EFFECTS OF LACTIC ACID ON BRONCHOMOTOR

TONE N THE NEWBORN

A thesis submitted to the Department of Physiology

in conformity with the requirements for the degree of

Master of Science

Queen's University

Kingston, Ontano, Canada

July, 1998

copyright O Michad A Nault

National Library Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services services bibliographiques

395 Weüingtm Street 395. rue Wellington OttawaON K 1 A W OttawaON KlAONLS Canada canada

The author has granted a non- exclusive licence allowing the National Library of Canada to reproduce, Ioan, distribute or sell copies of this thesis in microform, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or otherwise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la fome de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

Abstract

Stimulation of the pulrnonary C-fibre vanilloici receptor (VR 1) with capsaicin

(CAPS) evokes reflex bronchoconstnction in both the adult and newborn dog.

Subsequent studies have suggested that lactic acid (LA) acts as an endogenous ligand of

C-fibre ai3erents (Lee, Morton and Lundberg, 1996), although preliminq studies

suggested LA was unable to evoke bronchoconstriction in the newbom @ucros and

Trippenbach, 199 1). We tested the hypotheses that LA behaves as an endogenous C-fibre

stimulant in the newborn to cause bronchoconstnction and that LA causes reflex

bronchoconstriction via the same pulmonary C-fibrdVR1 receptor mechanisrn as CAPS.

Right hart injection of LA (0.4 mmol-kg") caused a s i d c a n t increase (49 I 11%) in

lung resistance (RL) that was atropine-sensitive (reduced by 80 1 1%; P < 0.05). Right

heart injections of CAPS (25 pg-kg-') caused a signifiant increase (41 f 8.5%) in I& that

was reduced by 83 * 5%, (P < 0.0 1) during infusion of the cornpetitive CAPS-antagonist

capsazepine (500 pg-kg*'-min). The CAPS response returned toward the baseiine

response (7 1 I 22%) 1 5 min post capsazepine infusion. In contrast to CAPS, LA-evoked

increases in & were unaBiected by CAZP (reduced by 16 * 12%, P > 0.05). We

conclude that: 1) C-fibre stimulation with LA causes reflex bronchoconstriction in the

newbom dog, 2) CAZP reversibly antagonizes reflex bronchownstriction elicited by right

hart injection of CAPS in the newbom dog, presumably by acting at the C-fibre

"capsaicin" receptor and 3) LA-induced reflex bronchoconstriction is not attenuated by

CAZP, suggesthg that CAPS and LA evoke reflex bronchoconstriction by stimulating

dinerent C-fibre receptor signal transduction mechanisms, or by stirndating different C-

fibre subpopulations.

Statement of Co-Authorship

Under the guidance of Dr. 3. T. Fisher, Michael A Nault designed, performed and

analyzed the experiments descnbed in this thesis. Original drafts of al1 text were written

by Michael Nault. The finished produa includes editorial suggestions by Dr. Fisher.

Aclcnowledgments

1 would like to extend a heart-felt thanks to the faailty and statfin the department

of physiology, both past and present. They have contributed to a memorable experience

in the department. Special thanks to Ila Dalcourt for al1 her help in and out of the

teaching labs. To the students and post doc 1 have worked dong side with in the Fisher

lab, thanks for the Company, conversation and good times. Thanks as well to the Fisher

clan for welwming me into their home and aiiowing me to monopolize John's time.

1 am indebted to Sandra Vincent for far more than ber technical expertise. Her

patience with, and contributions to, my progress in solution chemistry (what are the units

of molecular mass?) and Fnday morning surgeries were invaluable. Beyond lab support,

her fnendship and tolerance of my mid-aftemoon mental lows have contributed immensely

to the enjoyment of my graduate work.

Lastly, 1 would l i e to thank my supervisor John Fisher for g i v i ~ g me the

oppominity to pursue my research hterests in physiology. His mentorship, patience,

understanding, Cendship and confidence in my abilities over the past two years have

contributed to my graduate experience beyond masure. One last thought, what is it you

used to always Say about administration?

Financial support for this project was generously provided by the Ontario

Thoracic Society and by a Queen's Graduate Fellowship. The research upon which this

thesis is based was supported by a grant from the Medical Research Council to Dr. Fisher.

This thesis is dedicated in Ioving memory of Gwendolyn Nault (1 9 12- 1997).

Table of Contents

........................................................................................................................... Abstract i ... ....................... Staternent of Co-Authorship ........................................................... .... 111

..................................................... Acknowledgrnents .. iv Table of Contents ............................ .... .................................................................... vi

... List of Figures ............................................................................................................. viii

List of Tables ................................................................................................................. ix List of Abbreviations .................................................................................................... x

Chapter 1

Introduction

Introduction ................................................................................................................... 2

Anatomy and Development ............................................................................................ 4 The Tracheobronchial Tree ............................................................................... - 4 Airway Smooth Muscle .................................................................................... - 6 Innervation .................... .... 8 ..........................................................................

Efleerent Innervation ................................................................................ 8 A fferent Innervafion ............................................................................... 13

h a y Reflexes ........................................................................................................... 17 Protective and Defensive Reflexes .................................................................... 17 C-Fibre-Mediated Bronchomotor Reflexes .................................................. 19 The Capsaicin Receptor .................................... ... ..................................... 2 2

Chapter 2

Methods

Preparation .................................................................................................................. 30 Surgery ............................ .. .......................................................................... -30 Analysis of Pulmonary Mechanics ................................................................... -32

Protocois ..................................................................................................................... 34 ................................................... Effect of Lactic acid on Bronchomotor Tone 34

Effect of Osmolality on Bronchomotor Tone ..................................................... 36 Capsmepine Dehery Method .......................................................................... -36 E f f i of capsazepine infusion on capsaicin- and lactic acid-induced reflex

............................................................. bronchoconstriction in the newborn - 3 8

Chapter 3

Results

Lung Mechanics Response to Lactic Acid ..................................................................... -43

Effect of Osmolality on Bronchomotor Tone .................................................................. 43

Lung Mechanics Responses During Capsazepine ............................................................ 46

Chapter 4

Discussion

Discussion ..................................................................................................................... 60 Potential Mechanisms of Action ........................................................................ 66 Summary .......................................................................................................... 68

References .................................................................................................................... -69

VITA ............................................................................................................................ 79

List of Figures

. ...........................................................-............... Figure I Imemation to the airways 9

. Figure 2 Experimental set.up ................................................................................... 31

Figure 3 . Pulmonary mechanics andysis .................................................................. - 3 3

Figure 4 . Effect of dosage on the peak resistance response to lactic acid ................... 35

Figure 5 . Expenmental protocol .............................................................................. -39

Figure 6 . Breath-by-breath mechanics response to lactic acid pre and ............................................................................................. post atropine 44

Figure 7 . Average maximal mechanics response to lactic acid in 0.2 and 0.4 ml ....... -45

Figure 8 . Acute breath-by-breath response of mean lung resistance to capsazepine ............................................................................................... 48

Figure 9 . Cardiopulmonary response to capsaicin pre and post capsazepine .............. 49

Figure 10 . B reath-by-breath resp onse of puimonary mechanics to capsaicin ............... -50

Figure 1 1 . Mean peak mechanics responses to capsaicin ............................................ 5 1

Figure 12 . Cardiopulmonary response to lactic acid pre and post capsazepine ............. 52

Figure 13 . Breath-by-breath mechanics response tu lactic acid .................................... 53

Figure 14 . Average maximi response of pulrnonary mechanics to lactic acid ............. -54

Figure 15 . Possible mechanism of C-fibre activation by H+ ......................................... 67

vii

List of Tables

. Table 1 Baseline values of lung mechanics ....................................................... 5 5

. .........*. Table 2 Latency and time to peak of lung resistance response to lactic acid -56

........................... . Table 3 Latency and time to peak for respome to lung resistance 57

. ............ ....................... Table 4 Heart rate surnrnary: Capsazepine protocol .. 5 8

List of Abbreviations

ACh

ASIC

ASM

BPD

CUF

ca2+

CAPS

CGRP

DRG

ECG

FRC

5-hydrolsrtryptamine

acetylcholine

area postrema

acid-sensing ionic channel

airway smooth muscle

b!ood pressure

bronchopuimonary dysplasia

dynamic lung cornpliance

calcium ion

capsaicin

capsazepine

calcitonin gene-related peptide

carbon dioxide

dorsal motor nucleus of the vagus

dorsal root ganglion

electrocardiograrn

dynamic lung elastance

fiinctional residual capacity

hydrogen ion

hart rate

HRP

2.p-

i. v.

LA

MX

NA

Na'

NaCl

NANC

NKX

N ' A

NO

NTS

PTP

PaCOz

Pa02

PBG

PG's

PGE2

PGF2a

RL

RAR

horseradish peroxidase

intraperitoneai

intravenous

lactic acid

muscarinic receptor subtype O[ = 1,2, 3, 4, or 5)

nucleus ambiguus

sodium ion

sodium chloride

non-adrenergic non-choiinergic

neurokinin receptor subtype (x = 1 or 2)

neurokinin A

nitric oxide

nucleus tractus solitarius

transpulmonary pressure

arterial partial pressure of carbon dioxide

artenai partial pressure of oxygen

p heny lbiguanide

prostagiandins

prostaglandin E2

prostaglandin FZa

inspiratory lung resistance

rapidly adapting receptor

slowly adapting receptor

store-operated charnel

substance P

core temperature

inspiratory time

transient receptor potential

thromboxane A2

ventilation

vasoactive intestinal peptide

vanilloid receptor subtype 1

tidal volume

Units and Svmbols

bpm beats per minute

crnfi0 centimeter of water

hr hour

g

kg kilo gram

ml rnilhliter

m g millimeter of mercury

mm01 millirnole

mOsm milliosmoIe

second

degree Celsius

micro gram

CHAPTER 1

Introduction

Introduction

The rich innervation to the lungs and its fiuictiod significance have long been

appreciated. Bartholinus (1663) described nerve branches to human bronchi w&& were

ascnbed a role in coordinating the distribution of ventilation by Sir Thomas Wtilis (1 679).

Contemporary thought holds that bronchomotor reflexes are of physiological relevance in

rniniminng dead space, iimiting exposure of the sensitive respiratory membranes to

noxious stimuli and rnatching the distribution of ventilation to lung perfusion

(Widdicombe, 1963; as reviewed in Coleridge & Coleridge, 1994). Bronchomotor

reflexes have not only long been appreciated for their contribution to regdatory and

protective reflexes, but were also recognized for their potential role in the

pathophysiology of asthma as long ago as 1698 when Floyer suggested that an asthma

attack was caused when "nemes are filled with Wmdy Spirits" (as cited in Bames, 1986).

At the tum of the century, Brodie (1900) implicated fine bronchopulmonary

aEerents in the reflex control of airway smooth muscle (as reviewed in Coleridge &

Coleridge, 1984). Perhaps the most important class of a6erents that affect airway smooth

muscle is the C-fibres, which outnumber other receptors 10 to 1 (Coleridge et al., 1989).

Appreciation that the behaviour of at least certain pulmonary affierents differs

between the adult and newbom is more recent (see Fisher et al., 1991 for review).

Continued development of numerous organs and organ systems extends weil into post-

natal life, confemng a variable ability of many neonatal physiological systems to mount

responses similar to those of the mature system. As such, our understanding of the neural

control of ainvay smooth muscle and the potential therapeutic implications for the

management of respiratory diseases in the adult may not directiy address the unique

physiology of the newbom.

One potentially important difference between the adult and neonate is the possible

discrepancy in the reflex effects of bronchopulrnonary C-fibres on ainvay smooth muscle

tone. In the adult dog, activation of pulrnonary C-fibres reflexly increases ainvay

resistance, highlighting both the known regulatory, protective and defensive roles of C-

fibres, as weil as theû suspected pathophysiological role in inflarnmatory Iung disease

(Bames, 1 986; Coleridge et al., 1 989). Identification of C-fibre-mediated reflexes in the

newborn has been more controversial and initially raised questions regarding the adaptive

benefits of mature C-fibre-mediated reflexes in the neonate (Kalia, 1976; Ducros &

Trippenbach, 199 1 ; Haxhiu-Poskurica et al., 199 1).

The pungent extract of hot peppers, capsaicin, has proven to be a powefil tool for

investigating C-fibre-induced reflexes and receptor recordiigs. Nevertheless, as capsaicin

is not a physiological C-fibre stimulant, our understanding of C-fibre biology continues to

progress even as researchers endeavor to ident@ the endogenous C-fibre "capsaicin

receptor" ligand. Among the many candidates for the in vivo agonist, lactic acid is known

for its ability to stimulate pulmonary C-fibres and elicit respiratory reflexes in the adult rat

(Lee et al., 1996; Hong et al., 1997). Additionally, lactic acid is produced in pathological

conditions characterized by increased airway reactivity in both the adult (Canny et al.,

1993) and newbom (Abman & Groothius, 1994). The potential contribution of lactic acid

to the etiology andor pathophysiology of neonatal airway disease is accentuated by the

metabolic acidosis characteristic of parturition and imrnediate post-natal life (Koch &

Wendel, 1968; Koch, 1968). AIthough eariy studies Ied to the conclusion that lactic acid

was not capable of reflexiy increasing airway smooth muscle tone in the newborq

methodological as op posed to p hysiological limitations may explain this observation. In

this thesis, measurements of lung mechanics were used to assess the ability of lactic acid to

reflexly alter ainvay caliber in the newbom and to address the role of lactic acid as an

endogenous C-tibre "capsaicin" receptor ligand.

Anatomy and Development

The Tracheobronchial Tree

The primary function of the ainvays is to provide a pathway between the

atmosphere and the gas exchange surfaces of the lung. The airways are organized in a

dichotomously branching, tree-like structure with the tmnk represented by the trachea; 23-

27 branching generations of smaller ainvays ultimately distribute gas to the gas-

exchanging alveoli (Weibei, 1963). In humans, the trachea is characterized by 16-20 C-

shaped cartilaginous rings which, although incornplete dorsally, are joined by connective

tissue and a band of smooth muscle (JeBey, 1998, see below). The £irst bifiircation of the

tracheobronchial tree occurs intrathoracicaüy at the carina, giving rise to two primary or

"mainstem" bronchi. The next 3-7 branchings are termed "bronchi" and are

morphologicaily characterized by cartilaginous, highly elastic walls containing Little ainvay

smooth muscle (ASM) (Jefiey, 1998). The lower bronchi give rise to approxirnately 14

generations of bronchioles, distinguishable From the bronchi on the basis of relatively

greater smooth muscle content and the absence of cartilaginous structures. The first 10

generations of bronchioles, in combination with the trachea and bronchi, are termed the

'konducting airways" and do not display gas exchange properties. In contrast, the last 4-5

bronchiolar generations are characterized by increasingly fiequent alveolar outpouchings

which participate in gas exchange and are thus termed "respiratory bronchioles" (Staub &

Albertine, 1988). The final generation of bronchioles enters the alveolar ducts, whose

walls consist almost exclusively of alveolar outpouchings. The alveolar ducts variably

branch an additional 1-2 generations before terminating in alveolar sacs, the principle sites

of gas exchange (Staub & Albertine, 1988).

Basic tracheobronchial morphology is complete at birth in most species (human,

canine) and no further division of the conducting airways occurs post-natally (Jefsey,

1998). Canine airways continue to increase unifomily in diameter at a rate proportional to

the cube root of body weight until adulthood (Horsfield, 1977). in humans, post-natal

respiratory system development includes firther aiveolarizatioq which continues into the

eighth year of post-natal Me (Staub & Albertine, 1988), as well as an increase in

anatomical dead space volume which parallels somatic development (Polgar & Weng,

1 979).

AU airways contained within the confines of the thoracic cavity are termed

"intrathoracic airways". These ainirays are subject to mechanical deformation as pleural

pressure varies dunng respiration, expiratory manoeuvres, or during defensive airway

reflexes (see below). The highly cornpliant airways of the newborn (Bhutani et al., 198 1;

Panitch et al., 1989) are especially susceptible to the collapsing forces accompanying

forced expiratory manoeuvres (Penn et al., 1988) as well as the distending forces

accompanying positive pressure ventilation, the latter of which are instrumental in the

etiology of bronchopulmonary dysplasia @PD) (Bhutani et al., 198 1; Abman &

Groothius, 1 994).

Airway Smooth Muscle

Ainvay smooth muscle (ASM) distribution varies throughout the Iung, generally

contributhg more to ainvay waii thickness along the length of the tracheobronchial tree

(JeBey, 1998). ASM is present in the trachea and large airways as a band of transversely

oriented muscle fibres, trachealis, which runs along the posterior ainvay wall, completing

the tips of the C-shaped cartilaginous rings (Stephens & Kroeger, 1980). In addition to

the typical transverse fibres, human newbom trachenls is characterized by intermittent

bundles of longitudinaify oriented fibres beneath the transverse layer of smooth muscle in

an arrangement that is similar to the organization of smooth muscle in the gastrointestinai

tract (Hakansson el al., 1976). Despite their presence in the newborq the hc t iona l role

of these longitudinal fibres remains unknown.

ASM connects the edges of the cartilaginous plates in the smaller bronchi, fonniflg

a distinct layer under the mucosa in the remaining bronchi and bronchioles (Stephens &

Kroeger, 1980). This layer is organized in two helices which wind around the airways in

both directions (Miller, 1947; Stephens & Kroeger, 1980; Bates & Martin, 1990). ASM is

continuous into the smallest bronchioles where characteristic sphincter-like rings of muscle

surround the openings to aiveolar ducts (von Heyek, 1960). Human neonatal ASM

distribution is thought to be comparable to that of the adult in the large airways but

contributes relatively less to the waiis in more penpherai airways (Matsuba & Thurlbeck,

1972; JefEey, 1998). Furthemore, ASM is differentidy distributecf between the large

intra- and extrapulmonary ainirays (Ma et al., 1997), with relatively lower distribution in

intrapulmonary bronchi possibly confemng protection to these ainvays by limiting

constriction in vivo.

ASM contraction confers stability to the ainnrays (Oken et al., 1967; Bhutani et

al., 1986) and appears to participate in the subtle coordination of ventilation-perfusion

matching (Forkert & Fisher, 1992) as weil as in playing a supportive role in airway

reflexes (see below). In studies using cholinergie agonists to assess airway mechanics,

contraction of ASM decreased lung cornpliance in both the aduit and newborn (Bhutani et

al., 1986; Penn et al., 1 %8), thereby conferring stability in the face of supra-atmospheric,

collapsing forces. However, the smaller contribution of ASM to the bronchioles of

neonates (JeEey, 1998) prevents newbom airways from achieving comparable stability to

the adult during bronchoconstriction (Matsuba & Thurlbeck, 1 972; JeBey, 1 998). This

may account for the increased nisceptibility of newbom airway smooth muscle to

barotrauma-induced hyperplasia (Bhutani et al., 198 1; Panitch et al., 1992).

Innervation

Contemporary experimental techniques have substantidy advanced Our

understanding of sensory innervation and motor control of the ainvays. Current

respiratory biofogy recognizes bronchopulrnonary plasticity and the dynamic role the

airways play in an array of regulatory, protective and defensive renexes (for review see

Paintal, 1973; SantJArnbrogio, 1982; Barnes, 1986; Coleridge el al., 1989; ,Coleridge &

Coleridge, 1994).

Airway innervation arises fiom bo th the sympathetic and parasymp athetic

subdivisions of the autonomie nervous system (ANS). In the lung, nerves subserving the

ANS converge to form two distinct neural plexuses: the penbronchial plexus and the

periarterial plexus (Laitinen, 1 985). Functionai characterization of airway innervation

identifies efferent and afferent subdivisions.

Efferent Innenta~zon

Traditional concepts of the efferent control of ASM describe contributions fiom

the sympathetic and parasympathetic subdivisions of the ANS. Excitatory innervation to

the airways is primarily parasympathetic, within the vagus nerve QQ, while adrenergic

inhibito ry innemation to the airway s &ses nom the ceMcal sympathetic tnink (Russell,

1 980; B arnes, 1 986). Addi tionally, a vagal non-adrenergic non-cholinergie (NANC)

system of inhibitory efferent innervation has been described in severai species (human,

baboon, cat, guinea pig) while vagai cholinergie excitatory innervatioc is complemented by

an additional adrenergic source of excitatory innervation in some species (Russell, 1980;

Figure 1. Innervation of the airways Schematic representation of airway innervation. Merent vagal innervation is

composed of myeiinated axons consisting of rapidly adapting and slowly adapting receptors @AR' s and S AR'S, respectniely) and unmyelinated broncho pulmonary C-fibres. Efferent innemation consists of vagal cholinergic and non-adrenergic, non-choiinergic (NANC) fibers and extra-vagal sympathetic adrenergic fibers. C-fibres r w widely and are associated with glands, epithelium and vasculature. ACh, acetylcholine; NO, nitric oxide; NE, norepinephriae; VIP, vasoactive intestinal peptide.

SYMPATHETIC

VAGAL EFFERENTS

VAGAL AFFERENTS /'

Epithelium

Smooth mhscle

Bames, 1986).

Excitatoq efferent ainvay innervation consists of preganglionic eEerent fibres

originating from the nucleus ambiguus (NA) and the dorsal motor nucleus of the vagus

(Dm which project to ganglia in the airway walls (Bames, 1986). From these ganglia,

shon post-gangiionic fibres project to ASM (Barnes, 1986). Acetylcholine (ACh)

released from presynaptic and prejunctional fibres mediates its ainvay efTects through

pharmacornechanical coupling with muscarinic receptors. Acetylcholinesterase (AChE)

staining indicates that chohergic innervation extends to the level of the terminal

bronchioles in dogs (Richardson, 1979) and humans (Laitinen, 1985) with AChE-positive

fibres most numerous in the upper bronchi of humans. This characteristic distribution of

choiinergic fibres is similar to that in the dog, in which the distribution of AChE-positive

rnotor ganglia decreases with ainvay size (Laitinen, 1985; Richardson, 1979). To date, 5

muscarinic receptor subtypes have been identified, of which 3 (Ml-3) possess

bronchopulmonacy actions (Fisher et al., 1998).

Activation of post-ganglionic fibres by nicotinic and perhaps also Ml receptor

agonists stimulates the release of ACh korn pre-junctional terminais (Barnes, 1986; Fisher

et al., 1998). Pharmacornechaaical coupliig of ACh to post-junctional ASM muscarinic

subtype 3 (M3) receptors stimulates a cascade of intracellular events which ultimately

l ads to ASM contraction (Bames, 1986; Fisher et al., 1998). The remaining aimay

muscarinic receptor subtype (M2) is pre-junctional and autoinhibitory to ACh release

(Banes, 1986; Maclagan & Barnes, 1989). M2 receptors have also been localized to

ASM and have been tentatively identified as inhibitory to contraction (Fisher et al., 1998).

Receptor binding studies suggest that, in neonatal porcine airways, M2 receptor subme

dzerentiation may be lacking (Haxhiu-Poskurica et al., 1993)- This observation is

supported by research demonstrating reduced effects of selective blockade of airway M2

receptors in the newbom dog (Fisher et al-, 1995). A lack of M2 receptor function may

predispose the neonate to airway narrowing (Fisher et al-, 1995).

While the primary source of excitatory innervation is parasympathetic, inhibitory

ainvay innervation arises primarily fiom ceMcal divisions of the sympathetic nervous

system (SNS) ( S w k i et ai., 1 976; Russell, 1 980). Typically, post-ganglionic efferents

arising f?om the stellate ganglia innervate parasympathetic airway gangtia where P-

adrenergic sympathetics appear to inhibit parasympathetic transmission (Baker et al.,

1983). Although fiinctional sympathetic innervation in human airways is thought to be

weak or absent (Widdicombe, 1985; Laitinen, 198 9, direct adrenergic innervation of

ASM is present in the dog and represents the major source of inhibitory airway innemation

(Russell, 1980). In the presence ofvagal tone, stimulation of the steliate ganglia produces

bronchodilation and reduces the degree of bronchoconstriction during concurrent vagal

stimulation (Cabezas et al., 197 1). In dogs, pretreatment with P-adrenergic blockers such

as propranolol or isoproterenol abolishes the bronchodilatory effects of selective P-

adrenergic stimulation (Banies, l 986). In contrast, P-adrenergic blockers do not increase

resting airway resistance in healthy adult humans (Nadel & Barnes, 1984) suggesting

resting sympathetic efferent tone is not present. In contrast, P-blockade in asthmatics

increases airway resistance, unmasking the presence of significant constrictor tone

(Barnes, 198 6).

While predominantly thought of as inbibitory, the role of adrenergic innemation to

the airways has also received attention for the potential role that a-adrenorecepton may

play in the ainvay hypemeactivity of asthma (Bames, 1986). In the adult, contractile

effects of a-adrenergic stimulation on smooth muscle can be demonstrated only under

conditions of aggressive P-blockade (Barnes, 1986; Waldron & Fisher, 199 1). In contras<

epinephrine or norepinephnne (mixed a and P receptor agonists) evoke tracheal smooth

muscle contraction in the neonate, highhghting what appears to be a greater a-adrenergic

receptor density in neonatal M M (Waldron & Fisher, 1991). To date, this contraction

has only been demonstrated directiy in newbom canine airways although it has been

suggested to be present in human newborns (Koslo et al., 1986).

In addition to inhibitory B-adrenergic control, the ainvays are innervated by non-

adrenergic non-cholinergie (NANC) ùihibitory nemes. Evidence for NANC innervation

was initially based on the observation that preconstricted human aiway strips are relaxed

by a neural mechanism that is insensitive to P-blockade with propranolol (Richardson &

Beland, 1976). NANC ainvay innervation is now recogoized to be present across several

species including guuiea pig (Cobum & Tomita, 1 973), cat (Ir- et al., 1 980) and baboon

(Mddendorf & Russell, 1980) and has been suggested to be the only direct hctional

neural bronchodilator pathway in humans. The principle NANC neurotransmitter is nitric

oxide (NO) (Bames, 1995) aithough vasoactive intestinal peptide (VIP) is also a NANC

neurotransmitter and appears to be wlocalized with ACh çWaldron & Fisher, 1991).

NANC innervation is fûnctional in the neonatal gut (summarized in Waidron & Fisher,

1991) and has also been demonstrated in newbom cat airways (Wddron et al., 1989).

Although reflex recnùtment of NANC has been demonstrated in the human aduIt,

evidence for reflex activation of NANC in the newbom rests solely on midies in the

guinea pig (Waldron & Fisher, 199 1).

Afferent Innervation

The Lungs are richly hewated with sensory afferents whicb provide the CNS with

information regarding respiratory system status and initiate a number of

bronchopulmonary reflexes. Both myelinated and unmyehted lung Serents run in the

vagus nerve, relaying idormation from airways and parenchymal tissue to the nucleus

tractus solitarius ( N I ' S ) (Kubin & Davies, 1 99 5 ) . Bronchopulmonary afFerents are

classified on the basis of three difEerent receptor types: slowiy adapting or pulmonary

stretch receptors (SAR), rapidly adapting imtant receptors (RAR) and unrnyelinated

bronchopulmonary C-fibres.

SAR are large myelinated vagal afferents (conduction velocities ranging nom 14-

59 ma1; SantAmbrogio, 1982) and comprise the majority of myelinated pulmonary

afferents. Terminal S AR arborizations are associated with ASM (as reviewed in Coleridge

et al., 1989) where they appear to be bound to comective tissue eiements. Stimulation of

S AR aEerents mediates the Heuring-Breuer innation inhibitory reflex which play s a major

role in the resting breathing patterns of anesthetized animals and, to a lesser extent, adult

humans (Coleridge et al., 1989). SAR inhibit inspiration in response to circumferentiai

ainvay tension while their reflex effect on ainvay smooth muscle is bronchodilation

(Coleridge et al., 1989). SAR are insensitive to most f o m of chernical stimulation In

particular the chemicals used extensively to investigate C-fibre neurobiology @radyk-

prostaglandin, capsaicin, phenylbiguanide; see below) have v b a l l y no direct effect on

SAR (Coleridge et al., 1989). In contrat to the adult, SAR display little or no activity at

FRC in newbom (Fisher et al., 1983; SantfAmbrogio, 1987). Despite this, humm

neonates possess a HeuringBreuer reflex sirnilar to that in anesthetized animals (Cross et

al., 1976).

Like their larger counterparts, RAR are srnall myelinated vagal axons with average

conduction velocities of 16-37 m-s-' (Sant'hbrogio, 1987). RAR terminal arborizations

are located between columnar cells in the airway epithelium, where they respond prirnarily

to changes in transpulmooaxy pressure and are responsible for evoking augmented

inspiratory breaths (sighs) (Coleridge et al., 1989). so named for their rapid

adaptation to maintained lung inflation, also respond to decreased lung cornpliance,

puirnonary edema and histamine release (Coleridge et al., 1989). RAR are stimulated by a

variety of chemicals with b ronchoconstricto r actions (histamine, sero tonin, p rostaglandins

E2 and Fz, and muscarinic agonists) (Mïlls et aL, 1969; Coleridge et al., 1989).

Furthemore, they play a sigaificant role in defensive airway reflexes (see below),

mediating cough and reflex bronchoconsuiction. In the newbom, RAR contribute l e s to

the overall Serent receptor population than in the adult (Fisher et al., 1983;

Sant' Ambrogio, 1 98 7).

Despite the signiIicant role played by myelinated vagal afferents in airway reflexes,

unrnyelinated bronchopulmonary vagal fibres outnumber their rnyelinated counterparts by

as much as 10 to 1 in some species (Waldron & Fisher, 199 1). These sensory afTerents

contribute to the C-wave of the cornpound action potential and are thus termed C-fibres

(average conduction velocities ranging fiom 0.9-2.3 ml'; Coleridge et al., 1989). C-fibre

afTerents innervate al1 levels of the lower respiratory tract and rami@ extensively in airway

epitheliurn and subepithelial structures (Coleridge et al., 1989). Lung C-fibres were

initially thought to be juxta-alveolar capillary in location due to their stimulation by

pulmonary edema (Paintal, 1973). Termed '7-receptors", Paintal initially proposed that

these afEerents served as interstitial stretch receptors (Paintai, 1973); however, lung C-

fibres were subsequently classified as either pulmonary or bronchial on the buis of

vascular accessibility and response to injected stimuli (Coleridge et al., 1989). Pulmonary

C-fibres are preferentiaily stimulated by injection of noxious stimuli (capsaicin,

pbenylbiguanide) into the right atrium or pulmonary artery with latencies of 1-2 S.

Bronchial C-fibres are activated by injection of capsaicin into the right h a r t with latencies

of 6-9 s and with latencies of 3-5 s by lefi atrial injections of capsaicin. Pulmonary and

bronchial C-fibre populations differ subtly in their response to numerous stimuli, iocluding

autocoids, hyperidlation and certain chernicals (Coleridge et al., 1 989).

Pulmonary C-fibres are stimulateci by hyperinaation (2-3 times tidal volume) and

do not respond to most autocoids, displaying ody weak responses to high doses of

prostaglandins (specifically PGE2). In contrast, bronchial C-fibres are strongly stimulated

by prostaglandin, bradykinin and histamine but dispiay weak discharge to hyperinflation up

to 3-4 times Vr.

The C-fibre terminais of certain species, most notably the rodents, exhibit high

immunoreactivity to substance P (SP), neurokinin A @KA) and calcitonin gene related

peptide (CGRP) (Lundberg & Saria, 1987; Lundberg, 1995; Spina & Page, 1996). High

performance liquid chromatography has localized SP to human airway tissue while CGRP

and as well as other neuropeptides (neuropeptide Y), are CO-localized in human

airway sensory nerves (Spina & Page, 1996). These sensory neuropeptides presurnably

act as trophic factors or neuromodulators (Lundberg, 1995) but are also potent effectors

of neurogenic inflammation, a condition characterized by vascular leak, edema, infiltration

of idammatory cells and, in the airways, bronchoconstriction (Barnes, 1986; Lundberg &

Saria, 1987). The tachykinins (SP, NKA) effect their responses by activation of

neurokinin (Mo receptors. SP is preferential for neurokinin subtype I receptors (NICI)

and is primarily responsible for vascular le& while activation of neurokinin subtype 2

(NK2) receptors with NKA mediates bronchoconstriction (Lundberg & Saria, 1987). The

role of tachykinins in neonatal ASM reflex control differs from those of the adult; SP-

induced modulation of ACh release increases with post natal age in rabbits (Gninstein et

aL, 1984). Furthemore, Murphy et al. (1 994) demonstrateci that dry gas hyperpnea-

induced bronchoconstriction was markedly diminished in newborn guinea pigs des pite

adequate airway contracüity and sensorimo tor fùnction (Murphy et al., 1 994).

In the past 100 years, a host of stimuli have been described which activate

bronchopulmonary C-fibres. While early descriptions tied the activation of

bronchopulmonary C-fibres only to pulmonary edema (Paintal, 1973), contemporary

understanding of bronchopulmonary C-fibres couples their stimulation to a much broader

category of stimuli; those evoking protective and defensive aiway reflexes.

Ainiray Reflexes

Protective and Defensive Reflexes

Cornroe (1954) classified bronchopulmonary reflexes as either regdatory or

protectivddefensive (as reviewed in Coleridge & Coleridge, 1994). He defined regulatoy

reflexes as those subject to "continuous neural input fiom low threshold afferents which

mediate events tightiy centered around a control setpoint.". These reflexes typically exert

continuous coatrol over respiratory and cardiovascular homeostasis. ln contras& reflexes

characterized as protective and defensive are evoked by stimuli that threaten lung fùnction

Such reflexes are involved both in limiting exposure of the sensitive respiratory

membranes to noxious stimuli (protective reflexes) and in the active expulsion of

particulate irritants from the ainvay lumen (defensive reflexes) (as reviewed in Coleridge

& Coleridge, 1994).

Cough, a forced expiratory maneuver serving to dislodge mucus and particdate

irritants entrapped within with high flow, is the principle defensive airway reflex (Bames,

1986; Karlsson et al., 1988; Coleridge & Coleridge, 1994). Reflex bronchoconstnction

invariably accompanies the cou& reflex, playing a key role in edmcing its effectiveness

by establishg turbulent flow in lower airways and increasing laminar aidow velocity in

the larger conducting airways (Coleridge et al., 1989). Turbulent flow favours the

deposition of particuiate pollutants in the lower airway mucus lining while high flow rates

increase impaction of particulate imtants in the airway mucus lining at sites of upper

ainvay bifùrcation (Karisson et al., 1988). In addition to its complementary role in

enhancing the expulsive effectiveness of cough, reflex bronchoconstriction also serves to

stabüize the airways and msintain airway patency during the explosive, coilapshg forces

accompanying cou& (Karlsson et al., 1988).

Where cough serves to remove inhaled irritants fiom the respiratory mucosa,

apnea, laryngeal narrowing and bronchoconstriction are reflexes which limit e n w of

particdate imtants into the airway lumen. In this way, reflex bronchoconstnction can act

independently of cough to manifest as a protective ainvay reflex (Karisson et al., 1988;

Colendge & Colendge, 1994). Despite the autonomous and supportive roles reflex

bronchoconsviction plays in airway reflexes, reflex b ronchoconsEnction is best ap preciated

for its role in the puimonary chemoreflex Initidy described by Brodie (1 900), and later

named by Dawes and Comme (1954), the pulmonary chemoreflex consists of an initial

tnad of apnea, bradycardia and hypotension that is accompanied by tachypnea and reflex

bronchoconstriction (as reviewed in Coleridge et al., 1989; Coleridge & Colendge, 1994).

Pulmonary extravasation and tracheal mucous gland secretion have also been variably

described in this reflex (Coleridge & Coleridge, 1994). The pulmonary chemoreflex is not

only triggered by inhalation of exogenous irritants such as ozone, cigarette smoke or

sulphur dioxide (Coleridge et al., 1989; Lee & Lundberg, 1994), but also by locally

released autocoids or introduction of various chemicai stimulants into the pulmonary

circulation, including p henylbiguanide (PB G) and capsaicin (Coleridge et al., 1 989;

Coleridge & Colendge, 1994).

Capsaicin, the pungent extract of hot peppers, is a specific stimulant of pulmonary

C-fibres (Holzer, 199 1) and has been used extensively to investigate the role of pulmonary

C-fibres in the reflex control of ASM. Capsaicin evokes the characteristic

cardiorespiratory depression typicai of the puimonary chernoreflex in guinea pigs

(Lundberg & Saria, 1982; Biggs & Goel, 1985), dogs (Coleridge et al., 1989; Coleridge &

Coleridge, 1994) and monkeys (Ravi & Singh, 1996). Capsaicin mediates its effects on

bronchomotor tone via two independent mechanisms: a central cholinergic reflex and a

peripheral "axon" reflex (Coleridge et al., 1989). The relative importance of each

mechanism varies remarkably between species.

C-Fibre-Mediated Bronchomotor Reflexes

In dogs, inhalation or vascular delivery of capsaicin stimulates pulmonary C-fibres

(Coleridge et al., 1989; Coleridge & Colendge, 1994) and evokes an atropine-sensitive

reflex bronchoconstnction (Coleridge et al., 1989) that is abolished by bilateral vagotomy

or vagal cooling to block C-fibres (Coleridge et al., 1989). Anterograde degeneration

studies, amino acid autoradiography and horseradish peroltidase (HRP) histochemistry

have identified the primary vagal afTerent termination sites as the nucleus tractus solitarius

( N T S ) and adjacent area postrema (AP) (Kubin & Davies, 1995). The projections of

second order neurons uftimately synapse at the DmnX and NA (Kubin & Davies, 1995)

where they stimulate vagal cholinergic efferents to release ACh at airway ganglia. While

this central cholinergic mechanism describes the reflex activation of ASM in the dog, C-

fibre stimulation in rodents evokes reflex bronchoconstriction through a different

mechanism.

In the guinea pig, activation of pulmonary C-fibres stirnulates the release of the

bronchoactive tachykinins NKA and SP as well as the sensory neuropeptide CGRP fiom

C-fibre axon coiiaterals (Fox et al., 1993; Lundberg, 1995; Coleridge et al., 1989).

Release of these neuropeptides initiates a cascade of responses including vasailar leak,

mucus hypersecretion and, in the airways, bronchoconstriction which are collectively

temed neurogenic idammation (Bames, 1986; Coleridge et al., 1989; Lundberg, 1995).

NKA, SP and CGEW are potent activators of ASM (Lundberg & Saria, 1982; Barnes,

1986; Biggs & Goel, 1985; Karlsson et al., 1988). Although other rodents, such as rats,

display tachykinui innervation, the magnitude of its effects on airway reflexes is much

weaker than in guinea pigs.

Severai experimentd observations support a potentid roie for axon reflex-

mediated increases in ASM tone in the pathophysiology of human inflanmatory lung

disease. The relative inability of cholinergie antagonists to reduce non-speci£ic airway

hyperreactivity (Bames, 1986), the locaiization of CGRP, SP and NKA in pulmonary C- r

fibre afTerent terminais (Lundberg & Saria, 1987; Coleridge & Coleridge, 19841, and the

effeaiveness of SP and CGRP to excite human branchial smooth muscle in vitro (Saria et

al., 1985; Lundberg et al., 1984), support a d e for axon reflex regdation of ASM in

humans. Furthemore, increased CGRP-like immunoreactMty in the epitheiial iayer of

infants suEering from BPD (Johnson et al., 1988; Johnson & Wobken, 1987) implies a

role for the axon reflex in human inflammatory iung disease (Ghatei et al., 1987; Lundberg

et al., 1984; Bames, 1986). However, apart from identification of SP involvement in

neurogenic idammation in the rat (Baluk et ai., 1 992), responses of the axon reflex type

have not been identified in animais other than rodents, nor do SP or CGRP appear to

evoke reflex bronchoconstriction in humaos in vivo (Lundberg & Saria, 1987). Thus the

relative contribution of this mechanism to human bronchomotor reflexes remains unclear.

In contrast, others have challenged the emphasis on the role of tachykinins in reflex

bronchoconstriction. Coleridge et al. (1 989) argue that cholinergie reflex control of

airway smooth muscle remains an important contributor in larger species including humans

and indeed may be predominant.

Despite elucidation of C-fibre-rnediated reflexes in the adult, early attempts to

identify the existence of mature C-fibre-mediated respiratory chemoreflexes in the

newbom were inconclusive. Kalia (1976) repolted that the characteristic ventilatory

response evoked by C-fibre stimulation with right atrial injection of PBG was weak or

absent in 1-6 d old kittens. Additionally, initial attempts to quanti@ bronchomotor

cornpetence in newbom of other species (pi& rabbit) suggested that bronchoconstrictor

reflexes characteristic of the adult were not present in the neonate (Ducros &

Trippenbach, 199 1 ; Haxhiu-Poskurica et al., 199 1). Using measurements of isometric

tracheai tension in silu fiom segments of extrathoracic trachea, Haxhiu-Poskurica et al.

(1991) were unable to elicit a bronchomotor response to preswnptive C-fibre stimulation

with capsaicin in newborn piglets. This apparent absence of mature bronchoconstnctor

reflexes in the newbom was initially interpreted as beneficial since the highly cornpliant

ainvays of the newbom would be at an increased nsk of coiiapse (Bhutani et aï., 1986).

On the other hand, it was argued that the absence of bronchoconstrictor reflexes in the

new-bom could be detrimental if reflex bronchoconstriction was designed to stabiiize the

ainvays (Olsen et al., 1967) or reduce dead space (wddicombey 1963) during rapid

shallow breathing and forced expiratory maneuvers.

Radiographie studies have demoostrated that the magnitude of ainvay narrowing

varies with respect to airway caliber (Cabezas el ai., 1971) such that, in the aduit dog and

pig, small airways (1 - 1.5 mm diameter) exhibit relatively p a t e r narrowing than do larger

ainvay such as the main stem bronchi and trachea (Cabezas et al., 1971 ; Murphy et al.,

199 1). This suggests that the inabiiity of HaKhiu-Poskurica et ai. (199 1) to identify a

bronchomotor response of the trachea to presumptive C-fibre stimulation in newbom pigs

does not preclude the existence of the mature reflex. Indeed, others have since shown that

the newborn does mount a competent bronchomotor response to presumptive C-fibre

stimulation. Using the method of LaFramboise et al. (1993) to measure pulmonq

resistance, Anderson and Fisher (1993) demonstrated that capsaicin causes a robust,

atropine-sensitive increase in lung resistance in the newbom dog and pig comparable to

that evoked by capsaicin in the adult (Coleridge et al., 1982). Thus recent data indicate

that presumptive C-fibre stimulation with capsaicin evoker reflex bronchoconstriction in

the newborn .

The Capsaicin Receptor

Recent developments in Our understanding of the molecular mechanisms activating

C-fibre afTerents and the development of capsaicin antagonists have signifïcantly enhanced

our ability to study the physiologie mechanisms activating pulmonary C-fibres. The

selectivity of capsaicin for thin sensory nerves, cornbined with the similarity of the reflex

effects evoked by structural capsaicin analogues (resiniferatoxin, mustard oil), suggests a

specific capsaicin recognition site on C-fibre endings. Using ion flux and patch clamp

shidies, this putative "capsaicin receptor" was initiaüy identined as a relatively non-

seledive cation conductance permeable to both mono- and divalent cations (Marsh et al.,

1987, for review see Holzer, 199 1). Due to the high specificity of the capsaicin receptor

for vanilloid compounds the putative capsaicin receptor was temed "vanilloid receptof

(Szallasi et al., 1995). The vanilloid receptor has recently been cloned from DRG neurons

and identified as a non-specific cation conductance preferential for ca2' (Caterina et al.,

1997). Dubbed vanilloid receptor subtype 1 (WU), it is strucnirally related to the

Drosophila retinal TRP gene, a putative store-operated channel (SOC) that appears to

mediate extracellular ca2' entry in response to depletion of intracelMar ca2' stores

(Clapham, 1996).

Despite molecular, pharmacological and biophysical characterization of VRI, its

endogenous ligand has not been identified. Capsaicin aimulates neurogenic inflammation

(see above) and evokes C-fibre-mediated reflex bronchocoIlStriction, both conditions

characterizing idammatory lung disease (Barnes, 1986; Canny et al., 1993; Abman &

GroothRis, 1994). As such, the metabolic byproducts of inflammation (H?, lactic acid) as

weil as idammatory mediators themselves (histamine, sero tonin, prostanoids) have been

suggested as potential endogenous VRl Ligands (Bevan & Geppetti, 1994; Lee et al.,

1996; Hong et al., 1 997; Stahl & Longhurst, 1992).

Early evidence favouring R as an endogenous VRl ligand was based on the

finding that ischemically sensitive afîerents and smali, unmyelinated DRG neurons are

activated by acidic solutions (Stahl & Longhura, 1992; Fox et ai., 1995; Lou &

Lundberg, 1992; Bevan & Geppetti, 1994). Furthemore, extracellular acidification at

patho physiological concentrations excites aitaneous nociceptors in both rats (Steen et al.,

1996; Steen et al., 1995) and humans (Steen et al, 1996; Steen el al., 1995) and lactic

acid can establish comparable acidification at sites of tissue ischemia and idammation in

vivo (Hong et al., 1997). An endogenous source of H', lactic acid evokes the pulmonary

chemoreflex (Lee et al., 1 996) and activates broncho pulmonary Cfibres (Trenchard,

1986a; Trenchard, 1986b; Lee et al., 1996; Hong et al., 1997). Furthemore, while is

a potent activator of C-fibres and mediator of the pulmonaiy chernorefle~ the reflex

effects and C-fibre activation evoked by H+ are potentiated in the presence of the lactate

anion (Lee et al., 1996; Stahi & Longhurst, 1992). However, the lactate anion itself does

not appear to increase C-fibre activity, nor evoke the characteristic cardiorespiratory

depression that accompanies pulmonary C-fibre activation (Trenchard, l986b; Hong et al.,

1997). Acid-evoked reflex resporises are abolished by perineurd capsaicin treatment to

selectively block C-fibres (Lou & Lundberg, 1992; Lee et al., 1996) and blockade of the

capsaicin receptor inhibits the acid-induced pulmonary chemoreflex in vivo (Lee &

Lundberg, 1994) and reduces acid-evoked C-fibre activity in vitro (Urban & Dray, 1991;

Fox et al., 1 99 5 ; see below). Combined with the hding that H' inhibits the binding of the

capsaicin analogue r e s ~ r a t o x h to vanilloid receptors in vitro (SzaUasi et al., 1 995) the

above mentioned shidies imply that H' and capsaicin activate common C-fibre

populations, possibly through a cominon receptor transduction mechanism.

Despite identification of LA'S role as a C-fibre stimulant and mediator of the

pulmonary chemoreflex in the adult, there is no direct evidence that LA-induced C-fibre

stimulation evokes reflex bronchoconstriction in either the adult or the newbom.

AIthough newbom display a capsaicin-indud reflex bronchoconstriction attributed to

stimulation of pulmonary C-fibre afTerents (Anderson & Fisher, 1993), using dynamic lmg

compliance (CLdyn) as an index of bronchoconstricti~n, others have show that lactic acid

does not evoke bronchomotor responses in newbom rabbits (Ducros & Trippenbach,

199 1). However, lung compliance is an indirect index of bronchoconstriction and may be

insensitive to small changes in smooth muscle tone or resistance (Widdicombe & Nadel,

1963). Given these considerations, the evidence for the ability of lactic acid to evoke

reflex bronchoconstriction in the newborn rem* sntroversial.

Development of the cornpetitive capsaicin antagonist capsazepinel provided an

experimental means wit h which to investigate capsaicin-evo ked, C-fibre-rnediated reflexes

as weil as those attributed to other substances, including lactic acid (Dickenson & Dray,

199 1; Bevan et ai., 1992; Belvisi et al., 1992; Urban & Dray, 199 1; Lou & Lundberg,

1992). Capsazepine reversibly attenuates or abolishes the pattern of breathing response

and C-fibre aaivity evoked by capsaicin in the adult rat (Lee & Lundberg, 1994) and

guinea pig (Fox et al., 1995). Capsazepine also antagonizes the reflex contraction of

smooth muscle, inhibiting the contractile response evoked by capsaicin in isolated rat vas

deferens (Maggi et al., 1993). Biophysical studies dernonstrate that capsazepine

reversibly inhibits capsaicin- and resiniferatoxin-induced ca2' uptake in rat DRG and

reduces or abolishes the inward current response to capsaicin of voltage-clamped rat DRG

neurons (Bevan et al., 1 992). In addition, capsazepine reversibly antagonizes the inward

cation current evoked by capsaicin in Xenopus oocytes transfected with VRl (Caterina et

al., 1997). In addition to capsazepine, which is a cornpetitive capsaicin-antagonist, the

inorganic dye ruthenium red antagonizes the reflex effects of capsaicin in a cornplex, non-

corn petitive manner (Maggi et al., 1 993).

Studies of capsaicin and lactic acid-induced apnea, bradycardia and hypotension, as

weil as C-fibre recordings (Lee & Lundberg, 1994; Lee er al, 1996), suggest that

capsazepine abolishes both the capsaicin- and possible lactic acid-evoked bronchomotor

responses, indicating a common capsaicin and lactic acid mechanism of C-fibre activation.

In contrast, biophysical studies suggest that H' per se is not a strong agonist of the VR1

receptor (Caterina et al., 1997) and favour the hypothesis that lactic acid activates an

alternate C-fibre receptor or stimulates the release of an intermediary endogenous C-fibre

ligand to mediate its potential bronchomotor effects (Karia et al., 1992; Shams et al.,

1988; Vyklicky et al., 1998). Discovery of a proton-gated N a channel locaiized to

capsaicin-sensitive fiog and rat DRG neurons (Akaike et al., 1 990; Bevan & Yeats, 199 1)

supports the hypothesis that lactic acid andor mediate C-fibre reflex effects by

activating a receptor signal transduction mechanism independent of VRl . Additional

evidence supporthg separate pulmonw C-fibre receptor mechanisms involved in

capsaicin and lactic acid sensing cornes fiom the recent cloning of an acid-sensing ionic

channel (ASIC) from Xenopus DRG (Waldmann et al., 1997; Wddmann et al, L 997).

Uniike direct C-fibre stimulation with H', acid-induced release of an intermediaq

endogenous C-fibreMt1 receptor ligand, likely an inflammatory mediator (Steen et al.,

1999, is an altemate hypothesis. Slow infusion of HCI stimulates platelets to release

thromboxane A2 (TY&), an idammatory mediator produced in the cyclooxygenase

pathway of arachadonic acid metabolism (Campbel 1990). TxA2 is a potent C-fibre

stimulant (Shams et al., l988), and additional idammatory mediators produced in the

cyclooxygenase pathway (prostaglandins EZ (PGE2) and F h (PGFk)) ais0 stimulate

unmyelinated sensory aEerents and/or potentiate the effects of other C-fibre stimulants

(Coleridge et al., 1978; Lee & Morton, 1995; Steen et al., 1996; Steen et al., 1995).

Recent evidence that a cocktail of idammatory mediators (PGE2, bradyliinin and 5-HT)

potentiates the inward cation current evoked by low pH in cultured newbom rat sensory

neurons (Steen et al.., 1996; Steen et al., 1995) supports this hypothesis. However,

application of these idammatory mediators alone at physiological pH does not induce an

inward cation current in these cells. The potentiating effects of these inflammatory

mediators on the current response evoked by extracellular acidosis is blocked by

capsazepine (Vyklicky et al., 1998) and the potentiating effect of acidic pH o n the

inflanmatory mediator-evoked current in sensory neurons is similar to that evoked by acid

pH during submaximai capsaicin application (Caterina et al., 1997). Based on these

observations, idammatory mediators such as PGEz may represent endogenous ligands for

the capsaicin or VRl receptor (Vykiicky et al., 1998).

The lack of consensus regarding the iduence of lactic acidosis on the reflex

control of ASM tone in the newborn is apparent. The potential role of lactic acid and K

is particularly relevant in the newbom since rnetabolic acidosis is a normal cornponent of

parturition (Koch & Wendel, 1968; Koch, 1968) as well as an important constituent of

neonatai idammatory lung disease (Penn et al., 1988; Abrnan & Groothius, 1994). In

addition to questions concerning the actions of lactic acid on MM, biophysical studies

and in VIVO electrophysiological investigations raise questions regarding the mechanisrn of

action of lactic acid on pufmonary C-fibre receptors.

We tested two hypotheses regarding the actions of lactic acid on bronchomotor

tone in the newbom: 1) lactic acid behaves as an endogenous C-fibre stimulant which

evokes atropine-sensitive reflex bronchoconstriction; and 2) lactic acid acts via the same

capsaicioNRl C-fibre receptor mechanism to cause reflex bronchoconstriction.

CHAPTER 2

Methods

Preparation

s u w v

Al1 experimentai procedures confonned to the guidelines of the Canadian Coucil

on Animal Care and were approved by the Queen's University Animal Care Cornmittee.

Experiments were perfomed on 38 newbom dogs ranging in age from 1 to 13 d (mean

5.1 + 0.4 d) and ranging in weight from 256 to 1,063 g (mean 569 I 29.6 g). Animals

were anesthetized with an initial i.p. injection of chlordose (75-100 mg-kg-') and urethane

(0.75-1.0 g-kg-l) and supplemental anesthesia was administered intravenously at

approxirnately 1 h intervals to maintain abolition of a response to a painfiil stimulus or

acute changes in blood pressure and h a r t rate. The electrocardiogram @CG) was

monitored via needle electrodes connected to a preamplifier, osciiioscope and audio

speaker. Animals were tracheostomized and ventifated with 40% Oz/balance N2 at a

ventilator frequency of -40-45 bpm and a tidal volume -8- 10 ml-kg-' adjusted to provide

normal pH (blood gases: pH = 7.41 & 0.01, PaC02 = 32.3 f 1.2 m g , PaOÎ = 191.0 f

12.6 mmHg). A mid-line thoracotomy was performed and an end-expiratory load of -2.3

c r n w was established to provide a normal fùnctional residuai capacity (Fisher &

Mortola, 1981). Transpulmonary pressure fi) WU m e m e d via a differential pressure

transducer (Validyne MP45, + 2 c-O) comected to the side port of the tracheal

cannula. A femoral arterial line was inserted in most animais for blood gas sampling and

arterial blood pressure (BP) measurernent. A femoral venous line was established for

administration of supplemental anesthesia and inhision of capsazepine (CAZP) (Harvard

Apparatus Syringe Infusion Pump, mode1 22). The right jugufar vein was cannulated and

Figure 2. Experimental set-up Animais were placed in a fiow plethysmograph and mechaaicaiiy ventilated with

40% oxygedbalance nitrogen through a tracheostomy tube. A positive end-expiratory pressure (PEEP) of -2.3 cmHzO was established to maintain normal FRC. Flow (Y) and transpulmonary pressure (Pw) were acquired on-lioe for calculation of inspiratory lung resistance (RL) and dynamic lung cornpliance (Cu,). The electrocardiogram ('CG) was monitored with neede electrodes. Animais were instrumenteci with a right hart catheter for capsaicin and lactic acid administration (CAPS/LA), a femoral venous Line for capsazepine infusion (CAZP) and a femoral arterial line for arterial blood pressure (BP). Core temperature (Tc) was monitored with a rectai temperature probe.

a catheter (dead space 4 . 3 4 . 4 ml) advanceci into the right heart (location codumed pod

rnortem) for capsaicin (CAPS) and lactic acid (LA) injections. Inspiratory efforts were

abolished by bilateral phrenic nerve section or paraiysis with doxacurium chloride (0.025

mg-kg-' i.v.), a non-depolarizing muscle relaxant with no muscarinic side effects

(Okanlami et al., 1996). During paralysis, additional anesthesia was administered based

on the schedule used pnor to paralysis (approximately every hour). Core temperature (Tc)

was regulated with a heat lamp and a servo-controlled heating blanket (Harvard Apparatus

Animal Blanket Control Unit, model # 50-6956). Animals were placed in a body

plethysmograph and respiratory flow (Y) was measured with a pneurnotachograph (Ham

Rudolph, model 8300) connected to a differential pressure transducer çValidyne MP45,

cmH20). Flow, ECG, BP and PTp were acquired with a computerized chart recorder data

acquisition package (CODAS, DATAQ Instruments).

Analysis of Pulmonary Mecbanics

Transpulmonary pressure and respiratory flow were acquired on-he by an

additionai wmputer at a sampling fieqyency of 100 ~arn~les-s~~ per channe1 for breath-by-

breath calculation of inspiratory lung resistance (RL> crnH,~-d-'-s-') and dynamic lung

cornpliance (Cm ml-cm&~"). & was calculated based on the method of Hildebrandt,

as described by LaFramboise et al. (1983). This rnethod calculates & by dividing the

average pressure required to overwme the flow resistive properties of the lung by the

mean inspiratory flow (Waldron & Fisher, 1988). Cu, was calculated by dividing the

Figure 3. Pulmonary mechanics analysis Schematic illustrahg the calculation of mean inspiratory lung resistance (RL) and

dynamic lung cornpliance (Cm). The cornputer-acquired signals of respiratory flow (i3, transpufmonary pressure (Pm) and tidal volume CVT) were used to calculate puimonary mechanics. C ~ d p is caldated by dividing Vr by the Plp measured at end-inspiration (elastic pressure). The pressure above that required to overcome the elastic properties of the lung describes the puirnonary resistance to flow and is termed the "resistive area". Mean "resistive pressure" is calculated by dividing the resistive area by the inspiratory time (Ti) and Rt is then calculated by dividing the resistive pressure by the mean inspiratory flow (V~ni). Dynamic lung elastance (&dyn) was calculated as the reciprocal of Cu,.

"Area" 1 I 1 I I

RL= Resistive Pressure

Elastic Pressure

tidal volume by the transpuhonary pressure swing between points of zero flow during

inspiration Dynamic lung elastance (EL+) was calculated from the reciprocai of CL+.

Protocols

Effect of Lactic Acid on Bronchornotor Tone

To determine the effects of LA on neonatal bronchomotor tone, we recorded the

breath-by-breath pulmonary mechanics response to right heart injection of LA. In

preliminary experiments, we assessed the bronchomotor response to right heart injections

of various doses of lactic acid ranging from 0.02-2.0 mmol-kg-' (Fig. 4). On the basis of

these results we chose to use 0.4 mmol-kg" for the present study since it was twice the

threshold dose of 0.2 mmol*kg*', and it elicited a robust bronchomotor response on which

we could test the effects of pharmacological antagonisrn

In order to assess the reflex nature of the responses to LA & and ELdyn responses

to right kart injection of LA were measured before and after muscarinic receptor

blockade with atropine (2 rng.kg") in 17 newborn dogs (4.9 + 0.6 d). The protocol

consisted of 3 acquisition periods separated by 15 min. Each acquisition period was

preceded by flushing the jugular catheter with 37OC saline and inflating the lungs to 1 20

c-0 (4 x VT) to establish a constant volume history. Two min after the inflation a 5-8

minute acquisition period began. The first trial consisted of saline (0.2 or 0.4 ml) and ACh

(10 pg) control injections at 1 and 3 min, respeaively, to assess ASM responsiveness. Ln

the second trial, we measured the bronchoconstrictor response to right heart injection of

LA (0.4 mmol-kg"). In 11 animais, LA was delivered in 0.2 ml. Due to the elevated

Figure 4. Effect of Dosage on the peak resistance response to Iactic acid Average peak & response (% change fiom baseline) to laaic acid at 4 doses: 0.2,

0.4, 0.5, and 0.75 mol-kg". Lactic acid at 1 .O and 2.0 mmol-kg-' evoked severe cardiac dysrhythmias that proved termioal. Injections at 0.02 mol-kg-' did not cause any significant change in k.

LACTIC ACID DOSE

osmolaiity of this stimulus (-600-2300 m0sm-r', mûan: 1,464 + 166 rnosnd), an

additional 6 animais were studied in which LA was dissolved in 0.4 mi which reduced

osmolality to -285-950 m ~ s m l - ' (mean: 510 t 57 m~sm-r') . Solution osmolaiities were

measured using the freezing point depression method (Osmette A Automatic Analyzer,

model # 5002, Precision Instruments Inc.). The pH of 0.2 ml and 0.4 ml volume LA

injections was 2.04 + 0.03 and 2.22 f 0.02, respectively (Accumet model 8 15MP, Fisher

Scientific). A second LA challenge was delivered d e r atropine to assess the reflex

contribution of vagal cholinergie efferents to the bronchomotor response.

Effect of Osmolality on Bronchomotor Tone

To investigate the possible effects of solution osmolality on pulmonary mechanics

we measured the breath-by-breath puhonary mechanics response to injection of

hypertonic saline (500, 1000, 1500, 2000, 2500 and 5000 m~sm-1-') ia 3 newborn dogs

(13.0 d). The protocol consisted of a saiine and ACh control trial (as described for the

effects of lactic acid on bronchomotor tone) and 2-3 hypertonic saline triais in which

animais were randomly chdenged with right heart bolus injections (0.4 ml) of hypertonic

saline (500-5000 m0sm.1-') separated by 5 min.

Capsazepine Delivery Method

To determine the most effective method of delivering CAZP for VRl receptor

antagonism, we compared the effects of bolus injections (3 -5 or 5.0 mgkg-' iv.) versus

infusions (500 pg-kg-'min-' i.v.) of CAZP on the bronchoconstrictor response to 25

=kg-' capsaicin in 5 newborn dogs (mean age 3.8 I 0.5 d). These doses were chosen

based on the findhgs of Lee and Lundberg (1994) in which either bolus (2.0-3.5 mg-kg-')

or infusion (250-3 50 pg-kg-'-min*') of CAZP abolished the reflex apnea, bradycardia and

hypotension elicited by injection of 1 -2 pg-kg*L capsaicin in the adult rat (Lee & Lundberg,

1994). Our protocol consisted of 4 (bolus study) or 5 (infusion study) acquisition penods,

of which the first 2 were comrnon to both and consisted of an initiai triai of saline and ACh

to establish baseline bronchomotor responsiveness, and bronchoconstrictor response to a

control injection of CAPS (25 pg.kg-'), respectively. For the CAZP bolus study, the third

and fourth trials wnsisted of a CAZP block triai, in which we measured the pulmonq

mechanics response to CAPS 2 min after bolus injection of CAZP (3.5 or 5.0 mg-kg-' iv.),

and a recovery trial, in which we tested the ainvay response to CAPS 15 min following

CAZP. For the CAZP infusion study the third acquisition period consisted of a CAZP

trial in which we measured & and EL+ during the onset of the CAZP infusion (500

Clg-kg-l~min-'). The fourth or "blockade" trial measured the pulmonary mechanics

response to CAPS at the 10 min mark of CAZP infusion. In the "recovery" trial, we

measured the pulmonary rnechanics response to CAPS and ACh at 15 and 18 min

following CAZP, respectively .

Bolus CAZP injection reduced the CAPS-evoked E ~ L responses by 25% (3 -5

mg.kg-') and by 59% (5.0 mgkg-'). Although the reductions in & were significant (one

way ANOVA), it was difficult to determine whether the animals were in a steady-state or

how critical the timing of the CAPS response was following the CAZP bolus. For this

reason we pursued the CAZP-blockade studies using the CAZP infusion technique.

Effect of Capsazepine Infusion on Capsaicin- and Lactic Acid-Induced Reflex

Bronchoconstriction in the Newbom

In order to test the hypothesis that LA and CAPS activate the same C-fibre

receptor mechanisrns, we first had to determine whether CAZP was capable of abolishing

capsaicin-induced reflex bronchoconstriction. Experiments were performed on 1 1

newborn dogs (mean age 5.4 f 0.9 d) in which the bronchomotor responses to right heart

injections of CAPS were measured before, dunng and after CAZP infusion (Fig. 5). in a

second set of experiments (n=8, mean age 4.9 + 0.4 d) we tested the actions of CAZP on

LA-induced bronchoconstriction. Protocols for both the CAZP/CAPS and CAZPILA

experiments consisted of five acquisition periods separated by 15 min (Fig. 5). Pnor to

each triai, the jugular catheter was flushed with 37OC saline and the animai inflated to 2 20

c m 0 (4 x Vr) to establish a constant volume history. The fxst trial (saline and ACh)

was the same as that described above for the muscarinic blockade study. The second trial

measured the response to nght hart injection of capsaicin (25 pgg-kg-') or LA (0.4

rnmobkg" in 0.4 ml). The third triai measured & and ELdyn prior to the onset and during

the capsazepine infusion (500 pg-kg-lmin) into the femoral vein The fourth trial assesseci

the lung mechanics response to CAPS and LA 10 min after initiation of the CAZP

infusion. The 6ft.h triai was perfonned 15 to 20 min post CAZP infusion to assess the

reversibility of the CAZP blockade.

Figure 5. Experirnental protocol Baseline and control trials were designed to assess the bronchomotor responses to

saline and ACh (BASELINE) and CAPS or LA (CONTROL). The BLOCKA.DE trial consisted of challenge with CAPS or LA at 10 min into CAZP infusion. A RECOVERY trial was performed at the end of the experiment to assess the reversibility of the blockade on the response to CAPS or LA. Triais were separated by at least 15 min Injections, denoted by arrows, occurred at approximately 1 and 4 minutes into the 8 min trial. ACh injections were 10 pg and CAPS and LA injections were 25 pg-kg" and 0.4 mmoLkg-', respectively. Break in BLOCKADE trial represents -9 min.

CONTROL

BLOCKADE

+ J.

saline ACh 1 (10 PS)

5- CAPS (25 pgokg-')

or LA (0.4 mmolekg-l)

J. J. CAPS or LA ACh 2

$",/- CAZP 500 1

or Atropine

8 min

J- 4

RECOVERY CAPS or LA

ACh 3 i

Dmgs

The following drugs were used: Chlordose (ICN Biochemicals) and urethane

(Sigma) dissoived as a mixture (0.25 g chlordose, 2.5 g urethane) in 10 mi heated saline,

administered at doses of 2-2.5 dekg-' with supplementd injections of 0.5 dakg'' to induce

and maintain anesthesia, respectively. A stock solution of capsaicin (Sigma) was prepared

by dissolving 10 mg capsaicin in 1 ml ethanol, 2 drops Tween 80 and 9 ml saline to arrive

at a final concentration of 1 mg-ml-'. The stock was diluteci fùrther with saline to the

required concentration for injection. A stock solution of 3.3 M lactic acid (Sigm%

L(+)lactic acid) was diluted with distilled HzO to the required concentration for injection.

Acetylcholine chloride (Sigma) was dissolved in saline to a concentration of 50 pgd*l.

Atropine (Sigma) was prepared as a 10 mg.ml-' stock solution and diluted with saline to

the required concentration for injection. Capsazepine (RB0 was prepared as a 2.5 mg-ml-'

stock solution by dissolving 25 mg in dimethyi sulfoxide and diluting it in a solution of 1

ml Tween 80, 1 ml ethanol and 8 mi saline. Injection volumes for capsaicin and ACh were

0.2 ml. Injection volumes for saline were 0.2 ml or 0.4 ml. Injection volumes for LA

were 0.2 mi or 0.4 ml in the LNatropine study but 0.4 ml in the CAZP protocols. Saline,

ACh, capsaicin and lactic acid were injected into the jugular catheter dead space then

rapidly flushed with saline. Chloralosdurethane and capsazepine were administered via

the femoral venous catheter and flushed with saline. Al1 solutions were warrned to 37°C.

Baseiine 5 and Eup were calculated on the average of the 10 breaths pnor to

injection. The peak lung mechanics response for each animal was defined as the average

resistance and elastance caiculated for the two largest consecutive values following

challenge. & response latency was defined as the One from jugular line injection to the

omet of a maintained or contuiued increase in & above baseline. The time to peak was

caiculated as the tirne from jugular iine injection to the frst breath of the maximal

response. HR baselines were calnilated as the average values over the 10 seconds

preceding injection. Bradycardia was calculated as the lowest instantaneous HR following

injection. Statistical cornparisons between responses were based on one way ANOVA,

Mam-Whitney rank sum or paired f-test. Tukey posi hoc analyses were performed if the

ANOVA was sigdcant. A significant dEerence was defined as P < 0.05. Al1 resdts are

expressed a s mean SEM udess othenvise indicated.

CHAPTER 3

Results

Lung Mechanies Response to Lactic Acid

The average breath-by-breath response of & to 0.4 mmol-kg" LA is shown in Fig.

6 (fiUed symbols). The 0.2 ml LA injectate resulted in a peak increase in IQ of 52 f 6.7%

(P < 0.01, Fig. 7) and the 0.4 ml LA injectate caused a peak increase in & of 49 k 10.9%

(Fig. 7). Peak LA-evoked RL increases were not signincantly different for 0.2 and 0.4 ml

injectate volumes (P > 0.05). Latency and time to peak values for the response of & to

LA are presented in Table 2.

The average breath-by-breath response of & to LA during muscarinic blockade

with atropine is shown in Fig. 6 (open symbols). The maximal response of & to the 0.2

ml LA injectate was relatively atropine-resistant (Fig. 7), displayhg a reduction of only 33

13.5% fkom the control value (P < 0.05). In contrat, the maximal responses associated

with the 0.4 mi LA injectate (Fig. 7) were atropine-sensitive and were reduced by 80 * 10.9% compared to control (P < 0.01). The effect of atropine antagonism on the response

of & was sigdicantly different (P < 0.01) for the 0.2 and 0.4 ml LA injectates.

Effect of OsmoIality on Bronchomotor Tone

Inspiratory lung resistance was unaEected by right heart injection of solutions of

500 to 2500 m0sm.l-'. However, & displayed a small (8.4%) decrease to 5000 rn0sm-1-'.

Although 0.4 mmol-kg" LA delivered in a 0.4 ml injectate evoked atropine-sensitive reflex

bronchoconstriction, the atropine-resistance of the bronchoconstriction evoked by the 0.2

mi LA injectate suggests a non-cholinergie component . S ince hypertonie saline solutions

(2400 to 4800 mol-I-') injected into the pulmonary circulation of aduit

Figure 6. Breath-by-breath mechanies response to lactic acid pre and post atropine Average (+ SEM) breath-by-breath values of percent change in lung resistance

(&, upper panels) and elastance (Etdyn, lower panels) in response to right heart injections of lactic acid (0.4 rnmol- kg-') in 0.2 ml (hi& osmolality; lefk panels) or 0.4 ml (low osmolality; right panels) injectate before (closed symbols) and after (open symbols) atropine (2 mg-kg-'). Note that atropine abolished the response to the 0.4 ml injectate whereas the 0.2 ml injectate was atropine-resistant. Arrows indicate injection of lactic acid. Mean ventilator ffequency was approltimately 45 breaths per minute.

(% CHANGE FROM BASELINE)

Figure 7. Average maximal mechanies response to lactic acid in 0.2 and 0.4 ml Mean peak pulmonary resistance (k, top panels) and dynamic lung elastance

(Ems lower panels) responses to saline and lactic acid (0.4 mol-kg-') in 0.2 ml (lefi) or 0.4 ml (right) injectate volume before (INTACT) and after atropine (ATROPINE). Peak values are slightly greater than maximal values for breath-by-breath averages in Figure 6 due to minor variations between animais for the ventilatory cycle at which maximal response occurred. Note: atropine reduced the response to the 0.4 ml injectate such that it was significantly difFerent fiom the intact response but was not di£Ferent nom saline. +, significantly Merent from INTACT lactic acid response; *, significantly dserent Born SALINE control. P < 0.05 (ANOVA and Tukey post hoc).

(% CHANGE FROM BASELINE)

dogs stimulate puhonary C-fibres and cause reflex bronchoconstriction via cholinergie

mechanisms (Pisarri et ul-, 1 99 1 ), the atropine-resistant bronchoconstrictor effects of the

high osmolality LA injectate in Our studies were not likely due of C-fibre stimulation.

Furthemore, injection of 5000 m0sm-1'' saline (twice our highest LA osmolality) had no

bronchoconstrictor action in 12- 14 d newbom dogs. Therefore, high osmolality per se

likely did not affect ASM tone, but evoked an as yet undetermined non-muscarhic

mechanism of increasing airway tone.

Lung Mechanics Responses During Capsazepine

CAZP intirsion aione was associated with an increase in & of 23 + 6.7% at 8 min

(n = 19) (Fig. 8). Hyperidation (4 x VT) did not signifïcantiy reduce the CAZP infusion-

evoked elevation in RL, which was 1 8 * 0.6% (P < 0.05) 2 min after d a t i o n . Eight min

of tidal breathing following hyperinfiation without any other intervention was associated

with an increase in & of -1 1% relative to control. In preliminary studies, infusion of

CAZP vehicle done did not alter the & response to LA, which was 68.2% and 60.5%

pnor to and following CAZP vehicle, respectively.

The average breath-by-breath response of & to right heart injection of capsaicin

for 1 1 anirnals is shown in Fig. 10. Capsaicin caused a maximal increase of 41 f 8.5% in

& (Fig. 1 1, top panel). Dunng CAZP infusion, the maximal response of & to capsaich

was inhibited by 83 * 4.7% (P < 0.0 1), indicating that cornpetitive antagonism of the

capsaicinlçal receptor effectively blocks reflex bronchoconstriction. Fifteen min

following CAZP infusion the response of & to capsaicin had recovered to 71 * 2 1.9% of

the control trial (Fig. 1 1, top panel).

The average breath-by-breath response of & and Em to 0-4 mi LA injectate (low

osrnolality) during CAZP is shown in Fig. 13. LA caused a m e a i increase of 50 +

7.0% in & (Fig. 14, upper panel) but, in contrast to CAPS, the LA-induced increase in

& was unafEected by CAZP infusion (39 + 5.7%; P > 0.05). At 15 min following CAZP

infùsion the response of & to LA was 95 * 13.7% of the control response. Figure 14

illustrates the average of the group peak (upper panel) and Eup (lower panel)

responses during controi, attempted CAZP blockade and recovery LA injections. E ~ L

response latencies and tirnes-to-peak for CAPS and LA are summarized in Table 3 . Heart

rate responses are summarized in Table 4.

Figure 8. Acute breath-by-breath response of mean lung resistance to capsazepine infusion

Average lung resistance response to capsazepine (500 pgkg%nin i.v.) in 19 newbom dogs. Intiision of capsazepine (- 6:45 min) caused an increase in inspiratory lung resistance fi) that was not significantly reduced by hyperinflation.

BREATH NUMBER

Figure 9. Cardiopulrnonary response to capsaicin pre and post capsazepine Cardio pulmonary res ponse illustrating the ant agonistic effect of capsaze pine (5 0 0

pg-kg-l-min Î. v.) on reflex responses to right heart injection of capsaicin (CAPS, 25 pg- kg- ') in a newbom dog. Transpulmonary pressure (Pm), flow 0, ddal volume (V7) and art enal blood pressure (BP) before (u p per panel) and during (lower panel) capsazepine infusion. The bronchoconstridor response is indicated by the increase in PTP and is accompanied by hyp O tension and bradycardia. During capsazepine the PTp and wdiovascular response were abolished. Arrows represent capsaicin inject and flush, respectively; - indicates expiratory flow.

Figure 10. Breath-by-breath response of pulmonary mechanics to capsaicin Average (k SEM) breath-by-breath values of percent change in inspiratory lung

resistance &) and dynamic lung elastance (E-) in response to right heart injections of capsaicin (arrow) in 11 newbom dogs for three separate conditions: MTACT (lefi panels) - Right heart injection of capsaicin caused a brisk bronchoconstrictor response. CAPSAZEPINE (rniddle panels) - Capsaicin response was blocked by capsazepine infusion. RECOVERY (right paneis) - Partial recovery of the capsaicin response 15-20 min afler CAZP.

% CHANGE FROM BASELINE

Figure 11. Average maximal changes in the response of pulmonary mechanies to capsaicin

Mean peak response (+ SEM) of inspiratory lung residance (RL, top panel) and dynamic lung elastance (ELdyi>, bottom panel) plotted as % change fiom baseline for saline and three dierent capsaicin injections (INTACT, BLOCK and RECOVERY). +, sigaificantly ciifferent from MTACT capsaicin response; * significantly different fiom SALINE control. P < 0 .O5 (ANOVA with Tukey post hoc).

SALINE INTACT BLOCK RECOVER

Figure 12. Cardiopulmonary response to lactic acid pre- and post- capsazepine Cardiopulmonary response illustrating the effect of capsazepine on reflex

responses to right hart injection of lactic acid (0.4 mol-kg-') in a single newborn dog. Note that capsazepine did not abolish the increase in Pip or hypotension response to lactic acid. Abbreviations as in Figure 9.

Figure 13. Breath-by-breath mechanic. responses to lactic acid Mean responses of lung mechanics to lactic acid (0.4 mmol-kge1) in 8 newbom

dogs. Average (+ SEM) breath-by-breath values of percent change in inspiratory lung resistance (RL, upper panels) and dynamic lung elastance lower panels) in response to rïght h a r t injections of lactic acid (arrows) for three separate conditions. Right heart injection of lactic acid caused a robust bronchoconstrictor response (INTACT, left panels) that was maintained during capsazepine infusion (500 pg- kg-'-min; CAPSAZEPNE, middle panels) and post capsazepine (RECOVERY, nght panels).

% CHANGE FROM BASELINE

Figure 14. Average maximal responses of pulmonary mechanics to laetic acid Mean peak response (+ SEM) of inspiratory lung resistance &, top panel) and

dynamic lung elastance (Etdyn, bottom panel) piotted as % change from baseline to saline and three Merent lactic acid responses (INTACT, BLOCK and RECOVERY). Lactic acid responses were ail sigoificantly different fiom saline (P < 0.05) but were not different from each other (P > 0.05). *, significantly different from SALINE control.

SALINE INTACT BLOCK RECOVERY

TABLE 2.

Lactic Acid INTACT ATROPINE

Latency T h e to peak Latency Time to peak

0.2 ml injectate 6.8 I 1.2 18.7 * 2.7 9.5 & 1.6 21.9 & 2.0

0.4 ml injectate 8.4 f 2.1 26.3 f 6.0 * n/a n/a

* different From 0.2 ml injectate time to peak (P > 0.05); d a not applicable.

TABLE 3 .

Protocol INTACT CAPSAZEPINE RECOVERY

Latency Time to peak Latency Time to peak Latency Time to peak (s) (s) (s) (s) (s) (s)

CAPS 2.3 I 0 . 4 6.4 I 1 .O nla nla 1.8 k 0.5 6.2 I 1 .O

ACh 3.2 î 1 .O 1 0.5 î 0.9 4.0 î 1.1 12.5 I 2.2 2.8 î 0.6 9.7 k 0.9

LA 5.2 I 1.7 17.8 * 3,1 8.2 k 1.8 21.6 k 3.7 4.8 k 1.8 19.2 * 4.5

da not applicable.

TABLE 4.

Challenge INTACT CAPSAZEPINE RECOVERY

Baseline % Decrease latency Baseline % Decrease latency Baseline % Decrease latency (bpm) (s) (bpm) (s) (bpm) (s)

saline 185 f 4.4 411.1* 4 I 0 . 3 nla nla nla nla nla nla

CAPS 206 f6.3 8 * p,g*t 5 f 1.4 208 k 5.8 6&2.4* 5 I 1 . 5 215 I 9.3 5 I 3 . 1 * 5 k 1 . 9

LA 181 f 5.6 31 * 7,8*+t 7 f 2.8 207 f 5.5 33 * l0,4*t 6 f 0.9 213 k 11.9 36 f 8.5*+ 9 * 3.8

* different from baseline value (P < 0.05); t different fiom capsaicin (P < 0.05); f different fiom saline (P < 0.05); da , not applicable.

CHAPTER 4

Discussion

Discussion

Our results show that LA evokes an atropine-sensitive increase in bronchomotor

tone that is consistent with that described for other stimulants of C-fibre afferents

(Coleridge et al., 1989). Our study also found that capsazepine, the cornpetitive

capsaicinNR 1 receptor antagonist, sigdïcantly attenuates the bronchomotor reflex

response to capsaicin, but not the response to LA. Taken together, these results indicate

that LA is not an endogenous VRI ligand but suggest that LA and CAPS stimulate reflex

bronchoconstriction via different receptor signal transduction mechanism.

In both the adult and neonate, activation of pulmonary C-fibres by the vanilloid

excitotoxin capsaicin evokes reflex bronchocoastriction (Coleridge et al., 1989; Fisher et

al., 1 998), which is variably accompanied by mucus hypersecretioq vascular exudation

and ainvay edema (Coleridge et al., 1989; Lundberg & Saria, 1982). Physiological

stimulation of puhonary C-fibres initiates airway reflexes aimed at iimiting exposure of

the ainvays to noxious stimuli (as in Coleridge & Coleridge, 1994; Paintai, 1973; Karlsson

et al., 1988); however, activation of pulmonary C-fibres has also been implicated in the

airway hyperreactivity accompanying idammatory lung diseases such as asthma or, in the

newborn, BPD (Barnes, 1986; Bames, 1995; Spina, 1996). The magnitude of the

bronchoconstriction we observed to both CAPS and LA is submaximal (Forkert & Fisher,

1992) and may reflect a mechanism to enhance gas exchange by reducing dead space

volume (Widdicombe, 1963) or to protect the highly cornpliant airways of the newbom

fiom dynamic compression (Olsen et al., 1967).

Despite characterization of the in vivo reflex effects of CAPS, the effects of

capsaicin on pulmonary C-fibres and elucidation of the molecular mechanism of action of

the putative C-fibre "capsaicin" receptor (Le. VRl ), the endogenous VRI ligand remains

to be identified. Likely activation of pulmonary C-fibres during idammatory conditions

has led to the proposal that biological compounds released in the i~arnmatory process

may represent endogenous agonists for the capsaicin receptor (Bevan & Geppetti, 1994;

Franco-Cereceda et al., 1994; Longhurst et aL, 199 1). Among these compounds, lactic

acid (LA) has received considerable attention. Acidic pH has been shown to evoke a

capsazepine-sensitive bronchoconstriction in the isoiated pettùsed guinea pig lung,

presumably by acting directly on C-fibre endings to cause release of tachykinins (Lou &

Lundberg, 1992). The ability of LA to act as an endogenous stimulant of reflex

bronchoconstriction in vivo however, has not been tested in the adult despite its excitatory

actions on C-fibres. The newbom is particularly interesting with respect to the actions of

K on C-fibres since metabolic and respiratory acidosis are normal components of

parturition as well as in certain pathologicai complications of birth (Koch & Wendel,

1968; Koch, 1968; Penn et a!., 1988; Abman & Groothius, 1994). While C-fibre

recordings have not been made in the newbom, C-fibre type reflexes have been examined

(Kalia, 1976; Ducros & Trippenbach, 199 1 ; Haxhiu-Poskunca et ai., 199 1 ; Anderson &

Fisher, 1993).

Initial studies in the newbom led to the conclusion that LA (0.25 mol-kg-') does

not evoke a bronchoconstnctor response in 1-6 d old rabbit pups @ucros & Trippenbach,

1991). In this study, we directly measured total pulmonary resistance following

presumptive C-fibre stimulation with comparable LA doses and found that LA caused a

robusf cholinergie, reflex broncho constriction in the newbom dog. Whiie the discrepancy

between Ducros and Trippenbach (1 99 1) and our findings could be attributable to species

dserences, other factors are equaliy as likely. Ducros and Trippenbach used the ratio of

tidal volume to eosophageal pressure swing (Le. Cum) to monitor changes in

bronchomotor tone. However, CUyn is an indirect index of bronchoconstriction and may

be insensitive to small changes in ASM tone or resistance (Widdicombe, 1963).

Furthermore, CLdp is constrained in its relative response since it can decrease only in the

range of 100 to 0% of the control value, whereas 5 can increase several fold above

control. Indeed, this is why we evaluated EU, in the present study. In preliminary

experiments we found that right heart injection of 0.2 mmol-kg-' LA evoked modest but

quantifiable bronchoconstrictor responses in the newbom dog (8.3% increase in & versus

a 5% decrease in CLdyn). The small CLdyn response associated with the response of to

0.25 mmol-kg-' LA may make bronchoconstriction more difficult to detect with dynamic

cornpliance measurements. Altematively, 0 -2-0 -2 5 mrnol-kg-' may be inadequate to

sufficiently stimulate C-fibres in the newborn rabbit, which is more immature at birth than

the dog.

Development of the cornpetitive C-fibre receptor antagonist CAZP allowed us to

specifically investigate the putative mechanism of C-fibre activation with LA. In vivo,

CAZP attenuates the cardiorespiratory depressor effects evoked by CAPS in the adult rat

(Lee & Lundberg, 1994) and both in vivo (rat) and in vitro (guinea pig) recordings of C-

fibre responses to CAPS support this observation (Lee et al., 1996; Fox el al., 1995).

Furthermore, CAZP abolishes the CAPS-evoked inward cation current in neonatal rat

DRG cells (Bevan et al., 1992). Nevertheless, to our knowledge the effect of CAZP on

CAPS-evoked reflex bronchoconstriction had not been investigated prior to Our study.

We found that CAZP effectively antagonized CAPS-evoked bronchoconstriction in vivo

(Fig. IO), which supports the notion that therapeutic targeting of sensory afferents in the

newbom may be a beneficid strategy for reducing both the cholinergie reflex component

as well as potential tachykinin components of the bronchomotor response associated with

C-fibre activation in idammatory lung disease (Spina, 1996; Spina & Page, 1996; Bames,

1986; Bmes , 1995; Szallasi & Blumberg, 1996).

Ln contrast to CAPS-evoked bronchoconstriction, LA-induced bronchomotor

refiexes were essentially unaEected by CAZP (Fig. 13). The CAZP-resistance of the LA-

evoked bronchomotor response indicates that LA and CAPS stimulate reflex

bronchoconsuiction either via different receptor signal transduction mechanisms withui the

same pulmonary C-fibre afTierent, or via different pulmonary C-fibre subpopulatiom. The

former explanation is the most likely, as single fibre recordings from adult rat have shown

that the same C-fibres are stimulated by CAPS and LA (Lee et al., 1996; Hong et al.,

1997). Descriptions of a proton-evoked cation current in frog and rat DRG neurons

(Akaike et al., 1990; Bevan & Yeats, 199 l), the latter localized in fibres sensitive to

CAPS, combined with the recent cloning of an acid-sensing ionic channel (ASIC) from

Xenopus DRG (Waldmann et al., 1997; Waldmann et al., 1997) provide additional

evidence in support of separate pulmonary C-fibre receptor mechanisms mediating CAPS-

and LA-evoked reflex bronchoconstriction. Altematively, the finding that acidic

extracellular pH below the threshold required to activate C-fibres facilitates capsaicin-

induced cation current (Caterioa et al., 1997) is consistent with Vycklicky et aL's (1 998)

later suggestion that the facilitatory actions of protons on the capsaicin-evoked cation

current may be mediated by protonation of VRl . While this latter speculation addresses a

possible mechanism by which protons potentiate the activation of VRI with capsaicin, its

mechanistic focus does not offer an alternative endogenous VRI ligand candidate. In

order for the facilitatory actions of protons to explain Our results, H* would have to

unmask a reflex, C-fibre-rnediated response to a cirnilating endogenous ligand.

Others have alluded to the potential role of additional innammatory compounds as

endogenous stimulants of VRl (Karla et al., 1992; Shams et al., 1988; Vyklicky et al.,

1998). The CAZP-insensitivity of the LA response, combined with the longer latency and

time-to-peak of the bronchomotor response to LA (Tables 2 and 3) compared to CAPS,

could reflect a LA-induced metabolic intermediary that activates pulrnonaiy C-fibre

receptors. Based on Our data, it is not possible to state whether the tirne course of the RL

response supports either a direct protonnA-mediated mechanism of C-fibre activation or

the generation of an intermediary endogenous C-fibre stimulant released by LA. Slow

infusion of hydrochlonc acid stimulates platelets to release thromboxane Az (TxA*), an

arachadonic acid cyclooxygenase metabolite and C-fibre stimulant (Sharns et al., 1988).

Other products of the arachadonic acid cyclooqgenase pathway, such as PGE2 and

PGF*,, also stimulate unmyelinated sensory afFerents andlor potentiate their activation and

reflex effects (Coleridge et cd, 1978; Lee & Morton, 1995; Steen el al., 1995; 1996).

While application of PGE2, bradykinin and 5-HT together does not evoke the inward

cation current characteristic of VRl activation in cultured newborn rat sensory neurons,

the acidic pH-evoked cation curent is greatly potentiated in the presence of i n f f t o r y

mediators (Steen et al, 1995; 1996). Furthemore, capsazepine appears to block the

potentiating effect of acid pH on idammatory mediator-evoked current but does not

reduce the underlying acid-evoked current itself (Vyklicb et al., 1998). The potentiating

effect of acidic pH on the infiammatory mediator-evoked current in sensory neurons is

similar to that evoked by acid pH during submaxirnal capsaicin application ( C a t e ~ a et al.,

1997) and has prompted the hypothesis that infiammatory mediators, specifidy PGE2,

may represent endogenous ligands for the "capsaicin" or VRI receptor (Vyklicky et al.,

1998). Whether these mechanisms play a role in the newbom dog's response to LA that

we observed is unclear, although they appear unlikely. Lee and colleagues found that

cyclooxygenase intermediaries were not involved in the LA-induced pulmonary

chernoreflex in the rat in which reflex apnea, bradycardia and hypotension evoked by LA

were unaffected by pretreatment with the cyclooxygenase enzyme antagonist indomethacin

(Lee & Morton, 1995; Lee et al., 1996). In a single animal, we assessed the effect of

indomethacin (15 mg-kg-'); it did not attenuate the bronchomotor response to LA

supporting Lee and colleague's conclusion that an LA-evoked cyclooxygenase

intermediary mechanism of C-fibre activation is unlikely .

Potential Mechanisms of Action

Based on the avaiiable data in the literature and Our study, it appears that C-fibres

are stimulated by at least 2 paralle1 receptor transduction mechanisms (Fig. 15). The first,

VRl, is a non-specific cation conductance which specifically binds vanilloid compounds

(Le. capsaicin, resiniferatoxin) and displays a secondary putative recognition site for H+

that facilitates or amplifies the response to CAPS. While aione appears to be only a

weak stimulant of this receptor (Caterina et al., 1997), it potentiates the inward cation

current evoked by both vanilloids and idammatory mediators (specincaiiy l'GEÎ), the

latter of which may represent an endogenous VRl agonist (Vyklicky et al., 1998). The

second receptor transduction mechanism (ASIC) is a H'-gated Na' conductance. Since

the lactate anion potentiates the acide-evoked pulrnonary chemoreflex, ASIC might also

display a secondary recognition site for the lactate anion (Fig. 15). Our hdings show a

clear separation of the mechanisms with respect to in vivo bronchomotor reflexes. C-fibre

recordings in the newbom are required to further clar@ the hypothesized parallel C-fibre

receptor transduction mechanisms by quantdjmg activation Iatencies and afferent

discharge fkequencies. Biophysical studies of the vanilloid receptor-induced cation current

and its pH sensitivity wiII also be cnticai in elucidating the mechanisms activating C-fibre

afferents.

Figure 15. Possible mechanisms of activation of C-fibre receptors by EI+ Hypotheses for the activation of pulmonary C-fibres with capsaicin and lactic acid

based on the present study and the literature. Capsazepine abolished the "capsaicin"NR1 receptor-mediated response to capsaicin but not the LA-evoked response. The present study indicates that CAPS and LA act via different sensory mechanisms, consistent with the 2 paraiiei activation mechanisms shown: VRI, a non-specific cation conductance with a primary activation site which binds capsaicin and a secondas, recognition site for H' that enhances the response to CAPS (Caterina et al., 1997); and ASIC (Acid-Sensing Ionic Channel), a Kgated conductance (Waldmann et al., 1997) with a secondary recognition site for the lactate anion that reflects the reported ability of lactate anion to enhance the H' response (Hong et al., 1997).

Surnrnary

In summary, we conclude that LA behaves a s an endogenous C-fibre stimulant to

cause reflex bronchoconstriction. Further, we conclude that capsazephe reversibly

antagonizes the reflex bronchoconstnction eiicited by right heart injections of capsaicui,

presumably by acting at the C-fibre "capsaicin"NR1 receptor. Finally, the CAZP-

resistance of the LA-induced airway response indicates that LA acts via an independent

mechanism to that of CAPS to evoke reflex bronchoconstriction, possibly by activation of

an acid-sensing ionic channel-like C-fibre receptor transduction mechanism.

References

ABMAN, S. H. and GROOTHIUS, J. R (1994) Pathophysiology and Treatment of Bronchopulmonq Dysplasia Respir. Med 4 1 (2), 277-295.

AKA[KE, N., KRISHTAL, O. A. & MARUYAMA, T. (1 990). Proton-induced sodium m e n t in fiog isolated dorsal root ganglion cells. J. NeMophysioL 63, 805-8 13.

ANDERSON, J.W. & FISHER J.T. (1993). Capsaicin-induced reflex bronchoconstriction in the newbom. Respir. Physiol. 93, 13-27.

BAKER, D.G., BASBAUM, C.B., HERBERT, D A & MITCHELL, RA. (1983). Transmission in airway ganglia of ferrets: inhibition by norepinep hrine. Neuroscz. Lett 4 1, 139-143.

BALUK, P., NADEL, J.A. & MCDONALD, D.M. (1 992). Substance P-immunoreactive sensory axons in the rat respiratory tract: a quantitative study of their distribution and role in neurogenic inflammation. J. Comp. Neurol. 319, 586-598.

BARNES, P.J. (1986). Asthma as an axon reflex. Lancer 242-245.

BARNES, P.J. (1986). Neural control of human airways in heaith and disease. Am. Rev. Respir. Dis. 134, 1289-1314.

BARNES, P.J. (1995). 1s asthma a nervous disease? Chest 107, 119s-125s.

BATES, J.H. & MARTIN, J.G. (1990). A theoretical study of the effect of ainvay smooth muscle orientation on bronchoconstriction. J. Appl. Physiol. 69, 995- 100 1.

BELVISI, M.G., MIURq M., STRETTON, D. & BARNES, P.J. (1992). Capsazepine as a selective antagonist of capsaich-induced activation of C-fibres in guinea- pig bronchi. Eur. J. Pharmacol. 215, 34 1-344.

BEVAN, S. & GEPPETTI, P. (1994). Protons: s m d stimulants of capsaicin-sensitive sensory nerves. Trendii Nemsc i . 17, 509-5 12.

BEVAN, S., HOTHI, S., HUGHES, G., JAMES, I.F., RANG, H.P., SHAH, K., WALPOLE, C.S.J. & YEATS, J.C. (1992). Capsazepine: a cornpetitive antagonist of the sensory neurone excitant capsaicin. Br. J. Phannacoi. 107, 544-552.

BEVAN, S. & YEATS, J. (1991). Protons activate a cation conductance in a sub- population of rat dorsal root ganglion neurones. J. Physzol. (Lund.) 433, 145- 16 1.

BKUTAM, V.K., KOSLO, RJ. &: SHAFFER, T.H. (1986). The effects of tracheal smooth muscle tone on neonatal ainvay collapsibility. Pediafr. Res. 20,492-495.

BHUTANI, V.K, RUBENSTEIN, S.D. & SHAFFER, T.H. (198 1). Pressure induced deformation in immature airways. Pecüatr. Res. 15, 829-83 2.

BIGGS, D.F. & GOEL, V. (1985). Does capsaicin cause reflex bronchospasm in guinea- pigs? Eur. J. Phmucol. 115, 71-80.

CABEZAS, G. A, GRAF, P.D. & NADEL, J.A (1 97 1). Sympathetic versus parasympathetic nervous regulation of ainvays in dogs. J. Appl. Physzol. 3 1, 65 1-65 5.

CAMPBELL, W. B. (1 990) Lipid-Derived Autacoids: Eicosanoids and Platelet-Activating Factor. In 7he Phmmacol~~cul Baris of 7herapeufics., eds. A. Goodman Gilmaq T.W. Rall, AS. Nies and P. Taylor. Permagon Press, New York, pp 600-6 17.

CANNY, G. J., BOHN, J. G., REISMAN, J. J, AND LEVISON, H. (1993) Childhood Asthma In Bronchiul Asthma. , eds. E.B. WEISS AND M. STEIN. Little, Brown and CO., Bostoq pp1062-1084.

CATEFUNA, M.J., SCHUMACHEq M.A., TOMINGA, M., ROSEN, T.A., LEVINE, J.D. & JULNS, D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nahrre 389,8 16-824.

CLAPHAM, D.E. (1 996). TRP is cracked but is CRAC TRP? Neuron 16, 1069- 1072.

COBURN, RF. & TOMiTA, T. (1973). Evidence for nonadrenergic inhibitory nerves in guinea pig trachealis muscle. Am. J. Physiol. 224, 1072- 1080.

COLERIDGE, H.M. & COLERIDGE, J.C.G. (1994). Pulmonary refiexes: neural mechanisms of puhonary defense. Anm. Rev. Physzol. 56,69-9 1.

COLERIDGE, H.M., COLERIDGE, J.C.G., BAKER DG., GINZEL, K.H. & MORRISON, U A . (1978). Cornparison of the effects of histamine and prostaglandin on aEerent C-fibre endhgs and irritant receptors in the intrapulmonary airways. In m e reguiation of respiration during sleep cmd anesthesia. , eds. FITZGERALD , R S., GAUTIER, H. & LAHIRI, S., Plenum Press, New York.

COLERIDGE, H.M., COLERIDGE, J.C.G. & SCHLILTZ, H.D. (1989). AfEerent pathways involved in reflex regulation of ainvay smooth muscle. Phannacol. Ther. 42, 1- 63.

COLERIDGE, J-C. & COLERIDGE, H.M. (1 984). Anerent vagal C fibre innervation of the lungs and ainvays and its fùnctional significance. Rev. Physiol. Biochem. Phmaco l . 99, 1-110.

COLERIDGE, J.C.G., COLERIDGE, H.M., ROBERTS, A.M., KAUFMAN, M.P. & BAKEK D.G. (1982). Tracheal contraction and relaxation initiated by lung and somatic at3erents in dogs. J. Appl. Physol. 52, 984-990.

CROSS, B A , GUZ, A, MIN, S-K, ARCHER, S., STEVENS, J. & REYNOLDS, F. (1976). The effect of anaesthesia of the airway in dog and man: a study of respiratory reflexes, sensations and lung mechanics. Clin. Sci. Md. Med 50, 43 9-454.

DICKENSON, A-H. & DRAY, A. (1991). Seledve antagonism of capsaicin by capsazepine: evidence for a spinal receptor site in capsaicin-induced antinociception. Br. J. Pharmacol. 104, 1045- 1049.

DUCROS, G. & TRIPPENBACY T. (1991). Respiratory effects of lactic acid injected into the jugular vein of newbom rabbits. Pediair. Res. 29, 548-5 52.

FISHER J.T., BRUNDAGE, K.L. & ANDERSON, J. W. (1 995). Cardiopulmonary actions of muscarinic receptor subtypes in the newbom dog. C m J. Physiol. Phmmacol. 74: 603-613.

FISHER, J. T., HAXHIU, M. A , AND MARTIN, R J. (1998) Regulation of Lower Airway Function. In Fetal and Neonatal Physioiogy., eds. RA. POLIN AND W. W. FOX, W.B. Saunders and Company, Philadelphia, pp. 1060- 1070.

FISHER J.T., MATHEW, O.P. & SANT'AMBROGIO, G. (199 1). Morphologicai and neurophysiologicai aspects of airway and pulmonary receptors. In Deveiopmental neurobiology ojbreaihing, eds. HADDAD, G.G. & FARBER, J.P., pp. 2 19-244. New York: Marcel Dekker, Inc.

FISHER J.T. & MORTOLA J.P. (1 98 1). Statics of the respiratov system and growth: an experimental and dometric approach. Am. J. Physiol- 241, R336-R341

FISHER, J.T., SANT'AMBROGIO, F.B. & SANT'AMBROGIO, G. (1983). Stimulation of trached slowly adapting stretch receptors by hypercapnia and hypoxia. Respir. Physiol. 53, 325-339.

FORKERT, L. & FISHER, J.T. (1 992). The effect of vagal stimulation on the distribution of inspired gas in the lungs. Respir. Physiol. 88, 277-287.

FOX, A.J., BARNES, P.J., URBAN, L. & DRAY, A (1993). An in vitro study of the pro perties of single vagal Serents innervating guinea- pig ainvays. J. Physioi. @and.) 469,21-35.

FOX, AJ., URBAN, L., BARNES, P.J. & DRAY, A (1995). Effects of capsazepine against capsaicin- and proton-evoked excitation of single airway C-fibres and vagus nerve from the guinea-pig. Neurosci. 67, 74 1-75 2.

FRANCO-CERECEDq A., KALLNER G. & L W B E R G , J.M. (1994). Cycle- oxygenase products released by low pH have capsaicin-iike actions on sensory nerves in the isolated guinea pig heart. Cardiovmc. Res. 28, 365-369.

GHATEI, M.A, SPRINGALL, D.R, RICHARDS, LM., OOSTVEEN, LA, GRIFFIN, RL., CADIELJX, A., POL& J.M. & BLOOM, S-R (1 987). Regdatory peptides in the respiratory tract of Macaca fàsciiculcrris. or^ 42,43 1-439.

GRUNSTELN, M.M., TANAKA, D.T. & GRUNSTEIN, J.S. (1984). Mechanisrns of substance P-hduced bronchoconstriction in maturing rabbits. J. Appl. PhysioC. 57(4), 1238-1246.

HAKANSSON, CH., MERCKE, U., SONESSON, B. & TOREMALM, N.G. (1976). Fundional anatomy of the musculature of the trachea. Acta MorphoII Neerl. S c d 14, 29 1-297.

HAXHTU-POSKURICA, B., CARLO, W.A., MILLER, M.J., DIFIORE, J.M., HAXIIIU, M A & MARTIN, RJ. (1991). Maturation of respiratory reflex responses in the piglet. J. Appl. PPhysd. 70,608-616.

WAXHIU-POSKURICA, B., ERNSBERGER P., EIAxHIU, M.A., MILLER, M.J., CATTAROSSI, L. & MARTIN, R J. (1 993). Development of cholinergic innervation and muscarinic receptor subtypes in piglet trachea. Am. J. Physiol. 264, L606-L6 14

HOLZER P. (1991). Capsaicin: Cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Phannacol. Rev. 43, 143-20 1.

HONG, J.L., KWONG, K. & LEE, L.Y. (1997). Stimulatinn of pulmonary C fibres by lactic acid in rats: contributions of H-+ and lactate ions. J. Physiol. (zond) 500, 3 19-329.

HORSFIELD, K. ( 1 977). Postnatal growth of the dog's branchial tree. Respir. Physiol. 29, 186-191.

IRW, CG., BOILEAU, R, TREMBLAY, J., MARTIN, RR & MACKLEM, P.T. (1 980). Bronchodilatation: noncholinergic, nonadrenergic mediation demonstrated in vivo in the cat. Science 207, 79 1-792.

JEFFREY, P. K. (1998) The development of large and small airways. Ani. J Reqir. Crit. Care Med 157, S174-S180.

JOHNSON, D.E. & WOBKEN, J.D. (1 987). Calcitonin gene-related peptide imrnunoreactivity in ainvay epithelial ceils of the human fetus and infant. Cell T ' e Res. 250, 579-583.

JOHNSON, M.D., GRAY, M.E. & STAHLMAN, M.T. (1 988). Calcitonin gene-related peptide in human fetal lung and in neonatai lung disease. J Histochem. Cytochem. 36, 199-204.

KALIA, M. (1976). Viscerai and somatic reflexes produced by J pulmonary receptors in newbom kittens. J. Appl. Physiol. 41, 1-6.

=A, W., SHAMS, H., O R . J.A & SCHEID, P. (1992). E f f ~ of the thromboxane Az mimetic, U46,6 19, on pulmonary vagal afferents in the cat. Respir. Physiol. 87, 383- 396.

KARLSSON, LA, SANT'AMBROGIO, G. & WIDDICOMBE, J.G. (1 988). AfZerent neural pathways in cough and reflex bronchoconstriction. J. Appl. PhyszoL 65, 1007- 1 023.

KOCH, G. (1 968). Lung fbnction and acid-base balance in the newbom infant. Acta Paediab. S c d Suppl. 181.

KOCH, G. & WENDEL, H. (1968). Adjustment ofarteriai blood gases and acid base balance in the nomal newborn infant during the first week of Me. Biol. Neonat. 12, 136- 161.

KOSLO, RJ., BHUTANI, V.K. & SHAFFER, T.H. (1986). The role of tracheal smooth muscle contraction on neonatal tracheai mechanics. Pediu*. Res. 20, 1216-1220.

KUBIN, LL. AND DAWES, R 0. (1 995) Central Pathways of Pulmonary and Ainvay Vagal Merents. In Replation of Breathing, eds. J.A DEMPSEY AND A.I. PACK Marcel Dekker, Inc., New York, pp. 2 19-284.

LAITINEN, A. (1985). Autonornic innervation of the human respiratory tract as reveded by histochemicd and ultrastructural methods. Eur. J. Respir. Dis. 66(Supp. 140), 1-42.

LEE, L.-Y. & LUNDBERG, J.M. (1994). Capsazepine abolishes pulmonary chemoreflexes induced by capsaicin in anesthetized rats. J. Appl. Physiol. 76, 1 848- 1 8 5 5.

LEE, L.Y. & MORTON, RF. (1995). Pulmonary chexnoreflex sensitivity is enhanced by prostaglandin Ez in anesthetized rats. J. Appl. Physiol. 79, 1 679- 1 6 86.

LEE, L.Y., MORTON, RF. & LUNDBERG, J.M. (1996). Puimonary chemoreflexes elicited by intravenous injection of lactic acid in anesthetized rats. J. Appl. Physiol. 82, 2349-2357.

LONGHURST, J-C., ROTTO, D.M., KAUFMAN, M.P. & STAHL, G.L. (1991). Ischemicdy sensitive abdominal visceral aerents: response to cyclooxygenase blockade. Am. J. Physiol. 261, t-8 1

LOU, Y.-P. & LUNDBERG, J.M. ( 1 992). inhibition of low pH evoked activation of airway sensory nerves by capsazepine, a novel capsaicin-receptor antagonist. Biochem. Biophys. Res. Commun. 189, 53 7-544.

LUNDBERG, J.M. (1 99 5). Tachyklliins, sensory nerves, and asthma-an overview. Ccm. J. Physiol. Phmacol . 73, 908-9 14.

LUNDBERG, J.M., HOKFELT, T., MARTLING, C.-R, S w A & CUELLO, C. (1984). Substance P-immunoreactive sesory nemes in the lower respiratory tract of various mammals including man. CeZZ Tissue Res. 235, 25 1-26 1.

LUNDBERG, J.M. & SARIA, A. (1 982). Bronchiai smooth muscle contraction induced by stimulation of capsaicin-sensitive sensory neurons. Acta Physid. S c d 1 16, 473 -476.

LUNDBERG, J.M. & SARI4 A. (1 987). Polypeptide-containing neurons in airway smooth muscle. Anm Rev. Physiol. 49, 557-572.

MA, X., LI, W. & STEPHENS, N.L. (1997). Heterogeneity of ainvay smooth muscle at tissue and cellular levels. Cm. J. Physiol. P h a c o l . 75, 930-935.

MACLAGAN, J. & BARNES, P.J. (1 989). Muscarinic phannacology of the airways. 77PS Suppl. Dec., 88-92.

MAGGI, C.A., BEVAN, S., WALPOLE, C.S., RANG, H.P. & GIULIAM, S. (1 993). A cornparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens. Br. J. Pharmacol. 108, 80 1 -805.

MARSH, S.J., STANSFELD, C.E., BROWN, D.A., DAVEY, R & MCCARTHY, D. (1987). The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro. Neurosci 23,275-289.

MATSUBA, K. & THIJRLBECJS, W.M. (1972). A morphometric study of branchial and bronchiolar walls in children. Am. Rev. Respir. Dis. 105, 908-9 13.

MIDDENDORF, W.F. & RUSSELL, I.A. (1980). Innervation of airway smooth muscle in the baboon: evidence for a nonadrenergic inhibitory systern. J. AppL Physzol. 48, 947- 956.

MILLER, W. S. (1 947). The Lung. Springfield, Ii. : Charles C. Thomas.

MILLS, J.E., SELLICK, H. & WIDDICOMBE, J.G. (1969). Activity of lung imtant receptors in pulmonary microembolism, anaphylaxis and drug-induced bronchoconstrictions. J. Physzol. (Zond) 203,3 3 7-3 57.

MURPHY, T.M., RAY, D. W., ALGER L.E., PHILLIPS, 1. J., ROACH, J-C., LEFF, A R & SOLWAY, J. (1994). Ontogeny of dry gas hyperpnea-induced bronchoconstriction in guinea pigs. J. Appl. Physiol. 76, 1 1 50- 1 1 55.

MURPHY, T.M., ROY, L., PHILLIPS, LI., MITCHELL, RW., KELLY, E.A., MUNOZ, N.M. & LEFF, A.R (1991). Effect of maturation on topographie distribution of bronchoconstnctor responses in large diameter airways of young swine. Am. Rev. Respir. Dis. 143, 126-131.

NADEL, J . A & BARNES, P.J. (1 984). Autonomie regdation of the airways. Anm. Rev. Med 35, 45 1-467.

OKANLAMI, O.A, FRYER, AD. & HIRSHMAN, C . k (1996). Interaction of nondepolarizing muscle relaxants with M2 and M3 muscarinic receptors in guinea pig heart and lung. Anesrheszology 84, 1 55- 16 1.

OLSEN, C.R, DEKOCK, M.A. & COLEBATCH, H.J.H. (1967). Stability of airways during reflex bronchoconstnction. J. Appl. Physiol. 23(1), 23-26.

PAINTAL, A. S. (1 973). Vagal sensory receptors and their reflex effects. Physiol. Rev. 53, 159

PANITCH., H.B., ALLEN, J.L., RYAN, J.P., WOLFSON, M.R & SHAFFER, T.H. (1989). A cornparison of preterm and adult airway smooth muscle mechanics. J. Appl. Physiol. 66, 1 760- 1 765.

PAMTCH, H.B., DEORAS, K.S., WOLFSON, M.R & SHAFFER T.H. (1 992). Mahirational changes in ainvay smooth muscle structure-function relationships. Pediatr. Res. 31, 151-156.

PENN, RB., WOLFSON, M.R & SHAFFEq T.H. (1988). Effect of ventilation on mechanical properties and pressure-flow relationships of immature ainvays. Pediatr. Res. 23, 5 19-524.

PISARN, T.E., JONZON, A., COLERIDGE, H.M. & COLERIDGE, J.C. (1 99 1). Intravenous injection of hypertonie NaCl solution stimulates pulmonaq C-fibers in dogs. Am. J. Physol. 260, H1522-H1530.

POLGAR, G. & WENG, T.R (1979). The finctional development of the respiratory system. Am. Rev. Respir. Dis. 120, 625-695.

RAVI, K. & S INGH, M. (1 996). Role of vagal lung C-fibres in the cardiorespiratory effects of capsaicin in monkeys. Respir. Physiol. 106, 1 3 7- 1 5 1.

RICHARDSON, J. & BELAND, J. (1976). Non-adrenergic inhibitow nervous system in human airways. J. Appl. Physzoi. 4 1, 764-77 1.

RICHARDSON, J.B. (1979). Nerve supply to the lungs. Am. Rev. Respir. Dis. 119, 785- 802.

RUSSELL, J.A. (1980). Innervation of ainvay smooth muscle in the dog. Bull. Eur. Physiopa fhoL Respir. 16 , 67 1 -692.

S ANT'AMBROGIO, G. (1 982). Mortnation arising from the tracheobronchiai tree in marnxnals. Physiol. Rev. 62, 53 1-539.

SANT'AMBROGIO, G. (1987). Nervous receptors of the tracheobronchial tree. Anm. Rev. Physd. 49,6 1 1-627.

S u A., MARTLING, C R , DALSGAARD, C.J. & LUNDBERG, J.M. (1985). Evidence for substance P-immunoreactive spinal afferents that mediate bronchoconstriction. Acta Physiol. S c d 125,407-4 14.

SHAMS, H., PESKAq B . A & SCHEID, P. (1 988). Acid infusion elicits thromboxane A2-mediated effects on respiration and puhonary hemodynamics in the cat. Respir. P~YSZOL 71, 169-183.

SPINA D. (1 996). Ainvay sensory nerves: a burning issue in asthma? irhorm 51, 335- 337.

SPINA, D. & PAGE, C.P. (1 996). Airway sensory nerves in asthma-targets for therapy? PuZm. Phannacol. 9, 1 - 1 8.

STAHL, G.L. & L O N G W T , J.C. (1992). Ischemidy sensitive visceral afferents: importance of I-I+ denved from lactic acid and hypercapnia. Am. J Physiol. 262, H748- H753.

STAUB, N. C. AND ALBERTINE, K. H. (1988) The Structure of the Lungs Relative to Their Principal Function. in Textbook of Respirufory Medicne, eds. J.F. MURRAY AND J.A. NADEL, W.B. Saunders Company, Phiiadelphia, pp. 12-36.

STEEN, K.H., STEEN, A.E., KREYSEL, H.W. & REEH, P.W. (1996). Inflammatory mediators potentiate pain induced by experimental tissue acidosis. Pain 66, 163-1 70.

STEEN, K.H., STEEN, A.E. & REEH, P.W. (1995). A dominant role of acid pH in inflarnmatory excitation and sensitization of nociceptors in rat &in, in vitro. J Neurosci. 15, t-9

STEPHENS, N.L. & KROEGEK E. A (1 980). Ultrastructure, biop hysics and biochemistry of airway smooth muscle. In Lung Biology in Health and Disease. Physiology and Phmmacology of the azrways, ed. NADEL, J. A., Marcel Dekker Inc., New York.

SUZUKI, H., MORITA, K. & KURIYAMA, H. (1976). Innervation and properties of the smooth muscle of the dog trachea. Jq. J Physiol. 26, 303-320.

SZALLASI, A. & BLUMBERG, P .M. (1 996). Vanilloid receptors: new insights enhance potentid as a therapeutic target. [Review] [172 refs]. Pain 68, 1 95-208.

SZALLASI, A., BLUMBERG, P.M. & LUNDBERG, J.M. (1995). Proton inhibition of [3~resiniferatoxin binding to vanilloid (capsaicin) receptors in rat spinal cord. Eur. J. Phannacol. 289, 181-187.

m C H A R D , D. (1 986b). Chemoreflexes in rabbits. Reqir. Physid. 66, 3 67-3 86.

TRENCHARD, D. (1 986a). C O z M receptors in the lungs of anaesthetized rabbits. Respir. Physiol. 63, 227-240.

URBAN, L. & DRAY, A. (1 991). Capsazepine, a novei capsaicin antagonisf selectively antagonises the effects of capsaicin in the mouse spinal cord in vitro. Neurosci. Lett. 134, 9-11.

VON HEYEK, H. (1960) The Human Lung. Hafner Publishing Company, h c . pp. 206- 212.

VYKLICKY, L., KNOTKOVA-URBANCOVA, H., VITASKOVA, Z., VLACHOVA V., KRESS, M. & REEH, P.W. (1998). Inflammatory mediators at acidic ph activate capsaicin receptors in cultured sensory neurons fkom newbom rats. J. Neurophysioi. 79, 670-6.

WALDMANN, R, BASSILANA, F,, DE, W.J., CHAMPIGNY, G., HEURTEAUX, C. & LAZDUNSKI, M. (1997). Molecular cloning of a non-inadvathg proton-gated Na' channel specific for sensory neurons. J. Biol. Chem. 272,20975-20978.

WALDMANN, R, CHAMPIGW, G., BASSILANq F., HEURTEAUX, C. & LAZDUNSKI, M. (1 997). A pro ton-gated cation channel involved in acid-sensing. Nature 386, 173-177.

WALDRON, M.A., CONNELLY, B.J. & FISHER J.T. ( 1 989). Nonadrenergic inhibitory innervation to the ainvays of the newbom cat. J. Appl. Physiol. 66, 1995-2000.

WALDRON, M.A. & FISHES J.T. (1988). Differential effects of CO2 and hypoxia on bronchomotor tone in the newbom dog. Reqpir. Physiol. 72, 271-282.

WALDRON, M.A. & FISHER, J.T. (199 1). Neural control of airway smooth muscle in the newbom. In Developmental neurobzology of breafhing, eds. HADDAD, G. & FARBER, I.P., pp. 483-5 18. New York: Marcel Dekker, Inc.

WEIBEL, E. R (1 963) Morphometry of the Human Lung. Springer-Verlag, Berlin.

WIDDICOMBE, J.G. (1 963). Regulation of tracheobronchial smooth muscle. Physiol. Rev. 43, 1-37.

WDDICOMBE, J.G. (1985). Control of aiway caliber. Am. Rev. Respzr. Dis. 131, S33- S35

WIDDICOMBE, J.G. & NADEL, J . A . (1963). h a y volume, ainvay resistance, and work and force of breathing: theory. J. Appl. Physiol. 18, 863-838.

IMAGE EVALUATION TEST TARGET (QA-3)

APPLIED INLAGE . lnc - - - 1653 East Main Street - -. - Rochester. NY 14609 USA -- -- - - Phone: 716/482-0300 -- -- - - Fax: 716/288-5989

O 1993. Appiied Image. inc.. All Rights Resemd