated with not only the severity of pancreatic injury, but also the changes in

oxidative status in acute pancreatitis.

91

4.1.2 Differential effects of losartan and PD123319

Inhibition of the Ang II synthesis by the non-specific Ang II receptor antagonist,

saralasin, has been demonstrated in previous section to provide protective effects

against pancreatic injury and oxidative stress in acute pancreatitis; therefore, the

synthesis of Ang II appears to contribute in the respective alterations. It is

acknowledged that Ang II exerts its physiological functions by binding to its cognate

receptors, designated ATi and AT2 [de Gasparo et al 2000]. In the present study,

losartan, the specific ATi receptor antagonist, and PD123319, the specific AT2

receptor antagonist, were applied to define the differential roles of both ATi and AT2

receptors on the changes in tissue injury and oxidative status in cerulein-induced

pancreas.

With respect to the acute pancreatitis-induced pancreatic injury, both

prophylactic and therapeutic treatments with losartan at 200 |ig/kg showed marked

improvements. They were implicated by the significant decreases in the plasma

levels of a-amylase and lipase along with the reduction in the pancreatic water

content. However, prophylactic and therapeutic treatments with PD 123319 did not

provide any effect on the severity of cerulein-induced pancreatic injury (Fig. 3-12, 3-

14, 3-15). The differential effects of the ATi and AT2 receptor antagonists would

probably be due to their diverging or contrasting roles for Ang II [Sohn et al 2000].

The ATi receptor has been suggested to oppose some ATi receptor mediated-effects

on cell growth, water and electrolyte balance [Hein et al. 1995; Munzenmaier and

Greene 1996]. The results in the current study showed that the ATi receptor could

contribute to the alterations of tissue injury and histology of the pancreas. In other

words, losartan would be the suitable RAS inhibitor in the aspect of ameliorating the

severity of pancreatic injury induced by acute pancreatitis

92

With reference to the protective effects of saralasin against oxidative stress,

the specific Ang II receptor blockers are inferred to provide same positive influences.

The differential actions of losartan and PD123319 on changes of oxidative status in

cerulein-induced pancreas were confined in terms of their suppressive abilities in

protein oxidation and NAD(P)H oxidase activation in the present study. In fact, Ang

II has been suggested to stimulate intracellular formation of reactive oxygen species

generation, such as superoxide anion, hence leading to oxidative stress [Wolf 2000].

The superoxide anion generation induced by Ang II was found mainly via the

mediation of NAD(P)H oxidase system [Di Wang et al. 1999]. In this study, the

NAD(P)H oxidase activity was reduced significantly in rats either pre-treated or

post-treated with losartan at 200 |dg/kg when compared to the enhanced NAD(P)H

oxidase activity in the cemlein-treated control (Fig. 3-18). However, no positive

change in NAD(P)H oxidase activity was observed in treatments with PD 123319

(Fig. 3-18). Indeed, the activation of NAD(P)H oxidase was demonstrated in

endothelial cells via the regulation of ATi receptor for Ang II [Zhang et al 1999;

Rueckschloss et al 2002]. In addition, Ang II has been also demonstrated to cause

generation of superoxide anion via the NAD(P)H oxidase system in vascular tissues

[Griendling et al. 1994]. On account of the protective effects of ATi receptor

blockade, the linkage between the pancreatic RAS and the generation of reactive

oxygen species was further supported in the current study.

In parallel to the activation of NAD(P)H oxidase system, the development of

oxidative stress was also reflected by the increase of protein oxidation. The DNPH-

reactive protein carbonyls are relatively more stable than other oxidative markers

such as protein sulfide and disulfides. The generation of protein carbonyls could be

caused by a number of oxidative processes related to the action of reactive oxygen

93

species [Reinheckel et al 1999]. Other than protein oxidation, reactive oxygen

species could also mediate activations of cellular macromolecules, inflammatory

response and tissue injury. In the present study, the elevated pancreatic level of

protein carbonyls was significantly reduced by both prophylactic and therapeutic

treatments with losartan, but not by the treatments with PD123319 (Fig. 3-19). Thus,

the RAS in pancreas was shown to participate in the regulation of protein oxidation,

in turn of oxidative stress, in the course of acute pancreatitis. Similarly, damage to

proteins was reduced by the blocking of ATi receptor, but not the ATi receptor, in

chronic kidney disease [Agarwal 2003]. Again, losartan appeared to be an effective

RAS inhibitor in attenuating the acute pancreatitis-induced NAD(P)H oxidase

activation as well as the protein oxidation. On the contrary, PD 123319 was not

demonstrated with any effect in these aspects. Taken together, the ATi receptor, but

not AT2, is believed to be responsible for the majority of the physiological effects of

Ang II in cerulein-induced oxidative stress. However, some recent studies have

suggested that the AT2 receptor may play a counter-regulatory role against the

actions of Ang II mediated by the ATi receptor [Horiuchi et al 1999; Siragy et al

1999].

Taken together, it is therefore plausible to speculate that ATi and AT2

receptors may have differential roles and mechanisms in the regulation of changes in

the severity of tissue injury induced by pancreatitis observed in the present study. In

this regard, blockade of the ATi receptor by losartan could inhibit NAD(P)H

oxdiase-dependent superoxide formation induced by cerulein. Consequently, this

reduced oxidative modification of cellular macromolecules, inflammatory response

and cellular injury in the pancreas.

94

4.2 Potential functions of RAS in pancreatic acinar secretions

The pancreatic exocrine secretions are regulated at multiple levels, generally by

hormonal factors including CCK, secretin, gastrin and intracellular calcium, as well

as the cholinergic and adrenergic neural inputs. Pancreatic acini are mainly

responsible for the exocrine secretion of digestive enzymes. Apart from the classical

regulatory controls, many other mediating factors such as cytokines, prostaglandin

and vasoactive peptides are found to stimulate the acinar secretion [Chey and Chang

2001]. As reported by many studies, CCK agonist has been demonstrated for its

regulatory role in the pancreatic acini as evidenced by the highly stimulated a-

amylase output [Niederau et al 1994; Saluja et al. 1999]. The vasopeptide, Ang II,

is thought to be a potential stimulus. Major components of RAS have been

discovered in the AR42J acinar cell line [Chappell et al 1995]. The existence of the

intrinsic RAS would be of importance in the regulation of acinar exocrine functions

including the secretion of digestive enzymes. In the present study, the pancreatic

acini were freshly isolated from rat pancreas by enzymatic digestions followed with

mechanical shearing. The isolated rat pancreatic acini were testified with

secretagogue CCK-8 to preliminary check whether the acinar cells were functional

after a series of digestion and washing procedures [Palmieri et al. 1990]. In order to

examine the role of RAS, the pancreatic acini were exposed to stimulating

concentrations of Ang II. The responsiveness of acinar cells to exogenous Ang II

was evaluated in terms of the stimulation in release of digestive enzymes.

The acinar secretion was assessed with the releases of both a-amylase and

lipase in this study. The double measurements were taken to balance out the

variations in the degree of synthesis and secretion of pancreatic enzymes in

experimental cell suspension samples [Kim and Kim 1996]. The enzymatic activities

95

were further normalized with acinar protein and DNA contents of individual sample.

Nevertheless, the proteolytic digestive enzymes, such as trypsin and chymotrypsin

[Hirschi et al 1994; Saluja et al 1999], were not assessed in the current study. It

was because the incubation medium contained protease-inhibiting reagents, such as

PMSF and EDTA, which might influence the activities of the proteolytic enzymes.

The RAS in the exocrine pancreas is thought to play an important role in

regulating the cellular responses, such as secretion, growth and tissue damage of the

acinar cells. In this study, the role of RAS in rat pancreatic acini was evaluated by

observing the secretory responsiveness of acinar cells to the exogenous Ang II and

the specific Ang II receptor antagonists in terms of the stimulated digestive enzyme

secretions.

4.2.1 Potential role of ATi receptor

The ATi receptor for Ang II has been evidenced for its presence throughout the

pancreas [Serina 2001]. Its existence has been further investigated in specific cell

types of the pancreas. The mRNA expression of ATi receptor has been reported in

the AR42J acinar cells [Chappell et al 1995]. In the present study, the RT-PCR

analysis demonstrated that the isolated rat pancreatic acini expressed the mRNA for

ATI receptor as well (Fig. 3-27). Similar to the findings in other tissues, the ATia

was identified in this study as the dominant isoform for ATi receptor in rat

pancreatic acini [de Gasparo et al. 2000].

The results in current study showed that exogenous Ang II (0.1 nM to 100

nM) could evoke acinar secretion of digestive enzymes, including a-amylase and

lipase, in a concentration-dependent manner (Fig. 3-22, 3-23). This stimulatory

effect of Ang II suggested a physiological role of RAS in the pancreatic acinar

96

functions, which was consistent with the study in the AR42J acinar cells [Cheung et

al 1999]. The RAS inhibitors, such as losartan and PD123319, would be useful to

further evaluate the potential roles of Ang II, which is the major effector of a RAS.

In the AR42J acinar cells, Ang II has been reported to dose-dependently induce the

calcium signaling as shown by the elevation of free cytosolic calcium under fura-2-

based microfluorimetry [Bamhart et al 1999]. The blockade of ATi receptor by

losartan, the specific ATi receptor antagonist, resulted in a significant reduction in

the Ang II-stimulated cytosolic calcium in the AR42J acinar cells [Barnhart et al.

1999; Chappell et al 2001]. Similar inhibition of losartan to Ang Il-evoked

intracellular calcium was also found in the human endocardial endothelial cells

[Jacques et al. 2003]. The ATi receptor, therefore, was responsible for the mediation

of Ang Il-dependent intracellular calcium signaling. In this study, as the pancreatic

acini were pre-incubated with losartan, the Ang Il-evoked a-amylase elevation was

inhibited almost to the non-Ang Il-evoked basal level. Losartan alone was shown

with no alteration in the basal a-amylase output (Fig. 3-24). This experimental result

agreed with the finding as shown in the AR42J acinar cell line [Cheung et al 1999].

The effect of losartan supported the presence of ATi receptor on pancreatic acini as

shown by the RT-PCR analysis; besides, it also implicated the role of ATi receptor in

the Ang Il-stimulated release of digestive enzymes in pancreatic acinar cells.

Consistent with previous results, ATi receptor seems to mediate most of the

physiological functions of Ang II in cardiovascular, renal, hepatic and other target

cells [de Gasparo et al. 2000]. Therefore, the ATi receptor may play a critical role in

regulating the acinar functions including secretion of digestive enzymes and

probably pancreatic injury in pancreatic acini.

97

4.2.2 Potential role of AT2 receptor

Similar to the ATi receptor, AT2 receptor is also distributed throughout the pancreas

[Serina 2001]. Apart from the detection of mRNA for ATi receptor, the isolated

pancreatic acini also expressed the mRNA for AT2 receptor in the present study (Fig.

3-28). The expression of ATi receptor is found predominant in majority of tissues

including adrenal cortex, liver, vascular smooth muscle [Chiu et al. 1989;

Whitebread et al. 1989]; however, the ATi receptor subtype has been reported to

express in a significant amount in the AR42J acinar cell line [Chappell et al 2001].

Unlike the result of ATi receptor antagonism, the blockade of AT2 receptor by

PD 123319 did not influence the Ang Il-evoked secretion of a-amylase from

pancreatic acinar cells in the present study (Fig. 3-25). Although the AT2 receptor

was found present in the isolated pancreatic acini, the AT2 receptor might not take

part in regulating the acinar secretion of digestive enzymes. However, the AT2

receptor could promote production of bradykinin and prostaglandin [Siragy and

carey 1999], neuronal differentiation [Laflamme et al. 1996] and apoptosis [Yamada

et al 1996] in cultured cells, and may counteract to ATi receptors. In addition, the

AT2 receptor has been shown to negatively regulate the activation of tyrosine

phosphatase activity and the phosphorylation of insulin receptor kinase in the AR42J

acinar cells [Elbaz et al. 2000; Chappell et al. 2001]. Nevertheless, the role of AT2

receptor for pancreatic acinar exocrine secretion in the pancreas needs to intensive

investigations in the future.

4.3 Regulation of RAS in acute pancreatitis-induced acini

Gene expression of major RAS components in target tissues have been regulated by

various conditions including hypertension and athersclerosis [Singh and Mehta 2003],

98

PET [Lam and Leung 2002], experimental acute pancreatitis [Leung et al. 2000],

chronic hypoxia [Chan et al 2000], and bile-duct ligation [Paizis et al 2002]. These

results suggested a role for the tissue RAS in the disease states. The regulation of

RAS by acute pancreatitis in pancreatic acini was in particular interest of the present

study.

4.3.1 Regulation of RAS components in acinar cells

According to the semi-quantitative RT-PCR analysis, there was a significant effect of

acute pancreatitis on the expression in mRNA levels of major RAS components

including angiotensinogen, ATi and ATi receptors in acute pancreatitis-induced

pancreatic acini (Figs. 3-26, 3-27, 3-28). The increase in the relative mRNA

expression of RAS components by acute pancreatitis in the isolated acinar cells

found in the present study was consistent with the results obtained from whole

pancreatic tissue [Leung et al. 2000]. When the acute pancreatitis-induced

pancreatic acinar cells were exposed to exogenous Ang II, the a-amylase secretion

was stimulated by 2-fold when compared to the stimulation in normal acinar cells

(Fig. 3-29). Therefore, there may be a linkage between the changes in the Ang II-

stimulated secretory response and the up-regulation of RAS as evidenced by the

molecular analyses. The up-regulation of angiotensinogen might contribute the most

to the significant elevation of a-amylase secretion from the acute pancreatitis-

induced acinar cells. Angiotensinogen, which is the mandatory component for a

local RAS, precedes the synthesis of Ang I as well as the consequent product Ang II.

This series of conversions is believed not limiting to the circulating hormonal

cascade [Lavoie and Sigmund 2003]. Recent studies suggested that the angiotensin

products could be synthesized intracellularly within an intrinsic RAS. The local up-

99

regulation of ACE activity in infracted tissue has been resulted in an increase of local

Ang II generation, which in turn to stimulate fibrogenesis, in the course of

myocardial infarction [Voors et al 1995]. An increase in pulmonary Ang II

concentration was found in the course of bleomycin-induced lung injury; this result

also suggested that the local generation of Ang II, which might play a possible role in

the fibrotic response to acute lung injury [Marshall et al 2003]. In the present study,

not only the mRNA of angiotensinogen was demonstrated with a significant increase

in cerulein-induced acute pancreatitis, the Ang II cognate receptors were also

subjected to up-regulations accordingly. Thus the increase in endogenous Ang II

together with the up-regulated Ang II receptors might probably be responsible for the

extra elevation of a-amylase secretion found in the AP-induced acinar cells. The

resultant elevation of a-amylase output was in agreement with the up-regulated gene

expressions of RAS components in the cerulein-induced pancreatic acini.

4.3.2 Differential actions of losartan and PD123319

In those AP-induced acinar cells, pre-incubation with the selective ATi receptor

antagonist, losartan, was found to significantly inhibit the Ang Il-evoked elevation of

a-amylase secretion (Fig. 3-29). The inhibitory effect of losartan in acute

pancreatitis-induced acinar cells further supported the up-regulation of RAS, in

particular the ATi receptor, by AP. However, when the same concentration of

losartan (1 [iM) was applied to both acute pancreatitis-induced and normal pancreatic

acini, the action of losartan was not effective enough to completely hold down the

Ang Il-evoked elevation of a-amylase in acute pancreatitis-induced acini back to the

basal level as it was observed in the normal acini (Fig. 3-24, 3-29). In fact, the basal

a-amylase release has been reported to be significantly higher in the pancreatic acini

100

from rats with cerulein-induced acute pancreatitis [Bragado et al 1996]. Thus, there

might come with the insufficiency of antagonist in blocking the extensive increase of

AT] receptors present in the acute pancreatitis-induced acinar cells. The increase in

endogenous Ang II was taken into account as well. Apart from the effect of the up-

regulated RAS, the elevation of basal a-amylase secretion in acute pancreatitis-

induced acini could be due to calcium toxicity as well as integrity damage of acinar

cells [Bragado et al 1996]. In addition, the intracellular activation of pancreatic

digestive enzymes, and hence the hypersecretion, might occur as an early event in the

acute pancreatitis development [Gorelick et al. 1992; Saluja et al 1999]. Therefore,

losartan was shown to be an appropriate antagonist for inhibiting the Ang Il-evoked

acinar secretion of a-amylase in pancreatic acini; however, its application was not

sufficient to restrain the resultant outcome from the complicated events of acute

pancreatitis.

On the other hand, the Ang II-stimulated a-amylase secretion was not altered

by the pre-incubation with selective ATi receptor antagonist, PD 123319, in the acute

pancreatitis-induced pancreatic acini (Fig. 3-29). Although the mRNA expression of

AT2 receptor was demonstrated with an up-regulation in acute pancreatitis, the AT2

receptor might not have a role in the regulatory functions of acinar secretion. This

result was consistent with the previous experimental data. Therefore, PD 123319 was

inefficient in attenuating the Ang Il-evoked acinar secretion, in both normal and

acute pancreatitis-induced pancreatic acini. Nevertheless, the counter-acting

relationship of the ATi and AT2 receptors was not clearly revealed in this study with

respects to the regulation of acinar secretion of digestive enzymes.

101

4.4 Potential role of RAS in acute pancreatitis

Interleukins, platelet activating factor and tumor necrosis factor-a, the well-

established inflammatory mediators, are activated in the circulation as a systemic

feature of acute pancreatitis [Heath et al. 1993; Grewal et al 1994]. However, an

elevation of plasma renin observed in acute pancreatitis patients implicated the

involvement of RAS in the development of acute pancreatitis [Greenstein et al 1987].

Furthermore, the circulating RAS has been shown to mediate inflammation and has

been associated with the generation reactive oxygen species and reactive nitrogen

species [Fernandez-Alfonso and Gonzalez 1999]. Contemporary research studies

suggested that an intrinsic RAS could be related to the circulating RAS directly or

indirectly through the inflammatory mediators. A recent study has reported that

ACE inhibitor could attenuate pancreatic inflammation and fibrosis in chronic

pancreatitis [Kuno et al 2003]. In the present study, specific RAS inhibitors were

administrated to assess the role of RAS in the pancreas in terms of pancreatic injury

and oxidative stress induced by acute pancreatitis.

4.4.1 Regulation of RAS components by acute pancreatitis

Cemlein-induced acute pancreatitis could up-regulate the gene expression of major

RAS components including angiotensinogen, ATi receptor, AT2 receptor and the

ACE activity in pancreatic tissue [Leung et al. 2000; Ip et al 2003] as well as in

acinar cells. The up-regulation of RAS components could play an important role in

the regulation of inflammatory responses, microcirculation, oxidative stress and

tissue injury. The generation of reactive oxygen species may establish the linkage

between the activation of RAS and the development of oxidative stress and

pancreatic injury in the course of acute pancreatitis. Cemlein-induced acute

102

pancreatitis has been also reported to up-regulate the gene expression of

inflammatory cytokines (IL-lb, IL-6) in pancreatic acini [Yu et al. 2002]. Ang II has

been regarded as a potent vasoconstrictor, and it has also been suggested to mediate

the induction of inflammatory cytokines and the intracellular formation of reactive

oxygen species [Wolf 2000]. Elevation in endogenous angiotensinogen has been

observed in acute pancreatitis-induced pancreatic tissue as well as in the acinar cells.

Taken together, the up-regulation of RAS components by acute pancreatitis could

mediate the production of reactive oxygen species, e.g. superoxide anion via the

NAD(P)H oxidase system, and the oxidative modifications of protein and lipid;

hence leading to the development of oxidative stress and finally the pancreatic injury.

4.4.2 Differential functions of ATi and AT2 receptors in acute pancreatitis

The actions of RAS on the changes in severity of pancreatic injury and oxidative

stress were examined by the specific ATi and AT2 receptor antagonisms in acute

pancreatitis. According to the experimental results, losartan, the specific ATi

receptor antagonist, provided protective effects against the AP-induced tissue injury

and oxidative stress. In other words, ATi receptor was responsible for the mediation

of the effects of the activated RAS in acute pancreatitis. The ATi receptor could

regulate the secretion of digestive enzymes from pancreatic acini as shown in the

present study and from AR42J acinar cell line [Cheung et al. 1999], as well as from

the gland. In terms of tissue injury, ATi receptor could also regulate the formation of

tissue edema. Losartan has been reported to reduce the formation of cerebral edema

[AsieduGyekye and Antwi 2003] and the pancreatic edema in the present study. In

terms of oxidative stress, losartan has been demonstrated to suppress the pancreatic

NAD(P)H oxidase activity in the current study, and the same suppression was also

103

found in vascular endothelial cells [Zhang et al 1999] and isolated rat heart [Oudot

et al 2003]. The membrane-bound NAD(P)H oxidase is primarily responsible for

the induction of reactive oxygen species. In addition, the generation of reactive

oxygen and nitrogen species has been shown to be mediated by RAS, probably via

the ATi receptor [Wolf 2000; Mihm et al 2003]. The results from current study

were in consistent with the previous findings as losartan provided protective effects

against the AP-induced lipid peroxidation, protein oxidation and activation of

NAD(P)H oxidase system. On the other hand, the AT� receptor was not

demonstrated with a distinguished role in mediating the pancreatic injury in acute

pancreatitis. In the present study, no alteration was found in pancreatic secretion of

digestive enzyme and formation of tissue edema when PD 123319 was applied to

acute pancreatitis-induced pancreas or acinar cells. However, the AT2 receptor has

been suggested to regulate apoptosis [Weidekamm et al 2002]. Besides, the AT2

receptor may not have a role in the regulation of oxidative stress in the pancreatitic

pancreas. Indeed, no change in the activity of NAD(P)H oxidase, protein oxidation

and lipid peroxidation was observed when PD 123319 was administrated to acute

pancreatitis-induced pancreas in the present study.

4.5 Conclusion

The present study firstly addressed the effects of cerulein-induced acute pancreatitis

on the changes of pancreatic injury, histopathology and oxidative status of pancreatic

tissues. Secondly, the association between the activation of RAS in the pancreas and

the acute pancreatitis-induced pancreatic injury/oxidative stress were further

demonstrated. The potential role of the pancreatic RAS was revealed by the

significant reductions in the severity of acute pancreatitis-induced pancreatic injury

104

and oxidative stress by RAS inhibitions. The up-regulation of RAS in the pancreas is

believed to mediate a number of events in acute pancreatitis, such as the activation of

NAD(P)H oxidase system, glutathione depletion, protein oxidation as well as lipid

peroxidation. Through the application of specific Ang II receptor antagonists, ATi

receptor was demonstrated to mediate the physiological functions of Ang II in acute

pancreatitis. In the exocrine pancreas, the RAS in pancreatic acini was suggested to

have a regulatory role in acinar secretion of digestive enzymes. Through the specific

antagonism, ATi receptor was shown to mediate the Ang II-stimulated release of

digestive enzyme. Besides, the RAS in the exocrine pancreas was also subjected to

an up-regulation by acute pancreatitis as observed in pancreatic tissue; the acinar

secretion of digestive enzyme was enhanced accordingly. Regulation of exocrine

secretions could be mediated via the ATi receptor on pancreatic acini as

demonstrated in the present study. Taken together, the RAS in the pancreas could

have diverse actions in regulating functions of the gland, as well as its significance

on changes in the severity of acute pancreatitis-induced tissue injury and oxidative

stress. Therefore, future strategy for the inhibition of the pancreatic RAS may be of

importance in the management of acute pancreatitis-induced pancreatic injury.

4.6 Further studies

Although acute pancreatitis could be a mild self-limiting disease, occasionally it

could also lead to systemic complications, such as systemic inflammatory response

syndrome and multi-organ dysfunction syndrome. Apart from the hypersecretion of

pancreatic digestive enzymes, the release of various inflammatory mediators into the

circulation is followed. The pathogenic mechanisms leading from localized

pancreatic injury to system inflammation and multi-organ dysfunction are of

105

particular importance. However, they are not fully understood. Our preliminary

results demonstrated the involvement of RAS in acute pancreatitis-associated

systemic inflammation. Acute pancreatitis-induced pulmonary injury could be

attenuated by RAS inhibition, in particular the ATi receptor blockade, as evidenced

by the significant reduction in pulmonary MPO activity. Therefore, the pancreatic

RAS could have important implications not only limited for the pancreatic diseases,

but also for several acute pancreatitis-associated dysfunctions. To this end, further

work on the association between the activation of pancreatic RAS and systemic

inflammatory response syndrome and multiple organ dysfunction syndrome is being

undertaken.

106

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