Effects of ANG II and its receptor blockers on nasal salt gland secretion and

arterial blood pressure in conscious Pekin ducks (Anas platyrhynchos).

Roham Zandevakili

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Zoology

University of Toronto

@ Roham Zandevakili 1998

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TABLE OF CONTENT

TABLE OF CONTENTS .................................................................................................... i

............................................................................................... ACKNOWLEDGEMENTS iv

............................................................................................................. LIST OF FIGURES v

..* ........................................................................................... LIST OF TABLES ............. .: vil1

............................................................................................ LIST OF ABBREVIATIONS ix

INTRODUCTION .......................................................................................................... 1

............................................................................................................. Objectives. 2

The Renin Angiotensin System .......................................................................... 3

...................................................................................... Blood Pressure Regulation 7

................................................................................. Life in a Marine Environment 9

Vertebrate Osmoregulation ................................................................................... 1 1

............................................................................ Avian Osmoregulation.. 12

a) The avian renal-cloaca1 system ........................................................... 12

......................................................................... b) Avian nasal sait glands 13

MATERIALS AND METHODS ................................................................................... 1 6

........................................................................................................................ RESULTS -23

.................................................................................................. Nasal Salt Glands 24

................................................................................. Blood Pressure Experiments 4

................................................................................................................... DISCUSSION 52

....................................................... Nasal Salt Gland Response to ANG II and III 54

.............................................. Ionic Concentrations of the Nasal Salt Gland Fluid 55

..................................................................... Characteristics of ANG 11 Receptors 58

............. Effect of SARILE on Nasal Salt Glands and the Cardiovascular System 59

Effect of Mammalian ATI and AT2 Receptor Blockers ........................................ 61

..................................................................................................... FUTURE WSEARCH 65

..................................................................................................... LITERATURE CITED 66

ABSTRACT

The vertebrate renin-angiotensin system (RAS) controls cardiovascular, rend and

osmoregulatory functions. Angiotensin II (ANG Ii) is the most potent hormone of the RAS.

However in some vertebrates, Angiotensin IQ (wa14]-AN~ IiI) also has significant activity. The

effects of angiotensins with different amino acid sequences and mamrnalian ANG II receptor

antagonists on nasal salt gland function and arterial blood pressure in conscious white Pekin

ducks were investigated in the present shtdy.

There was a positive linear correlation: between nasal fiuid [Na7 and osmolality, between

[Na+] and [KCI and between the rate of nasal salt gland fluid secretion (NFS) and [Na+] and

[K"]. [ A ~ ~ ' , V ~ I ~ ] - A N G II (1 nrn01.k~" i.v.) inhibited NFS but did not change ionic

concentrations. [v~~~]-ANG III (1 or 5 nrn01.k~-') and ANG I (1-7) (20 nrn01.k~-') had no effect

on NFS. SARI[LE acted as an ANG II receptor agonist and resulted in a prolonged and complete

inhibition of NFS. The ATl receptor antagonist-losartan (DuP 753) and the AT2 receptor

antagonist-PD 1233 19 both failed to block the inhibitory effect of [ A S ~ ~ , V ~ ~ ~ ] - A N G II on the

nasal salt glands.

AS^' ,v~~']-ANG II (2 nmol.kg" i.v.) increased mean arterial blood pressure by about

40 % (MABP) whereas the sarne dose of [ A S ~ ' , V ~ ~ ~ ] - A N G II (teleost) had only 30% of the

pressor effect of [ A S ~ ' , V ~ ~ ~ ] - A N G II . Neither 1 nor 5 nrn01.k~-' of [V~I~I -ANG III i.v. nor 20

nrn01.k~" of ANG I (1-7) had any measurable effect on MABP. SARILE completely blocked

the pressor response to [ A S ~ ' , V ~ ~ ~ ] - A N G II, but the ATI antagonists losartan and CGP 48933

and the AT2 antagonist PD 1233 19 al1 failed to block the pressor response to [AS~ ' ,V~~~] -ANG

Ir.

iii

AOWLEDGEMENTS '

1 am very thankful to my parents, Ehteram and Gholamali, whose moral support and

understanding of the demanding life of a graduate student was invaluable. It has been an

unforgettable experience to work in Dr. Butler's lab. The arnount of knowledge gained was

immense. I take great pride to have leamed my surgical skills from one of the best in the field

of animal surgery, Dr. Butler. The past two years taught me that hard work usually results in

success. It has been the hard work that made it possible for me to travel to Spain for a

conference and meet the keynote speaker Prof. Knüt Schmidt-Neilsen who discovered the

function of nasal salt gland. It was the highlight of my years in the Department of Zoology.

During the ups and downs in the lab, it h& been the support of my good colleague,

Donald Bang, that would always lift my spirit. He was always ready to help and gave fieely his

insightful ideas. Thank you Don. 1 am thankful to many people in the Department who went

beyond their duties to help me, and they include: Terry, Sonia, Kim, Ed, Louisa, Liz, Susan,

Marie, Peter, Diana, Rosana, Jim, Ziggy, Lu, Eric, Rudy, Fred, Ray, Norman, Daniel, Scott,

Steve S., Steve C., Janet, also Sharon and Christie for guiding me in the right direction, al1 the

way from my undergraduate years.

Finally I thank Dr. D.G. Butler for his training and patience. 1 greatly appreciate Dr. R.

Stephenson's, Dr. L. Buck's, and Dr. H.H. Harvey's criticism and useful advice. Lastly, but not

least, Dr. Gavin Oudit and Ms. Cadinouche for having prepared me for research in Dr. Butler's

lab. Special thanks to Ms. Anoush Migirdicyan and Ms. Sun-Hee Pak for helping me with the

long and tedious analytical procedures and proof-reading my thesis.

LIST OF F'IGUIRES

Figure 1. Avian angiotensin biosynthesis ..................................................................... 4,5

Figure 2A. Positive linear correlation between [Na+] and osmolaiity of fluid secreted by the

nasal salt glands (Y = 1.61X + 153.1; r = 0.87; n = 334; Pe0.001). .......................... 25, 26

Figure 2B. Positive linear relationship between the rate of nasal fluid secretion (d.kg-'.5min-')

.............................. and the [ ~ a + ] (Y = 170.5X + 5 1 1.3; r = 0.48; n=334; Pc0.001). 2 5 26

Figure 3A. Positive linear correlation between the rate of nasal fluid secretion (rnl.kg". 5 min")

................................ and the [K+] (Y = 4.83X + 12.25; r = 0.41; n = 334; P<0.001). 27, 28

Figure 3B. Positive linear correlation between the nasal fluid mat] and [K*] (Y = 0.025X -

0.45; r = 0.76; n = 334; P<û.001) ............................................................................... 27, 28

Figure 4. Effects of [ A S ~ ' , V ~ ~ ~ ] - A N G II and [ V ~ ~ ~ J - A N G III on nasal salt gland secretion in

freshwater Pekin ducks during an i.v. infusion of hypertonie saline (1000 mosmol.kg-l) at a rate

of 0.97 ml.rnin 'l. Values arerneans andS.E.M. n = 5. ........................................... 3 31

Figure 5. Effects of ANG 1 (1-7) and [AS~',V~I~]-ANG II on nasal salt gland secretion during

an i.v. inhision of hypeaonic saline (1000 rnosm01.k~-') at a rate of 0.97 ml.min 'l. Values are

..................................................................................... means and S.E.M. n = 5. . . . . . . , 34

Figure 6A. Effects of the mammalian angiotensin antagonist SARZLE on nasal salt gland

secretion and the response by the nasal sd t glands to two i.v. injections of [ A S ~ ' , V ~ ~ ~ ] - A N G

II given before and after the antagonist. Values are means and S.E.M. n=5. ........... 35, 36

Figure 6B. Pressor response to [ A S ~ ' , V ~ ~ ~ ] - A N G II before and after an i.v. infusion of the

........................................................... mammalian angiotensin antagonist SARILE. 3 7 , 3 8

Figure 7. Nasal salt gland secretion following injections of [ A S ~ ' , V ~ ~ ~ ] - A N G II i.v. which

were administered both before and after a single i.v. injection of the mammalian ATi antagonist

losartan (n = 1). .................................................................................................... O 41

Figure 8. Nasal salt gland secretion following injections of [ A S ~ ~ , V ~ ~ ~ ] - A N G II i.v. which

were administered both before and &er a single i .v. injection of the marnmalian AT2 antagonist

PD 123319 ................................................................................................................ 4 43

Figure 9. Mean arterial blood pressure in Pekin ducks before and after random i.v. injections

of [ ~ s n ' , valS]-AN~ II, [AS~',V~?]-ANG II, [ V ~ I ~ - A N G ICI, and [ A ~ ~ ' , V ~ ~ ~ ] - A N G I (1 -7).

Values are means and S.E.M. n = 6. NS = not significant compared with vehicle-injected

control. A P c 0.01 cornpared with vehicle-injected control and [ ~ s n ' , V&]-ANG II. * P <

0.05 compared with vehicle-injected control. ......................................................... 4 , 46

Figure 10. Pressor responses to AS^' ,val'>]-ANG II (1 nmol .kg-') injected i.v. before and after

blockade with [ s ~ ~ ' , I ~ ~ ~ ] - A N G II (SARILE). Values are mean artenal blood pressure and S.E.M.

n = 6. NS = not significant compared with vehicle-injected control. A P < 0.01 compared with

vehicle-injected control. ............................................................................................ 7 , 48

Figure 11. Mean arterial blood pressure in Pekin ducks following a series of i.v. injections

of AS^', V~I~] -ANG II (2 nrn01.k~-l) adrninistered before and after successive i.v. injections

of the mammalian angiotensin antagonists Losartan (AT,), PD 123319 (AT2) and CGP 48933

(ATI). NS = not significant compared with vehicle-injected control. A P < 0.01 compared

with vehicle-injected control. .................................................................................. O , 5 1

Figure 12. Proposed mechanism of cellular transport of ions in the avian nasal salt gland

tissue ......................................................................................................................... 6 , 57

vii

LIST OF TABLES

Table 1 . Average concentrations of major ions in different bodies of water ................... 10

Table 2 . Plasma ionic composition of Pekin ducks ......................................................... 10

viii

LIST OF ABBREVIATIONS

............................................................................ AS^', val5]-ANG II A i angiotensin II

....................................................................... [~sn', V~I'J-ANG II T e l o s t angiotensin II

[ v ~ ~ ~ ] - A N G III.. .................................................................................. Avian angiotensin III

............................................................................................. [ V ~ ~ ~ I - A N G IV A n i o t e s n IV

.................... ........................................................... ACE : Angio t e s i converting enzyme

ATl ....................................................................................... Anotensn receptor subtype 1

AT2 ..................................................................................... A n o t e s receptor subtype 2

ADH ................................................................................................... Ani diuretic hormone

............................................................................................................ CGP 48933 Valsartan

............................................................................................................... Ca*.. Ionic calcium

Cl' ................................................................................................................... Ionic chloride

.............................................................................................................. O C D e e e s Celsius

.......................................................................................... ECFT ExtracelIular fiuid tonicity

ECFV ........................................................................................ Extracellular fluid volume

................................................................................................................. HCRT Hematocri t

.................................................................................................. i.c.v. Intra cerebroventricular

.............................................................................................................. IU International Unit

K+ ................................................................................................................. Ionic potassium

mOsm ........................................................................... Milliosmoles per kilogram of water

............................................................................................. mmHg, Millimetres of mercury

mM ......................................................................................................... Millimoles per litre

.......................................................................................................... Mg*. Ionic magnesium .

......................................................................................................................... mg Milligram

............................................................................................................................ ml Millilitre

MW ........................................................................................................... Molecular weight

...................................................................................... MABP M e arterial blood pressure

NFS ...................................................................................... Nasal salt gland fluid secretion

............................. .............,.....,.,...,.,..............,.,...............*........................ Na' ; Ionic sodium

NaCI ........................................................................................................ Sodium Chloride

...................................................................................................................... nmol Nanomole

N.S ................................................................................................................. Not Significant

............................................................... OVLT Organum vasculosum of lamina terminalis

PE .................................................................................................................... Poly Ethylene

........................................................................................................................... pg Picogram

....................................................................................................................... pmol Picomole

RAS .............................................................................................. Renin angiotensin system

................................................................................................. SARILE.. [ ~ a r ' - I l e ~ ] - f W ~ II

soi2 ........................................................................................................... Ionic sulphate

........................................................................................................... SFO Subfornical organ

S.E.M. ......................................................................................... S t a n d error of the mean

pg . ... ... .. .... . ... ..... ..... . .... . . .. ... ... . . ....... . . . . . ........... . . . . . . . . . . . . . . Microgram

VSM ....................................................................................... Vascular smooth muscle

INTRODUCTION

(Adapted from D.G. Butler, R. Zandevakili, G.Y. Oudit (1998). Effects of ANG II and Ili and

angiotensin meptor blockers on nasal salt gland secretion and arterial blood pressure in conscious

Pekin ducks (Anasplatyrhynchos). J Comp Physiol B 168: 213-224.)

OBJECTIVES

To learn more about the Na+/K* CO-transport system in the nasal salt glands.

To learn more about the way the renin-angiotensin system controls the nasal salt glands

in the Pekin duck.

To test the effects of ATr and AT2 blockers on the attenuation of nasal salt gland

secretion by Angiotensin II.

To show if Angiotensin III attenuates the nasal saIt gland function.

To show if the pressor response to Angiotensin II is blocked by ATi and AT2 blockers.

Renin Angiotensin Svstem

The renin-angiotensin system (RAS) is an enzyrnatic cascade of reactions. The enzyme

renin, produced by the juxtaglomerular cells of the kidney, is released into the blood Stream

and hydrolyses angiotensinogen (a large liver protein) to form angiotensin 1 (ANG I), a

decapeptide. Angiotensin Converting Enzyme (ACE), located primarily in pulrnonary blood

vesse1 walls, hydrolyses ANG 1 by removing the last two C-terminal arnino acids. The

product of this reaction is the most potent pressor peptide of the system, ANG II (Ganong

1997) an octapeptide which has the following sequence in the avian species: ~ s ~ ' - A r ~ ~ -

~ d ~ - ~ ~ r ~ - ~ a l ~ - ~ i s ~ - ~ r o ~ - ~ h e ~ . The physiological range of ANG II concentration in Pekin

ducks is between 35-1 00 p g h l (Kobayashi and Takei 1996, Gerstberger and Gray 1993).

ANG II is further hydrolysed by aminopepidases which remove the N-terminal AS^' residue

producing [va14]-AN~ III (Ganong 1997). This process is represented in Fig. 1.

Figure. 1. Avian angiotensin biosynthesis.

ANGIOTENSINOGEN Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Ser-Leu---Protein (renin substrate)

Renin

ANGIOTENSIN I Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Ser-Leu + Protein Angiotensin Converting

Enzyme

ANGIOTENSIN II

ANGIOTENSIN m Aminopeptidases

Smaller fragments [e.g. ANG IV(3-8)]

Circulating and local angiotensin II (ANG IL) maintain homeostasis through a highly

CO-ordinated series of actions on the cardiovascular, rend, endocrine and peripheral and

central nervous systems (Peach 1977; Nishimura 1980; Wilson 1984; Simon et al., 1992). In

birds, ANG II increases drinking (Evered and Fitzsirnons 198 1 ; Kasuya et al., l985), inhibits

salt gland secretion (Butler et al., 1989; Simon et al., 1992) and mediates cardiovascular

responses (Fregly et al., 198 1 ; Moore et al., 198 1 a; Nishimura et al., 1982; Wilson and West,

1986; Takei and Hasegawa 1990). ANG II dso increases aldosterone, but not corticosterone

secretion from the adrenal steroidogenic cells (Vinson et al., 1979; Gray et al., 1989; Kocsis

et al., 1994) and ANG II stimulates the release of catecholamines from peripheral

sympathetic nerve endings and adrenal chromaffin tissue (Wilson 1984).

ANG III has been known to have vasopressor effects of approximately 30 % that of ANG

II in rnammals and only about 1-2 % in most avian species (Nishimura et al., 1982; Takei and

Hasegawa 1990; Evered and Fitzsimons 1981). In mammals, ANG Ili is also involved in

osmoregulation by increasing the plasma levels of aldosterone. This response to ANG III is not

observed in birds (Gray et al., 1989). There has been a great deal of scientific interest in the

effects of ANG II receptor (ATi and AT2) antagonists in submarnrnalian vertebrates, following

the recent cloning of ANG II receptor subtypes and the development of specific nonpeptidergic

receptor antagonists (Timmermans et al., 1993; Smith and Timmermans 1994). Recently, in vivo

and in vitro studies of ATI and AT2 antagonists in the fowl and turkey have shown that the

vascular and adrenal steroidogenic ANG II receptors in birds differ from those in mammals d

(Nishimura et al. 1994; Kocsis et al., 1994, 1995). It was the intent of this study to shed some

light on the physiological and functional characteristics of the ANG II receptor subtypes. This

was done by testing the effects of ANG II molecules with different sequences and mammalian

AT1 and AT2 receptor blockers on the avian system.

BZood Pressure Remidon

Mean arterial blood pressure (1/3 Pulse Pressure + Diastolic Pressure) remains relatively

constant in the face of fluctuations in local blood flow. This rneans that if blood flow to an organ

requires an increase, then the cardiac output or vascular resistance in another organ must also

increase. This will avoid large fluctuations in the mean arterial pressure (Gordon1972). In the

mature Pekin duck, the mean arterial pressure has been reported to be around 150 mmHg

(Sturkie 1986). Changes in nomal blood pressure are sensed by sensory receptors within the

blood vesse1 walls of the aortic arch and the carotid sinuses. Several systems regulate blood

pressure by different means. The sympathetic system releases catecholamines (epinephrine and

norepinephrine), potent vasoconstrictors (Ganongl997). Conversely, the parasympathetic

system releases acetylcholine, a vasodilator. The renin angiotensin system is characterised by

ANG II, a vasoconstrictor, which increases the blood pressure by either direct or indirect

actions. It acts directly on the smooth muscle of the blood vessels and induces constriction

(Ganong 1997) or indirectly it increases aldosterone secretion from the adrenal glands, which

will promote water retention by increasing sodium uptake thus increasing blood volume. It also

stimulates the release of anti-diuretic hormone (ADH) from the posterior pituitary gland, which

increases water retention (Ganong1997). Al1 of these regulatory activities have been well

investigated in mamals. However, in avian species, there are some important differences that

have not been investigated thoroughfy. For example, ANG Ei has only 2 9% pressor activity in

fowl, quail (order Galliformes: Nishimura et al., 1982; Takei and Hasegawa 1990) and pigeon

(order Columbiformes: Evered and Fitzsimmons 198 1). Moreover, [ v ~ ~ ~ - A N G IiI has virhially

no dipsogenic effect in the pigeon (Evered and Fitzsimmons 1981) and budgerigars (order

Psittaciformes) (Kasuya et al., 1985). Another difference is that there is no corticosterone

secretion in response to ANG II in birds (Vinson et al., 1979; Gray et al., 1989; Kocsis et al.,

1994).

The vasopressor response to systemic ANG II in birds is mediated largely via

catecholamines released from peripheral sympathetic nerve endings and chromaffin cells

(Moore et al., 1981a; Nishimura et al., 1982; Wilson and Butler 1983 a, b; Butler et al., 1989).

Even though a relatively large arnount of data has been gathered for the RAS in ducks (order

Anseriformes) (Wilson 1984; Wilson and West 1986; Simon et al., 1992) there is relatively little

. information about the function of [ v~~~] -ANG III and the characteristics of the ANG II

receptors.

Takei et al., (1988) have used radioligand ( U ~ ~ - A N G II) binding techniques to identify

vascular ANG II receptors in fowl aortic smooth muscle. These receptors have similar agonist

binding properties to those of marnmalian vascular ANG II receptors. Fowl atrial myocardial

ANG II binding sites are saturable, reversible and rnodulated by divalent cations and guanine

nucleotides (Baker and Aceto 1989). However, others have shown that fowl vascular ANG II

receptors have different specificities and regulatory characteristics than rnammalian vascular

ANG II receptors (Nishimura et al., 1994; Stallone et al., 1989). This study examines the effects

of various angiotensin peptides on the nasal salt gIands and on blood pressure in white Pekin

ducks (Anas platyrhynchos). It also examines the effects of mammalian peptidergic and

nonpeptidergic AN% Il receptor antagonists on vasopressor and nasal gland secretory responses.

This will help in understanding the importance of different ANG II peptide sequences and their

specificity for the ANG II receptors in the Pekin duck.

Life in a Marine Environment

Of al1 the major groups of animals that live on Earth, only insects and desert animais live

independently of the oceans. The majority of animds live within close proximity to different

bodies of water with varying ionic compositions similar to those in Table 1. It is clear that

during their evolution animals have had to cope with very unusual water compositions.

Although these animals employ a wide variety of strategies, a basic tenet is that the range of

internal solute concentrations compatible with Iife is much narrower than the external media,

particularly sodium, potassium, calcium and magnesium (Gordon 1972). AnimaIs can be

divided into two main groups with respect to the way they regulate the osmotic pressure of their

body fluid in relation to the outside environment. If the animal's intemal composition changes

with fluctuations in ionic concentration in its environment, the animal is an Osmoconfomer.

If the internal ionic concentration (see Table 2) remain fairIy constant regardless of changes in

the external environment, the animal is an Osmoregulator (Gordon 1972). Al1 organisms living

in close proximity to sea water will inevitably ingest excess arnounts of salts if they consume

marine invertebrates some of which are iso-osmotic (osrnoconformers) with sea water, or by

ingesting the sea water itself. The sea water is three tirnes higher than the plasma osmolality of

most marine birds (compare Tables 1. and 2). This results in the osrnotic loss of water and an

increase in intemal salt concentrations. Since pure water is not available to balance the ingested

salts, the organisms must conserve water and actively excrete salts.

The ducks in this study are osmoregulators. They maintain a very constant plasma

concentration of solutes regardless of the osrnotic pressure of their drinking water. Therefore,

mechanisms must be in pIace which would enable the birds to excrete the excess ions. This will

be discussed in the following section.

Table 1. Average concentrations of major ions in different bodies of water

(Gordon 1972).

Little Manitou Lake, Sask.,

Canada

Great Salt Lake, Utah

Table 2. Plasma ionic composition of Pekin ducks (Personal observations).

2000

6000

lm7

(mmol.1-')

145

780

3000

[K*3

(mmo1.l")

3.46

28

90

[Cu

(rnrnol.~'')

111.2

14

9

[Cal

(mmo1.l")

2.8 1

500

230

660

3100

HCRT

(9%)

42.2

M d

(mrno1.1'~)

1.10

Osmolality

(mosm.kg-'water)

298

Vertebrate Osmoremrlution

To maintain cellular function, both the intracellular volume and solute concentrations

must be held within narrow lirnits. In lower life forms, such as multiceIlular organisms, sodium

and chloride are the major osmoregulatory ions interchanging with the outside environment

(Gerstberger and Gray 1993). However, in the vertebrate kingdom, severd mechanisms exist

to regulate extracellular fluid volume (ECFV) and extracellula. fluid tonicity (ECl?ï). The

kidneys are the major osmoregulatory organs in mammalian salt and water reguiation. In the

lower vertebrates, novel and highly specialized transport mechanisrns have evolved to cope with

this stress. These include the gills of teleost fish (Foskett 1987), the rectal glands of

elasmobranchs (Solomon et al., 1984a, 1985a), the skin of amphibians (Lindemann and Voute,

1977), the intestinal system of fish and birds (Skadhauge, 1981; Kirsch et al., 1985) and the

nasal salt secreting glands of birds and reptiles (Peaker and Linzell, 1975; Schmidt-Nielsen et

al., 1958)

A vian Osmoremclation

a) The avian renaLcloacaZ system

The avian urinary organs consist of a pair of symmetrical kidneys and ureters that

transport urine to the urodeum of the cloaca. Each kidney is divided into three lobes; cranial,

rniddle and caudal (Sturkie 1986). Although the kidneys contain both cortical and medullary

regions, the two are not easily distinguishable. The cortical region is rnainly cornposed of

nephrons of the reptilian type, which lack the loop of Henle. These nephrons do not possess the

same urine concentrating ability as the mamrnalian type, which possess long loops of Henle. The

concentrating ability of the kidneys depends on its ability to establish a countercurrent multiplier

system with the aid of meduI1ar-y loops of Henle, the vasa recta and the collecting ducts (Sturkie

1986). The lower part of the cortex contains some mammalian type nephrons, which arise from

efferent arterioles (for more information on the anatomy of the avian kidney refer to Sturkie

1986). Therefore, the kidneys in birds inhabiting freshwater regions and terrestrial areas are

involved in salt conservation, mainly sodium chloride, due to the shortage of salts in their

natural diet. But for birds that drink sea water, their main osrnoregulatory chailenge is to excrete

the high amounts of the ingested sodium chloride and other ions (see Table 1). The kidneys of

birds with nasal salt glands cm only excrete salts at concentrations near their plasma osrnolality,

(see Table 2), with urine/plasma osrnolality ratios of about 1.2 (Sturkie 1986). This leaves

approximately 2/3 of the ingested sea water ions unexcreted in the blood Stream. Since the avian

kidneys lack loops of Henle and are involved in the excretion of uric acid, the end product of

protein metabolism in birds (c.f. urea in mamrnals), marine birds have evolved the nasal salt

glands also known as supraorbital glands which cope with the excess salt load.

Another organ in birds that is involved in osmoregualtion is the cloaca. This structure

dong with the rectum constitutes the lower intestine of birds (Goldstein 1989). The cloaca

receives urine from the ureters. It has been shown that once the urine reaches the cloaca it is

refluxed by reverse peristalsis into the upper regions of the lower intestine. Hence urine mixes

with the chyme and the resulting fluid undergoes modification in the rectum, cloaca and caeca

(Goldstein 1989). The osrnoregulatory role of the cloaca has received a great deal of attention

in the past ten years. It has been shown that an important portion of the ureterally excreted

sodium is reabsorbed, dthough against a concentration gradient (Skadhauge 1989). This process

at first does not seem advantageous to birds that want to get rid of the excess salt in their

plasma. But Schmidt-Nielsen et al., (1963) proposed that there may exist a reno-cloacal-salt

gland interaction whereby sodium and water of rend ongin is reabsorbed in the cloaca/lower

intestine and NaCl is subsequently excreted by the nasal salt glands, therefore conserving free

water. The rational for this loop is that if salts were excreted directly from the lower intestines,

they would carry too much water with them and thus lead to water loss. By absorbing the salt

and then excreting it through the salt glands, water conservation is enhanced.

b) Avian nasal salt glands

A generaI description of the nasal salt glands was published as early as 1664, by Nicolaus

Steno (Fiinge et al., 1958). But their existence in birds was not confirrned until 1813 by

Jacobson (Butler et al., 1991). These glands also exist in reptiles and mamrnals (dthough in

mammals, they are non-functional and extrernely small) (Fange et al., 1958). The function of

these glands was not established until 1957, when Knut Schmidt-Nielsen, C. Barker Jorgensen

and Humio Osaki published the first account of their work on the double-crested corrnorant

(Phalacrocorax auritus) (order pelecaniformes) as an abstract in Federation Proceedings

(Peaker and Linzell 1975). Over the past two decades, a great deal of research has been done

on many bird species possessing these glands, among them are the orders of Charadriiformes

(gulls and plovers), Procellariiformes (aibatrosses and petrels), Pelecanifonnes (pelicans and

comorants), Sphenisciformes (penguins), Gaviifonnes (divers), Falconiformes (eagles) and

Anseriformes (ducks and geese) (Gerstberger and Gray 1993).

Avian sait glands secrete a hypertonie fluid composed predorninantly of Na+, Cl', Kf and

some Ca* and Mg *, thereby conserving osmotically free water (Butler 1985). The glands are

under neural and hormonal control (Butler 1984; Holmes and Phillips 1985; Butler et al., 1989;

Gerstberger and Gray 1993). Activated salt glands are strongly inhibited by both peripheral

(intravenous) (Hamrnel and Maggert 1983; Butler 1984) and central (intracerebroventricular)

(Gerstberger et al., 1984) injections of ANG Il[ at levels not known to evoke cardiovascular

changes. However, [val4]-ANG III has never been evaluated as a potentiai regulator of nasal salt

gland. Gerstberger (observation cited by Simon et al. 1992) reported that [ V ~ ~ I - A N G III failed

to stimulate ANG II-sensitive or ANG LI-insensitive neurons in duck subfornical organs.

However, this does not preclude the presence of other neurons in the area postrerna or OVLT,

which may be responsive to [ V ~ ~ ~ J - A N G III.

From a phylogenetic standpoint, it is important to study different species of birds because

of the novel responses to ANG II. Often these responses are govemed by unique ANG II

receptor subtypes, and also their tissue specific distribution. The recent advent of specific

nonpeptide ANG II receptor antagonists have opened up new avenues for the investigation of

these receptors. However, they have been tested onIy in species within the Order Galliformes

(fowl, quail and turkey). These species show an atypical biphasic cardiovascular response to

ANG II; an initial vasodepressive followed by a secondary vasopressive effect. Therefore it is

important to investigate nonpeptide ATI and AT2 antagonists in a non-gallinaceous species

which displays a typical pressor response to ANG 11, such as the Pekin duck. Moreover,

vasopressor responses to ANG 1 (1-7), [ v ~ ~ ~ ] - A N G III and [ A S ~ ~ , V ~ ~ ~ ] - A N G II have been

measured in some birds but not in the white Pekin duck nor has W ~ ~ ~ J - A N G III been tested for

its possible hormonal regdation of duck nasal salt glands.

It is the aim of this study, to investigate the association between the renin angiotensin

system and the secretory functions of the avian nasal salt gland, using ANG II, ANG III, ANG

I (1-7), and the angiotensin receptor antagonists (CGP 48933, Losartan, PD1233 19, Sade). This

was done by measuring the concentrations of sodium and potassium in the nasal fluid following

salt loading. The composition of the nasal fluid is thought to remain constant with the functional

state of the glands (see Figs. 2 and 3). Since the dmgs used have easily measurable

cardiovascu1ar effects in the avian mode1 (Matsumura and Simon 1990, Nishimura et al., 1994),

their effect on blood pressure was used as an unarnbiguous indicator of physiological activity.

By measuring the secretory and the cardiovascular parameters, 1 hope to shed light on the

identity of ANG II receptor subtypes mediating the observed changes.

MATERIALS AND METHODS

Animak

White Pekin drakes (Anas platyrhynchos) were purchased from King Cole Ducks Ltd.,

Aurora, Ontario, and housed under 12:12hr light-dark cycle in the communal animal care

facilities of the Department of Zoology. They were fed commercial duck grower feed and tap

water ad libitum. Food was withheld for 12 hours prior to each experiment. The ducks were 14.2

4 2.1 weeks old and weighed 3.79 t 0.17 kg (n=12) for the nasal salt gland experiments, and

10.2 f 1.8 weeks old and 3.25 A- 0.27 kg (n=12) for the blood pressure experiments.

Salt gland experiments: A total of 334 nasal fluid samples were collected, using the

methods described below, from a total of 17 ducks that were maintained on duck grower food

and freshwater ad libitum. Some of the ducks were sampled twice following a three day interval

(Figures 2A and B and 3A and B). Five new ducks were used for an experiment in which 1 and

5 nrn01.k~-' doses of [va14]-AN~ III were injected i.v. to determine if the peptide would affect

secretion. These injections were followed by an i.v. injection of 1 nmol.kghl of [ ~ s ~ ' , v a l ~ ] -

ANG Ii, to verify that we salt glands were responsive to the peptide (Figure 4). After a three-

day recovery period the same five ducks were used for testing the response of the nasal salt

glands, to ANG 1 (1 -7) (20 nm01.k~" i-v.). Again it was verified that the glands were responsive

to 1 nmol.kg -' of [ A S ~ ' , V ~ ~ ~ ] - A N G II (Figure 5).

Five new ducks were primed with an i.v. injection of 10 rnl.kg-' of hypertonic NaCl (1000

rnosm01.k~-' water) two days before they were used in the experiment. This was carried out

to increase slightly the rate of nasal fluid secretion. On the day of the experiment, the effect

of 1 nm01.k~" of [AS~' ,V~~]-ANG II i.v. on nasal fluid secretion and on arterial blood pressure

during an i.v. infusion (5 pg.kg%nin") of the mammalian ANG II antagonist, SARILE

([sa' , I l e 8 ] - ~ h J ~ II ) was measured (Figure 6A). Arterial blood pressure was recorded to show

that SARILE had blocked completely the pressor response to [AS~' ,V~~~]-ANG II (Figure 6B).

One new duck was injected with [ A S ~ ~ , V ~ ~ ~ ] - A N G II (2 nm01.k~-' i-v.) (Figure 7) to show that

the nasal salt glands were responsive, that is, that they would stop secreting fluid. Then, after

a 40-minute interval, 20 mgkg-' of the rnammalian ATl antagonist losartan was injected i.v.

Two separate injections of 2 nm01.k~" of [ A S ~ ~ , V ~ ~ ~ ] - A N G II were given 30 and 120 min. after

losartan to show if the response to the peptide had been blocked. (Figure 7). One new duck was

given an i.v. injection of 20 mg.kg-' of the mammalian AT2 antagonist PD 123319. That was

followed 5 and 30 min. later by two successive injections of 2 nm01.k~-' i.v. of [ ~ s ~ ' , V a l ~ ] -

ANG II to show that the response to the peptide had been blocked (Figure 8).

Bloodpressure experiments: Arterial blood pressure was measured in six new ducks

before (baseline), and after each duck had been given an i.v. injection of 0.7 ml of 0.9% saline

(vehicle) (Figure 9). This was followed, in each duck, by the random (in time) i.v. injection of

the following peptides: I nm01.k~-' of [ ~ s p ' , ~ a l ~ 1-ANG II, [ A ~ ~ ' , v ~ ~ ~ ] - A N G II and [Val4]-

ANG m, dso 5 nm01.k~-' of [ V ~ ~ ~ J - A N G III, and 20 nrn01.k~-' of [ A S ~ ' , V ~ ~ ~ ] - A N G 1 (1 -7)

(Figure 9). Six new ducks were used for this experiment which tested the pressor response to

1 nm01.k~'' i.v. of [ A S ~ ' , V ~ ~ ~ ] - A N G 11 before and after a single i.v. injection of SARILE; then

5 minutes and 30 min. after the start of a continuous i.v. infusion of SARILE at a rate of 5

pg.kg-l.min" (Figure 10). Two ducks were used for a second experiment in which the dose of

SARILE was reduced by half (not shown). That is, the birds were given a bolus injection of 25

pg.kgal of SARILE, followed irnmediately by a continuous i.v. infusion of 2.5 pg.kg-'. Test

injections of [ A S ~ ' , V ~ ~ ] - A N G II were identical to those used for the higher dose of SARILE

(Figures 6A ,6B and 10).

The six ducks were allowed to rest for a period of three days before they were used for the

next experiment. Arterid blood pressure was measured before (baseline) and after the injection

of 0.7 ml of saline (vehicle) (Figure 1 1). This was followed by an i.v. injection of 1 nm01.k~-'

of [ ~ s ~ ' , ~ a l ~ ] - ANG II to demonstrate a clear pressor response to the peptide. Next, losartan (10

mg.kg-l i.v.) was injected. 5 and 30 min. later two single injections of 1 nm01.k~-' of

[ A S ~ ' , V ~ ~ ~ - A N G II were administered to show if losartan had blocked the pressor response to

the peptide. After an interval of 45 min., the duck was given an i.v. injection of 10 mg.kg1 of

PD 1233 19. The dmg injection was followed 5 and 30 min. later by two single injections of 1

nm01.k~-' of [ A S ~ ' , V ~ ~ ~ ] - A N G II. Finally, after an additional 45 min. interval, the duck was

injected with CGP 48933 (10 mg.kgS1 i.v). The effectiveness of this marnrnaiian AT2 blocker

was tested by two separate injections of 1 nrn01.k~-' of [ A S ~ ' , V ~ I ~ ] - ANG II given 5 and 30

min. after the drug (Figure Il).

The injection of a dmg or peptide was followed by 0.5 ml of 0.9% saline to clear the

catheter prior to the next injection. Al1 solutions were equilibrated to room temperature (26OC)

before they were injected.

A heparin-f'illed (50 IU heparin per ml of 0.9% saline) dmg delivery catheter (PE 50,

Intrarnedic, Clay Adams, New Jersey, U.S.A) was inserted into the left ulnar vein under

local anaesthesia (2% lidocaine in 0.9% saline). A second heparin-fïlled catheter (PE 50)

was inserted about five centimetres into the right brachial artery. It was used to measure

mean arterial blood pressure. Both catheters were coiled and tucked under the wing where

they were covered with a sterile gauze pad and wrapped with surgical tape to prevent the

ducks from tearing them out. The ducks were then placed in separate holding cages supplied

with food and water ad libitum for a day before they were used for the experiments.

Procedure for collecting nasal fluid

Each duck was placed on the holding board and given an i.v. injection of 10 ml.kg-' of

hypertonic saline solution (1000 rnosm01.k~-' water) to produce an onset of nasal fluid

secretion. Secretion cornrnenced within 5 minutes, and was sustained by a continuous i.v.

infusion (0.97 rnl.min-') of hypertonic saline (1000 rnosrn01.k~-'). Nasal fluid was collected

for 5 minute periods seriatirn as it dripped from the nostrils into a clean 100 ml beaker. At the

end of each 5-min. collection period the nasal fluid was withdrawn into pre-weighed 1 or 3 ml

hypoderrnic syringes (depending upon the volume of fluid). The syringe was re-weighed, and

then the fluid volume was estimated by the difference. Nasal fluid sarnples were then

transferred to 1.5 ml Eppendorf tubes and stored at -20 O C until analysed.

Procedure for measurina arterial blood messure

Systolic and diastolic brachial arterial blood pressures were monitored continuously using

a mode1 1050 BP pressure transducer (UR, Morro Bay, CA, U.S.A) and a Linear Model 1200

single-pen recorder (Linear Instrument, Reno, N V , U.S.A). The transducer and the zero

pressure level were adjusted to the level of the duck's heart. Mean arterial blood pressure

(MABP) was calculated as the sum of the diastolic pressure and one-third of pulse pressure.

Measurement of osrnolality und electrolvte concentrations

Nasal fluid osmolaIity was deterrnined by freezing point depression (Advanced

Instruments micro-osmometer, Model 3MO) and [Na*] and [K*] by flame photometry

(Instrument Laboratories, Mode1 943).

Druas and neptides

Fowl Angiotensin II = [ A S ~ ~ , V ~ ~ ] - A N G II (MW=103 1 S); teleost (eel) Angiotensin II =

[ A S ~ ' , V ~ ~ ~ ] - A N G II (MW=1030.5); Angiotensin Iü (2-8) = [V~I~J-ANG IiI (MW-916.5) ;

Angiotensin 1 (1-7; ~ s n ' , val5, pro7) = ANG 1 (1-7) (MW=883.5) and SARILE = [saJ ,Iles]-

ANG II (MW = 967.6) were al1 supplied by Penninsula Laboratories, Belmont, CA, U.S .A . The

ANG II receptor blockers were supplied as follows: Losartan potassium (DuP 753, MK 954;

MW = 461)-Dupont Merck Research and Development, Wilrnington, DE, U.S.A; PD 1233 19

(MW = 749.4)-Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI, U.S.A; and

CGP 48933 (valsartan; MW = 433)-Ciba-Geigy Ltd, Basel, Switzerland. Xylocaine (2%

lidocaine hydrochloride in 0.9% isotonic NaCl; Astra Pharrnaceuticals, Mississauga, Canada;

Ampicillin sodium, 50 mg per ml isotonic saline, Novopharrn, Toronto, Canada; Heparin

solution (Hepalean-Organon Tekita, Toronto, Ontario, Canada). Al1 peptides and dnigs were

dissolved in 0.9% NaCl except CGP 48933 which was dissolved in a 20% ethanoV isotonic

NaCl (v/v).

Values are means and S.E.M. Linear regressions in Figures 2 and 3 were calculated by

the method of Ieast squares using Statgraphics plus (Version 3.0; Manugistics, hc., Rockville,

MD, U.S.A.). Data in Figures 9, 10 and 11 were compared using an ANOVA followed by

Duncan's multiple range test (Statgraphics). The fiduciary limit was set at P ~ 0 . 0 5 .

Nasal salt alands

Figure 2A shows that there is a positive linear correlation between nasal fluid ma+] and

osrnolality (Y = 1.61X + 153.1; r = 0.87; n = 334; Pcû.001) and between the rate of nasal fluid

secretion and the ma7 (Y = 170.5X + 5 1 1.3; r = 0.48; n = 334; P4.001; Figure 2B). There was

also a positive linear correlation between the nasal fluid secretion rate and [K'] (Y = 4.83 +

12.25; r = 0.41; n = 334; Pe0.001; Figure 3A) and between the [Na+] and [K+] concentrations

in nasal fluid (Y = 0.025X - 0.45; r = 0.76; n = 334; Pd.Qû1; Figure 3B).

Figure 2A. Positive linear correlation between [Na'] and osmolality of fluid secreted by the

nasal salt glands (Y = 1.61X + 153.1; r = 0.87; n = 334; P<O.ûûl).

Figure 2B. Positive linear relationship between the rate of nasal fluid secretion (ml.kg-'Smin-')

and the [Na'] (Y = 170.5X + 51 1.3; r = 0.48; n=334; P-eO.001).

Nasal fluid secretion ( ml.kg -'.5 min -')

Figure 3A. Positive iinear correlation between the rate of nasal fluid secretion (ml.kgh'. 5 min-')

and the [K'] (Y = 4.83X + 12.25; r = 0.41 ; n = 334; P<0.001).

Figure 3B. Positive linear correlation between the nasal fluid [Na'] and [Kf] (Y = 0.025X -

0.45; r = 0.76; n = 334; P4.001) .

Nasal fluid secretion ( rnl.kg -'.5 min -')

Nasal fluid secretion rates usually became stable within 30 min. after commencing the

infusion of 1 0 mosm.kg'' NaCI. Neither the rate of nasal fluid secretion nor its ionic

concentration changed following i.v. injections of 1 and 5 nmol.kg-l of V ~ ~ - A N G III (Figure

4). However, an intravenous injection of 1 nm01.k~-l [ A S ~ ' , V ~ ~ ~ ] - A N G II was followed by a

rapid and statistically significant decrease in nasal fluid secretion frorn 0.23 + 0.06 to 0.05 -1-

0.01 ml.kgml during the 5 min. collection period imrnediately after the peptide was injected

(Figure 4). There was some variation in the response to [AS~ ' ,V~~~] -ANG II. In three ducks the

glands shut off completely and there was no secretion during the 5 min. period following the

[ A S ~ ' , V ~ ~ ~ ] - A N G II injection. Then secretion restarted and gradually increased to the pre-

injection rate (Figure 4). Neither the 1 nor 5 nrn01.k~-' doses of [va14]-AN~ III nor the 1

nrn01.k~-' doses of ANG II had any significant effect on the [ ~ a q or [Kf] or osmolality of the

nasal fluid samples.

Figure 4. Effects of [AS~' ,V~I~]-ANG II and [V~I~I-ANG III on nasal salt gland secretion in

freshwater Pekin ducks during and i.v. infusion of hypertonie saline (1000 rnosrn01.k~-l) at a

rate of 0.97 m h i n -'. Values are means and S.E.M. n = 5.

Infusion of 1OOO mOsm NaCl (0.97 mlsdn*' i.v.)

20 40 60 80 I O 0 120 140

Time (min)

Twenty nmol.kge' of ANG 1 (1-7) i.v. had no measurable effect on the either the rate of

nasal fluid secretion or its electrolyte concentrations (Figure 5) whereas there was a clear

response to 1 n r n o l . ~ ' of [ A S ~ ~ , V ~ ~ ~ ] - A N G II. The latter peptide lowered the secretion rate

but ciid not affect ionic concentrations (Figure 5).

The effectiveness of the mammalian ANG II blocker SARILE on'the response by the nasal

salt glands to [AS~ ' ,V~~~] -ANG II is shown in Figure 6A. D u h g an i.v. infusion of 1000

rnosrn01.k~-' of hypertonie saline the injection of 1 nm01.k~" of [AS~ ' ,V~~] -ANG II virtually

shut off NFS. Then, following a single i.v. injection of 50 pg.kg-l of SARILE, and immediately

after the start of an i.v. infusion of 5 pg.kg-'.min" of SARILE (Figure 6A) the nasal salt glands

shut off and remained in this state of inactivity for as long as the SARILE i.v. infusion

continued. When the infusion of SARILE was stopped, the glands began to secrete fluid at a

progressively increasing rate (Figure 6A). Figure 6B shows that, during the infusion of

SARILE, there was a total block of the pressor response to [ A S ~ ~ , V ~ I ~ ] - A N G II. In the two

ducks in which the dose of SARILE was halved, the pressor response to [AS~ ' ,V~~~] -ANG II

was reduced by about 60%, yet there was still no NFS during the continuous i.v. infusion of

SARILE.

Figure 5. Effects of ANG 1 (1 -7) and [AS~',V~I~]-ANG II on nasal salt gland secretion during

and i.v. infusion of hypertonic saline (1000 rnosrn01.k~-') at a rate of 0.97 ml-min -'. Values

are means and S.E.M. n = S.

ANG 1 (1-7) ANG II

Figure 6A. Effects of the mammalian angiotensin antagonist SARILE on nasal salt gland

secretion and the response by the nasal salt glands to two i.v. injections of [ A ~ ~ ' , V ~ I ~ ] - A N G

Il given before and after the antagonist. Values are means and S.E.M. n=S.

. Time (min)

Figure 6B. Pressor response to [ A S ~ ~ , V ~ ~ ~ ] - A N G II before and after an i.v. infusion of the

mammalian angiotensin antagonist SARILE. n=l

Arterial Blood Pressure (mmHg)

Figure 7 shows that the mammalian ATl receptor antagonist, losartan failed to block the

inhibitory effect of [AS~',V~I~]-ANG II on the rate of fluid secretion. The initial ANG II

injection (2 nrn01.k~-' i.v.) was followed by an immediate 80% reduction in nasal flow, leading

gradually to a complete shut-off for 10 minutes. After a further 10 minutes the flow rate

returned to the baseline level. Then losartan (20 mg.kgm1 i.v.) was injected. At this dose if the

dmg was to have any meaningful effect it would have had, therefore no other ducks were used

the same reasoning goes for PD 1233 19. The second (30 min post-losartan) and third (1 20 min

post-losartan) injections of ANG II (2 nrn01.k~-') also stopped nasal fluid secretion, with no

evidence of receptor blockade (Figure 7). Moreover, losartan had no effect of nasal fluid Na+,

Kf or osmolal concentrations.

The effect of the marnrnalian AT2 receptor antagonist, PD 1233 19, on the NFS is shown

in Figure 8. In the prelirninary experiment, 2 nrn01.k~-' of [ A S ~ ' , V ~ ~ ~ ] - A N G II i.v. shut off

secretion by the nasal salt glands. After a recovery period, the duck was given an i.v. injection

of 20 mgkg-' of PD 1233 19. Two subsequent injections of 2 nmol .kg-' of ( A S ~ ' , V ~ ~ ~ ] - A N G

II were administered 30 and 120 min. after the initial injection of ANG II shut off secretion in

the usual manner (Figure 8). The response to [ A S ~ ~ , V ~ ~ ~ ] - A N G II had not been blocked by PD

,123319.

Figure 7. Nasal salt gland secretion following injections of [ A S ~ ~ , V ~ ~ ~ ] - A N G il i.v. which

were administered both before and after a single i.v. injection of the marnmalian ATl antagonist

losartan (n = 1).

Osmolality (mosmol. kg" water) [K'] (mm01.1'~) [Na'] (mmol.1-')

Nasal fluid secretion (ml. kg-' 9 m i d )

I----

m m . A W l f (2 nmobkg-1

ANG m.

i nmol

Figure 8. Nasal salt gland secretion following injections of [AS~' ,V~I~]-ANG II i.v. which

were administered both before and after a single i.v. injection of the mamrnalian AT2 antagonist

PD 123319.

Infusion of lûûû mOsm NaCl (OS7 ml&-' i.v.) .

T h e (min)

Blood pressure exaeriments

The resting (baseline) mean arterial blood pressure (MABP) was 137 + 5 mmHg in

conscious ducks (Figure 9). Intravenous injections of 1 nmol.kgml of bird angiotensin

AS^' ,valS J-ANG IQ and teleost angiotensin ( [ ~ s n ' ,v~~']-ANG 11) increased the MABP by

about 32% and IO%, respectively (P<0.01 and P d 0 5 compared with vehicle injected controls;

ANOVA followed by Duncan's multiple range test). Note also that the difference between

mamrnalim and bird angiotensins is statisticdly significant (P~û.05). In contrast, neither 1 nor

5 nrn01.k~-' doses i.v. of [ V ~ ~ I - A N G III nor a higher dose of 20 nm01.k~-' of ANG 1 (1 -7) i.v.

had any measurable effect on MABP (Figure 9).

The effect of the mammalian angiotensin blocker SARILE on the pressor response to

[AS~' ,V~I~]-ANG I I is illustrated in Figure 10. The pressor response to 2 nrn01.k~" of

[AS~ ' ,V~~~] -ANG II i.v. was significant before but not after the injection of the blocker. When

[ A S ~ ' , V ~ ~ ~ ] - A N G II was injected immediately after the i.v. injection of 50 pg.kg-' of SARILE

the pressor response was blocked completely (Figure 10). Two further injections of 1 nrn01.k~-'

of [ A S ~ ' , V ~ ~ ] - A N G II given 5 min. and 30 min. &ter the start of an i.v. infusion of SARILE

(5 pg.kg-'.min-') failed to increase MABP due to the continued blockade (Figure 10). It is

noteworthy to mention that there was a characteristic agitation of the duck 2-4 min. following

the i.v. injection of the SARILE (Figure dB).

Figure 9. Mean arterial blood pressure in Pekin ducks before and after random i.v. injections

of [ ~ s n ' , v ~ ~ ~ ] - A N G II, AS^' ,V~']-ANG II, [val4]-ANG III, and [ ~ s n ' ,v~~']-ANG 1 ( 1-7).

Values are means and S.E.M. n = 6. NS = not significant compared with vehicle-injected

control. A P c 0.01 compared with vehicle-injected control and [ ~ s n ' , v ~ ~ ~ ] - A N G II. * P c

0.05 compared with vehicle-injected control.

Figure 10. Pressor responses to [ A S ~ ~ , V ~ ~ ~ ] - A N G II (1 nrn01.k~-') injected i.v. before and afler

blockade with [ s ~ ~ ' , I ~ ~ ~ ] - A N G II (SARILE). Values are mean artenal blood pressure and

S.E.M. n = 6. NS = not significant compared with vehicle-injected control. A P < 0.01

compared with vehicle-injected controI.

MEAN ARTERIAL PRESSURE (mmHg)

The failure of mamrnalian ATi and AT2 blockers to block the vasopressor response to bird

ANG II is illustrated in Figure 11. Losartan, PD 123319 and CGP 48933 dl failed to dampen

the full pressor response to single i.v. injections of 2 nmol.kg-i of [AS~',V&]-ANG II at 5 and

30 minutes post-blocker injection.

Figure 11. Mean arterial blood pressure and S.E.M. in Pekin ducks following a series of i.v.

injections of AS^', v ~ ~ ~ ] - A N G II (2 nrnol .kgm1) administered before and after successive i.v.

injections of the mamrnalian angiotensin antagonists Losartan (AT1), PD 123319 (AT2) and

CGP 48933 (AT,). n=6. NS = not significant compared with vehicle-injected control. A P <

0.0 1 compared with vehicle-injected control.

MEAN ARTERIAL PRESSURE (mmHg)

DISCUSSION

The renin-angiotensin system (RAS) embodies a cascade of enzymes (renin and

angiotensin-converting enzyme (ACE), aminopeptidases), protein and peptide substrates

(angiotensinogen, ANG 1 and E) leading to the biosynthesis of active peptides including ANG

1 (1-7), ANG II, ANG III (2-8) and ANG IV (3-8). The biochemistry and physiology of the RAS

has generated tremendous interest in comparative endocrinology (Wilson 1984; Nishimura

1980; Simon et al., 1992). In birds, ANG 1 (1- 10; Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Ser-Leu)

is converted enzyrnatically @y ACE) &O ANG II (1-8; Asp-Arg-Val-Tyr-Val-His-Pro-Phe)

which, in tum, is converted by aminopeptidase to [ v ~ ~ ~ J - A N G IU (2-8; Arg-Val-Tyr-Val-His-

Pro-Phe) (Nakayarna et al., 1973; Peach 1977; Takei and Hasegawa 1990).

1 have studied the in vivo effects of some of these peptides and their putative receptor

antagonists in an avian model, the white Pekin duck (Anas plaîyrhynchos). It is important to

study different species of birds because of the interesting dichotomous response to ANG II in

Class Aves. For example, gallinacious birds such as fowl and quai1 have a pronounced initial

vasodepressor response to ANG II followed by the typical vasopressor response. This

vasodepressor response may be due to a unique ANG II receptor. However, specific nonpeptide

ANG II receptor antagonists have been tested only in species within the order Galliformes (fowl,

quail and turkey). Therefore it is important to investigate nonpeptide ATi and AT2 antagonists

in a non-gallinaceous species which displays a typical pressor response to ANG II. Moreover,

vasopressor responses to ANG 1 (1-7), [ v ~ ~ ~ ] - A N G III and [ ~ s n l , ~ a l ~ ] - A N ~ II have been

measured in some birds but not in white Pekin ducks nor has [ v ~ ~ ~ ] - A N G III been tested for

its possible hormonal regulation of duck nasal salt glands.

Nasal salt ghnd response to Angiotensins II and III:

There was rapid and pronounced inhibition of NFS following the i.v. injection of ANG

II (Figure 4) which is in line with the early experirnents which showed that NFS is attenuated

by ANG JI (Hamme1 and Maggert 1983; Butler 1984; Butler et al., 1989; see Simon et al., 1992

for review). In some lower vertebrates, for example, freshwater eels (Anguilla rostrata) the

pressor response to an i.v. injection of [val4]-ANG Iü is significant compared to that of

[ ~ s n ',V~I~]-ANG II (Butler and Oudit 1995). Morwver, [ V ~ ~ J - A N G III effectively stimulated

ANG II-responsive neurons in the circumventricular region in cats (Felix and Schlegel 1978)

and rats (Harding and Felix 1987) but not ducks (Matsumura and Simon 1990). Finally, both

ANG II and ANG ID induced a pressor and dipsogenic response in rats (Wright et al., 1985).

[va14]-AN~ III has never been tested for its possible attenuation of nasal salt gland

secretion in ducks until now. Figure 4 shows that [V~~~I -ANG III did not stop NFS in the ducks.

Nevertheless, this finding is compatible with the absence of increased electrical activity

following the in vitro application of [ V ~ ~ ~ J - A N G III (100 pmol.1-l) to a duck hypothalamic

neuronal preparation (Gerstberger, unpublished observation, cited by Simon et al., 1992). In

addition, [val4]-ANG III did not affect either ANG &sensitive or ANG II-insensitive neurons

within the circurnventricular region (subfornical organ) of the duck brain (Matsumura and

Simon 1990). It appears that [ A S ~ ' , V ~ ~ ~ ] - A N G II may be the only peptide hormone of the RAS

which reduces nasal salt gIand activity in birds.

Ionic concentrations and osmolality of nasalfluid..

There was a positive linear relationship between the nasal fluid [Na7 and its osmolality

extending over wide range of [Naq (400-750 rnmol.l~') and osmolal concentrations (830-1 330

mosrn01.k~") (Figure 2A). This was not an unexpected finding since Na* and Cl- ions are the

main osmolytes in duck nasal fluid (Butler et aI., 1989) and their concentrations determine

negative free water clearance (Butler 1985), the rate at which water is gained following the

ingestion of a salt load. A sirnilar positive Iinear correlation between m a 7 and [CI-] and nasal

fluid secretion rates was observed in domestic geese which were reared on freshwater then given

a stomach load of hypertonic saline (Hanwell et al., 1971). In contrat there was a negative

linear correlation between flow rates and m a 7 and [Cl1 within individual birds (Hanwell et al.,

197 1). In the present experiments there was also a positive Iinear correlation between the nasal

fluid secretion rate and the [KT (Figure 3A) and the ma? and [Kq (Figure 3B) which implied

CO-transport of Na* and K*.

The most recent hypothesis argues that ion secretion by cells within the compound tubular

salt glands (Butler et al., 1993) involves the transcellular active transport of CI', wliich is

associated with a basolateral electroneutral furosemide-sensitive Na+-K+-2CI- cotransporter

driven by basolateral electrogenic N~+-K+ ATPase (Lowy et al., 1989; Torchia et al., 1992;

Gerstberger and Gray 1993) and an apical cystic fibrosis transmembrane conductance regulator

(CFïR)-like Cl' channel (Ernst et al., 1994; Martin et al., 1994). Lowy et al., (1989) also found

that application of ]Ba++ on the apical side of the ce11 inhibited the outflux of ions, which

suggested that there rnight be a K' channel on the apical membrane (Figure 12).

Figure 12. Proposed mechanism of cellular transport of ions in the avian nasal salt gland tissue.

Represented are two principal cell. (Modified from Lowy et al., 1989 and Gerstberger and Gray

1993)

Basolateral membrane Apical

membrane

Furosernide- 2 Cl

Bau, TEA

Oubain A

In these experiments the positive linear relationship between the [Na'] and [Kr in the

nasal fluid (Figure 3B) is compatible with the hypothesis that the cellular action of a Na%'-

2C1- cotransporter is fundarnentally important for Salt gland secretion. This theory is also

consistent with the idea of a fixed relationship between the Na%+ ratios of plasma and salt

gland fluid in the Pekin duck (Simon and Gray 1991). In addition, it was proposed that the

paracellular pathway might have a similar permeability (leakage) to Na+ and K+. Various

hormones including ANG II, aidosterone, atriai natriuretic peptide and arginine vasotocin are

not involved in the short term regulation of rend K+ transport which has been shown to be

independent of a distal tubular mechanism (Simon and Gray 199 1). The salt glands may be an

alternative and important hormonaily controlled extrarend transport site for the regulation of

extracellu~ar K+ concentrations.

Characteristics of ANG II receptors, responses tu ANG 1 (1-7):

The reason behind using a high dose of ANG 1 (1-7) (20 nm01.k~-') was so that the

reduced potency of asparagine subs'titution would not be responsible for a lack of pressor effect

should it occur. ANG 1 (1-7; des-phes ANG II) has been shown to be an inactive peptide in d l

vertebrate species tested so far: freshwater eels (Butler and Oudit 1995), duck (Figures 5 and

9), pigeon (Evered and Fitzsimons 1981) and marnmals (Peach 1977). This commondity

illustrates an important phylogenetically conserved structural requirement for the activation of

the ATI receptor, namely the presence of phenylalanine as the C-terminal residue, in al1

vertebrates. MolecuIar studies have identified the -COOH functional group as the interacting

species with a positively charged amino acid residue in the agonist binding pocket of the AT1

receptor. Every angiotensin I has a phes residue therein providing strong biochemical evidence

for the importance of this residue in mediating the agonist activity of ANG II and III. In

mammals, locally produced ANG I (1-7) has been shown to have important effects in the

brainstem but these responses are predominantly mediatecl by the AT2 subtype (Diz and Ferrario

1 996).

Effect of SARZLE on the response by the nasal salf glands and cardiovascuhr system tu

AS^: V&ANG II:

SARILE was selected as an antagonist of [ A S ~ ' , V ~ ~ ] - A N G II because it is known to be

one of the more potent analogues of ANG II in birds (Murphy et al., 1993) where angiotensin

receptors may be structurally related to but pharmacologically distinct from those of the

Marnmalia. A bolus injection of 50 pg.kg-' of SARILE inhibited completely the pressor

response to [AS~ ' ,V~~~] -ANG II (Figures 6B and 10). However, it acted concumntly as an

ANG II agonist and shut off completely the salt glands (compare Figures 4,s and 6A) during

an i.v. infusion of hypertonie saline. This observation was unexpected because earlier in vitro

recordings of discharge rates in duck subfornical organ (SFO) neurones showed that neuronal

activity increased in response to superfused ANG II (10'~ M). These SFO neurons lost

responsivity when ANG II and SARILE ( 1 0 ~ M) were CO-infused implying that receptors in the

SFO had been blocked by SARILE (Matsumura and Simon 1990).

Other studies have shown that ANG II acts on the central nervous system and induces

drinking in birds. For exampIe, ANG II-induced drinking in pigeons was inhibited by

intracerebroventricular injections of both SARILE and [ S ~ ~ ' , L ~ U ~ ] - A N G II (De Caro et al.,

1982) whereas a third analogue, [s~~',A~z?]-ANG II failed to block the dipsogenic response.

SARILE partially blocked the ANG II-induced drinking in turkeys (Denbow 1985). These

findings in other birds, led us to expect that SARlLE would act as an antagonist and block the

inhibition of nasal salt gland that is known to follow intravenous injections of ANG JI (Hamme1

and Maggert 1983; Butler 1984). Instead, SARILE acted as an agonist and its administration

(Figure 6A) was followed by a cornplete and prolonged inhibition of the nasal salt glands for

the entire period of SARlLE infision (Figure 6A). When the SARILE infusion was stopped,

nasal fluid secretion started again and the flow rate increased gradually to the preinjection level

(Figure 6A). The dose of SARILE was selected because (a) it had been used successfully to

nearly block the pressor response to ANG If in chickens (Nishimura et al., 1982) and (b)

because it blocked fully the pressor response to ANG II in the present experirnent (Figures 6B

and 10). When the dose of SARILE was reduced by 50% in two ducks, that is, an initial bolus

i.v. injection of 25 yg.kg-' followed immediately by a constant i.v. infusion of 2.5 pg.kg-l.min-'

using the same time course as described in Figure 6A. The pressor response to an i.v. injection

of. 1 nrnol.kg*l of ANG II was reduced by about 60% yet the nasal glands shut off completely

during SARILE perfusion as with the higher dose.

The use of the earlier SARILE-based ANG II receptor antagonists has also confirrned

distinct binding praperties of avian ANG II receptors. In birds, SAIClLE is an effective

antagonist of the in vivo pressor and depressor responses to ANG II (Nishimura et al., 1982;

Khosla et al., 1983; Takei and Hasegawa 1990) and in vitro binding of ANG II to myocardial,

VSM and adrenal steroidogenic celluIar ANG II receptors (Baker and Aceto 1989; Stallone et

al., 1989; Kocsis et al., 1995). Other closely related analogues such as [ ~ a r l , ~ l a ~ ] - and

[s~~ ' ,T~~' ] -ANG II were much less effective in biocking the pressor/depressor and dipsogenic

responses in birds (Moore et al., 198 1 b; Evered and Fitzsimons 198 1 ; Nishimura et al., 1982;

Takei and Hasegawa 1990). These responses in birds are different than in mammals where

[~ar',Ala']- and [s~~' ,T~~*]-ANG Il are the most potent saralasin-related ANG II receptor

antagonists known (see Peach 1977 and Stallone et al., 1989 ; Evered and Fitzsimons 198 1).

Avian ANG II receptors are apparently more sensitive to the nature of the arnino terminal

residue. [AS~',V~I~]-ANG lI has less than 25% of [ A S ~ ~ , V ~ ~ ~ ] - A N G II depressor/pressor action

in the duck (Figure 9), fowl (Khosla et al., 1977; Nishimura et al., 1982), quail (Takei and

Hasegawa 1990) and pigeon (Evered and Fitzsimons 1981). Replacement of AS^' by Sar is

followed by a 70% loss of pressox activity (Nakamura et al., 1982). Third, unlike the situation

in the rat, [ I l e 8 ] - ~ ~ ~ II competes directly with the endogenous agonist ( [AS~ ' ,V~~~]-ANG II)

for the ANG II receptor in the fowl since the respon'se was unaffected by prior ACE inhibition

(Nishimura et al., 1982).

The differential inhibition of the ANG II-mediated vasopressor effect (antagonistic effect)

in cornparison with the central inhibition of the nasal salt gland response (agonistic effect) using

SARLE is significant because it illustrates a pharmacological dimorphism of the ANG II

receptors controlling these two distinct physioIogical responses in the white Pekin duck.

Eflect of mammaliun ATl and AT2 receptor anhzgonists on the response by the nasal salt

glanas und cardiovusculrv system to [ A S ~ ' , V ~ ~ ] - A N G ZZ:

The prototypical antagonists for the ATI receptor are biphenylimidazole compounds,

which possess a tetrazole ring. DuP 753 (losartan) is such a compound. AT2 antagonists are

tetrahydroimidazopyridhes, represented by PD 123 177 and PD 1233 19 (see Timmermans et al.

1993; Smith and Timmermans 1994 for reviews). CGP 48933 (valsartan) is one of the first

nonhetereocyclic ANG II receptor antagonist to be synthesised and is a potent long-acting ATi

antagonist (Criscione et al., 1992) with a rapid onset of action. The experirnents here are the

first to test CGP 48933 in a non-marnmalian vertebrate animal (Figure 1 1). In addition, ATi

receptors are inhibited by the sulphuryl reducing agent, dithiothreitol, while AT2 receptors are

potentiated by dithiothreitol and inhibited by the peptide CGP 421 12A. ATI receptor activation

can account for virtudly dl of ANG II effects (Timmermans et al., 1993; Smith and

Timmermans 1994). Sulphuryl reducing agents were able to dissociate the binding of ANG II

in the duck's hypothalamus and amygdala-cornplex preparation indicative of ATl-like binding

sites (Gerstberger unpublished observation; cited by Simon et al., 1992). However, the

marnrnalian ATl and AT2 antagonists, described above, do not block the physiological responses

to ANG II in ducks or they are largely ineffective. For example, DuP 753 had only a feeble

inhibitory effect on ANG II sensitive subfomical organ neurones in ducks (Schafer et al., 1993).

The lack of antagonism of ANG II actions cannot be due to inadequate access of the antagonist

since the central areas that are responsible for ANG II action lack a blood-brin barrier.

Moreover, the ANG Ii binding sites in turkey adrenal steroidogenic cells were inhibited by

dithiothreitol but were blocked poorly by losartan (Kocsis et al., 1994).

Mammalian ATI and AT2 receptor antagonists not only failed to block the actions of

ANG II in ducks but also in fowl. Neither the in vivo vasodepressor or vasopressor actions of

the peptide nor its in vitro aortic relaxation were blocked following exposure to 10 mg.kgi of

losartan and PD 1233 19 (Nishimura et aI., 1994). Binding studies using losartan, PD 123 177

and PD 123119 in the fowl and turkey have shown that the ANG II receptors are

pharmacologically distinct from the typical mammalian ANG II receptors (Nishimura et al.,

1994; Kocsis et al., 1994). It is unlikely that the unique nature of avian ANG II receptors are

related to ontogenic andfor postnatal changes in the receptors. Both functional and radioligand

binding studies suggest that the angiogenic (growth prornoting) action of ANG II in the chick

embryo was unaffected by losartan and PD 1233 19 but were inhibited by the peptidergic AT2

antagonist CGP 421 12A (Le-Noble et al., 1993). A study of the signdling pathways induced by

ANG II is further evidence to support the novel form of the avian ANG II receptors. In turkey

adrenocortical cells, the ANG II-mediated elevations in intracellular [Cau] and aldosterone

production are not coupled temporally but are regulated by distinct ANG II receptor subtypes

or isornorphs (Kocsis et al., 1995). There is evidence for a heterogeneous pattern of the ANG

II receptor system in the fowl. ANG II receptors in the vaccular smooth muscle (VSM; aortic)

membrane displayed responses to cations and a guanine nucleotide that were different from the

typical mamrnalian ANG II receptors (Stallone et al., 1989). In contrast, Baker and Aceto (1989)

failed to demonstrate an atypicd binding pattern for the myocardial (atdal) ANG II receptors

in the fowl. ANG II binding to myocardial receptors was modulated by cations and a guanine

nucleotide consistent with the mammalian pattern. These receptors were coupled to both

mechanical activity (inotropic effect) and phosphoinositide hydrolysis (Baker and Aceto 1989).

As such, the significance of the contrasting effects of divalent cations and guanine nucleotide

on the avian VSM and myocardial ANG XI receptors may be simplyan indication of a tissue-

specific variation in the biochemical nature of the ANG II receptor in the fowl. Indeed, the VSM

ANG Ii receptors in birds have not been ascribed a definite function (Nishimura et al., 1994)

but may be invoIved in the endothelium-dependent vascular relaxation (Stallone et al., 1989).

In addition, although [II~']-ANG II (non-native) is twice as potent as [V~']-ANG II (native) at

the VSM ANG II receptors in the fowl (Stallone et al., 1989), both peptides are equipotent

mediators of the pressoddepressor responses in the fowl and quail (Nishimura et al., 1982;

Khosla et al., 1983; Takei and Hasegawa 1990) and in binding to ANG II receptors on adrenal

steroidogenic cells (Kocsis et al., 1994).

My experiments may be summarized as follows:

1. The present observations support the theory that duck nasal salt glands possess a N~+/K+

CO-transport system.

2. Marnrnalian AT1 and AT2 blockers did not attenuate the pressor response to ANG II

which suggested that bird ANG II receptors differ from those of marnrnals.

3. SARILE blocked the pressor response to ANG II but shut off the nasal salt glands

irnplying that it was an agonist in the CNS. These observations supported the theory that

ANG II receptors in duck sympathetic nerve endings and chromaffin (adrenal) cells

differ from those in the circumventricular organs of the brain.

FUTURE RESEARCH

It has been shown in the present study that the ANG II receptors responsible for the

attenuation of nasal salt gland secretion may be distinct fiom the ANG II response mediating

the vasopressor response in the Pekin duck. The mechanisms underlying the inhibiting effect

of ANG II on the glands and the exact identity of the receptor subtype responsible for this

phenomenon are not completely understood. Future research should be devoted to understanding

more preciseIy the biochemical characteristics of circumventncular ANG II receptors, including

their amino acid sequences. The developrnent of specific antagonists for these receptors could

lead to the research for similar receptors in a mammalian model.

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