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(7‘. ”,1 ‘ R1,. .2 '4: , . 5 » 3111;“ u. u... , ,. -v ' I“ n.11l-w...~ .m'w 1...:11'!.w. 11.1.3.4“ w .‘V‘ELrn' ”II-nu THESlS minimi‘miflinnmiliiu 93 99 8225 This is to certify that the dissertation entitled Cholinergic reactivity of feline tracheal smooth muscle in vivo presen.- Cheryl Rae Killingsworth has been accepted towards fulfillment of the requirements for PhD degreein Physiology Major professor Date 9/10/90 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 b. l' ——~. Llfikmar “Milan State 1 University L“ v——— ~—~——- PLACE IN RETURN BOX tcfd -we this checkout from your record. TO AVOID FINES return on or before date due. ’ DATE DUE DATE DUE DATE DUE i MSU Is An Affirmative Action/Equal Opportunity Institution czblmWMS-u: THE CHOLINERGIC REACTIVITY OF FELINE TRACHEAL SMOOTH MUSCLE IN VIVO By Cheryl Rae Killingsworth A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1990 Mb :3“ W ABSTRACT CHOLINERGIC REACTIVITY or FELINE TRACHEAL SMOOTH MUSCLE IN VIVO By Cheryl Rae Killingsworth Atropine sulfate aerosol can reverse or prevent virus-induced airway hyperreactivity, suggesting that post-ganglionic, cholinergic pathways cause the enhanced smooth muscle contraction that follows acute respiratory infection. Although parasympathetic efferent pathways appear to play a role in virus- induced airway hyperreactivity, the exact mechanism is unknown. This dissertation describes: 1) the use of an animal model to investigate the effect of acute respiratory viral infections on airway responses to cholinergic stimuli, and 2) the role of muscarinic inhibitory receptors in limiting airway constriction. Airway responsiveness was studied in cats 3 or 6 days following exposure to feline herpesvirus—I. Airway smooth muscle contraction was produced using vagal stimulation and intra-arterial acetylcholine (ACh) Changes in the external diameter of tracheal ring 4, pulmonary resistance (RL), and dynamic compliance (Cdyn) were measured. There was more tracheal ring constriction during vagal stimulation at 3 days post-inoculation (PID3) than at 6 days (PID6) or in control cats. There was no difference in RL or Cdyn during vagal stimulation in control and infected cats, nor was there a difference in any of the variables measured when control and infected cats were compared following postsynaptic muscarinic receptor stimulation with ACh. Tracheal hyper— responsiveness to vagal stimulation at PID3 was completely blocked by iv atropine and correlated with virus isolation tests. These data suggest that virus- induced tracheal hyperresponsiveness is mediated by prejunctional muscarinic receptors. To investigate the role of prejunctional muscarinic M2 inhibitory receptors in limiting airway constriction, airway responses during cholinergic stimulation were measured before and after cumulative iv doses of the M2 receptor antagonist gallamine. The changes in RL and Cdyn during prejunctional vagal stimulation were enhanced by iv gallamine, but gallamine had no effect on tracheal ring 4 constriction. This suggests that M2 receptors may play an important role in limiting intrathoracic airway constriction during vagal stimulation, but a similar role of M2 receptors in the cervical trachea could not be demonstrated. Dedicated to my husband, James E. McMinn, and to my parents, Patricia L. and Gordon W. Killingsworth, for their constant love and encouragement. iv 3| ACKNOWLEDGMENTS I gratefully recognize the support and education that I have received under the tutelage of Dr. N. Edward Robinson. He is a dedicated scientist, and certainly one of the finest teachers that I have encountered. I appreciate his continuous support, advice, understanding, and his thoughtful critiques during victories as well as disappointments. I also wish to thank other individuals in the pulmonary laboratory who never failed to listen to my ideas and offer valuable input, including Drs. Richard V. Broadstone, Victoria M. Kingsbury, Frederick J. Derksen, Peter R. Gray, Mingfu Yu, and Jacqueline S. Scott. Their careful evaluations of my work, as well as their friendships, will continue to influence my scientific career. In addition, I would like to thank Dr. Roger K. Maes for his expertise in virology and for providing the virus necessary for these studies. I received considerable assistance in the laboratory from a number of conscientious, bright students and technicians. I particularly appreciate the help of Cathy Berney, Elizabeth Rozanski, Stephanie Rubie, Melinda Wilkins, and Carol Kuzma. It would have been less enjoyable and much more difficult to complete my research without the contributions of these individuals. Finally, I wish to gratefully acknowledge the other members of my guidance committee, including Drs. Tom Adams, Greg Fink, John Chimoskey, Harvey Sparks, and Patrick Dillon. Their help with developing instrumentation and scientific techniques, statistical advice, computer training, and their evaluation and counsel regarding seminars, experimental protocols, manuscripts, and this dissertation have been sincerely appreciated. These studies have been supported in part by NIH Grant #HL01900. vi TABLE OF CONTENTS Page LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii INTRODUCTION 1 CHAPTER 1 4 LITERATURE REVIEW 4 Airway smooth muscle innervation 4 Introduction 4 Parasympathetic nervous system 6 Sympathetic nervous system 8 Nonadrenergic, noncholinergic nervous system 10 Afferent nerves 14 Airway ganglia 17 Muscarinic receptors on airway smooth muscle 18 Introduction 18 Ml receptors in airways 22 M2 receptors in airways 25 ' M3 receptors in airways 27 Viral infections and airway hyperreactivity 29 Introduction 29 Measurement of airway hyperreactivity 31 Potential mechanisms of airway hyperreactivity 34 Genetic factors 34 Decreased base-line airway caliber 36 Increased responsiveness of airway smooth muscle 37 Neuropeptides in airway hyperreactivity 39 Abnormalities in autonomic control of airway smooth muscle 42 vii TABLE OF CONTENTS (continued) Viral respiratory disease in the cat Introduction Feline herpesvirus-I infection in the cat SPECIFIC AIMS OF STUDIES MATERIALS AND METHODS Animals in herpesvirus-I studies Animals not infected with herpesvirus-I Surgical preparation Tracheal caliper and calibration Measurement of pulmonary resistance and dynamic compliance CHAPTER 2 IN VIVO AIRWAY CALIBER MEASUREMENT USING MICROFOIL STRAIN GAGE TRANSDUCERS Introduction Experimental design Results Calibration Airway caliber measurements Discussion CHAPTER 3 CHOLINERGIC REACTIVITY OF TRACHEAL SMOOTH MUSCLE FOLLOWING INFECTION WITH FELINE HERPESVIRUS-I Introduction Experimental protocol Statistics Results Clinical signs and histopathology Virus isolation and quantitation Tracheal ring 4 Intrathoracic airways Discussion viii 8&& 52 55 55 57 63 3 TABLE OF CONTENTS (continued) CHAPTER 4 MUSCARINIC M2 RECEPTORS MODULATE THE INCREASE IN PULMONARY RESISTANCE AND DYNAMIC COMPLIANCE IN CATS WITH TRACHEAL HYPERRESPONSIVENESS TO VAGUS NERVE STIMULATION Introduction Animal groups and experimental protocol Statistics Results Time control cats Effect of gallamine on airway responses during vagal stimulation in control and TH cats Effect of gallamine on airway responses to ACh in control and TH cats Discussion CHAPTER 5 MUSCARINIC INHIBITORY RECEPTORS DO NOT LIMIT FELINE TRACHEAL CONSTRICTION DURING VAGAL STIMULATION IN VIVO Introduction Animal groups and experimental protocol Statistics Results Time control cats Tracheal ring 4 measurements in control cats and cats with tracheal hyperresponsiveness Change in RL and Cdyn in control cats and cats with tracheal hyperresponsiveness Discussion CHAPTER 6 SUMMARY AND CONCLUSIONS LIST OF REFERENCES ix 93 94 94 98 98 107 110 111 112 112 116 120 127 132 a“ Table LIST OF TABLES Virus isolation, quantitation, and clinical signs in virus- infected and control cats. Pulmonary mechanics testing of virus-infected and control cats pre- and post-atropine injection. Effect of repeated vagal nerve stimulation on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats. Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats. Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in control and hyperresponsive cats before and after gallamine treatment. Effect of repeated vagal nerve stimulation on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in cats before and after gallamine injection 78 83 95 103 113 114 119 il Figure 10 11 LIST OF FIGURES Schematic representation of airway with major neural control mechanisms to smooth muscle Ventral view of the anatomy of the right side of the feline cranial cervical trachea Diagram of the device used for isotonic measurements of tracheal diameter Representative static calibration of the device used for isotonic measurements of tracheal diameter Changes in the tracheal diameter and tracheal pressure during spontaneous breathing in an anesthetized cat Changes in the diameter of tracheal ring 4 as a function of time during bilateral vagal stimulation (24 V, 0.5 msec duration, 05 -16 Hz) Mean frequency-response curve of 15 anesthetized cats Mean frequency-response curves of control, virus- infected PID3, and PID6 cats before and after atropine administration Mean ACh dose-response curves of tracheal ring 4 in control, virus-infected PID3, and PID6 cats before and after atropine administration Effect of gallamine on tracheal ring 4 constriction in control and tracheal hyperresponsive cats during bilateral vagal nerve stimulation Changes in total pulmonary resistance from base-line in control and tracheal hyperresponsive cats during bilateral vagal nerve stimulation prior to gallamine injection and following increasing doses of iv gallamine xi 35 59 61 62 69 71 81 82 97 ii I LIST OF FIGURES (continued) 13 14 15 16 17 18 Correlation of the change in pulmonary resistance and the change in diameter of tracheal ring 4 in cats during vagal stimulation prior to gallamine treatment Correlation of the change in pulmonary resistance and the change in diameter of tracheal ring 4 in cats during vagal stimulation after gallamine treatment Changes, in dynamic compliance from base-line in control and tracheal hyperresponsive cats during bilateral vagal nerve stimulation prior to gallamine injection and following increasing doses of iv gallamine Effect of gallamine on tracheal ring 4 constriction during bilateral vagal nerve stimulation (05-16 Hz) in 10 cats Effect of gallamine on tracheal ring 4 constriction during bilateral vagal nerve stimulation (05-16 Hz) in control cats and cats with tracheal hyperresponsiveness Changes in pulmonary resistance during vagal stimulation (05-16 Hz) prior to gallamine and following gallamine injection in 10 cats Changes in dynamic compliance during vagal stimulation (0.5-16 Hz) prior to gallamine and following gallamine injection in 10 cats xii 100 101 102 115 117 118 121 ia iv NANC PID ARL SP TCID,o TH VIP LIST OF ABBREVIATIONS acetylcholine dynamic compliance change in dynamic compliance from pre-stimulation value excitatory postsynaptic potential feline herpesvirus-I intra-arterial intravenous nonadrenergic, noncholinergic nervous system post-inoculation day pulmonary resistance change in pulmonary resistance from pre-stimulation value substance-P concentration required to produce infection in 50% of the cells in culture cat group with tracheal hyperresponsiveness to vagal stimu- lation vasoactive intestinal peptide xiii THE! INTRODUCTION Viral infections of the respiratory tract are the most common naturally occurring insult that produces airway hyperreactivity to bronchoconstricting stimuli (Golden et al. 1978, Richardson et a1. 1981). The common cold, or upper respiratory infection, is also the most frequent infection in humans, and is responsible for approximately 50% of time lost from work and school (National Center for Health Statistics 1975). Normal children infected with common respiratory viruses can develop bronchiolitis, airway hyperresponsiveness, asthma, or persistent pulmonary dysfunction (Gurwitz et al. 1981, Pullan and Hey 1982, Wright et al. 1989). Pulmonary function abnormalities are regularly a component of nonpneumonic influenza infection in humans. Several studies have suggested that in the self-limiting form of influenza virus infection, significant abnormalities in gas exchange and pulmonary mechanics are frequent and prolonged well beyond clinical illness (J chanson et al. 1969, Homer and Gray 1973, Hall et al. 1976, Little et al. 1976). There is also evidence that acute viral respiratory infections may initiate the development of asthma in normal persons (McIntosh 1976, Halperin et al. 1985). Parasympathetic efferent nerve fibers are supplied to the airways by the vagus nerves. Preganglionic vagal fibers travel from the central nervous system to parasympathetic ganglia located in the walls of the airways, and 2 postganglionic fibers from these ganglia extend to airway smooth muscle. Because atropine sulfate can prevent exaggerated bronchomotor responses associated with viral respiratory infections, investigators have implicated the parasympathetic nervous system in virus-induced airway hyperreactivity (Empey et al. 1976, Laitinen et al. 1976). My goal has been to study cholinergic regulation of the tracheobronchial system during acute upper respiratory infection. The cat frequently suffers from a viral respiratory disease that could prove useful in understanding the effects of viral respiratory disease in humans. My experimental plan included the study of tracheal smooth muscle segments in vivo plus measurements of intrathoracic resistance and dynamic compliance during cholinergic stimulation in control and virus-infected cats. The literature review is divided into five sections. The first section describes the anatomy and physiology of airway smooth muscle innervation, with particular emphasis on the cat. Reviews of the excitatory parasympathetic nervous system and the sympathetic and nonadrenergic, noncholinergic inhibitory systems, afferent neural pathways to respiratory smooth muscle, and airway ganglia are also included in this section. The second section provides information on muscarinic receptor subtypes that have been described in the airways. The third section describes measurements and potential mechanisms of airway hyperreactivity, particularly in regard to virus-induced hyper- responsiveness. The fourth section discusses the epidemiology of upper respiratory disease in the cat. Finally, the specific aims of the protocols described in this dissertation are outlined. Early in the tenure of my study, Dr. Tom Adams designed a device that employs matched microfoil strain gages to quantify airway diameter. The 3 instrument is attached to the external surface of the airway wall in situ with minimum surgery and allows the airway to retain its normal innervation and perfusion. A typical application leaves tracheal rings intact and limits exposure and drying of airway linings. The instrument construction and calibration is presented in the body of this proposal. The second group of experiments described in this dissertation have been designed to investigate airway responsiveness to cholinergic stimulation in cats infected with herpesvirus-I compared to sham-infected cats. Cats were studied at either post-inoculation day 3 or 6. Tracheal and intra-thoracic responses produced by intra-arterial ACh injection and bilateral vagal stimulation were measured before and after intravenous injection of atropine. Having discovered that atropine blocks the airway hyperresponsiveness detected in virus-infected cats studied at post-inoculation day 3, I began in vivo investigations of muscarinic receptor subtypes. Studies to examine the role of presynaptic muscarinic M2 inhibitory receptors in the control of airway caliber are described in chapters 4 and 5. Airway constriction was produced by presynaptic vagal stimulation and by postsynaptic muscarinic receptor stimulation with local infusions of ACh. Changes in tracheal and intrathoracic airway caliber were quantitated before and after injection of the M2 receptor antagonist gallamine. The final chapter is a summary of the results from the experiments completed during my tenure of study. ill CHAPTER 1 LITERATURE REVIEW Airway smooth muscle innervation Introduction Ultrastructural and fluorescence histochemical studies have shown that the airway smooth muscle of the cat receives extensive autonomic innervation (Silva and Ross 1974). Nerve fasciculi pass between bundles of smooth muscle as well as between the muscle cells to innervate the trachea, bronchi, and bronchioles. The trachea of the cat consists of a series of C-shaped cartilage rings connected to the trachealis muscle by dense fibroelastic tissue that is attached on the outer surface of the cartilage. The trachealis is composed of about 30 layers of smooth muscle that run uniformly parallel to the tracheal rings (Silva and Ross 1974) The trachea is surrounded by connective tissue that supports blood vessels, nerves, and lymphoid tissue. The nervous tissue consists of numerous large and small nonadrenergic and adrenergic nerve bundles, nonadrenergic ganglia, and occasional adrenergic ganglia (Silva and Ross 1974) At the lower end of the trachea, the trachealis muscle divides into two branches that continue on the dorsal aspect of the main bronchi. The proximal THE! 5 parts of the main bronchi are structurally similar to the trachea, consisting of C-shaped cartilaginous rings connected posteriorly to extensions of the trachealis muscle. Anatomic changes occur in the distal part of the main bronchi where plates of bronchial cartilage enclose a well-developed network of bronchial smooth muscle, the thickness of which does not decrease in proportion to the reduction in the lumen diameter (Towers 1953, Silva and Ross 1974). Ganglion cells are scattered along the peribronchial plexus down to the small bronchi, and are mainly in the extrabronchial plexus, which is external to the cartilage (Knight 1980). Nerves are present on the surface of the muscle as well as between muscle bundles and muscle cells to the level of the terminal bronchioles. The small respiratory bronchioles consist of approximately three layers of muscle where the nerves are seen mainly on the outer surface of the muscle, and less frequently, between the muscle and epithelium. Airway smooth muscle in the cat has three different neural inputs that are divided into excitatory and inhibitory pathways. In addition to classic cholinergic and adrenergic mechanisms, there is a third component of neural control, the nonadrenergic, noncholinergic pathway, which has been described more recently (Diamond and O’Donnell 1980, Irvin et al. 1980). Although the neurotransmitter for this inhibitory system remains uncertain, the neural nature of this response is confirmed by the fact that it can be abolished by the nerve toxin tetrodotoxin (Ito and Takeda 1982). THE! Parasympathetic nervous system In 1819, Krimer galvanized the vagus nerves and observed "contraction' of the lungs (Quoted by Widdicombe 1963). Similar experiments and a review of those reports has been published (Macklin 1929) A nerve-mediated excitatory cholinergic response is the dominant bronchoconstrictor mechanism in the airways of all species investigated and plays an important role in the regulation of airway tone (Widdicombe 1963). Cholinergic efferent nerves arise in the vagal nuclei of the brain stem and form the jugular ganglion at the level of the jugular foramen. A short distance beyond the foramen the nerve fibers form a second ganglion, the nodose ganglion, which lies dorsocaudad to the superior cervical sympathetic ganglion. The ganglia and the vagus and sympathetic trunks are closely bound together by connective tissue until their separation just before entering the thorax (Reichard and Jennings 1925). At the level of the first rib, the vagus usually receives one or two branches from the middle cervical ganglion of the sympathetic trunk (Reichard and Jennings 1925). Efferent fibers travel in the vagus nerve to synapse in ganglia situated in the airway wall and from these ganglia, short postganglionic fibers pass to airway smooth muscle cells. All nerves leading into the trachea and bronchi are mixed and contain a combination of afferent and efferent fibers and a mixture of parasympathetic preganglionic and sympathetic postganglionic fibers. Most of the approximately 30,000 fibers of the vagus nerve in the cat are nonmyelinated (Agostoni et al. 1957). Of 4900 myelinated fibers, approximately 40 percent are efferent motor fibers to cat airways. i | 7 Excitation of the vagus nerve results in the release of ACh from agranular vesicles in cholinergic nerve terminals and the ACh rapidly diffuses the relatively short distance to cholinergic receptors on the target cell. Vagal nerve stimulation causes constriction from the trachea to the small airways, which is potentiated by cholinesterase inhibitors and blocked by the muscarinic receptor antagonist atropine (Nadel and Barnes 1984). Maximal constriction during vagal stimulation occurs in cat airways with a resting diameter of 0.8 to 2.0 mm and minimal constriction occurs in airways less than 08 mm in diameter (Nadel et al. 1971). Bronchoconstriction occurs rapidly and is readily reversed. Rapid freezing of the airways in cats during vagal stimulation has confirmed that muscle contraction is responsible for the airway narrowing rather than airway wall edema or luminal obstruction with mucus (Olsen et al. 1965). In many species, there is resting bronchomotor tone that can be abolished by section of the vagus nerve and by administration of atropine (Severinghaus and Stupfel 1955, Kilburn 1960, Widdicombe et al. 1962). In cats, this tone is restored by electrical stimulation of the transected vagal nerves at 4 Hz (Olsen 1965). Direct recording from vagal efferent fibers in cats confirms an irregular tonic firing at rest (Widdicombe 1961). In this dissertation, presynaptic will be used as a general term to refer to any location on the nerves, preganglionic will refer specifically to the fibers extending from the central nervous system to the ganglia, and prejunctional will be used for the postganglionic fibers that innervate smooth muscle and glands in the lung. Presynaptic modulation of cholinergic nerves by neurotransmitters or by inflammatory mediators has been studied most extensively in the dog. Norepinephrine reduces cholinergic nerve-mediated THE! 8 contraction of canine tracheal smooth muscle at concentrations that have little effect on ACh responses, suggesting an inhibitory effect on ACh release from postganglionic nerve terminals (Vermiere and Vanhoutte 1979) This inhibition is probably mediated by presynaptic beta-receptors, and is one of many examples of the presynaptic modulation seen in other nerve pathways (Barnes 1986). Prostaglandin E2 has a similar inhibitory effect on cholinergic neurotransmission in dogs (Walters et al. 1984) Serotonin, prostaglandin Fwy“, substance-P (SP), and adenosine appear to potentiate ACh release in airways (Hahn and Patil 1972, Sheller et al.1982, Tanaka and Grunstein 1984, Sakai et a1. 1989) Sympathetic nervous system The sympathetic system originates from the upper six thoracic segments of spinal cord and consists of a network of nerve fibers and a chain of ganglia on either side of the ventral surface of the vertebral column, interconnected by longitudinal nerves, and extending from the base of the skull to the tail. Complicated plexuses are formed as ganglia connect to spinal nerves by communicating branches, with numerous branches passing to thoracic and abdominal viscera (Reichard and Jennings 1925) The large superior cervical ganglion lies on the ventrocranial aspect of the nodose ganglion of the vagus at the level of the jugular foramen. From the superior cervical ganglion the sympathetic trunk passes caudad, closely bound with the vagus adjacent to the surface of the carotid artery on the lateral aspect of the trachea. Cranial to the first rib, sympathetic postganglionic fibers synapse in the middle cervical ganglion, the larger inferior cervical ganglion, and the upper four thoracic 9 ganglia. Postganglionic fibers run from these ganglia to the lung, and enter at the hilum with cholinergic nerves that form a dense plexus around airways and vessels (Richardson 1981) Fluorescence studies in the cat have demonstrated extensive adrenergic innervation to the pulmonary and bronchial vessels, as well as the airway smooth muscle from the level of the trachea to the respiratory bronchioles (Silva and Ross 1974, Richardson 1979). Stimulation of the sympathetic nerves to the lung in the cat releases catecholamines into the venous effluent (Locket 1957) and generally produces bronchodilation (Dixon and Ransom 1912). Both alpha- and beta-adrenoceptors increase mucus secretion into the cat trachea, but only the beta-adrenoceptors appear to respond to sympathetic nerve stimulation (Peatfield and Richardson 1982). In addition, although betal- adrenoceptors are generally believed to predominate in the heart and betaz- adrenoceptors predominate in the bronchi and peripheral blood vessels (Lands et al. 1967), this separation of beta-adrenoceptor function by organ is inconsistent in some species (Carlsson et al. 1972). There appears to be no clear separation of betal- and betaz-mediated responses in organs of the cat, including the airways (Lundgren et a1. 1979, Letts et al. 1983) Relaxation of the cat trachea appears to be mediated by both betal- and betaz-adrenoceptors and the predominant sub-type is beta1 (O’Donnell and Wanstall 1983) Sympathetic nerves may play a modulatory role in cholinergic neurotransmission. Stimulation of pulmonary sympathetic nerves causes bronchodilation by activation of beta-adrenoceptors on airway smooth muscle. In addition, there is evidence that the sympathetic nerves may inhibit parasympathetic neurotransmission at the ganglia (Partanen et al. 1982) and at THE! 10 the prejunctional cholinergic nerve terminals (Vermeire and Vanhoutte 1979). Low doses of pirenzepine, a muscarinic antagonist considered to be selective for M1 facilitatory receptors, can increase vagally-induced bronchoconstriction in guinea pig lung (Maclagan et 31.1989). In the same study, propranolol abolished the pirenzepine-induced enhancement of vagal stimulation, suggesting that these pirenzepine-sensitive M1 receptors must be located in the sympathetic nervous pathway. Therefore, blockade by pirenzepine of M1 receptors which facilitate neurotransmission in the sympathetic nerves could reduce norepinephrine release and indirectly increase the bronchoconstriction produced during vagal nerve stimulation. A similar facilitatory role for M1 receptors in the rat and rabbit superior cervical ganglia of the sympathetic nervous system has been described (Brown et al. 1980, Ashe and Yarosh1984). Investigators have also demonstrated that norepinephrine inhibits the firing of airway ganglion cells in cat and ferret via alpha-adrenoceptors (Baker et al. 1983). Therefore, the sympathetic nervous system can regulate cholinergic neurotransmission in some species through activation of alpha- or beta-adrenoceptors. Nonadrenergic, noncholinergic nervous system Nonadrenergic, noncholinergic (NANC) inhibitory neural responses have been demonstrated in vivo in cat airways. Following alpha- and beta- adrenoceptor and muscarinic blockade, and after restoration of smooth muscle tone with agents such as S-hydroxytryptamine, vagal stimulation causes a reduction in flow resistance and an increase in lung compliance (Diamond and O’Donnell 1980, Irvin et al. 1980). After injection of the nicotinic receptor blocking agent hexamethonium, relaxation is inhibited during preganglionic THES 11 vagal stimulation (Diamond and O’Donnell 1980). These pharmacological studies provide evidence of a N ANC neuronal pathway in the cervical vagus nerves of the cat that can produce dilation of pulmonary airways. Although NANC inhibitory pathways have been demonstrated in vitro in both the cervical (Ito and Takeda 1982) and thoracic (Altiere et al. 1984) trachea of the cat, NANC pathways have not been demonstrated in vivo in cat trachea (Matsumoto et al. 1985, Don et al. 1988). In situ studies of the cervical trachea in the cat showed that vagal stimulation evoked contraction followed by relaxation of smooth muscle of trachea and lower airways and sympathetic stimulation evoked relaxation only. After muscarinic blockade and restoration of smooth muscle tone with 5-hydroxytryptamine, vagal stimulation produced no change in tracheal segment tension, whereas sympathetic-evoked relaxation was preserved. Vagal stimulation did reduce flow resistance and increase lung compliance. The investigators concluded that NANC pathways were present in the lower airways of cats but the studies did not demonstrate NAN C-related relaxation in the cervical trachea in vivo (Don et al. 1988). Similarly, roentgenograms obtained during insufflation of tantalum into cat airways revealed insignificant changes in tracheal diameter after vagal stimulation in the presence of muscarinic and beta-adrenergic blockade (Matsumoto et al. 1985). The diameter of the intrathoracic trachea and peripheral airways less than 1 mm in diameter was only slightly affected by either inhibitory system (Matsumoto et al. 1985). Tantalum bronchography in cats has established that NANC bronchodilation occurs predominantly in midsized airways (Matsumoto et al. 1985). Following beta-adrenoceptor blockade 12 with propranolol, the amount of relaxation produced during vagal stimulation was comparable to that obtained with sympathetic nerve stimulation in the cat. The NANC nervous system acts to regulate secretion of airway mucus. Stimulation of vagus nerves promotes mucus secretion in cat trachea and secretion is reduced but not abolished by cholinergic and adrenergic blockade (Peatfield and Richardson 1983). Electrical field stimulation of ferret tracheal segments in vitro can also produce NANC-induced mucus secretion (Borson et al. 1984). The neurotransmitter in NANC inhibitory airway nerves has not been identified. Purines have been implicated as neurotransmitters in the gastrointestinal tract, genitourinary tract, and the respiratory system (Souhrada et al. 1980, Satchell 1982). Current evidence argues against a purine as neurotransmitter of NANC nerves in the airways. Exogenous ATP relaxes airway smooth muscle, but the antagonist quinidine does not block NANC relaxation either in vitro or in vivo (Ito and Takeda 1982). Exogenous adenosine fails to produce relaxation, and its antagonist theophylline does not block nonadrenergic relaxation (Karlsson and Persson1984). Purine uptake inhibition with dipyridamole does not enhance nonadrenergic bronchodilation (Irvin et al. 1982, Ito and Takeda 1982). Vasoactive intestinal peptide (VIP) is well established as a neurotransmitter in the gastrointestinal tract (Costa and Furness1982, Said 1984). A large amount of evidence suggests that VIP is at least one of the neurotransmitters of nonadrenergic inhibitory nerves in airways (Said 1982). Vasoactive intestinal peptide has been localized to neurones and nerve terminals in mammalian airway smooth muscle (particularly in the upper 13 airways), around submucosal glands, and in bronchial and pulmonary vessels (Uddman and Sundler 1979, Dey et al. 1981, Laitinen et al. 1985). Prolonged relaxation produced by VIP in vitro in airway smooth muscle is unaffected by adrenergic and cholinergic blockers (Ito and Takeda 1982, Cameron et al. 1983, Altiere and Diamond 1984) In human bronchial smooth muscle, VIP is the most potent endogenous bronchodilator reported, being approximately 50 times more potent than isoproterenol (Palmer et al. 1986) In addition, VIP mimics the electrophysiologic changes in airway smooth muscle produced by N ANC nerve stimulation (Ito and Takeda 1982, Cameron et al. 1983). Vasoactive intestinal peptide is released from nerve terminals and stimulates specific receptors that have been demonstrated in lung membranes by radioligand binding with 124I-labeled VIP (Robberecht et al. 1981) Autoradiographic studies have shown that VIP receptors are widely distributed in airway epithelium and submucosal glands (Robberecht et al.1981) Bronchial smooth muscle is also labelled, but not bronchiolar smooth muscle, consistent with the finding that VIP relaxes bronchi but not bronchioles (Palmer et al. 1986). The VIP-immunoreactive nerves are frequently distributed with cholinergic nerves. Ultrastructural studies suggest that VIP may coexist in the same nerve terminals as ACh, functioning as a cotransmitter (Laitinen et al. 1985). In addition to a direct effect on VIP receptors, VIP may also have a prejunctional ganglionic effect on ACh release (Kawatani et al. 1985), or an effect on post junctional cholinergic receptors, as demonstrated in submaxillary glands (Lundberg et al. 1982). 14 At present, it cannot be concluded whether a separate type of VIP nerve exists and/or if several transmitters coexist in the same nerves (Laitinen and Laitinen 1990). Neural fibers can contain two or more transmitters that may have more than one action on effector tissues (Laitinen and Laitinen 1990). It is possible that physiologically VIP acts as a neuromodulator in airway smooth muscle, and it may be preferentially released under certain conditions (Barnes 1986). Although the NANC nervous system is predominantly inhibitory in its action on airway smooth muscle, it has also been shown to have an excitatory action under certain circumstances. This excitatory action has been demonstrated in the guinea pig tracheobronchial tree, and can be inhibited by alphaz-adrenergic receptor activation (Grundstrom et al. 1984). The putative mediator of the NANC excitatory pathway in guinea pig airways is SP. A similar excitatory NANC nervous system has not been described in cat airways, but SP-like immunoreactive nerves occur in the lower respiratory tract of many species, including rats, mice, cats, and humans (Lundberg et al. 19843). Afferent nerves Several types of afferent nerve terminals have been described in the airways (Larsell 1923). Their purpose is to send sensory information up the vagus nerve so that appropriate changes in breathing pattern and bronchomotor tone can occur. Afferent or sensory fibers from the airways terminate in the vagal nuclei and the nerve cell bodies are localized in the nodose ganglia (Richardson and Ferguson 1979). 15 The main afferent nervous pathway in airway smooth muscle is from the bronchopulmonary stretch receptors. These slowly adapting receptors are myelinated nerve terminals localized primarily to smooth muscle of conducting airways (Guz and Trenchard 1971) The irritant and pulmonary J-receptors are found primarily on bronchial epithelium and in the alveolar wall. They may have an effect on airway smooth muscle through reflex action causing broncho— constriction, cough, and hyperpnea (Widdicombe 1981, Sant’Ambrogio 1982). The common link between viral respiratory infections and airway hyperresponsiveness could be epithelial damage with exposure of sensory nerve receptors. Sensory receptors from myelinated and nonmyelinated afferent nerves are located below the epithelium and between epithelial cells. These receptors are sensitive to mechanical stimulation and chemical stimuli including capsaicin, bradykinin, histamine, prostaglandins Fulpm, E2, 12, and sulfur dioxide (Coleridge et al. 1965, Kaufman et al. 1980, Coleridge and Coleridge 1984). The link between epithelial disruption with exposure of sensory nerve endings and virus-induced airway hyperreactivity are uncertain but there are several possible mechanisms whereby epithelial damage could cause bronchial hyperresponsiveness. One mechanism linking afferent nerve pathways and virus infections could be damage and shedding of airway epithelium with subsequent inflammation and release of sensory neuropeptides. Stimulation of sensory receptors by inflammatory mediators such as bradykinin may result in an axon (local) reflex with antidromic conduction down afferent nerve collaterals and release of sensory neuropeptides such as SP, neurokinin A, and calcitonin gene- related peptide. These peptides are potent inducers of airway smooth muscle 16 contraction, bronchial edema, extravasation of plasma, and mucus hypersecretion (Barnes 1986, Borson et al. 1989) Respiratory virus infections in rats, guinea pigs, and ferrets can cause enhanced contractile response of airway smooth muscle to SP (Jacoby et al. 1988, Borson et al. 1989, Dusser et al. 1989). These studies have demonstrated that increased airway constriction to SP is caused by decreased enkephalinase activity in infected tissues The enzyme enkephalinase is found in airway epithelium, glands, and smooth muscle, and degrades SP. Substance-P is localized in nonmyelinated vagal afferent nerve endings. Therefore, these investigators have concluded that respiratory infection can result in epithelial damage with increased contractile response of airway smooth muscle to SP due to decreased enkephalinase activity in infected tissues (Jacoby et al. 1988). A second proposed hypothesis linking afferent vagal pathways and virus- induced airway hyperreactivity involve cholinergic pathways. Upper respiratory viral infections may cause damage and shedding of airway epithelium with subsequent exposure of afferent nerve endings (Hers 1966). Afferent nerve endings are found in the airway mucosa of several species, including man, and inhalation of atropine can both reverse and largely prevent virus-induced airway hyperreactivity in humans (Empey et al. 1976). Other investigators have demonstrated that airway constriction produced by cold air and exercise in subjects with upper respiratory infection can be prevented by previous administration of atropine (Aquilina et al. 1980). Therefore, the primary abnormality associated with virus-induced airway hyperreactivity in humans may involve the afferent side of the vagal reflex, resulting in reflex bronchoconstriction via cholinergic pathways. 17 'rwa an lia Neural inputs to airway smooth muscle can be controlled and modulated at the level of the central nervous system, the peripheral airway ganglia, and at the neuromuscular junction. Although it was previously believed that airway ganglia were simple relay stations for cholinergic neurotransmission, the complexity of their structure suggests an immense number of possible interactions that act to locally regulate smooth muscle tone and glandular secretion. Ganglia are found within the airway wall, usually external to smooth muscle and cartilage, and occasionally under epithelium (Richardson and Ferguson 1979). In trachea and major bronchi, ganglion cells are found primarily in the posterior wall, but they are more evenly distributed within the wall of smaller intrapulmonary airways. Ganglia in human airways generally consist of several nerve bodies with a complex ultrastructure. Ganglia can contain cholinergic nerves and cell bodies as well as adrenergic nerves (Richardson and Ferguson 1979). Some cells within ganglia contain dense core vesicles, which are believed to contain catecholamines or neuropeptides (Polak and Bloom 1982, Hakanson et al. 1983). Current evidence suggests that airway ganglia process cholinergic excitatory, and sympathetic and nonadrenergic inhibitory inputs (Coburn 1987). To date, there is no evidence that peripheral airway ganglion neurons can generate signals independent of input from the central nervous system, in contrast to the myenteric plexus, where central input modulates rather than controls neural input (Wood 1984). Afferent input from the epithelium or from 18 stretch receptors may influence ganglionic integration of neural inputs, although conclusive evidence is lacking (Coburn 1987). Evidence is increasing regarding the importance of airway ganglia in regulation of airway tone and secretion in health and disease. A study of the effects of norepinephrine on prejunctional release of ACh in ferret paratracheal ganglia was the first to demonstrate presynaptic inhibition of neurotransmission in airway peripheral ganglia (Baker et al. 1982, Baker et al. 1983). Direct recording from ganglionic neurons demonstrated that norepinephrine inhibits neurotransmission via alpha-receptors similar to its effects on parasympathetic ganglia of the gastrointestinal tract (Baker et al. 1983, Wood 1984). Similarly, beta-receptor agonists can modulate neurotransmission through ganglia by inhibiting ACh release from preganglionic nerve terminals (Danser et al. 1987). Various inflammatory mediators and neuropeptides may also influence ganglionic neurotransmission. Airway ganglia are often in close proximity to mast cells that contain histamine or serotonin (Coburn 1989). Histamine has been shown to inhibit nicotinic neurotransmission in enteric ganglia (Tamura et al. 1988). Neuropeptides isolated from peripheral airway ganglia include VIP and SP (Coburn 1989) and calcitonin gene-related peptide (Lundberg and Saria 1987). Muscarinic receptors on airway smooth muscle Introduction Over 70 years ago, Dale suggested that the effects of ACh could be differentiated into a "muscarine action" and a "nicotine action" and that atropine 19 abolished the muscarinic effects of the agonist (Dale 1914). Muscarinic receptors have been identified in the lungs by receptor binding techniques with radiolabelled muscarinic antagonists such as [3H] quinuclidinyl benzilate (Cheng and Townley 1982, Murlas et al. 1982). The density of muscarinic receptors in peripheral lung of all species is low, and in marked contrast to the high density found in tracheal smooth muscle (Cheng and Townley 1982, Murlas et al. 1982, Barnes et al. 1983) The muscarinic receptors belong to a larger family of proteins, which includes rhodopsin and a number of seven-helix drug receptors, including beta- adrenergic, alphaz-adrenergic, 5HT1cserotonergic, and substance K receptors (Goyal 1988). In common with other family members, the muscarinic receptor molecule crosses the plasma membrane seven times with its NHz-terminus outside the cell and COOH-terminus in the cytoplasm of the cell. Muscarinic receptors have at least two binding sites, one for a ligand on the cell surface, and the other for a guanosine-S ' -triphosphate binding protein (G protein) inside the cell (Goyal 1988, Peralta et al. 1988a). Like many other neurotransmitter and hormone receptors, muscarinic ACh receptors transduce agonist signals by activating G proteins to regulate ion channel activity and the generation of second messengers via the phosphoinositide and adenylate cyclase systems. Muscarinic receptors are coupled to different post-receptor mechanisms, which include inhibition of adenylate cyclase and the stimulation of phosphatidylinositol turnover with formation of inositol triphosphate. Inhibition of adenylate cyclase decreases intracellular cyclic AMP content, which leads to contraction of airway smooth muscle (Jones et al. 1986) Formation of inositol triphosphate results in the release of calcium ions from 20 intracellular stores and subsequent smooth muscle tension deveIOpment (Berridge and Irvine 1984). There is increasing evidence that M1 and M3 receptors are predominantly linked to phosphatidylinositol turnover and that M2 receptors act mainly by inhibiting adenylate cyclase (Grandordy et al. 1986, Peralta et al. 1988a, Yagisawa et al. 1988). It is also possible that a specific muscarinic receptor subtype couples to each of the second-messenger response systems with varying degrees of efficiency. The ability of a muscarinic receptor subtype to couple to various effector systems through different second messengers may be differential, rather than exclusive (Peralta et al. 1988b, Harden 1989). In addition, stimulation of muscarinic receptors in the lung increases cyclic GMP content by activation of guanylate cyclase. The increase in cyclic GMP appears to be secondary to the increase in cytosolic calcium, and may be a mechanism for turning off further calcium release (Schultz et al.1973, Kaliner 1977). The first indication that muscarinic receptors did not form a homogeneous class came from the observation that gallamine, a neuromuscular blocking agent regarded as a nicotinic drug, blocked the action of ACh on the heart but did not alter the effect of ACh elsewhere (Riker and Wescoe 1951). At least five functional muscarinic receptor subtypes exist with substantial differences in amino acid sequence, ligand-binding properties, and patterns of tissue-specific expression (Peralta 1988a, Burgen 1989). Unfortunately, muscarinic receptor subtype nomenclature has not been standardized. Receptors were classified as M1 and M2 receptors based on early pharmacological studies using the antagonist pirenzepine to characterize the M1 subtype described in cerebral cortex and the M2 subtype found in myocardium. More recent pharmacological 21 studies utilizing other selective muscarinic ligands have suggested the existence of additional subtypes besides M1 and M2 receptors (Peralta 1988b). Muscarinic receptor subtypes have also been named based on molecular cloning studies that have shown differences in molecular masses and amino acid compositions. Thus, muscarinic receptor subtypes have also been termed ml, m2, m3, m4, and m5 (Bonner et al. 1987, Bonner et al. 1988) By comparing antagonist binding affinities towards the gene products when expressed in cell lines and the tissue distribution of their mRNAs, it has been possible to correlate the M1, M2, and M3 receptor subtypes to the m1-, m2—, and m3—gene products; the m4- and mS-gene products clearly displaying different profiles (Maeda et al. 1988, Buckley et al. 1989). For example, m1 binds pirenzepine with 25-fold greater affinity than m2, a difference comparable to that observed between M1 receptors of rat brain and M2 receptors of rat atria (Peralta 1988b). Investigators have also shown that m2 and m4 receptors efficiently inhibit adenylate cyclase activity but poorly activate phosphatidylinositol hydrolysis (Peralta 1988a). In contrast, the m1 and m3 receptors strongly activate phosphatidylinositol hydrolysis, but do not inhibit adenylate cyclase, and in fact can substantially elevate cAMP levels. A high degree of amino acid identity occurs in the seven transmembrane domains and short connecting loops common among the muscarinic receptor subtypes. By contrast, the large cytoplasmic region found in each subtype is highly diverged with closer similarity between the m1 and m3, and the m2 and m4 subtype pairs (Peralta 1988b). Therefore, the muscarinic receptor subtypes that are functionally similar also appear to be more similar in amino acid sequence (Peralta 19883). It is uncertain whether further muscarinic receptor subdivision will occur. 22 Specific muscarinic agonists and antagonists have identified three muscarinic receptor subtypes in the airways. The M1 receptors are pirenzepine- sensitive and act as facilitatory receptors in parasympathetic and sympathetic neural pathways (Barnes 1989, Maclagan et al. 1989) Inhibitory autoreceptors (M,) are blocked by gallamine, AF-DX 116, and methoctramine, and are located on cholinergic nerves (Barnes 1989, Minette and Barnes 1988) The classic muscarinic receptors (M3) mediate smooth muscle contraction and mucus secretion in the airways and are antagonized by Miphenyl acetoxy N-methyl piperidine (4-DAMP) and hexa-hydrosiladifenidol (Minette and Barnes 1988, Barnes 1989, Maclagan et al. 1989). M1 receptors in airways The M1 receptors were first demonstrated in brain and sympathetic ganglia (Hammer and Giachetti1982, Birdsall and Hulme1983). The M1 receptor antagonist pirenzepine is used clinically to reduce gastric acid secretion. Pirenzepine acts predominantly on parasympathetic ganglia by inhibiting neurally mediated gastric secretion (Hirschowitz et al.1983). Innervation of the airways is derived embryologically from that of the gut, so it is not surprising to find M1 receptors in airway ganglia, although there appear to be considerable species differences in the location of muscarinic receptors in pulmonary nerves (Barnes 1989). As an M1 receptor antagonist, pirenzepine is very effective in blocking bronchoconstriction due to vagal stimulation in rabbits and dogs (Beck et al. 1987, Bloom et al. 1987a). Because in vivo vagal stimulation activates the cholinergic nerves preganglionicly, and methacholine and exogenous ACh 23 activate muscarinic receptors on the smooth muscle directly, investigators hypothesized that the pirenzepine-sensitive muscarinic receptor might be associated with parasympathetic nerves rather than smooth muscle. Because pirenzepine has a low potency against ACh-induced contraction of rabbit airways but can completely abolish contraction produced by vagal stimulation, investigators have concluded that M1 receptors are not present in rabbit airway smooth muscle (Bloom et al.1987a). In addition, an in vitro preparation of rabbit mainstem bronchus compared contraction produced by preganglionic vagal stimulation and postganglionic electric field-stimulation (Bloom et al. 1988). Pirenzepine was more potent in inhibiting vagally stimulated contraction than field-stimulated contraction, and was only slightly less potent than atropine in inhibiting vagally stimulated contraction. In contrast, pirenzepine was 102- to 178-fold less potent than atropine in inhibiting smooth muscle contraction produced by electric field-stimulation. In summary, pirenzepine is more effective in blocking pre- than post-ganglionic vagal nerve stimulation in rabbit airways in vitro (Bloom et al. 1988). Since muscarinic receptors on airway smooth muscle have a low affinity for pirenzepine, excitatory M1 receptors must be located on the vagal pathway controlling airway caliber, most likely on parasympathetic ganglia rather than in smooth muscle (Eglen and Whiting 1986, O’Rourke et al.1987, Roffel et a1. 1988). Similarly, in vitro binding studies with the selective M1 receptor antagonist pirenzepine and the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate suggest that Ml receptors are not present in bovine airway smooth muscle (Grandordy 1986, Madison 1987). 24 The M1 receptors make up more than half of the muscarinic binding sites in human lung. Recent autoradiographic mapping of muscarinic receptor subtypes in human airways has shown that M1 receptors are present on submucosal glands but not on airway smooth muscle. They are also found on alveolar walls, which would account for the high denSity of M1 receptors in peripheral lung (Bloom et al. 1987a, Casale and Ecklund 1988) The function of M1 receptors in alveoli is unknown (Barnes 1989) The physiologic role of M1 receptors in autonomic ganglia remains speculative. The primary receptors responsible for transmission through parasympathetic ganglia are the nicotinic cholinoceptors. These receptors mediate the fast, excitatory postsynaptic potential (EPSP). The EPSP can be followed by a slow excitatory postsynaptic potential and by a slow inhibitory postsynaptic potential (Gallagher et a1. 1982, North and Tokimasa 1982). These slow responses are blocked by the muscarinic receptor antagonist atropine and are mediated by M1 and M2 receptors, respectively. The slow inhibitory postsynaptic potential has been well described in pelvic ganglia of the cat (Gallagher et al. 1982). This slow muscarinic response is elicited at stimulus frequencies in the physiological range of 2-10 Hz. The slow inhibitory postsynaptic potential is induced orthodromically or by ionophoretic application of ACh, and is capable of inhibiting the firing of spontaneously active neurons. These observations suggest that hyperpolarization mediated by muscarinic receptors may occur under physiological conditions and has sufficient magnitude to inhibit neuronal activity. Therefore, these facilitatory and inhibitory muscarinic receptors probably regulate ganglionic transmission and have a role in long-term regulation of cholinergic tone (Barnes 1989). THE 25 M2 receptors in airways Muscarinic receptor subtypes that are not pirenzepine sensitive were initially called M2 receptors, but it is now clear that there is heterogeneity among the original M2 receptors. The M2 receptors in the atria ("cardiac type") that mediate tachycardia are selectively activated by pilocarpine and blocked by gallamine (Hammer et al. 1986). By contrast, the muscarinic receptors on smooth muscle and glands are sensitive to 4-DAMP and have been reclassified as M3 receptors (Barnes et al. 1988). A prejunctional receptor that is effected by the output of its own neurotransmitter is called an autoreceptor, in contrast to receptors on nerve terminals that are effected by other neurotransmitters, autacoids, local hormones, and neuropeptides. Muscarinic autoreceptors have been identified in guinea pig (Fryer and Maclagan 1984), cat (Blaber et a1. 1985), dog (Ito and Yoshitomi 1988), human (Minette and Barnes 1988), and rat (Fryer and El- Fakahany 1990) airways. The M2 receptors have not been demonstrated in rabbit airways (Maclagan and Faulkner 1989). In binding studies of human lung homogenates, no appreciable population of M2 receptors has been identified (Mak and Barnes 1989). Cholinergic nerves probably make up a trivial proportion of lung membranes so their contribution to a lung homogenate might be undetectable (Barnes 1989) However, in vivo studies in humans indicate that autoreceptors have a role in regulation of airway caliber. The M2 receptor agonist pilocarpine has an inhibitory effect on cholinergic reflex bronchoconstriction induced by inhalation of sulfur dioxide in healthy atopic volunteers (Minette et al. 1988). In asthmatic subjects, pilocarpine has no such inhibitory action, suggesting a possible dysfunction of the autoreceptor, which 26 would result in exaggerated cholinergic reflex bronchoconstriction (Minette et al.1988). The M2 receptors appear to be located before the neuromuscular junctions on postganglionic parasympathetic nerves, where they can have a powerful inhibitory effect on ACh release and, thus, act to limit airway constriction. In both guinea pigs and cats gallamine potentiates bronchoconstriction due to vagal nerve stimulation, while having no effect on exogenous ACh-induced smooth muscle contraction (Fryer and Maclagan 1984, Blaber et al. 1985). Blockade of M2 autoreceptors could cancel feedback inhibition of acetylcholine and augment neurally-mediated bronchoconstriction (Blaber et a1. 1985). The cholinergic antagonists that are available for medical use, atropine and ipratropium bromide, are not selective for muscarinic receptor subtypes and may block M2 inhibitory receptors as well as postjunctional M3 receptors. An antimuscarinic agent that was selective for smooth muscle receptors without blocking M2 receptors might be more effective in inhibiting cholinergic bronchoconstriction. Recently, a possible relationship between increased vagally induced bronchoconstriction seen in viral respiratory infections and alteration of M2 receptors in the lung has been reported (Fryer et al.1990, Fryer and J acoby 1990). In sham-infected control guinea pigs, pilocarpine attenuated vagally-induced bronchoconstriction by stimulating inhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. Blockade of M2 receptors with the antagonist gallamine produced a substantial increase in vagally-induced bronchoconstriction. However, in guinea pigs infected with parainfluenza virus, pilocarpine did not inhibit vagally-induced bronchoconstriction and gallamine THE 27 did not potentiate bronchoconstriction produced by vagal stimulation. There was no increase in base-line pulmonary inflation pressure in the infected animals compared to the controls, indicating that parainfluenza infection did not alter the initial airway caliber. Receptors on the airway smooth muscle did not appear to be affected because large doses of pilocarpine, which can stimulate M3 receptors on smooth muscle at high concentrations, caused the same amount of bronchoconstriction in both groups of animals. Virus-induced changes in M2 receptor function were specific to pulmonary parasympathetic nerves, and not part of a generalized decrease in M2 receptor function because gallamine inhibited the vagally-induced fall in heart rate equally in control and infected animals. Therefore, M2 receptor mediated inhibition of acetylcholine release from parasympathetic nerves in the lungs was decreased in parainfluenza-infected guinea pigs. The investigators hypothesized that damage to M2 inhibitory receptors could result in the loss of negative feedback inhibition to ACh release. The increased release of acetylcholine from the parasympathetic nerves could activate M3 receptors on airways smooth muscle and might explain virus-induced airway hyperresponsiveness (Fryer and J acoby 1990). M3 receptors in airways The M3 muscarinic receptor subtype is located on airway smooth muscle and mediates contraction (Minette and Barnes 1990, Roffel et al. 1990) There appears to be a greater density of M3 receptors in central than in peripheral airways in human, bovine, and guinea pig airway smooth muscle (Roffel et al. 1990). Binding studies in guinea pig lung membranes indicate a preponderance 28 of M3 receptors, whereas M3 receptors make up less than half of the total muscarinic receptors in human lung (Mak and Barnes 1989). Autoradiographic mapping studies have shown that M3 receptors are also found on submucosal glands in the airways (Barnes 1989). Submucosal glands have both M1 and M3 receptors, which are both presumed to activate mucus secretion. Functional studies with selective antagonists in isolated cat trachea suggest a response intermediate between M1 and M3 receptors, suggesting that both receptor subtypes play a part in airway mucus secretion in the cat (Gater et al. 1989). Functional and autoradiographic mapping of muscarinic receptors in guinea pig and human airways consistently demonstrate M3 receptors on smooth muscle in all airways from trachea to peripheral bronchioles (Minette and Barnes 1990). By contrast, binding studies with selective M2 antagonists AF-DX 116 and methoctramine suggest the existence of both M2 and M3 receptors in bovine tracheal membranes (Roffel et al. 1988). However, functional studies reported by the same group were consistent with other species in which tracheal smooth muscle had low affinity for M2 antagonists and high affinity for M3 antagonists. If M2 receptors are present in bovine tracheal smooth muscle, their functional significance is unknown. Data from other species indicate that activation of target cells such as smooth muscle, submucosal glands, and endothelial cells is dependent upon M3 receptor occupation, whereas M1 and M2 receptors appear mainly to control parasympathetic neurotransmission (Minette and Barnes 1990). 29 Viral infections and airway hyperreactivity Introduction Airway hyperreactivity can be defined as extreme sensitivity of airway smooth muscle to physical, chemical, and pharmacologic stimuli resulting in an acutely enhanced bronchospastic response. The rapid time course and the rapid reversal of bronchospasm by beta-adrenergic agonists that relax airway smooth muscle suggest that the exaggerated constriction is primarily due to constriction of smooth muscle rather than to mucosa] edema or obstruction of the airway lumen by mucus (Boushey et al. 1980). Airway hyperreacivity is a common response to lung injury. A variety of oxidant gases in the environment induce transient airway hyperreactivity, but acute viral infections of the respiratory tract are the most common naturally occurring insult that causes airway hyperresponsiveness (Golden et al. 1978, Richardson et al. 1981). Airway hyperreactivity to non-specific stimuli is a hallmark of asthma and is considered the underlying abnormality in the pathophysiology of this disease (Hay 1989). In addition, airway hyperreactivity is commonly manifest in other pulmonary conditions, including chronic bronchitis (Klein and Salvaggio 1966, Laitinen 1974), cystic fibrosis (Mellis 1978), allergic rhinitis (Laitinen 1974, Cockcroft et al. 1977), and emphysema (Klein and Salvaggio 1966, Laitinen 1974). Airway hyperreactivity can be considered a form of supersensitivity. Supersensitivity occurs in the lungs, or in other body systems, when the amount of substance needed to elicit a set response under certain conditions is less than normal (Fleming et 31.1976, Westfa111981). There is a leftward shift in the dose- response curve and, in some instances, with this increase in sensitivity, there 30 may be an increase in the maximum response produced by the stimulant and/or a change in the slope of the dose-response curve (Fleming et al. 1976, Westfall 1981). Supersensitivity has been classified into two types: type I, which has also been termed deviation supersensitivity, presynaptic or prejunctional supersensitivity; and type II, which has been called nondeviation supersensitivity, disuse, nonspecific, or postjunctional supersensitivity. Type I supersensitivity is due to an increase in the amount of agonist interacting with specific receptors. There is no inherent alteration in the properties and responsiveness of the effector tissue (Hay 1989). Type I is specific for a few agonists and has been characterized primarily for the adrenergic system (Fleming et al. 1976, Westfall 1981). Type II supersensitivity is nonspecific and occurs as a result of an increase in the responsiveness of the effector tissue rather than an alteration in the drug concentration reaching the receptor (Hay 1989). In smooth muscle, type II supersensitivity is usually demonstrated experimentally as a consequence of a chronic suppression in the normal communication between the neurotransmitter and its associated effector cell. Type II supersensitivity probably involves multiple mechanisms, and the relative involvement of these mechanisms may depend on such factors as the particular effector tissue and the agonist under study. Postulated mechanisms to account for increased tissue responsiveness include an alteration in receptor density and/or receptor affinity, a morphological change in the effector tissue, including a change in the membrane electrophysiological properties, or a post-receptor biochemical change, such as a change in the levels and activity of cyclic nucleotides (Fleming et al. 1973, Fleming 1976, Westfall 1981, Hay 1989). A more popular 31 postulated mechanism to explain type II supersensitivity is alteration in the permeability and transport of ions, and there is some evidence for this hypothesis in Ca++ homeostasis of smooth muscle (Hay 1989). Measurement of airway hyperreactivity In vitro airway studies allow quantitative assessment of smooth muscle contraction under highly controlled conditions (e.g., dose of agonist, composition of bathing medium, anatomic selection of study segment). Important information on the mechanisms of increased responsiveness has been provided by examining the effect of stimuli on the tension developed by the muscle during contraction at a constant length. Studies performed at muscle lengths that produce maximal response to stimulation yield a reproducible sigmoid curve relating the dose of agonist to the tension developed (Stephens et al. 1968). Three characteristics of this curve are useful in assessing the behavior of the muscle: 1) the threshold of dose for deve10pment of active tension; 2) the slope of the mid-portion of the curve; and 3) the maximal active tension developed in response to the stimulus. An increase in the effective concentration of agonist at drug receptor sites produces a parallel shift to the left of the dose-response curve with no change in slope or maximal active tension achieved. This parallel shift in the dose-response curve is characteristic of type I supersensitivity that does not involve alterations in the responsiveness of the smooth muscle itself (Fleming et al. 1973). Changes in the excitation-contraction coupling of the muscle after drug-receptor binding can also affect the dose-response curve. These changes 32 can result in an increase in slope and an increase in the maximal active tension achieved, and are typical of type II supersensitivity (Fleming et al. 1973). The results of in vitro smooth muscle studies frequently do not correlate with the results of in vivo airway studies (Vincenc et al.1983, Armour et al. 1984, Woolcock and Permutt 1986). In vitro investigations generally fail to consider the smooth muscle in situ length-tension relationships, and the preload and afterload stresses on the muscle. In vitro techniques also deprive the muscle of its normal nerve and blood supplies and its relationship to neighboring tissues. Also, in vitro tests customarily report developing isometric tension, whereas increases in airway smooth muscle tension in vivo result from isotonic bronchial and tracheal smooth muscle contractions (Woolcock and Permutt 1986). Most in vivo studies of bronchial reactivity in humans infer smooth muscle contraction by measurements that indirectly reflect changes in airway caliber. Common functional measurements, such as forced expiratory volume or airway resistance, provide information about the net effects of airway narrowing along respiratory passages but cannot determine the specific sites of narrowing (Boushey et al. 1980). These indirect measurements cannot differentiate airway smooth muscle contraction from other changes, such as laryngeal narrowing, airway wall edema, or mucus accumulation (Hyatt and Wilcox 1961, Macklem and Mead 1967, Szereda-Przestaszewska 1973). Most investigators study airway hyperreactivity in vivo by delivering drugs directly to the airways by topical or aerosol administration or by injection into the circulation to produce bronchoconstriction. There are advantages and disadvantages to each route of administration. For example, parenteral injection may produce undesirable systemic effects. Bronchoactive drugs 33 delivered intravenously may cause catecholamine release from the adrenal - medulla that can alter airway smooth muscle responses (Staszewska-Barczak and Vane 1965, Colebatch and Engel 1974). The arterial hypotension that follows injection of some drugs can lead to a baroreceptor-mediated increase in sympathetic efferent activity, which can also influence airway smooth muscle (Diamond 1972). In addition, some bronchoactive drugs such as prostaglandin Fm”, are inactivated during their passage through the lungs (Ferreira and Vane 1967). Other bronchoactive drugs such as ACh are rapidly destroyed by circulating and tissue esterases following intravenous injection (Colebatch et al. 1966). Many in vivo studies of bronchial reactivity use histamine or a cholinergic agonist delivered as an aerosol, and the response is measured by the change in maximal expiratory flow or in airway resistance and thoracic gas volume. Differences in the type of nebulizer and delivery system used have made standardization of protocols difficult (Chai et al.1975). Other factors that can influence aerosol deposition include the size of the inhaled particle, the time of delivery during the respiratory cycle, and airway narrowing present prior to inhalation (Dolovich et al. 1976, Ruffin et al. 1978, Juniper et al. 1978, Boushey et al. 1980). Methods that analyze changes in maximal flow at high lung volumes are complicated by inhalation to total lung capacity, which affects airway smooth muscle tone (Nadel and Tierney 1961). Measurements of airway resistance or conductance provide a more sensitive, specific index of the caliber of central airways but are insensitive to changes in small, peripheral airways (Boushey 1980). These variables need to be considered and controlled in order to obtain reproducible measurements of airway reactivity. 34 Potential mechanisms of airway hyperreactivity Clinical and experimental evidence suggests that several processes are involved in airway hyperreactivity. Possible causes for the exaggerated smooth muscle contraction in airway hyperreactivity include: 1) genetic factors; 2) decreased base-line airway caliber; 3) increased responsiveness of the smooth muscle; 4) induction of airway inflammation and release of neuromediators; and 5) abnormality in autonomic nervous control of the smooth muscle. Figure 1 is a schematic diagram of the airway, including the major neural control mechanisms of smooth muscle. Arabic numbers in the figure represent sites where changes in the airway may occur to produce exaggerated smooth muscle contraction associated with airway hyperreactivity. Genetic factors The importance of genetic predisposition in the development of airway hyperreactivity remains to be elucidated. A recent report described the increased bronchial reactivity that existed in 19/20 healthy individuals prior to the deve10pment of asthma (Hopp et al.1990). Subjects were part of a larger on- going study of the Natural History of Asthma. These individuals either had no asthma symptoms or minimal symptoms on initial visit, but were from "asthma families" and subsequently developed asthma. There was a significant difference in airway reactivity between "normal family" subjects and the study subjects or the "asthmatic family" control subjects. These authors concluded that enhanced airway reactivity is nearly always present in subjects who do not have well-recognized symptoms of asthma, but who will subsequently develop asthma. 35 Nonadrenergic noncholinergic Parasympathetic Sensory Sympathetic ’ preganglionic afferent nerves post ganglionic .Mz Receptors e4cartilagj M1 Receptors Figure 1. Schematic representation of airway with major neural control mechanisms to smooth muscle. The possible causes for exaggerated smooth muscle contraction associated with airway hyperreactivity include: 1) decreased baseline airway caliber; 2) increased responsiveness of the smooth muscle; 3) induction of airway inflammation and release of neuromediators, such as substance-P (SP), neurokinins (NK), and calcitonin gene-related peptide (CGRP); 4) abnormalities in autonomic nervous control of the smooth muscle; and 5) genetic factors. 36 A unique murine model has been described in which a single autosomal recessive gene on the muscarinic ACh receptor regulated ACh-associated airway reactivity (Levitt and Mitzner 1988). The hyperreactive and hyporeactive phenotypes were easily distinguished in the progeny of two strains of inbred mice based on airway responses to ACh. These authors concluded that genetic variation in any of the mechanisms of hyperreactivity could be important in determining airway response, and that a mendelian or simple genetic basis should not be excluded. Decreased base-line airway caliber The caliber of the airways in the base-line state can influence the response to agents that induce bronchoconstriction. Most in vivo tests of airway narrowing depend on changes in airflow resistance. Because resistance is inversely proportional to the fourth power of the radius when flow in the airways is laminar, any decrease in the radius of a narrow airway causes a greater change in airway resistance than does the same decrease in the radius of a dilated airway (Boushey et al. 1980). Therefore, hyperreactivity in disease could be due to airways that are narrower prior to bronchial provocation (Benson 1975). Folding of the mucosa during bronchoconstriction, mucosal edema, and smooth muscle hypertrophy could further magnify the differences in airway caliber (Freedman 1972, Boushey et al. 1980). Although changes in base-line airway caliber may be important in some conditions, they do not explain the differences in bronchial reactivity in several studies where base-line values of airway caliber were similar among subjects. Asthmatic subjects in clinical remission (Townley et al. 1971, 1975, Cockcroft et 37 al. 1977) and normal subjects recovering from a viral upper respiratory infection (Empey et al. 1976) or brief exposure to ozone (Golden et al. 1978, Holtzman et al. 1979) have no evidence of airflow obstruction in the base-line state but have increased bronchial reactivity. Factors besides the relationship between radius and resistance to flow must underlie bronchial hyperreactivity. An increased amount of muscle is capable of developing greater tension and could play a role in decreasing airway caliber and causing airway hyper- reactivity (Figure 1). Hypertrophy and hyperplasia of airway smooth muscle occur in asthmatic patients and in some patients with chronic bronchitis (Dunnill 1960, Hossain and Heard 1970, Takizawa and Thurlbeck1971). Increases in the amount of airway smooth muscle and increased wall thickness probably contribute to exaggerated airway responsiveness of severely asthmatic subjects. However, they cannot be the cause of the hyperreactivity that occurs transiently during viral infections (Empey et al. 1976) or after exposure to oxidizing pollutants (Lee et al. 1977, Golden et al. 1978, Holtzman et al. 1979) because the mass of smooth muscle is unlikely to change in such a short time period. Increased responsiveness of airway smooth muscle Bronchial hyperreactivity may reflect a change in the behavior of airway smooth muscle rather than a change in the amount of smooth muscle present in the airways. For example, inhalation of ozone results in transient airway hyperirritability (Golden et al. 1978, Holtzman et al. 1979). Exposure to ozone has been shown to cause a decrease in acetylcholinesterase concentration in circulating red blood cells (Goldstein et al. 1968). Similarly, ozone may inhibit 38 smooth muscle acetylcholinesterase (Boushey et al. 1980) Other possible mechanisms of ozone-induced hyperreactivity are damage to drug-receptor binding sites and epithelial injury, which could add to airway hyperreactivity (Lee et al. 1977) Alteration in the myosin content might also affect airway smooth muscle contractility in hyperreactive states. Differences in the contractile response of isolated canine airways to ACh have been demonstrated based on the relationship of myosin content and total smooth muscle thickness (Mapp et al. 1989). These authors concluded that active force generated was dependent upon the content of myosin in the muscle strips studied. Myosin content in pulmonary diseases characterized by airway hyperreactivity has not been reported. Other changes in airway smooth muscle that could alter airway responses without differences in muscle thickness would be post-receptor mechanisms, such as changes in smooth muscle Ca++ release. A decrease in cAMP or cAMP- dependent kinase activity would result in increased smooth muscle tension development. An inverse relationship between methacholine sensitivity and cAMP or cAMP-dependent kinase activity has been demonstrated in canine tracheal smooth muscle at various anatomic sites (Jensen et al. 1986), but a relationship between airway hyperreactivity and cAMP levels has not been investigated. It remains uncertain whether airway hyperreactivity is characterized by changes in the intrinsic properties of airway smooth muscle. Dose-response studies in subjects with asthma show there are wide variations in the dose of carbachol needed to decrease specific airway conductance and in the slopes of 39 the curves (Orehek et al.1977). This could be due to an increase in the smooth muscle mass, or to changes in the contractile mechanisms or their regulation in airway smooth muscle (Boushey et al. 1980). Too few studies have been performed in subjects with diseases associated with hyperreactivity to make conclusions about the role of airway smooth muscle. Neuropeptides in airway hyperreactivity The roles of cholinergic and adrenergic nerves in regulating contraction of smooth muscle have been extensively studied. More recent studies of neural control of airway caliber have been directed toward understanding the roles of less well characterized neurotransmitters, the neuropeptides. To date, SP is the most commonly studied neuropeptide in the lung (Borson et al. 1989). Substance—P, and conceivably other tachykinins, increase inflammation by a variety of mechanisms including neutrophil chemotaxis (Marasco et al. 1981). However, the effect on neutrophils is seen at relatively high concentrations and is therefore of questionable significance. Neutrophil and macrophage phagocytosis and lysosomal enzyme release are potentiated by SP (Bar-Shavit et al. 1980) as is macrophage thromboxane release (Hartung and Toyka 1983). The effects of SP on human lung mast cells have not been studied. However, human skin mast cells release histamine upon stimulation by SP (Foreman and Jordan 1983). Substance-P is also a specific T-lymphocyte mitogen (Payan et al. 1983, 1984). Thus, by all these stimulatory effects, as well as by increasing vascular permeability (Lundberg et al. 1984b), SP tends to amplify the inflammatory response. 40 Both capsaicin and antidromic stimulation of the vagus cause broncho- spasm by release of SP (Lundberg et al. 1983). Depletion of SP from C-fiber nerve endings with capsaicin blocks the atropine-resistant component of bronchospasm caused by vagal stimulation (Martling et 31.1984) Exogenous SP causes bronchospasm in the guinea pig and contraction of ferret and human airway smooth muscle in vitro, although the latter effect is not seen at concentrations less than 1 am (Lundberg et al. 1983, Sekizawa et al. 1987). At lower doses, SP facilitates cholinergic neurotransmission in rabbit (Tanaka and Grunstein1986) and ferret (Sekizawa et al. 1987) airways, leading to an increased response to electrical field stimulation. The stimuli responsible for SP release in the airways under physiological conditions are unknown. Enkephalinase is an enzyme that degrades SP and other tachykinins into inactive metabolites (Skidgel et al. 1984). The discovery of enkephalinase in human airway tissues provides a mechanism for limiting the effects of released SP (Johnson et al. 1985). When the SP-degrading activity of enkephalinase in virus-infected ferret airways is inhibited using leucine-thiorphan, a contractile effect is seen with concentrations of SP as low as 5 x 10'7 M as compared with >1045 M in the absence of leucine-thiorphan (Sekizawa et al. 1987). This contraction is partially attenuated by atropine, suggesting that release of ACh is responsible for part of the effect of SP. Furthermore, while leucine- thiorphan does not cause contraction, treatment with leucine—thiorphan does lead to an increased response to electrical field stimulation. This effect can be blocked by pretreatment with a tachykinin receptor antagonist, suggesting that endogenous tachykinins (possibly SP) augment cholinergic transmission when freed from the controlling influence of airway enkephalinase. Tachykinins are 41 also degraded by other enzymes, including angiotensin-converting enzyme and serine proteases, but inhibition of these enzymes does not potentiate electrical field stimulation, suggesting that enkephalinase is the enzyme responsible for regulation of tachykinin activity. A decrease in airway enkephalinase activity may be one mechanism leading to airway hyperresponsiveness associated with viral infections (Figure 1). Infection of ferret tracheal rings with human influenza A virus in vitro leads to an exaggerated response to SP (J acoby et al. 1988) Treatment of tissues with thiorphan increases the SP—contractile response of infected and control tissues to the same level. The enkephalinase activity of the infected tissues is markedly decreased. The authors hypothesized that removal of the airway epithelium accounts for this decrease in enkephalinase activity, as histological examination revealed extensive epithelial denudation of infected tracheal rings. However, the response to exogenous ACh was not enhanced, excluding an increase in epithelial permeability or loss of a nonspecific epithelial-derived relaxant factor as the mechanism of the increased substance-P response (J acoby et al. 1988). Other possible excitatory mediators in the airways are calcitonin gene- related peptide and neurokinin A (Figure 1). Calcitonin gene-related peptide is a product of alternative processing of messenger RNA transcribed from the calcitonin gene (Rosenfeld et a1. 1983). It has been identified in the airways of guinea pigs (Lundberg et al. 1985), where it coexists with SP in C-fiber nerve endings, as well as in human airways (Palmer et al. 1987), and is a potent bronchoconstrictor in both species. Neurokinin A is a tachykinin that is also a bronchoconstrictor (Lundberg et al. 1985) The physiological regulation of the 42 effects of these substances has not yet been worked out, but neurokinin A, like SP, can be degraded by enkephalinase. Abnormalities in autonomic control of airway smooth muscle Airway hyperreactivity could be a consequence of an alteration in one or more of the membrane receptors in the respiratory tract. Changes in receptor affinity and/or number, or in receptor signal transduction coupling could account for’increased airway responsiveness. An imbalance between alpha- and beta-adrenoceptors has been proposed, particularly in regard to hyperreactivity in asthmatics. Szentivanyi proposed originally that airway hyperreactivity was due to an abnormality in the bronchodilatory betaz- adrenoceptor system (Szentivanyi 1968). He further postulated an increased number or activity of alpha-adrenoceptors, which mediate bronchoconstriction (Szentivanyi1979, 1980). Radioligand studies in human lung membrane fractions indicated a slight decrease in beta-adrenoceptor number concomitant with a marked increase in the numbers of alpha-adrenoceptors in asthmatics compared with control patients (Szentivanyi 1980). An apparent 10-fold increase in the number of alphal-adrenoceptors in parenchymal tissue from nine patients with chronic obstructive airway disorders compared to nonasthmatic patients has also been reported (Barnes et al.1980). However, these studies were conducted using a tissue containing many different cell types, and no attempt was made to correlate the alteration in receptor number with changes in the responsiveness of airway smooth muscle (Goldie et al. 1985). Respiratory virus infections can exacerbate bronchial asthma and enhance airway reactivity to smooth muscle contractile substances. Antagonism 43 of beta adrenergic or other inhibitory responses as a component of virus- induced changes in lung function has been proposed (Buckner et al. 1981), but evidence for beta-mediated changes in airway smooth muscle reactivity is generally lacking. There is some evidence to suggest impaired beta- adrenoceptor function in human granulocyte responses during upper respiratory infections and after in vitro incubation of granulocytes with live influenza vaccines (Busse 1977, Busse et al. 1979) An animal model using parainfluenza 3 infection in guinea pigs demonstrated a selective blockade of beta adrenergic- mediated relaxation of antigen-induced contraction of airway smooth muscle (Buckner et al. 1981). The authors concluded that this guinea pig model had some similarities with virus-associated effects in humans and might be useful to explore further mechanisms of virus-induced asthma. Further evidence for a role of alpha- and beta-adrenoceptors in virus-induced airway hyperreactivity in the guinea pig, human, or other mammals has not been found. Airway hyperreactivity could be due to increased parasympathetic or alpha-adrenergic activity, or to decreased beta-adrenergic or NANC inhibitory activity (Figure 1). However, there are few data to support the postulate that dysfunction in the N ANC nervous system contributes to virus-induced airway hyperreactivity. A recent preliminary report demonstrated an alteration in the balance between the cholinergic and NANC nervous systems in patients with asthma but not in those with chronic obstructive lung disorders (De Jongste et al. 1987). Other investigators (Lammers et al. 1989) have been unable to demonstrate a defect of the NANC nervous system in mildly asthmatic patients. 44 Normal resting airway tone is maintained by vagal efferent activity and is abolished by blocking conduction in the vagal nerves (Olsen et al. 1965, Karczewski and Widdicombe 1969) or by atropine (Severinghaus and Stupfel 1955, Nadel and Widdicombe 1963) In patients with asthma (Cropp 1975), cystic fibrosis (Larsen et al. 1979), and chronic bronchitis (Klock et al. 1975), atropine is reported to be equivalent in potency of bronchodilation to beta-adrenergic agonists, suggesting that the increased airway smooth muscle tone in these diseases is due to increased vagal activity. In a study of acute upper respiratory infection, isoproterenol hydrochloride or atropine sulfate aerosol each reversed and prevented the increase in airway resistance produced by histamine (Empey et al. 1976). These authors concluded that the enhanced bronchoconstriction in normal subjects after upper respiratory tract infection was caused by smooth muscle contraction and that cholinergic pathways were involved. Although parasympathetic efferent pathways appear to play a role in airway reactivity in some diseases, the exact mechanism(s) is unknown. Because the various stimuli that cause exaggerated bronchospasm (e.g., chemicals, dusts, and histamine) all stimulate sensory receptors in the airways with subsequent vagal reflex bronchoconstriction in animals, it has been suggested that damage to the airway epithelium might sensitize the sensory receptors and cause exaggerated reflex responses (Widdicombe et al. 1962, Nadel et al. 1965) Viral respiratory tract infections cause reversible damage to the airway epithelium (Hers and Mulder 1961) Empey and coworkers showed that histamine aerosols produced a greater degree of bronchoconstriction in subjects with colds than in a control group (Empey et al. 1976). This exaggerated response returned to normal spontaneously during several weeks. Similar findings were reported 45 after successful inoculation of live, attenuated influenza virus (Laitinen et al. 1976), suggesting that viral infections increased the accessibility of the histamine aerosol to the effector cells. Because atropine sulfate prevented the exaggerated bronchomotor responses, these authors concluded that the effect was not simply an increased accessibility of the histamine aerosol to the airway smooth muscle due to epithelial damage, but implicated the parasympathetic nervous system in the exaggerated responses (Laitinen et al. 1976). The above studies implicate the cholinergic nervous system in the increased airway responsiveness detected during viral respiratory infections, but they do not pinpoint the specific sites in the vagal system that are affected. Furthermore, the relationship between acute viral infection and airway epithelial damage remains uncertain. Studies designed to determine the importance of afferent and efferent cholinergic pathways in virus-induced airway hyperreactivity need to be completed in experimental animals with respiratory infections. Changes in the efferent pathway of the vagal nerves could cause virus- induced airway hyperreactivity, and modification of prejunctional cholinergic influences could be an important mechanism that would alter the airway response. Presynaptic modulation of cholinergic nerve activity by neurotransmitters or by inflammatory mediators has been reported in several species, including the dog, cat, guinea pig, and man (Vermiere and Vanhoutte 1979, Fryer and Maclagan 1984, Blaber et al. 1985, Ito and Yoshitomi1988, Minette and Barnes 1988). Norepinephrine reduces cholinergic nerve-mediated contraction of canine tracheal smooth muscle at concentrations that have little effect on exogenous ACh-induced smooth muscle contraction, suggesting an 45 inhibitory effect on endogenous ACh release proximal to the end organ (Vermiere and Vanhoutte 1979). This inhibition is presumably mediated by presynaptic beta-receptors. Prostaglandin E2 has a similar inhibitory effect on cholinergic neurotransmission in dogs (Walters et al. 1984). Serotonin, prostaglandin FZalpha’ SP, and adenosine appear to potentiate ACh release in airways (Hahn and Patil 1972, Sheller et al. 1982, Tanaka and Grunstein 1984, Sakai et al. 1989). However, the major limiting component to cholinergic neurotransmission may be the inhibitory feedback effects of ACh on prejunctional muscarinic autoreceptors. In the guinea pig, blockade of muscarinic autoreceptors with gallamine caused a sixfold potentiation of vagal- induced bronchoconstriction at a stimulation frequency of 15 Hz (Fryer and Maclagan 1986). The autoreceptors are likely to play an important role in modulating parasympathetic neurotransmission at rest and during reflex activation of the nerves in vivo because 15 Hz is close to the natural firing rate reported for parasympathetic postganglionic fibers in ferrets and cats (Baker et al. 1982, Mitchell et al. 1984). The evidence for these muscarinic autoreceptors was first obtained in experiments using the selective muscarinic antagonist gallamine (Fryer and Maclagan 1984). When the autoreceptors were blocked with this drug, transmitter output increased and the effect of vagally-induced bronchoconstriction was potentiated in a dose-dependent and frequency- dependent fashion. Consistent with previous reports, the effect on the heart was quite different because the bradycardia produced by vagal stimulation was abolished by gallamine following blockade of receptors on the atrial cell (Riker and Wescoe 1951). In the heart, the effect of gallamine was unrelated to 47 frequency of stimulation because the cardiac M2 receptors are located postjunctionally and do not modulate neurotransmitter output. Gallamine had little effect on the bronchoconstrictor response to injected ACh, indicating that gallamine was not a potent antagonist for the postjunctional muscarinic receptors in airway smooth muscle. In addition airway responses to vagal stimulation following gallamine injection were not related to any involvement of the sympathetic nervous system because pretreatment with guanethidine did not affect the results. Neither could the results be explained by an alteration of airway contractility as histamine-induced bronchoconstriction was unaltered by gallamine (Fryer and Maclagan 1984). As described previously in the section of this literature review on M2 receptors in the airways, a possible relationship between increased vagally induced bronchoconstriction seen in viral respiratory infections and alteration of M2 autoreceptors in the lung has been recently reported (Fryer et al. 1990, Fryer and Jacoby 1990). The M2 receptor-mediated inhibition of acetylcholine release from parasympathetic nerves in the lungs was decreased in parainfluenza-infected guinea pigs. The investigators hypothesized that damage to M2 inhibitory receptors could result in the loss of negative feedback inhibition to ACh release. The increased release of acetylcholine from the parasympathetic nerves could activate M3 receptors on airway smooth muscle and might explain virus-induced airway hyperresponsiveness (F ryer and J acoby 1990). Viral respiratory disease in the cat Introduction Viruses that produce respiratory disease in cats were devastating before the advent of effective vaccines. Presently, diseases caused by respiratory viral infections are most commonly seen in situations of poor husbandry or overcrowding, such as pet stores, kennels, and catteries (Moise 1985). Age is a critical factor predisposing cats to respiratory tract infection (August 1984). High titers of colostrally derived maternal antibody protect the young kitten from challenge early in life. As passive immunity wanes, the young kitten becomes highly susceptible to challenge from exposure to cats undergoing primary infections, or to carrier cats. Most fatal cases of respiratory disease occur in 8- to 10-week-old kittens (August 1984). Cats with immunologic compromise, which can occur with feline leukemia infection or immunosuppressive chemotherapy, are also more susceptible to the morbidity and/or mortality of respiratory viral infections (Bech-Nielson et al. 1981). The majority of respiratory isolates have been associated with the upper respiratory tract rather than with the lungs, and although agents may be cultured from the lungs, there is as yet no well defined viral pneumonia of cats (Povey 1969). Feline herpesvirus-I (feline viral rhinotracheitis) and feline calicivirus are the two major causes of respiratory disease in cats (Gaskell 1988). Both viruses are widespread throughout the world. They are of approximately equal importance in causing disease, and together account for at least 80% of viral respiratory disease in cats (Gaskell 1988). The feline strain of Chlamydia psittaci (previously called the feline pneumonitis agent) can also cause mild respiratory signs, but in general the major feature of C. psittaci infection is persistent 49 conjunctivitis (Povey 1969, Gaskell 1988). Feline reovirus has been shown to produce a mild, predominantly conjunctival disease experimentally, but it is probably not a significant cause of naturally occurring respiratory disease (Gaskell 1988). Bacteria, such as staphylococci, beta-hemolytic streptococci, Pasteurella spp. and coliforms, are probably important as secondary invaders. The role of Bordetella bronchiseptica, which has been detected in some laboratory colonies, has yet to be determined. M ycoplasma spp. are also important mainly as secondary invaders, although a more primary role, particularly for Mycoplasma felis in conjunctivitis has been suggested by some investigators (Gaskell 1988). The significance of mycoplasmas is difficult to assess because of the frequency with which they are also isolated from apparently normal cats. While feline herpesvirus-I and feline calicivirus are not the viruses most commonly infecting humans, they do produce primarily respiratory lesions that are similar to the lesions induced by common human respiratory infections. There is only one recognized serotype of feline herpesvirus and it is of reasonably uniform pathogenicity in susceptible cats (Crandell et al. 1961) Feline cell-associated herpesvirus was isolated initially from clinically normal kittens, a kitten with feline calicivirus, and a kitten with concurrent urethral obstruction and calicivirus upper respiratory tract disease (Fabricant et al.1971). However, feline cell-associated herpesvirus is antigenically distinct from feline herpesvirus type I and does not appear to cause respiratory tract disease (Fabricant 1977). Although there is only one primary feline calicivirus serotype, there are a number of different strains that vary slightly antigenically and are of varying pathogenicity (Povey and Hale 1974, Hoover and Kahn 1975). In 50 general, feline calicivirus infections are milder than those caused by feline herpesvirus and do not cause consistent clinical disease in the airways. Feline herpesvirus-I infection in the cat Feline herpesvirus-I is highly infectious to the susceptible cat. Respiratory disease tends to appear wherever cats are congregated together, with infection often introduced by a clinically normal carrier. Once present in a colony, the disease rapidly becomes endemic and there are chronically affected animals with recurrent or persistent signs. The virus is present in large amounts in the secretions from ocular, nasal, and oral discharges of infected cats. The major mode of transmission is via the intranasal, intra-oral, and con junctival routes. Limited experimental evidence suggests that transplacental herpesvirus infection probably does not occur following natural routes of infection, although it can be induced following intravenous inoculation of virus (Hoover and Griesemer 1971). Feline herpesvirus-I (FHV) is relatively fragile outside the cat, surviving for less than 18 hours. It is susceptible to heat, drying, and most common disinfectants (Povey and Johnson 1970). Continued survival of the virus depends primarily on its ability to survive inside the cat. Firstly, the virus spreads from acutely infected clinical cases to susceptible cats. Secondly, the virus can induce an immune carrier state in recovered animals (Gaskell and Wardley 1978). Studies have shown that at least 80 percent of FHV-recovered cats remain as viral carriers (Gaskell and Povey 1973, 1977). During acute infection, viral replication occurs predominantly in the turbinates, soft palate, conjunctivae, tonsils, and, to a lesser degree, in the 51 trachea and bronchi (Gaskell and Povey 1979). Active shedding of FHV in oculonasal and oropharyngeal discharges continues for 1 to 3 weeks after primary infections (Gaskell and Wardley 1977). During acute epizootics, transmission occurs predominantly through cat-to-cat contact. Early signs in susceptible cats include depression, paroxysmal sneezing, anorexia, and fever, which progress rapidly to a marked mucopurulent oculonasal discharge with conjunctivitis, salivation, and, on occasion, coughing and ulcerative keratitis. Most fatalities are due to fluid and caloric deficiencies, respiratory failure, and opportunistic bacterial infections. The acute stage usually lasts from 2 to 3 weeks, and unvaccinated cats may become susceptible to reinfection within 6 months. Adequate vaccination does not eliminate the possibility of viral respiratory disease, because current vaccines are not completely effective in preventing infection or disease (Arnett and Greene 1984). Vaccines have been moderately successful in controlling feline respiratory disease in the majority of healthy previously unexposed animals. Both modified live and inactivated systemic vaccines are available, as well as modified live vaccines given by the intranasal route. There is evidence that a previously unexposed cat vaccinated intramuscularly for FHV may subsequently become a virulent field virus carrier following challenge, without ever having shown any clinical signs (Gaskell 1984). Thus vaccination should be regarded as protection against disease for the individual rather than protection against infection. Although herpesvirus-I is one of the most common causes of respiratory 52 disease in the cat, there have been no studies to date on airway responses to cholinergic stimuli following acute viral infection. SPECIFIC AIMS OF STUDIES Viral respiratory infections in humans can cause transient abnormalities in airway function and a period of airway hyperreactivity to inhaled irritants, chemical mediators, and cold air (Empey et al.1976, Aquilina et al.1980, Stempel and Boucher 1981). Upper respiratory infections increase airway reactivity in normal subjects and in patients with asthma (Empey et al.1976, Busse 1985). In asthmatics, the consequence of respiratory infection is often a severe attack of asthma. Although this relationship is a frequent medical occurrence, mechanisms to explain virus-associated changes in airway reactivity are not fully established. I developed an animal model to investigate the role of the cholinergic nervous system in virus-induced airway hyperreactivity. The cat is the natural host of feline herpesvirus-I. This virus is of a single antigenic type and is specific for the respiratory tract of the cat. In addition, the autonomic regulation of cat airways is well described so there is a good data base for comparison of my findings. My primary purpose was to determine if there is hyperresponsiveness to cholinergic stimulation in vagotomized cats following acute infection with feline herpesvirus-I, and if so, to determine if the hyperresponsiveness is pre- or postjunctional. I recorded changes in cervical tracheal caliber and intrathoracic airway caliber during vagal nerve stimulation and following exogenous ACh injection. Because I found it difficult to obtain cats that were respiratory virus-free prior to each experiment, I have also 53 investigated the role of inhibitory muscarinic receptor subtypes in limiting airway responsiveness in a group of cats that were not infected with herpesvirus-I in the laboratory. Specifically, I have designed protocols to: 1 Investigate a new technique for measuring dimensional changes in selected airway segments. 2. Determine the effect of acute infection with feline herpesvirus-I on intra- and extrathoracic airway responsiveness during cholinergic stimulation. Two methods of muscarinic receptor stimulation were used in virus-infected and sham-inoculated (control) cats. 3. Exogenous ACh was delivered intra-arterially at tracheal ring 4 to stimulate postsynaptic muscarinic receptors on the effector muscle. b. Receptors were activated presynaptically by vagus nerve stimulation. 3. Compare airway responses to cholinergic stimulation measured at three days post-inoculation to measurements obtained at six days post- inoculation in virus-infected and control cats. 4. Determine the effect of muscarinic receptor blockade with atropine on airway responses measured during vagal nerve stimulation and following exogenous ACh injection. 54 Determine if the results from herpesvirus-I isolation and quantitation testing at different sites in the airways would parallel the sites where airway hyperreactivity was detected during vagal stimulation. Determine whether dysfunction of the prejunctional muscarinic inhibitory (M2) receptors were responsible for the exaggerated tracheal response to vagal stimulation by using the M2 receptor antagonist gallamine. As in the previous studies, cholinergic stimulation was induced by presynaptic vagal stimulation (24 V, 05 msec, 4 Hz) and by postsynaptic receptor stimulation on the effector organ with ia ACh. Determine if M2 inhibitory receptors could be demonstrated in the cervical trachea during vagal stimulation at frequencies other than 4 Hz (05 to 16 Hz). Determine whether tracheal hyperresponsiveness to vagal stimulation was accompanied by hyperresponsiveness in intrathoracic airways, and if so, whether intrathoracic airway hyperresponsiveness was mediated by M2 receptor dysfunction. MATERIALS AND METHODS Introduction The materials and methods used in all protocols are described in this section. The cat groups and the specific experimental design used in each protocol are described in the separate chapters. The first experiments were designed to study the responses of cat airways following acute infection with herpesvirus-I. Cats used in the virus studies were tested prior to the experiment and shown to be free of parasites and common infectious agents that might interfere with analysis of our results. Data was analyzed only from cats that were either infected in the laboratory with a known amount of herpesvirus or herpesvirus free (control). Subsequent experiments were designed to determine the role of inhibitory muscarinic receptors in limiting constriction of cat airways during cholinergic stimulation. These cats were not tested for parasites or infectious diseases, and they were not infected with herpesvirus. Animals in herpesvirus-I studies Adult, mixed breed cats were seronegative for heartworm antigen, feline leukemia antigen, chlamydia, and feline herpesvirus-I (feline rhinotracheitis virus). Hemagglutination inhibition tests for feline panleukopenia virus and calicivirus antibody were performed. Virus isolation for feline herpesvirus-I 55 56 was negative in all cats. Cats were randomly assigned to infected or control groups. Infected cats were studied at post-inoculation day (PID) 3 or 6; control cats 3 days after sham infection. Virus-challenged cats (PID3, n = 15; PID6, n = 5) were exposed to 10‘5 TCID50 (the concentration required to produce infection in 50% of Crandell- Rees feline kidney cells) of the C27 prototype strain of feline herpesvirus-I (Burgener and Macs 1988) distributed topically on the oral, conjunctival, and intranasal mucus membranes. Control cats (11 = 12) were sham infected with tissue culture medium. Nasal, oral, and conjunctival swabs were collected to confirm the presence or absence of feline herpesvirus-I immediately prior to inoculation and at PID3 or PID6 before beginning the experimental protocol. In addition to virus isolation, dilutions of swab extract were made in 10 additional cats (PID3 infected, 11 = 5; PID3 control, n = 1; PID6 infected, n = 3; PID6 control, n = 1) in order to quantitate the amount of virus present at various levels of the respiratory tract. Swabs from these 10 cats were collected from conjunctival, nasal, and oral mucus membranes as well as cranial cervical trachea, mainstem bronchi, and peripheral lung. Techniques used for virus propagation and titration have been described previously (Burgener and Maes 1988) To maintain the integrity of the viral culture, swabs were transported to the laboratory on ice and extracted into 2 ml of Eagles’s Minimum Essential Medium containing 10 times the concentration of the antibiotics used in growth medium. These procedures assured that the cats were either infected with a known viral agent or herpesvirus free. 57 Animals not infected with hegpesvirus-I I attempted to obtain virus-free cats from three sources. First, I purchased random-source adult cats from the University Laboratory Animal Resources facility at Michigan State University, tested them for the diseases listed above, and conditioned the "disease-free" cats for 30 days in the Clinical Center. This process proved to be labor intensive, costly, and unsatisfactory as the cats began to develop enteritis caused by Salmonella spp. and could not be used in my experiments. Next, I purchased "minimal disease" adult cats from Liberty Laboratories in New Jersey. These cats were very expensive and difficult to handle. In addition, there was a long delay of four months between ordering and obtaining the cats. Finally, I decided to purchase random source cats from the university, test for diseases as described above, perform the experiments, and eliminate the data if, subsequently, the cats proved to be positive for any of the diseases listed above. This resulted in elimination of 34 to 56 percent of my data, depending upon the time of year. Therefore, the virus studies were discontinued and random-source cats were obtained from the University Laboratory Animal Resources facility at Michigan State University for in vivo investigation of the role of muscarinic receptor subtypes in the control of airway caliber. Surgical preparation Cats were anesthetized with thiamylal (15-20 mg/kg iv) administered to effect to maintain a corneal reflex and prevent withdrawal response following paw pinch. The trachea was exposed via a midline ventral cervical incision and an endotracheal tube was placed via tracheostomy at the thoracic inlet so that 58 the cranial cervical tracheal segment to be studied was undisturbed. Following intubation, anesthesia was maintained with urethan (500 mg/kg iv) and chloralose (100 mg/kg iv). Cats were paralyzed with pancuronium bromide (0.6 mg/kg iv) and connected to a fixed volume ventilator. In unpublished experiments completed during the initial M2 receptor studies, I established that pancuronium had no effect on the response of tracheal ring 4, pulmonary resistance, or dynamic compliance to vagal stimulation. However, because pancuronium has been described as a potential muscarinic receptor antagonist, I did not use the drug in the studies described in Chapter 5. Respiratory frequency was adjusted to maintain PaCO2 between 28 and 32 mm Hg and PaO2 between 85 and 100 mm Hg. Sodium bicarbonate was infused intravenously as necessary to maintain arterial pH between 7.360 and 7.440. Cats were placed on a thermostatically controlled heating pad. Lactated Ringer’s solution was administered by continuous infusion (10 ml/kg/hr iv). A catheter was placed in the left femoral artery for continuous measurement of blood pressure (P23 ID physiological pressure transducer, Gould) and for collection of arterial blood samples to monitor blood gases. The vagus nerve was bilaterally isolated from the sympathetic trunk in the mid to distal cervical region (Figure 2). The sympathetic trunk was transected bilaterally after the observation of mydriasis following electrical nerve stimulation. The vagus nerves were transected bilaterally at the angle of the mandible. In the mid- cervical region, both vagus nerves were placed over platinum electrodes connected to a dual output square pulse stimulator. The right common carotid artery was ligated cranial to the cranial thyroid artery and caudal to tracheal ring 4. An indwelling catheter was introduced so that its tip reached the level 59 /—————‘ common ca rotld arte ry ,/——————— vagus (transected) ,r—r sympatheuc trunk cranial thyroid artery figature CT 1;) 1 trachealrflng i I , 3 £/ I} C J2 ,1: i z! )3 ": H j L plane of —:: :: —-€j— --_'-—-- :i) —“._.-- ment _:: :i ‘ E :ji measure —‘—‘ is E C gs E E! a j C _. j; u C DE :— 1': j C " 5': j C H C )8 "' 3 L C )9 j C C D /.—-——-‘ r). Hgature (transected) §;—\\\__ sympathetk: trunk Figure 2 Ventral view of the anatomy of the right side of the feline cranial cervical trachea. Changes in tracheal diameter were measured by a calibrated electromechanical caliper placed on the fourth tracheal ring ("plane of measurement"). Tracheal constriction was produced by local unilateral intra- arterial injections of acetylcholine or bilateral vagal stimulation. The vagus nerves were transected at the angle of the mandible and the sympathetic trunks were transected in the midcervical region. Acetylcholine was injected through a catheter in the right common carotid artery. The tip of the catheter was at the level of the cranial thyroid artery. The cranial thyroid arteries are the primary blood supply to the cranial cervical trachea and the vagal nerves contain the efferent motor nerves to the cervical trachea. 60 of tracheal ring 4 adjacent to the origin of the cranial thyroid artery for local intra-arterial infusion of drugs (Figure 2). Tracheal caliper and calibration The device for quantitating dimensional changes in the airway is diagrammed in Figure 3. The tips of the device were connected to the external surface of the trachea with single sutures. Forces applied at these points deform a thin, flexible, elastic, steel strip (nom. 8 mm wide, 0.05 mm thick) to which two microfoil strain gages (model 6/120LY13; Omega Engineering Inc.) were bonded on either side. Using two gages for each device, each of which is represented as one element in a bridge-balanced voltage divider, increases sensitivity and provides temperature compensation for the instrument. The surface of the instrument is covered with a thin layer of epoxy or lacquer so that neither the gages, their electrical connections, nor the steel strip are in contact with tissue fluid. The instrument and its connecting wires are adequately light in weight (nom. 1.4 gm) so that they are easily supported above the tissue and do not deform it during measurements. Fine wires from the strain gages connect to a solid state resistance bridge (or voltage divider), two sides of which are formed by the gages themselves. An analog DC voltage from the bridge is sent to a solid state amplifier and to an analog strip chart, X-Y recorder and/or computer. The instrument was calibrated using a digital micrometer (Mitutoyo NO. 293-765) with 1.0 micron resolution for both compression and extension of the measuring device. A representative calibration is shown in Figure 4. Subsequent statistical curve- fitting techniques using calibration data generate an equation for each device. 61 heat shrink microfoil tubing strain gages \ ' fie ib e s ee # plate 4 L # 1 ‘_/ connections \/ . to bridge sits? rigid / / support cartilaginous epithelial ring lining /\ \.../ trachealis muscle J :11 measured ‘— _ —.. \ distance suture suture Figure 3. A diagram of the device used for isotonic measurements of tracheal diameter. Two microfoil strain gages were bonded to opposite sides of a thin, flexible, steel strip. Anchored firmly to the proximal end of this strip was a bent metal rod that served as a binding site for the gages’ electrical connections. A similarly stiff metal rod was firmly attached to the distal end of the steel strip. The distance over which an isotonic measurement was made was defined by the configuration of the ends of the 2 metal rods that were attached to the external surface of the airway. Isotonic measurements were made by bonding the strain gages to a more flexible steel plate. Isometric measurements can be .made by bonding the strain gages to a less flexible steel plate and calibrating the device gravimetrically. 62 I 1 extension lrlfiiri' A 57—wrrs VV’VYVV O rrI I I I I II C H s N 4 ~ compressmn VJ : 3- E L L. 2- 0 0H E 1~ 3) b 0 Q. / 4.) s Q) . 2.0 -2~ no '5 > -3- O . 330 _4_ E 0 LI m _5.Ltl.l.lLL.lrlml.l. I l I l . l.l.l.liL -10 -9 -8 -7 -6 -5 -4 —3 —2 —1 O 1 2 3 4 5 6 Displacement (microns x 100) 10 Figure 4. A representative static calibration of the device used for isotonic measurements of tracheal diameter. Matching levels of bridge voltage output (ordinate) are shown as steady state functions of thirty randomly ordered displacements (abscissa) of the device shown in Figure 3. Calibration of the robe on seven separate occasions resulted in an r value of 0.9950 :i; 0.003 (mean j; SE) and slope = 0.50 i 0.014. 63 Measurement of pulmonary resistance and dynamic compliance Respiratory air flow was measured using a Fleisch 00 pneumotachograph attached to the endotracheal tube. This system was calibrated before each experiment by forcing known volumes of air with a 60-ml calibrated syringe through the pneumotachograph. A 6-cm incision was made in the right lateral thoracic wall so that pleural pressure was equal to ambient air pressure. Transpulmonary pressure was equal to the pressure at the distal end of the endotracheal tube and was measured with a differential pressure transducer (Model MP 45; Validyne). Pressure and air flow data were processed by a pulmonary function computer and data processor (Model DL—12; BUXCO Electronics, Inc.), displayed on a 12-channel physiograph and recorded on light- sensitive paper. The responses of the catheter-transducer systems were matched to 15 Hz. Tidal volume was obtained by electronic integration of the flow signal, and dynamic compliance and pulmonary resistance were measured from simultaneous recordings of transpulmonary pressure, flow, and volume. Dynamic compliance (Cdyn) was calculated as the volume change compared to the change in transpulmonary pressure at zero flow. Pulmonary resistance (R1) was calculated by the isovoiume technique (Amdur and Mead 1958). Accuracy of the lung function computer data was verified regularly by hand calculation from the physiograph trace. CHAPTER 2 In Vivo Airway Caliber Measurement Using Microfoil Strain Gage Transducers Introduction There are several direct and indirect techniques for measuring respiratory smooth muscle contraction (Macklem and Mead 1967, Bass and Wiley 1972, Souhrada and Dickey 1976, Beinfield and Seifter 1980, Woolcock and Permutt 1986). Direct measurements of smooth muscle tension are commonly made in an organ bath in which the tissue is deprived of normalianatomic relationships. Indirect measurements such as airway resistance, dynamic compliance, and maximal expiratory flow yield information about airway caliber along the respiratory passages. However, these tests can determine neither the specific site of obstruction nor the mechanism of airway narrowing such as smooth muscle contraction, mucus accumulation, or airway wall edema (Woolcock and Permutt 1986). Several devices have been developed to measure tracheal dimensions in situ using either microfoil strain gages (Bass and Wiley 1972, Souhrada and Dickey 1976, Beinfield and Seifter 1980) or inductance (Fouke et al. 1981) technologies. Typically, one or more strain gages are bonded to elastic metal base strips that are sutured transversely and/or longitudinally to the outer 64 65 tracheal wall. Depending on the physical characteristics of the gages themselves and the matrix to which they are bonded, these small and extremely sensitive devices yield valuable information about both static and dynamic changes in airway dimensions. Similar data about inner tracheal diameters can be obtained when the instrument is configured to be inserted into the airway, not attached to its outer surface (Fouke et al. 1981). Common difficulties in using these devices include accurately relating deformation sensed by the measuring device to airway dimensions because of the changing geometry of the tissue and the nonlinear calibration characteristics of the instrument itself. The stiffer the metal base to which the gage is bonded, the more it transduces the isometric forces developed in the tissue rather than the dimensional changes. For the trachea, deducing. how tissue forces change airway dimension and resistance is impossible unless tissue compliance is known. A solution is to use an instrument which itself is sensitive, hysteresis-free and one which is calibrated in units of measured distance in the tissue to which it is affixed, not in terms of the forces developed by the tissue. Also, instruments are most useful when methods for their attachment are simple, and when the device itself interferes minimally with normal air flow, lining epithelium, and the blood and nerve supply of the study segment. There are important bonuses if the device is easy and inexpensive to make, holds its calibration for long periods, and is innocuously secured to the tissue under study. This chapter describes a new technique for measuring dimensional changes in selected airway segments and for quantifying the strength and time course of respiratory smooth muscle contraction. The device, which 66 incorporates 2 microfoil strain gage transducers, measures changes in the outer diameter of the airway and is unaffected by airway wall edema or mucus accumulation. Depending on the configuration of the device, it can measure either isometric or isotonic muscle contractions. It is attached to the external surface of the airway wall in situ with minimum surgery and allows the airway to retain its normal innervation and perfusion. A typical application leaves tracheal rings intact and limits exposure and drying of airway linings. I will describe construction and calibration of the instrument and present data on isotonic contraction of tracheal smooth muscle induced by electrical stimulation of vagus nerves in the anesthetized cat. Experimental design Adult, mixed breed cats were anesthetized with thiamylal (15-20 mg/kg iv) to a level which maintained corneal reflex but not a withdrawal response to paw pinch. The trachea was exposed by a midline ventral cervical incision and the tips of the electromechanical caliper were attached to either side of tracheal ring 4 at its widest diameter with single 4—0 silk sutures (Figure 3). Changes in tracheal diameter were observed during respiration and during vagal stimulation. To measure tracheal diameter changes during spontaneous breathing, a short endotracheal tube was inserted so that the inflatable balloon was within the larynx and cranial to tracheal ring 4. Tracheal pressure was measured by inserting a 20-gauge needle into the tracheal lumen immediately caudad to the probe. The needle was attached to a catheter and pressure transducer so that 67 pressure and tracheal diameter could be recorded simultaneously on a physiograph. To study changes in tracheal diameter during vagal stimulation, an endotracheal tube was placed by tracheostomy at the thoracic inlet so that the cranial cervical tracheal segment to be studied was undisturbed. Following intubation, maintenance of anesthesia and instrumentation was performed as described in the Materials and Methods section of this dissertation. A cumulative frequency-response curve (24 V, 05 msec duration continuous stimulus, 05-16 Hz) was generated in 15 cats. Results Calibration The microfoil strain gage transducer system generated a linear voltage output as a function of incremental and decremental displacements, as shown in Figure 4. The equation of the line of best fit for a single calibration was: bridge output (microvolts) = 0.56 (distance; microns) - 26.85; r = 0.9933. Because the natural frequency of the instrument was determined to be approximately 15 Hz, it is assumed that its time response is suitable for most animal tests in which smooth muscle responses are considerably slower, as shown by data reported in Figures 5 and 6. The instrument was drift-free and unaffected by thermal transients within a range of physiological temperatures. Airway caliber measurements Figure 5 shows the changes in tracheal diameter during spontaneous breathing. As expected during inhalation, tracheal diameter decreased as SECS illitlttimittliitiItiiiiiitillitiiii Tracheal Diameter 10°“ W +10— cmHZO 0- W -10.... ‘ Tracheal Pressure Figure 5. Changes in the tracheal diameter (upper trace; upward deflection represents tracheal narrowing) and tracheal pressure (lower trace) during spontaneous breathing in an anesthetized cat. Tracheal diameter decreased concurrent with the decrease in tracheal pressure during inhalation and increased as tracheal pressure increased during exhalation. 69 850 800 750 — 700 650 600 550 500 450 400 350 300 250 200 150 100 50 Constriction (microns) —50 Time (minutes) Figure 6. Changes in the diameter of tracheal ring 4 of an anesthetized cat are shown as a function of time, as constant supramaximal voltage stimuli (24 V, 05 msec duration continuous stimulus) were applied bilaterally to vagal nerves at increasing frequencies of 05-16 Hz. 70 tracheal pressure decreased by about 25 cm H20. During exhalation, tracheal diameter increased as tracheal pressure increased by approximately 35 cm H20. The net change in tracheal diameter was in the order of 35 microns. The base-line outer diameter of tracheal ring 4 post-vagotomy was 0.9 i 0.03 cm (mean i SE). Bilateral vagal stimulation caused a decrease in the diameter of tracheal ring 4 that was a nonlinear function of stimulus frequency, as shown in Figure 6. Airway caliber returned to the pre-stimulation diameter when nerve stimulation ceased. The mean frequency-response curve from 15 animals is shown in Figure 7. Discussion Several different techniques have been used to measure airway smooth muscle activity. In vitro, smooth muscle can be studied directly in an organ bath (Woolcock and Permutt 1986). The results of these studies, however, frequently do not correlate with in vivo studies (Vincenc et al.1983, Armour et al. 1984, Woolcock and Permutt 1986). In vitro investigations generally fail to consider the smooth muscle in situ length-tension relationships, and the preload and afterload stresses on the muscle. These techniques necessarily deprive the muscle of its normal nerve and blood supplies and its relationship to neighboring tissues. Also, in vitro tests customarily report deve10ping isometric tension, whereas increases in airway smooth muscle tension in vivo result from isotonic bronchial and tracheal smooth muscle contractions. Measurements of airway resistance can be made on anesthetized animals and unanesthetized, cooperative subjects and the state of respiratory smooth muscle can be inferred (Woolcock and Permutt 1986). Indirect measurements 71 650 - 600 l . 550 i i 500 — 450 L ./.\s 400 l 350 L 300 L 250 1 200 - T 150 - e 100 l /l 50 - _50 . I . I . I . I . I . I . I . I . I . I -2 0 2 4 6 8 10 12 14 16 18 Change in Distance (microns) Stimulus Frequency (Hz) Figure 7. Mean frequency-response curve of 15 anesthetized cats. The base-line outer diameter of tracheal ring 4 post-vagotomy was 0.9 3; 0.03 cm (mean i SE) Vagal nerves were stimulated as described in Figure 6, and the change in tracheal diameter was recorded with the tracheal caliper. Data are mean i SE. 72 of smooth muscle contraction, such as airway resistance, provide information about the net effects of airway narrowing along respiratory passages and cannot determine the specific sites of airway narrowing nor differentiate smooth muscle contraction from airway wall edema or mucus accumulation (Hyatt and Wilcox 1961, Macklem and Mead 1967, Szereda-Przestaszewska and Widdicombe 1973). A force transducer attached directly to a tracheal (Brown et al. 1980, Mitchell et al. 1985, Baker and Don 1987) or bronchial (Lef f and Munoz 1981) test site provides a more direct method for studying respiratory smooth muscle in vivo in anesthetized animals. This technique targets a specific airway segment in situ but provides information only about isometric smooth muscle contraction. The technique also tends to stretch the airway smooth muscle beyond normal physiologic lengths in order to obtain optimal contraction. Normal anatomic relationships are disrupted when the study segment is transected proximally and distally and incised on midline for stabilization and attachment to a transducer. During the experiment, the inner surface of the airway segment is directly exposed to air at room temperature and humidity, which can induce cooling and drying of the tissue. Other devices employing strain gage technology have been used to measure isometric force development in airway smooth muscle (Souhrada and Dickey 1976, Beinfield and Seifter 1980). The major difference between these devices and the instrument described in this report is the ability of this instrument to quantitate isotonic smooth muscle shortening in a specific part of the trachea. The ability to measure isotonic changes is important because in vivo airway narrowing is related primarily to airway smooth muscle shortening 73 (James et al. 1987). Data reported in Figure 4 indicate that response characteristics of the instrument are linear and free of hysteresis and that the instrument responds predictably to both compressive and extensive forces. This system allows measurement of tracheal caliber with minimal surgical intervention at the selected tissue site and permits the study of the airway smooth muscle at its normal length in the tissue. The bonded metallic foil grid resistance strain gages we use for our instrument are made from electrically conductive alloys rolled to a thin foil. The alloys are processed to optimize mechanical properties and temperature coefficient of resistance. A grid configuration for the strain sensitive element is used to allow higher values of gage resistance while maintaining short gage lengths. The foil strain gages used on this device had a 63 x 12.8 mm carrier with grid dimensions of 2.8 x 6 mm. The instrument we describe is small and inexpensive to construct. This study demonstrates that this instrument can be used to quantify dimensional changes in a selected tracheal ring during tidal breathing and in response to electrical stimulation of the vagus nerves. As expected, tracheal diameter decreased concurrent with the decrease in tracheal pressure during inhalation and increased as tracheal pressure increased during exhalation. These changes in diameter probably result from passive variations in tracheal transmural pressure. Vagal stimulation predictably decreased tracheal diameter as the frequency of stimulation increased. CHAPTER 3 Cholinergic Reactivity of Tracheal Smooth Muscle Following Infection with Feline Herpesvirus-I Introduction Acute upper respiratory viral infections in humans can increase airway reactivity in persons with or without asthma (Empey et al.1976, Little et 31.1978, Aquilina et al. 1980). Viral respiratory infections may also initiate the development of asthma in normal individuals or exacerbate attacks in asthmatic subjects (Little et al. 1978, Halperin et al. 1985). Because the causative mechanisms for virus-provoked airway hyperreactivity are unknown, I have used an animal model to investigate respiratory viral infections and their effect on airway function. Feline rhinotracheitis, designated herpesvirus-I, causes naturally occurring upper respiratory infection, and is of a single antigenic type specific to the cat (Hoover et al. 1970, Povey 1979). This chapter describes the study of tracheal and intrathoracic airway responses in cats acutely infected with feline herpesvirus-I. Uncomplicated viral respiratory infections in humans can cause airway hyperreactivity in response to nonspecific stimuli such as inhaled histamine or citric acid. Because inhalation of atropine sulfate both reverses and largely prevents virus-provoked airway hyperreactivity, it has been suggested that this 74 75 airway constriction is mediated by post-ganglionic, cholinergic nerves that innervate airway smooth muscle (Empey et al. 1976). However, atropine is a nonselective muscarinic blocker and can inhibit both prejunCtional receptors on nerves and postjunctional receptors on airway smooth muscle and mucus secreting glands (Fryer and Maclagan 1987) Therefore, in cats acutely infected with herpesvirus-I, I measured airway responses to muscarinic receptor stimulation that was produced by two different techniques. Exogenous acetylcholine was delivered intra-arterially at tracheal ring 4 to stimulate postsynaptic muscarinic receptors on the effector muscle. Secondly, receptors were activated presynaptically by vagus nerve stimulation. The vagus nerves were transected at the angle of the mandible to eliminate central nervous system influences on airways. These two types of stimulation were repeated following muscarinic receptor blockade with atropine. Experimental protocol Base-line measurements included pre- and post-vagotomy tracheal ring 4 diameter, RD and Cdyn. A noncumulative ACh dose response and cumulative frequency response curve to bilateral vagal stimulation were generated in each cat in random order. Acetylcholine was administered by bolus injection into the right common carotid artery with the catheter tip at the level of tracheal ring 4. Each cat received local intra-arterial (ia) infusions of 10‘s, 10”, 10", 10‘s, and 10“ molar ACh. The volume (ml) of each ACh concentration injected was 0.1 x body weight (kg). Injections were at 10-minute intervals. Mean arterial pressure, micron change in the diameter of tracheal ring 4, RD and Cdyn were measured at each ACh dose. 76 In pilot studies (11 = 7 cats), the voltage-response characteristics of the cervical tracheal smooth muscle to electrical stimulation of the vagus nerves were determined to identify supramaximal stimulation parameters. Cumulative voltage-response curves were generated by stimulating the nerves (24 Hz, 05 msec duration, 10 s continuous stimulation) at increasing voltages of 2 to 68 V. In the current study, constant supramaximal voltage stimuli (24 V, 05 msec duration stimulus) were applied bilaterally to vagal nerves. Frequency of stimulation was increased progressively from 05 Hz to 16 Hz. The same dependent variables were recorded as described for the ACh dose response. After the completion of the two dose-response curves, atropine was given (2 mg/kg iv) Fifteen minutes later, the ACh dose response and the frequency response were repeated in random order. The cats were euthanized with an iv overdose of sodium pentobarbital. Sections of major bronchi, peripheral lung, and cervical tracheal rings were fixed in 10% phosphate-buffered formalin. Sections of fixed tissues were embedded in paraffin, stained with hematoxylin and eosin (H & E) and trichome and examined by light microscopy. Stall-Sues Single factor randomized design analysis of variance was used to compare the outer diameter of tracheal ring 4 in control and infected cats. Single factor repeated measures analysis of variance with Tukey’s test of multiple comparisons was used to compare a stimulus-response curve in one group of cats. Split-plot analysis of variance was used to compare the stimulus-response curves in control and infected cats pre- and post-atropine. Student’s t test for 77 independent samples was used to compare pre- and post-atropine data and the micron change in diameter of tracheal ring 4 of control or infected cats at a specific frequency or ACh dose (Steel and Torrie 1980). Log transformation was used for data that were not normally distributed. All values are reported as mean i SE. Differences were considered significant when P < 0.05. Results Clinical signs and histopathology Clinical signs detected in virus-infected cats were similar to those reported previously (Burgener and Maes 1988), and included sneezing, conjunctivitis, oculonasal discharge, depression, and ptyalism. All cats inoculated with herpesvirus and studied at either PID3 or PID6 displayed one or more of these signs (Table 1). Control cats were free of any signs of respiratory tract infection. There were multifocal areas of epithelial cell erosion of mild to moderate severity in the trachea of most cats infected with herpesvirus-I. The erosive tracheobronchitis was accompanied by focal lymphocytic and plasma cell aggregates in the lamina propria and submucosa with few neutrophils and minimal inflammation in any section. Focal vacuolar epithelial cell degeneration was detected in some virus-infected cats. No changes in the smooth muscle were detected. Sections of lung were within normal limits. Tracheal sections in control animals varied from completely normal to a few focal sites of minimal epithelial erosion that were not accompanied by edema or inflammation, smooth muscle changes, or necrosis. Multifocal erosive tracheobronchitis of mild to moderate severity could be found in some control 78 TABLE 1. Virus isolation, quantitation, and clinical signs in virus-infected and control cats. Virus Quantitation (T Cleo/ml) Lung__ Mainstem Cat Number CS Oral Nasal Conjunctiva Trachea Bronchi PIDa (infected) 1 (+) 6.7 x103 6.7 x103 2.1 x103 2.1 x102 2.1 x102 (-) 2 (+) 2.1 x103 6.7 x103 2.1 x105 2.1 x103 2.1 x105 (-) 3 (+) 2.1 x103 2.1 x10‘ 2.1 x105 6.7 x101 6.7 x10? (-) 4 (+) 6.7 x102 6.7 x101 2.1 x10‘5 2.1 x105 6.7 x103 (-) 5 (+) 2.1 x103 2.1 x103 2.1 x105 2.1 x10’5 6.7 x105 (-) PIDS (control) 6 (-) (-) H (-i (-) (-) (-) PIDS (infected) 7 (+) 6.7 x 103 2.1 x 107 2.1 x 105 (-) (-) (-) 8 (+) 2.1 x 103 2.1 x 105 6.7 x 10" Q (-) (-) 9 (+) 2.1 x 103 2.1 x 10‘ 6.7 x 10‘ (-) (.) (-) PIDB (control) 10 (-) (-) (-) (-) (-) (-) (-) PIDS = post-inoculation day 3; PIDB = post-inoculation day 6; CS = clinical signs post- inoculation; (+) = present; (-) = absent. Virus quantitation results are reported as TCleo/ml swab extract (the concentration required to produce infection in 50% of Crandell-Rees feline kidney cells). herpesvirus-l at the three pre-inoculation isolation sites. All cats were negative for 79 cats without herpesvirus infection, making differentiation between the two groups difficult. Herpesvirus-induced inclusion bodies could not be conclusively demonstrated in the airways of either group of cats. Virus isolation and quantitation Oral, conjunctival, and intranasal swabs collected at PID3 or PID6 tested positive for herpesvirus-I in all infected cats and all swabs tested negative in the sham-infected control cats. Virus quantitation was completed in 10 cats and was calculated as the TCIDSO/ml swab extract. Herpesvirus was isolated and quantitated at the three sites of inoculation (conjunctiva, nasal and oral mucus membranes) as well as the cervical trachea and mainstem bronchi on day 3 in infected cats (Table 1). Virus was also present at the three sites of inoculation on day 6 in infected cats, but virus was not isolated from the trachea or bronchi. Virus was not detected in the peripheral lung in any of the cats. Two control cats were maintained under the same conditions as the infected cats. One cat was tested at day 3 and one cat tested at day 6 following sham-infection. These two control cats were virus-negative at all six sites (Table 1). Tracheal ring 4 There was no significant difference in the mean base-line outer diameter of tracheal ring 4 pre—vagotomy in PID3 or PID6 cats infected with herpesvirus- 1 compared to control cats (0.9 i 0.03 cm; 08 i 0.04 cm; 0.9 i 0.03 cm, respectively) Similarly, there was no significant difference in the outer diameter of tracheal ring 4 post-vagotomy in PID3 or PID6 infected cats and control cats (0.9 i 0.02 cm; 0.9 i 0.04; 0.9 j; 0.03 cm, respectively). Bilateral vagal stimulation caused a 80 progressive reduction in the diameter of tracheal ring 4 with increasing frequency of stimulation. At stimulus frequencies of 4, 8, 12, and 16 Hz, the constriction was significantly greater in PID3 infected cats compared to control cats (Figure 8; P < 0.05) By PID6, the tracheal response was not significantly different from that of control cats (Figure 8). The tracheal response to vagal stimulation was eliminated by atropine (Figure 8). Intra-arterial ACh decreased the diameter of tracheal ring 4, but there was no significant difference in control or infected cats (Figure 9). Atropine eliminated the tracheal response to ACh in all groups of cats. Intrathoracic airways Pulmonary resistance and Cdyn of control and infected cats pre- and post- atropine are shown in Table 2. Total pulmonary resistance increased with increasing frequency of vagal stimulation, but there was no difference between control and infected cats. Following iv atropine, the rise in RL was eliminated in both groups of cats. There was no significant change in RL with increasing doses of intra-arterial ACh pre- or post-atropine in control and infected cats. Cdyn tended to decrease with increasing frequency of vagal stimulation and increasing ACh doses, but there was no difference in control and infected cats. Discuss'on The effect of acute infection with feline herpesvirus-I on airway responsiveness to cholinergic stimulation was studied in the cranial cervical trachea (tracheal ring 4) and In the intrathoracic airways (RL and Cdyn). Tracheal response at the fourth ring was studied because herpesvirus-I can be 600 550 ”‘11:? 500 ,.. * .\ o 450 /*./ g L. . o 400 , 0H E 350 i v 300 /4 Cl 0 250 or-I '8 200 T 0—0 control '5'. 150 l 8 0—0 infected PID3 if: T/ /1 a—A infected PID6 C: 100 O/ . o A—A post—atropine /J_ o S- —A A A A A _50 . A l . I . l m I A J A l . l . l A o a 4 6 8 ' 10 , 12 14 16 18 Stimulus Frequency (Hz) Figure 8. Mean frequency-response curves of control (n = 12), virus-infected PID3 (n= 15), and PID6 (n = 5) cats before and after atropine administration. Vagal nerves were stimulated as described in Figure 6, and the decrease in tracheal diameter was recorded with the caliper shown in Figure 3. Tracheal constriction was significantly greater at stimulation frequencies of 4, 8, 12, and 16 Hz in virus-infected cats studied at PID3 compared to control cats. There was no significant difference between control cats and virus-infected PID6 cats. Increasing tracheal constriction with increasing frequencies of stimulation was eliminated in both control and infected cats after atropine administration. Data from all 3 groups of cats after atropine is represented by the closed triangles. Values are means 1 SE. *P < 0.05 compared with control cats. 82 600 ' r”? 500 L 0—0 control g: ‘ C 0—0 infected PID3 S 400 :- A—A infected PID6 a A—A post—atropine I v 300 »- 0 :1 l- O - . 3 200 T i l O _ O 'C - . A a; 100 - /A/1 U 0 - I———-. h A A A r _100 b l m I I . J I LR —8 —7 —6 —5 —4 ACh Concentration (log M x wt kg) Figure 9. Mean ACh dose-response curves of tracheal ring 4 in control (n = 12), virus-infected PID3 (n = 15), and PID6 (n = 5) cats before and after atropine administration. Increasing doses of ACh were injected intra-arterially every 10 minutes and produced increasing tracheal constriction. There was no significant difference between control and virus-infected cats. The response to ACh was eliminated in all 3 groups of cats after iv atropine (closed triangles). Values are means i SE. 83 TABLE 2. Pulmonary mechanics testing of virus-infected and control cats pre- and post-atropine Control n=12 PID3 n=15 PIDG n=5 Total Pulmonary Resistance (cm H20/L/sec) Nerve Stimulation (Hz) Pre Post Pre Post Pre Post X SE X SE X SE X SE X SE X SE Base 18.0 1.23 18.0 1.40 19.0 1.10 8.1 1.03 18.6 0.79 18.0 1.32 0.5 17.4 1.36 18.2 1.41 19.2 1.11 17.9 0.99 18.8 0.84 17.8 1.26 1.0 18.4 1.26 18.2 1.39 20.1 1.11 18.0 0.99 19.6 0.83 17.7 1.35 2.0 19.3 1.48 18.2 1.58 20.6 1.16 17.8 0.99 20.0 1.01 17.4 1.19 4.0 22.4 2.70 17.8 1.66 22.7 1.40 17.9 0.94 21.6 0.72 17.6 1.44 8.0 250* 3.59 18.0 1.38 25.0 2.24 17.8 0.95 22.9 0.82 17.8 1.13 12.0 257* 3.71 17.9 1.41 26.1 2.89 17.7* 0.96 22.2 3.22 17.6 1.16 16.0 265* 4.37 17.8 1.52 30.7* 7.10 17.7* 0.96 259* 3.19 17.9 1.14 ACh (M) Base 20.9 2.63 19.0 1.76 21.2 1.51 17.8 1.02 21.1 1.80 20.0 2.48 LacR 20.5 2.22 19.1 1.74 21.4 1.46 17.7 1.02 21.2 1.73 20.2 2.48 10"3 20.7 2.27 18.7 1.64 20.7 1.41 18.2 1.11 21.3 1.75 19.5 2.07 10'7 20.1 2.08 18.5 1.50 20.0 1.22 18.0 1.05 20.5 1.61 19.0 1.85 10“3 18.8 1.37 18.2 1.78 19.8 1.26 17.6 1.01 20.2 0.52 18.2 1.71 10* 18.5 1.28 17.9 1.69 19.1 1.17 17.3 0.92 19.1 0.75 18.5 1.34 10“ 19.0 1.26 19.0 1.69 20.2 1.34 16.9 0.86 18.9 0.72 17.9 1.39 Dynamic Compliance (ml/cm HZO/kg) WM Pre Post Pre Post Pre Post Base 2.9 0.21 2.8 0.25 2.8 0.14 2.8 0.17 3.7 0.52 3.5 0.53 0.5 2.8 0.21 2.8 0.25 2.8 0.14 2.8 0.17 3.6 0.52 3.5 0.53 1.0 2.8 0.21 2.8 0.24 2.7 0.13 2.8 0.16 3.6 0.50 3.5 0.53 2.0 2.8 0.20 2.8 0.25 2.7 0.14 2.8 0.17 3.6 0.50 3.5 0.53 4.0 2.7 0.18 2.8 0.24 2.7 0.14 2.8 0.17 3.5 0.48 3.5 0.55 8.0 2.6 0.18 2.8 0.24 2.7 0.14 2.8 0.17 3.4 0.49 3.5 0.53 12.0 2.6 0.18 2.8 0.24 2.7 0.15 2.8 0.18 3.4 0.50 3.5 0.52 16.0 2.6 0.19 2.8 0.25 2.6 0.16 2.8 0.16 3.4 0.53 3.5 0.51 LCM Base 2.9 0.26 2.9 0.27 2.7 0.18 2.8 0.18 3.4 0.58 3.7 0.68 LacR 2.9 0.26 2.8 0.27 2.7 0.19 2.8 0.18 3.4 0.56 3.7 0.66 10‘ 2.9 0.24 2.8 0.25 2.7 0.16 2.7 0.17 3.3 0.52 3.6 0.64 10'7 2.8 0.24 2.8 0.25 2.6 0.16 2.7 0.17 3.3 0.50 3.5 0.63 1043 2.9 0.26 3.0 0.35 2.6 0.17 2.6 0.16 3.2 0.28 3.5 0.62 10‘5 2.8 0.24 2.7 0.24 2.6 0.16 2.6 0.15 3.4 0.43 3.4 0.65 10“ 2.7 0.23 2.6 0.26 2.5 0.19 2.6 0.14 3.3 0.42 3.4 0.62 PID3 = post-inoculation day 3; PIDS = post-inoculation day 6; Pre = pro-atropine; Post = post- atropine; LacR = Lactated Ringer’s; * Significantly different from base-line values (P < 0.05). 84 recovered in larger amounts from the upper trachea than from the lower trachea (Gaskell and Povey 1979). Although herpesvirus-I infects primarily the upper respiratory tract, intrathoracic airway responses to cholinergic stimulation as measured by RL and C(1),“ were studied because upper airway viral infection can cause heightened lower airway reactivity in humans (National Heart, Lung, Blood Institutes Workshop, 1988). The pathophysiologic interrelationship between the upper and lower respiratory tracts is uncertain, but nasal pulmonary reflexes could produce smooth muscle contraction and increased release of mediators, or there could be subclinical infection with loss of a "relaxing" factor produced by epithelial cells of the lower respiratory tract. In previous studies, viral effects on airway reactivity have been eliminated by atropine, which suggests the involvement of cholinergic pathways (Empey et al. 1976). Because atropine does not differentiate enhanced muscle responsiveness from altered neural activity, airway constriction was produced in control and infected cats by two methods. Bilateral vagal nerve stimulation and intra-arterial ACh were used to separate the muscarinic responses of airway smooth muscle resulting from pre- and postsynaptic stimulation. The arterial blood supply to the cranial trachea is derived from the thyroid arteries that branch directly from the common carotid artery in the cranial cervical region (Nicholas and Swingle 1925). The arterial blood supply to the distal trachea is less consistent and can originate from multiple small branches of the bronchial artery (Nelson 1985). Consistent retrograde cannulation of the thyroid arteries for local injection of ACh proved difficult during this study because of the small size of the vessels. In order to deliver ACh at the level of tracheal ring 4, a catheter was placed into the right 85 common carotid artery with its tip ending at the arterial branches of the thyroid artery (Figure 2). The common carotid artery was ligated just cranial to the cranial thyroid artery. The left common carotid artery was undisturbed to assure maintenance of blood supply to the study segment. Blood could be withdrawn and ACh could be injected through the catheter without resistance in each cat throughout the studies. During pilot investigations, Evans blue dye was also injected through the carotid catheter at the end of the experiment to check the arterial blood supply to the cranial cervical trachea. Infusion of Evans blue uniformly stained tracheal ring 4. Local ia injection produced progressive constriction of tracheal ring 4 with increasing doses of ACh. The maximal constriction of tracheal ring 4 to ACh injection was not determined, because doses greater than those shown in Figure 9 produced severe hypotension and death in some cats. Marked vasodilation with a fall in blood pressure has long been recognized in anesthetized animals, even with small doses (1 to 5 jig/kg) of ACh (Hunt 1918, Koelle1975). Previous studies have shown that exogenous ACh must reach the pulmonary and bronchial circulation in order to produce airway constriction (Colebatch et al. 1966). A change in tracheal diameter without a change in RL and C(1)." were detected in my study because of the site of injection, the small volumes of ACh that I was injecting (less than 05 ml), and the rapid hydrolysis of the drug by local and circulating esterases. The purpose of this study was to investigate airway responses to cholinergic stimulation during the early stages of infection with herpesvirus-I. My decision to study airway responsiveness at PID3 and PID6 was based on previous studies in which cats were inoculated similarly to the cats in my 86 investigation by direct application of virus to oral, nasal, and conjunctival mucosa with the C-27 strain of herpesvirus-1(Burgener and Maes 1988). In these studies, signs of upper respiratory tract disease were apparent in 7/10 cats by PID3 and in 10/10 cats by PID6. Virus was recovered from 10/10 cats by PID3, but virus quantitation was not performed (Burgener and Maes 1988). Herpesvirus-I increased responsiveness to vagal stimulation in the cat trachea but not in the intrathoracic airways. There are several possible reasons for this difference in airway response. One possibility is that the customized electromechanical caliper attached to the outer diameter of tracheal ring 4 provides a very sensitive method to quantify isotonic forces developed by contracting smooth muscle in the upper trachea in situ, and is unaffected by airway wall edema and mucus accumulation. This technique allowed the smooth muscle to be studied with minimal surgical intervention without transecting tracheal rings and at physiologic length without stretching to obtain maximal contraction. The caliper system provided a more specific measurement of smooth muscle shortening than functional measurements such as RL and Cdyn, which produce a weighted average of lung response without information as to specific site or cause of airway narrowing. Another possible explanation for the difference in tracheal and lower airway responses is the site of viral infection. Virus isolation and pathologic studies following acute infection with feline herpesvirus-I suggest that this virus can be isolated most frequently and in greatest amounts from the nasal cavity, oro- and nasopharynx, and consistently but in smaller amounts from the conjunctiva, regional lymph nodes, and upper trachea (Gaskell and Povey 1979). The virus has been isolated inconsistently from salivary glands, mid- and lower 87 trachea, and lung lobes. When virus was quantitated in this study at PID3, herpesvirus was found associated with the tracheal study segment and mainstem bronchi, but virus was not found in swabs taken distal to the mainstem bronchi. By PID6, virus was no longer isolated from the trachea and bronchi but was still present at the sites of inoculation. Although isolation and quantitation of virus in the mainstem bronchi and peripheral lung were performed in a limited number of cats (11 = 10), results were consistent between cats at both PID3 and PID6. The absence of herpesvirus distal to the mainstem bronchi may account for the lack of difference in RL and Cdyn between PID3-infected and control cats. Hyperresponsiveness to vagal stimulation was detected in the trachea but not in the pulmonary airways. Furthermore at PID6, the trachea was no longer hyperresponsive and virus could not be isolated. Therefore, my physiologic studies at PID3 and PID6 largely parallel the virus isolation findings. The histopathological findings of erosive tracheobronchitis with cellular infiltration in the mucosa, submucosa, and lamina propria detected in virus- infected cats are consistent with the cytologic features detected in previous studies of herpesvirus infection in conventional and germ-free cats (Hoover et a1. 1970, Gaskell and Povey 1979) Early in the course of disease, focal necrosis is limited primarily to the nasal mucosa, respiratory sinuses, and conjunctiva, with neutrophilic infiltration occurring presumably as a result of secondary bacterial infection (Hoover et al. 1970). Experimental infection with herpesvirus-I in germ-free kittens can be surprisingly severe, but even so, inclusion bodies are found infrequently in the larynx and trachea, and never more than one-third of the tracheal epithelium in any section is destroyed (Hoover et al. 1970). 88 The increased tracheal response to vagal stimulation in infected cats studied at PID3 could be explained by an increased muscle response to endogenous ACh released from postganglionic fibers or by presynaptic events that increase the response to a given electrical stimulus. The first possibility is unlikely, because tracheal response to exogenous ACh was not significantly different between infected and control cats Presynaptic electrical nerve stimulation did result in tracheal ring 4 contraction that was significantly greater in virus-infected cats than in control cats. The enhanced contraction of tracheal ring 4 was completely blocked by the cholinergic antagonist atropine. Based on these data, I reasoned that at PID3, herpesvirus-I causes augmented tracheal ring 4 constriction in response to vagal nerve stimulation by a cholinergic, presynaptic mechanism that is absent at PID6 when virus is no longer detectable in the cervical trachea. I further theorized that the increased response to electrical stimulation in the absence of an increased response to exogenous ACh could be caused by co-release of a second mediator that enhances the release of ACh from cholinergic nerve terminals or the muscle’s response to Ach. Various mediators, including neuropeptides such as SP, may modulate airway smooth muscle contraction and facilitate cholinergic neurotransmission (Sekizawa et al. 1987) Low concentrations of SP can cause an atropine-sensitive enhancement of the response of tracheal muscle to electrical field stimulation but no enhanced response to ACh. These observations are comparable to the findings of the present study. Substance-P is normally metabolized by several peptidases, but thus far only neutral endopeptidase (NEP) has been shown to modulate SP- induced effects in airways (Umeno et al.1989). Following viral infections in rats, 89 NEP activity does appear to be reduced in the trachea of infected animals compared to pathogen-free animals. In the present study, neuropeptides such as SP may have been released during vagal stimulation by antidromic activation of sensory nerves and the action of these mediators prolonged at PID3 due to decreased NEP activity. Although inflammation and epithelial damage appeared to be mild in virus-infected cats, N EP activity may have been reduced. The histopathological findings of erosive tracheobronchitis with cellular infiltration in the mucosa, submucosa, and lamina propria are consistent with the cytologic features detected in previous studies of herpesvirus infection in conventional and germ-free cats (Hoover et al. 1970, Gaskell and Povey 1979). Inhibitory muscarinic autoreceptors (M2) have been described in association with parasympathetic nerves in normal feline airways (Blaber et al. 1985), and can inhibit ACh release during vagal stimulation of cat, guinea pig, and human airways (Blaber et a1. 1985, Minette and Barnes 1988). The potential role of muscarinic autoreceptors in virus-induced airway hyperresponsiveness is speculative, but down regulation of inhibitory receptors or up regulation of facilitating receptors during acute viral infection could result in increased ACh release and enhanced airway constriction to electrically or environmentally induced contraction. CHAPTER 4 Muscarinic M2 Receptors Modulate the Increase in Pulmonary Resistance and Dynamic Compliance but not the Change in Tracheal Diameter in Cats with Tracheal Hyperresponsiveness to Vagus Nerve Stimulation Introduction The parasympathetic nervous system regulates airway caliber through the vagus nerve, which releases a cholinergic neurotransmitter that binds to muscarinic receptors in airway smooth muscle (Barnes 1989). There are at least five different muscarinic receptor subtypes as confirmed through expression of cloned genes in frog and mammalian cells (Gross and Barnes 1988, Barnes 1989, Bonner 1989). In the airway, there are facilitatory receptors (M1) in parasympathetic ganglia, inhibitory autoreceptors (M2) on cholinergic nerves, and M3 receptors on airway smooth muscle and mucus secreting glands (Barlow et al. 1976, Birdsall and Hulme1985, Bloom et al. 1987c, Minette and Barnes 1988, Barnes 1989). While investigating the effect of acute infection with herpesvirus-I on airway responses, I detected a group of cats with tracheal hyperreactivity to vagal stimulation independent of upper respiratory infection. These cats were all adult, mixed breed cats of either gender, and the reason for the tracheal 90 91 hyperresponsiveness was unknown. The external diameter of the cervical trachea prior to vagal stimulation was the same in hyperresponsive cats and cats without tracheal hyperresponsiveness, indicating that the hyperreactivity was not due to airways that were narrower initially. The tracheal hyperresponsive cats were hyperresponsive only to presynaptic stimulation of the vagal nerves, and were not hyperresponsive to exogenous ACh delivered to postsynaptic muscarinic receptors on the airway smooth muscle. Knowing that prejunctional neuronal muscarinic receptors can decrease the response of airway smooth muscle to parasympathetic nerve stimulation by inhibiting ACh release in cats (Blaber et al. 1985), I hypothesized that dysfunction of the prejunctional inhibitory (Mg receptors could be responsible for the exaggerated tracheal response to vagal stimulation detected in some cats. I further hypothesized that the same cats would exhibit intrathoracic airway hyperresponsiveness during vagal stimulation. I also proposed that airway smooth muscle contraction induced directly by exogenous acetylcholine would be unaffected by blockade of the prejunctional inhibitory receptors. To test these hypotheses, I measured changes in airway caliber induced by bilateral vagal stimulation and exogenous ACh in control cats and in cats with tracheal hyperresponsiveness. Tests were made before and after cats received graded iv doses of the M2 receptor antagonist gallamine. I used gallamine because it is an antagonist for the M2 neuronal receptor at doses which have minimal postsynaptic M3 receptor action (Blaber et al. 1985). 92 Animal groups and experimental protocol Cats were divided into two groups, control (n = 11) and tracheal hyperresponsive (TH, 11 =16) based on their tracheal response to bilateral vagal stimulation (24 V, 05 msec, 4 Hz) Cats were placed in the TH group if their tracheal constriction was greater than 2 SE above the mean for twelve healthy, respiratory virus-free control cats that had been studied previously (Chapter 3) A third group of cats (11 = 11) served as time controls independent of their tracheal response to nerve stimulation. Airway smooth muscle contraction was produced using bilateral stimulation of the distal ends of the cut vagus nerves (24 V, 05 msec, 4 Hz) and by ia injection of 10‘1 M ACh, the maximal bolus of ACh that could be injected consistently without causing severe hypotension and death in some cats (Killingsworth et 31.1990). The volume (ml) of each ACh concentration injected was 0.1 x body weight (kg). Each cat received local ia infusions of the vehicle (lactated Ringer’s solution) as a control injection prior to receiving ACh. The same volume (approximately 0.5 ml) of vehicle and ACh was injected into the right common carotid artery with the catheter tip at the level of tracheal ring 4 (Figure 2). Following vagotomy, initial base-line measurements were made of tracheal ring 4 diameter, Ru and Cdyn. Secondly, with a 10-minute stabilization period between each stimulation, airway responses to bilateral vagal stimulation, vehicle, and ACh injections were recorded before giving cumulative doses of the M2 receptor antagonist gallamine. At 10-minute intervals after each dose of gallamine (0.1,1.0, 10.0 mg/kg iv), bilateral vagal nerve stimulation was repeated and the changes in the diameter of tracheal ring 4, RD and Cdyn 93 were recorded. The second vehicle and ACh injections were given following the last dose of gallamine and nerve stimulation to determine the effect of gallamine on ACh-induced airway constriction. Time controls received the same treatments before and after three injections of gallamine vehicle (0.9% sodium chloride) to determine the effect of repeated vagal stimulation, vehicle, and ACh injections. Cats were euthanized with an overdose of iv sodium pentobarbital. 5mm. All values are reported as mean i SE. Single factor randomized design analysis of variance was used to compare the outer diameter of tracheal ring 4, R1, and Cdyn in control, time control, and TH cats at base-line. Single factor repeated measures analysis of variance with Tukey’s test of multiple comparisons was used to determine the effect of gallamine within a group of cats. Split-plot analysis of variance compared the stimulus-response curves in control and TH cats, and Student’s L test for independent samples compared the two cat groups at a specific gallamine dose (Steel and Torrie 1980). Spearman rank order (rho) analysis was used to correlate the change in tracheal ring 4 diameter and RL during vagal stimulation before and after gallamine treatment. Differences were considered significant when P < 0.05. Results The outer diameter of tracheal ring 4 at base-line did not differ among control, time control, and TH cats (0.88 i 0.02 cm; 0.90 :t 0.02 cm; 0.94 i 0.03 cm, respectively). Control, time control, and TH cats also had similar base-line 94 measurements of RL (17.4 i 1.28 cm HZO/L/sec;17.7 i 1.15 cm H20/L/sec;18.9 i 1.80 cm HZO/L/sec, respectively) and Cdyn (2.6 i 0.22 ml/cm HZO/kg; 2.7 i 0.17 ml/cm HZO/kg; 2.6 i 0.17 ml/cm HZO/kg, respectively). Time control cats Because RL and Cdym measurements tended to vary between stimulations, the increase in RL and the decrease in Cdyn during stimulation are reported as the change from pre-stimulation measurements (ARD- ACdyn, respectively). Vagal stimulation increased RL and decreased Cdyn and the diameter of tracheal ring 4 in every cat. The ARL, AC and tracheal ring 4 responses did not change dyn’ significantly with repeated vagal stimulations (Table 3). Vehicle injections prior to ACh produced no change in RD Cdyn, or the diameter of tracheal ring 4. Exogenous ACh injection decreased the diameter of tracheal ring 4 in every cat but did not alter RL or Cdyn significantly (Table 4). In the absence of gallamine, the first and second ACh injections produced the same change in RU Cdyn, and the diameter of tracheal ring 4 (Table 4). Effect of gallamine on airway responses during vagal stimulation in control and TH cats Tracheal constriction produced by vagal stimulation was significantly greater in TH cats than control cats at every gallamine dose (P < 0.01), but there was no significant effect of gallamine in either group of cats (Figure 10). Gallamine significantly increased the ARL induced by vagal stimulation in both groups of cats. The ARL at the highest dose of gallamine (10 mg/kg) was significantly greater than the ARL measured prior to gallamine administration and following 01 mg/kg gallamine in both groups of cats (P < 0.01). The change 95 TABLE 3. Effect of repeated vagal nerve stimulation on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats Saline Injection Vagal Vagal Vagal Vagal Stimulation 1 Stimulation 2 Stimulation 3 Stimulation 4 Change in 406.3 3; 131.5 405.2 1 136.1 432.6 i 163.1 445.7 i 134.4 Tracheal Ring 4 diameter (microns) ARL 2.5 i: 0.79 2.6 :1; 0.49 2.7 i 0.73 3.6 i 1.15 (cm H20/L/sec) AC 0.12 i 0.02 0.12 ;I-_ 0.03 0.15 i 0.03 0.21 1:. 0.06 (mliizm H20/kg) Values are mean i SE; n = 11 cats. Change in tracheal ring 4 diameter = decrease in external diameter from base-line (base-line = 0.88 i 0.02 cm); ARL = change in pulmonary resistance from base-line (base-line = 17.7 i 1.15 cm H20/L/sec); AC,” = change in dynamic compliance from base-line (base-line = 2.7 -_i-_ 0.17 ml/cm HZO/kg). TABLE 4. Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats Vehicle ACh ACh Injection Injection 1 Injection 2 Change in Tracheal NR 311.8 _-l_-_ 440* 280.3 _-I_~_ 70.7* Ring 4 diameter (microns) ARL 0.07 _+_- 0.19 0.59;); 0.36 0.68;: 0.31 (cm HZO/L/sec) AC 0.05 i: 0.05 0.05 i 0.01 0.10 i 0.06 (mlicysin HZO/kg) Values are mean 1: SE; n = 11 cats. Change in tracheal ring 4 diameter = decrease in external diameter from base-line (base-line = 0.88 i 0.02 cm); ARL = change in pulmonary resistance from base-line (base-line = 17.7 i 1.15 cm HZO/L/sec); ACdyn = change in dynamic compliance from base-line (base-line = 2.7 i 0.17 mI/cm HzO/kg): NR = no response. *P < 0.05 compared to base-line and vehicle injection. There was no significant difference in any of the variables measured between ACh injection 1 and ACh injection 2. F.1l|l| ||l.| ‘- 97 800 Tracheal Constriction (microns) 0.0 0.1 1.0 Gallamine (mg/kg iv) ‘9‘ Control +TH Figure 10. Effect of gallamine on tracheal ring 4 constriction in control (n = 11) and tracheal hyperresponsive (TH; n = 16) cats during bilateral vagal nerve stimulation (24 V, 05(msec, 4 Hz). Constriction prior to gallamine injection (0.0 mg/kg) and that with increasing doses of gallamine were quantitated with the caliper shown in Figure 3. Vagal stimulation decreased the external diameter of tracheal ring 4 in both groups of cats; constriction was significantly greater in the TH cats compared to control cats. Increasing doses of gallamine, however, had no effect on the amount of tracheal ring 4 constriction produced by repeated vagal stimulation in either group of cats. Base = external diameter of tracheal ring 4 prior to Stimulation (Control = 0.9 i 0.02 cm; TH = 0.9 i 0.03 cm) * Significantly different from base (P < 0.01) 1' Significantly more constriction in TH compared to control cats (P < 0.01). 98 in R1. produced by vagal nerve stimulation was greater at gallamine doses of 1.0 and 10.0 mg/kg in TH cats than in controls (P < 0.05; Figure 11). There was only a weak correlation between ARL and tracheal ring 4 response to vagal stimulation prior to gallamine administration (P = 0.07; Figure 12), but there was a significant correlation following gallamine blockade of M2 receptors (P = 0.03; Figure 13). The vagally induced change in C,13m was not different between the two groups at any dose of gallamine (Figure 14). However, there was a significant increase in ACdyn at 10 mg/kg gallamine compared to 0.0 and 01 mg/kg gallamine in the TH group. Effect of gallamine on airway responses to ACh in control and TH cats Vehicle injection did not cause significant changes in tracheal ring 4 diameter, ARU or ACdyn in any cat. The change in RL, Cdyn, and the outer diameter of tracheal ring 4 induced by ACh injection were similar prior to gallamine administration and after the highest dose of gallamine (Table 5). There were also no differences in any of the measured variables when the airway responses to ACh were compared between control and TH cats. Discussion I investigated the in vivo role of muscarinic inhibitory (M7) receptors in control of airway caliber by inducing airway constriction in cats before and after injection of the M2 receptor antagonist gallamine. Airway smooth muscle contraction produced by exogenous ACh and by bilateral vagal nerve stimulation was studied in the intra- and extrathoracic airways. Previous studies 14Pulmonary Resistance (cm water/L/sec) * r l 12— 10- 4- ,,#/r ..—-—-*"""f * 2—t—fl‘TTT—fl O 0.0 0.1 1.0 10.0 Gallamine (mg/kg iv) '4‘ Control +TH Figure 11. Changes in total pulmonary resistance from base-line (ARL) in control (n = 11) and tracheal hyperresponsive (n = 16) cats during bilateral vagal nerve stimulation (24 V, 0.5 msec, 4 Hz) prior to gallamine injection (0.0 mg/kg) and following increasing doses of iv gallamine. At a gallamine dose of 10 mg/kg, ARL in control cats Was significantly greater than before gallamine injection or at 0.1 mg/kg gallamine C“ P < 0.05) In TH cats, ARL at 10 mg/kg gallamine was significantly greater than at all other points on the dose-response curve (* P < 0.05). At 1.0 and 10.0 mg/kg gallamine, TH cats had a greater change in ARL than control cats (1 P < 0.05) 100 30 Pulmonary Resistance 25* Pre - gallamine Injection O 1 ' 1 1 l 1 J 0 5 1O 15 20 25 30 Tracheal Ring 4 Constriction Figure 12. Correlation of the change in pulmonary resistance (ARL) and the change in diameter of tracheal ring 4 in cats (11 = 27) during vagal stimulation (24 V, 0.05 msec, 4 Hz) prior to gallamine treatment. The Spearman test required ranking of raw data to determine the correlation coefficient for any pair of variables. There was no significant correlation between ARL and tracheal constriction (P = 0.07). 101 30 Pulmonary Resistance 25- 5 ‘ . ' Post - gallamine Injection 0 ' 1 1 1 1 1 1 0 5 10 15 20 25 30 Tracheal Ring 4 Constriction Figure 13. Correlation of the change in pulmonary resistance (AR,) and the change in diameter of tracheal ring 4 in cats (11 = 27) during vagal stimulation (24 V, 0.05 msec, 4 Hz) after gallamine treatment. The Spearman test required ranking of raw data to determine the correlation coefficient for any pair of variables. There was a Significant correlation between ARL and tracheal constriction following M2 receptor blockade (P = 0.03). 102 [Dynamic Compliance (ml/cm water/kg) 0.3 * 0.2 ~ / / / 0.1 — i7 0 0.0 10.0 0.1 1.0 Gallamine (mg/kg iv) 4‘ Control +TH Figure 14. Changes in dynamic compliance (ACdyn) from base-line in control (n = 11) and tracheal hyperresponsive (n = 16) cats during bilateral vagal nerve stimulation (24 V, 0.5 msec, 4 Hz) prior to gallamine injection (0.0 mg/kg) and following increasing doses of iv gallamine. At gallamine doses of 10 mg/kg, ACd in TH cats was significantly greater than ACdyn before gallamine injection or at 0.1 mg/kg gallamine (* P < 0.05). 103 TABLE 5. Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in control and hyperresponding cats before and after gallamine treatment Vehicle Pre-Gallamine Post-Gallamine Injection ACh Injection 1 ACh Injection 2 Change in Tracheal Ring 4 Diameter (microns) Controls NR 311.1 _-1_-_ 51 .4* 228.0 _-1; 54.5* TH NR 390.2 i 57.6* 304.9 :1; 59.2" ARL (cm H20/L/sec) Controls 0.14 i 0.16 0.53 i 0.25 0.62 i 0.24 TH 0.24: 0.16 0.29:1; 0.10 0.15 i 0.15 A0,,” (ml/cm HZO/kg) Controls 0.001 3; 0.008 0.07 :1: 0.02 0.06 _-1; 0.02 TH 0.015 _-1; 0.009 0.06 _-_1-_ 0.01 0.05 ;1-_ 0.02 Values are mean _-1-_ SE; Controls (n = 11); TH = tracheal hyperresponsive cats (n = 16); NR = no response; Change in tracheal ring 4 diameter = decrease in external diameter from base-line in control and TH cats (base-line = 0.9 i 0.02 cm; 0.9 ;1-_ 0.03, respectively): ARL = change in pulmonary resistance from base-line in control and TH cats (base-line = 17.4 ;1-_ 1.28 cm HZO/L/sec; 18.91 1.80 cm H20/L/sec, respectively); AC”, = change in dynamic compliance from base-line in control and TH cats (base-line = 2.6 i 0.22 ml/cm HZO/kg; 2.6 i 0.17 ml/cm H20/kg, respectively). *P < 0.05 compared to base-line and vehicle injection. There was no significant difference in any of the variables measured between ACh injection 1 and ACh injection 2. 104 showed that the tracheal and intrathoracic airway constriction produced by these two methods is completely blocked by the non-selective muscarinic receptor antagonist atropine (Killingsworth et a1. 1990). As has been reported by others (Blaber et al.1985, Beck et al.1987), the M2 antagonist gallamine enhanced the response of the intrathoracic airways to vagal stimulation. The ARL induced by vagal stimulation was significantly greater following administration of 10 mg/kg gallamine than pro-gallamine in both groups of cats. Furthermore, in the TH group, vagal stimulation caused no change in Cdyn pre-gallamine but caused a significant decrease in Cdyn following 10 mg/kg gallamine. Based on the measurements in time control cats, the above responses are due to gallamine and not simply a result of repeated nerve stimulation. My data indicate that M2 receptors may act to restrict airway narrowing, because the effects of vagal nerve stimulation in the intrathoracic airways were enhanced by increasing doses of gallamine. I did not demonstrate a similar role for M2 receptors in the cranial cervical trachea. The magnitude of tracheal constriction during vagal stimulation following graded doses of gallamine was the same as that produced before gallamine injection in both groups of cats. Failure to demonstrate an effect of gallamine could be due to lower numbers of M2 autoreceptors associated with the cat cranial cervical trachea compared to those in the intrathoracic airways. Autoradiographic localization techniques using a non- selective muscarinic antagonist have demonstrated a nonuniform distribution of muscarinic receptors in central and peripheral airways (Cheng and Townley 1982, Murlas et a1. 1982, Barnes et al. 1983). Specific radioligand binding studies could determine the density and the distribution of M2 inhibitory receptors in 105 the airways, and might demonstrate a higher density of muscarinic inhibitory receptors on intrathoracic cholinergic nerves compared to the cervical tracheal nerves, regardless of the large population of M1 or M3 receptors that may be present on tracheal smooth muscle, glands, and ganglia of cat airways. A higher density of M2 receptors in central airways, lower numbers in peripheral airways, and absence of M2 receptors in the cholinergic nerves of the trachea would explain our physiologic findings. Alternatively, M2 receptor activation may be frequency dependent (Blaber et al. 1985, Wessler 1989) and a frequency of 4 Hz may be too low to trigger autoinhibition of ACh release in the trachea. This is highly unlikely because I was able to demonstrate an effect of gallamine in intrathoracic airways at a stimulus frequency of 4 Hz. In the original design of the study, I wanted to compare the effects of gallamine on the tracheal response to vagal stimulation and locally administered ACh. Because I anticipated that M2 receptors are located at a presynaptic site, I reasoned that gallamine would have an effect on smooth muscle contraction induced by vagal stimulation but not on contraction caused by ACh. However, gallamine had no effect on the tracheal response to either vagal stimulation or exogenous ACh. As expected, local administration of ACh contracted the trachea but had no effect on RL or Cdyn, presumably because ACh is hydrolyzed before reaching the pulmonary circulation (Colebatch et al. 1966). As I have previously reported, cats that are hyperresponsive to vagal stimulation are not hyperresponsive to locally administered ACh. This suggests that the hyperresponsiveness is presynaptic in origin and led to my hypothesis that the tracheal hyperresponsiveness to vagal stimulation is due to dysfunction 106 of M2 inhibitory receptors. The results of the present study do not support this hypothesis, because I was unable to demonstrate increased tracheal constriction following gallamine in the control cats. There was only a weak correlation between tracheal constriction and the rise in RL during vagal stimulation prior to muscarinic inhibitory receptor blockade. There was also no significant difference in the ARL caused by nerve stimulation pre-gallamine in the two groups of cats. These observations suggest that cats with tracheal hyperresponsiveness do not have hyperresponsive intrathoracic airways. However, following gallamine administration, there was a significant relationship between tracheal constriction and the increase in RL and there was a significant difference in ARL between the two groups of cats. In addition, ACdyn increased significantly during vagal stimulation only in cats with tracheal hyperresponsiveness following M2 receptor blockade. These data suggest that muscarinic inhibitory receptors may play an important physiologic role in inhibiting endogenous ACh release and thus limiting intrathoracic airway constriction, particularly in cats with tracheal hyperresponsiveness. A similar modulatory function for M2 receptors in cat cervical trachea could not be demonstrated. CHAPTER 5 Muscarinic Inhibitory Receptors Do Not Limit Feline Tracheal Constriction during Vagal Stimulation In Vivo Introduction Use of specific muscarinic agonists and antagonists has identified three muscarinic receptor subtypes in the airways: facilitatory receptors (M1) identified in parasympathetic ganglia that are pirenzepine-sensitive, inhibitory autoreceptors (M2) that are blocked by gallamine and located on cholinergic nerves, and the classic muscarinic receptors (M3) that mediate smooth muscle contraction and mucus secretion in the airways (Barlow et al. 1976, Birdsall et al. 1985, Hammer et al. 1986, Bloom et al. 19873). An uneven distribution of muscarinic receptors from trachea to peripheral airways has been confirmed by autoradiography using nonselective radiolabelled antagonists (Cheng and Townley 1982, Barnes et al. 1983). In all species studied to date, the density of muscarinic receptors in peripheral lung is low compared to a high density found in central airways. The results of competitive binding experiments using selective muscarinic receptor antagonists are beginning to appear in the literature (Bloom et al. 1987b, Fryer and El- Fakahany 1990, Mak and Barnes 1990). These studies suggest that the distribution of the different muscarinic receptor subtypes varies within the 107 108 lungs and among species. For example, autoradiography revealed that muscarinic receptors are widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and in airway smooth muscle of large and small airways (Mak and Barnes 1990). In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not (Mak and Barnes 1990). The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3—subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the Mz-subtype present. In human bronchial submucosal glands, MI- and M3-subtypes appeared to coexist. In human alveolar walls, the muscarinic receptors were entirely of the Mz-subtype and their functional significance is unknown (Mak and Barnes 1990). Studies of rat airways have demonstrated that the dominant muscarinic receptor subtype is M2 (Fryer and El-Fakahany 1990). In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3. In summary, these studies demonstrate that the type of muscarinic receptor found at various levels of the lungs differs among animals, and the distribution of muscarinic receptor subtypes varies between the central airways and peripheral lungs. The correlation between in vitro binding assays and the physiological function of these receptor subtypes at different locations in the lungs remain to be determined. In a separate study, I measured tracheal ring 4 constriction, pulmonary resistance, and dynamic compliance during bilateral vagal stimulation at 4 Hz. 109 Nerve stimulation caused significant narrowing of the cervical trachea, but the magnitude of constriction was not altered by intravenous injection of the M2 receptor antagonist gallamine at doses up to 10 mg/kg. Although I was unable to demonstrate M2 receptors in vivo in cat trachea during cholinergic stimulation, I measured significant changes in intrathoracic airway caliber following M2 inhibitory receptor antagonism. The change in pulmonary resistance induced by vagal stimulation was augmented following gallamine administration. In addition, there was a significant correlation between the degree of tracheal constriction and the increase in pulmonary resistance after gallamine injection. Cats with exaggerated tracheal constriction during vagal stimulation also had an augmented decrease in dynamic compliance following gallamine treatment. These data demonstrate that M2 receptors can limit intrathoracic airway constriction in response to vagal stimulation, but the in vivo studies did not reveal a physiological inhibitory role for M2 receptors in the cervical trachea. Receptor activation is frequency dependent during nerve stimulation (Blaber et al. 1985, Wessler 1989). A frequency of 4 Hz may not induce autoinhibition of ACh release in the trachea because insufficient ACh may be released to activate M2 receptors. The purpose of the studies described in this chapter was to determine whether the function of M2 inhibitory receptors is frequency dependent, and, therefore, whether M2 receptors might limit tracheal constriction during vagal stimulation at frequencies greater than 4 Hz. I also examined whether there might be a difference in muscarinic autoreceptor function in cats with tracheal hyperresponsiveness to vagal stimulation compared to a control population of cats. As described in Chapter 4, a group 110 of adult, mixed breed cats had been recognized with tracheal hyperresponsiveness to presynaptic vagal stimulation. My working hypothesis was that these cats might be hyperresponsive because of dysfunctional neuronal M2 inhibitory receptors. Ani a rou s a d x im ntal rotocol Cats received gallamine (n = 10) or vehicle (0.9% sodium chloride) without gallamine (time controls, n= 5). I subdivided the cats receiving gallamine into two groups to investigate further the change in tracheal ring 4 diameter and the role that M2 receptors might play in limiting tracheal constriction. The cats were considered control (n = 4) or to exhibit tracheal hyperresponsiveness (TH; 11 = 6) based on the change in tracheal ring 4 diameter during bilateral vagal stimulation (24 V, 05 msec, 4 Hz). Cats were placed in the TH group if their tracheal constriction was greater than 2 SE above the mean for twelve healthy adult cats that I had studied previously. Airway smooth muscle contraction was produced using bilateral stimulation of the distal ends of the cut vagus nerves (24 V, 05 msec, 0.5-16 Hz) and by ia injection of 10“ M ACh. The volume (ml) of each ACh concentration injected was 0.1 x body weight (kg). Each cat received local ia infusions of the vehicle (lactated Ringer’s solution) as a control injection prior to receiving ACh. The same volume (in the order of 0.4 ml) of vehicle and ACh was injected into the right common carotid artery with the catheter tip at the level of tracheal ring 4 (Figure 2). Following vagotomy, initial base-line measurements were made of tracheal ring 4 diameter, RL, and Cdyn. The changes in tracheal ring 4 diameter, 111 RD and Cdyn were measured during vagal stimulation at increasing frequencies and with ia injections of vehicle and ACh prior to gallamine administration, allowing a 10-minute stabilization period between each stimulation. Gallamine (10.0 mg/kg iv) was injected, and ten minutes later, the frequency response and ACh injection were repeated in random order as described above. Changes in the same variables were recorded. The effect of repeated vagal stimulation, vehicle, and ACh injections was determined in time control cats before and after injection with gallamine vehicle. Cats were euthanized with an overdose of iv sodium pentobarbital. Statistics All values are reported as mean i SE. Single factor randomized design analysis of variance compared the outer diameter of tracheal ring 4, R1; and Cdyn at base-line in cats receiving gallamine and in time control cats. Single factor repeated measures analysis of variance with Tukey’s test of multiple comparisons examined a frequency response curve within a group of cats. Student’s t test for independent samples tested the change in tracheal ring 4 diameter, RL, or Cdyn following ACh injection or at a specific frequency before and after gallamine (Steel and Torrie 1980). Differences were significant when P < 0.05. 390110 The outer diameter of tracheal ring 4 at base-line did not differ between cats in the time control and gallamine-treated groups (087 i 0.02 cm; 0.84 i 0.02 cm, respectively). Time control and gallamine-treated cats also had similar base- 112 line measurements of RL (242 :_l-_ 257 cm H20/L/sec; 22.4 i 2.67 cm H20/L/sec, respectively) and C (2.1 i 030 ml/cm HZO/kg; 2.7 -_l—_ 022 ml/cm HZO/kg, dyn respectively). Time control cats Because RL and C measurements tended to vary between stimulations, dyn the increase in RL and the decrease in C during stimulation are reported as dyn the change from pre-stimulation measurements as described in Chapter 4 (ARI; AC respectively). Increasing frequency of vagal stimulation increased RL and dyn’ decreased both C and the diameter of tracheal ring 4 in every cat. The ARD dyn ACdyn, and change in tracheal ring 4 diameter were not significantly different between the first and second frequency-response curve (Table 6). Vehicle injections prior to ACh produced no change in RI; Cdyn, or the diameter of tracheal ring 4. Exogenous ACh injection decreased the diameter of tracheal ring 4 in every cat but did not alter RL or Cdyn significantly (Table 7). ARD ACdyn, and the change in tracheal ring 4 diameter were not significantly different between the first and second ACh injections in the time control cats (Table 7). Tracheal ring 4 measurements in control cats and cats with tracheal h y perres ponsiveness Vagal stimulation caused a progressive reduction in the diameter of tracheal ring 4 as the frequency of stimulation was increased (Figure 15). The constriction was significantly greater at stimulus frequencies of 4, 8, 12, and 16 Hz compared to base-line pre- and post-gallamine injection. However, there was no significant difference in the amount of constriction at any frequency of 113 .0000 _o.E00 0E: 5 00.n0..0> 00.00000. 00.5 0:. m:oE0 5.0.050 6 3:030: .60 00 0 E00 9 00.00Eo0 0 E00 5 00:0.0...0 0:005:90 o: 003 0.05 .006 v a. 5.0.050 05.00 00000.05 >=:00....:m.0 u a .:o=00.5 0.0.:0> 0:0 0 5.0.0500 .0m0> .I. 0 56 60200.5 0.0.:0> 0.200 P 5.0.05.0 _0m0> n 0 5.0 ”60:00... E0\_E 00.0 H 0.0 u 05.0008 05.0000 E0: 00:0..0Eo0 0.5056 5 090:0 .1. 5004 2000350: E0 00.0 H 0.00 n 05.0000. 05.0000 E0... 00:00.00. E0552. 5 090:0 u 64. :80 No.0 .1... 00.0 n 05.0000. 05.0000 Eo.. 0.2.0.0 .0520 5 0000.000 u .000E0.0 v .05.. 00:00.... 5 090:0 .000 m u : ”mm H. 0:00E 0.0 00:.0> 0.30 .....1 «had mod H «vwd 00.: H .0000 00.0 H .0000 0.00 H .0000 0.00 H .0000 0.0. 00 H 00.0 8.0 H .000 00.0. H .0000 00.0 H .000. 0.2 H .0000 0.00 H .0000 0.0: 00.0 H 00.0 00.0 H .000 00.0 H 00.2 00.0 H 00.0: 0.00 H .0000 3... H .0000 0.0 00.0 H :0 00.0 .H. 00.0 00.0 H 00.0 00.. H 00.0 .30 H .0000 :00 H .000 0.0 5.0 H 5.0- 5.0 H 00.0 00.0 H 00.0 00.: H 00.. 0.00 H 0.03 0.0.. H .002 0.0 0.0 M 00.0 00.0 .+.. 00.0 00.0 H 00.0 00.0 H 00.0 :0. H :00 0.00 H 0.00 0.. 0.0 + 00.0 0.0 + 5.0 00.0 H 00.: 00.0 H 3.0 0.0: H 0.00 0.00 H 0.00 0.0 0 0.00 F :50 0 :50 P 0.00 0 .000 F 0.00 0.... 00.0. -256 0202 60.50... E0\_Ev 800200... E0. A0:0.0.E. .000E0.o .500 0:0 0 00.: .0200... c. 000000 .wuwo 63:00 05: 5 00:03:50 0.5050 0:0 00:00.00. 20:05:: 09080.0 0 05. 00:00.. :0 5.0.0500 0Z0: .0m0> 00.0000. .o 80cm .0 m..m<.r 114 TABLE 7. Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in time control cats. Vehicle ACh ACh Injection Injection 1 Injection 2 Change in Tracheal 12.2 i. 10.9 3328* .1: 39.6 2569* i 46.8 Ring 4 diameter (microns) ARL -0.16 i 0.21 0.97 i 0.42 0.31 i 0.19 (cm HZO/L/sec) ACM 0.00 i 0.01 0.04 _-_1-_ 0.02 0.01 i 0.01 (ml/cm HZO/kg) Values are mean _-1; SE; n = 5 cats. Change in tracheal ring 4 diameter = decrease in external diameter from base-line (base-line = 0.87 _-1_-_ 0.02 cm); ARL = change in pulmonary resistance from base-line (base-line = 24.2 i 2.57 cm Hzo/L/sec); AC6," = change in dynamic compliance from base-line (base-line = 2.1 i 0.30 ml/cm HZO/kg). ACh produced significant constriction of tracheal ring 4 compared to vehicle (*P < 0.05). There was no significant difference in any of the variables measured following ACh injection 1 and ACh injection 2. 115 Tracheal Constriction (microns) eoor * * / /L _— — _— X-—-l* 600 .._J. :1-1——4 400 200 o l l l L l l l l 0 2 4 6 8 10 12 14 16 Base Frequency (Hz) 4‘ Pre-gallamine +Post-gallamine Figure 15. Effect of gallamine on tracheal ring 4 constriction during bilateral vagal nerve stimulation (24 V, 05 msec, 0.5-16 Hz) in 10 cats. Constriction was quantitated with the caliper shown in Figure 3. Vagal stimulation decreased the external diameter of tracheal ring 4 significantly both pre— and post- gallamine injection at stimulus frequencies of 4, 8, 12, and 16 Hz compared to base (* P < 0.05). However, gallamine (10 mg/kg iv) had no effect on the amount of tracheal ring 4 constriction. Base = external diameter of tracheal ring 4 prior to stimulation (08 i: 0.02 cm). 116 stimulation when the first and second frequency-response curves were compared (Figure 15). Injection of gallamine did not potentiate tracheal constriction during vagal stimulation at frequencies of 05 to 16 Hz. Tracheal responses to vagal nerve stimulation prior to gallamine injection separated the cats into a control group and a group with tracheal hyperresponsiveness (TH). Although TH cats had significantly greater constriction of tracheal ring 4 compared to control cats at 4, 8, 12, and 16 Hz, there was no significant effect of gallamine on the change in tracheal ring 4 diameter at any stimulation frequency in either group (Figure 16). Intra-arterial ACh decreased the diameter of tracheal ring 4 compared to measurements recorded following vehicle injection, but the amount of tracheal constriction was not altered by gallamine treatment between ACh injections 1 and 2 (Table 8). Change in RL and C d”, in control cats and cats with tracheal h yperresponsiveness Vagal stimulation prior to gallamine injection produced a rise in RL that was significantly increased at 12 and 16 Hz (Figure 17). When the frequency- response curve was generated following gallamine administration, the ARL was significantly greater at 4, 8, and 16 Hz. Comparison of the two frequency- response curves generated before and after gallamine injection indicated significant augmentation of the ARL responses at 4, 8, and 16 Hz following gallamine injection. Gallamine administration produced no change in the ARL measurements following ACh injection 2 compared to ACh injection 1 (Table 8). 117 0Tracheal Constriction (microns) . 750 500 250 O O 2 4 6 8 10 12 14 16 Base Frequency (HZ) "‘- TH Pre + TH Post "*5" Con Pre 4 Con Post Figure 16. Effect of gallamine on tracheal ring 4 constriction during bilateral vagal nerve stimulation (24 V, 0.5_msec, 05-16 Hz) in control cats (11 = 4) and cats with tracheal hyperresponsiveness (TH; n = 6) Constriction was quantitated with the caliper shown in Figure 3. Vagal stimulation decreased the external diameter of tracheal ring 4 significantly both pre- and post-gallamine injection at stimulus frequencies of 4, 8, 12, and 16 Hz compared to base in both groups of cats (P < 0.05). Cats with tracheal hyperresponsiveness to vagal nerve stimulation (TH) had significantly greater tracheal ring 4 constriction at stimulus frequencies of 4, 8, 12, and 16 Hz both pre- and post-gallamine administration compared to control cats (P < 0.05) However, gallamine did not significantly alter the amount of tracheal ring 4 constriction in either group of cats. Base = external diameter of tracheal ring 4 prior to stimulation (Controls = 0.8 i 0.02 cm; TH = 0.9 i 0.03 cm. 118 Pulmonary Resistance (cm water/L/sec) 4O O 2 4 6 8 1O 12 14 16 Frequency (Hz) -* Pre-gallamine +Post-gallamine Figure 17. Changes in pulmonary resistance during vagal stimulation (24 V, 0.5 msec, 05-16 Hz) prior to gallamine and following gallamine injection in 10 cats. Because RL measurements tended to vary between stimulations, the increases in RL are reported as the change from pre-stimulation measurements (ARI; RL prior to stimulation = 22.4 i 2.67 cm H20/l/sec). Vagal stimulation increased resistance significantly at 12 and 16 Hz (* P < 0.05). Gallamine injection (10 mg/kg i.v.) augmented the rise in ARL during vagal stimulation with significant increases at 8, 12, and 16 Hz (" P < 0.05). Comparison of the two frequency- response curves generated before and after gallamine injection indicated significant increases in ARL responses at 4, 8, and 16 Hz (+ P < 0.05). 119 TABLE 8. Effect of vehicle and ACh injections on tracheal ring 4 diameter, pulmonary resistance, and dynamic compliance in cats before and after gallamine injection. Vehicle ACh ACh Injection Injection 1 Injection 2 Change in Tracheal 4.6 ;1-_ 4.4 3339* _-1; 70.3 191.6* :L 40.6 Ring 4 diameter (microns) ARL -0.58 i 0.25 1.26 i 0.63 1.71 i 1.37 (cm HZO/L/sec) A0,, 0.01 i 0.01 0.06* :1: 0.01 0.07 i 0.05 (ml/cum HZO/kg) Values are mean :1; SE; n = 10 cats. Change in tracheal ring 4 diameter = decrease in external diameter from base-line (base-line = 0.84 i 0.02 cm); ARL = change in pulmonary resistance from base-line (base-line = 22.4 _-1; 2.67 cm HZO/Usec); A0,”, = change in dynamic compliance from base-line (base-line = 2.7 :1: 0.22 mI/cm HZO/kg). ACh produced significant change in tracheal ring 4 and AC,” compared to vehicle (*P < 0.05). Gallamine was injected after ACh injection 1, but there was no effect on the variables measured when responses measured at ACh injection 1 were compared to ACh injection 2. 120 The AC,“m before gallamine injection was changed significantly during vagal nerve stimulation at 8, 12, and 16 Hz (Figure 18) Following gallamine injection, the change in Cdyn was enhanced and significantly different from pre- gallamine measurements at 4, 8, and 12 Hz. Injection of ACh produced a significant change in ACdyn compared to vehicle injection, but gallamine administration produced no change in the ACdyn measurements following ACh injection 2 compared to ACh injection 1 (Table 8). Discussion Most nerves have autoreceptors on the nerve ending that inhibit further release of neurotransmitter and act as a negative presynaptic feedback mechanism to reduce neurotransmission (Starke et al.1989). The primary role of inhibitory receptors may be in self-regulation of neuronal activity by limiting the neuron’s chemical signal. Since their discovery, there has been much speculation about the importance of autoreceptors in disease. Autoreceptors associated with cholinergic nerves supplying airway smooth muscle have been reported in a number of mammalian species (Fryer and Maclagan 1984, Blaber et al. 1985, Ito and Yoshitomi 1988, Minette and Barnes 1988). Previous in vivo studies in the cat have used the M2 receptor agonist pilocarpine and M2 receptor antagonist gallamine to demonstrate the existence of neuronal muscarinic receptors that inhibit parasympathetic transmission (Blaber et al. 1985) The authors reported changes in RL and Cdyn as indicators of central and peripheral airway constriction, respectively, and they concluded that inhibitory muscarinic receptors were present in parasympathetic nerves of the trachea, bronchi, and respiratory bronchioles. In 121 8Dynamic Compliance (ml/cm water/kg) 0 2 4 6 8 10 12 14 16 3"” Frequency (Hz) —x Pre-gallamine +Post-gallamine Figure 18 Changes in dynamic compliance during vagal stimulation (24 V, 0.5 msec, 05-16 Hz) prior to gallamine and following gallamine injection in 10 cats. Because Cd measurements tended to vary between stimulations, the decrease in Cdyn is reported as the change from pro-stimulation measurements (ACdyn; Cd 0 prior to stimulation = 2.7 i 022 ml/cm H20/kg). Vagal stimulation changed Addy“ significantly at 8, 12, and 16 Hz (* P < 0.05). Gallamine injection (10 mg/kg i.v.) augmented ACdyn during vagal stimulation with significant increases at 4, 8, 12, and 16 Hz (* P < 0.05). Comparison of the two frequency-response curves generated before and after gallamine injection indicated significant increases in ACdyn at 4, 8, and 12 Hz (+ P < 0.05). 122 Chapter 4, I described studies in which I demonstrated increased intrathoracic airway constriction as measured by RL and Cdyn during vagal stimulation following gallamine administration, but I was unable to demonstrate inhibitory receptors in the cervical trachea. My previous gallamine dose-response studies (n = 27 cats) demonstrated modulation of intrathoracic airway caliber without gallamine-induced changes in tracheal constriction during vagal stimulation at 24 V, 05 msec, and 4 Hz. A customized electromechanical caliper capable of measuring isotonic tracheal constriction or relaxation in microns was attached to the external surface of tracheal ring 4. Bilateral vagal stimulation caused significant constriction of tracheal ring 4, but gallamine had no effect on the amount of tracheal constriction at any dose (0.1, 1.0, and 10.0 mg/kg iv). One possible explanation for my failure to demonstrate tracheal M2 receptors was frequency-dependent receptor activation (Blaber et al. 1985, Wessler 1989), in which a frequency of 4 Hz might be too low to produce autoinhibition of ACh release in the trachea. In the present study, vagal stimulation (24 V, 0.5 msec duration) caused airway constriction that increased with greater frequencies of stimulation (05-16 Hz) Following gallamine administration, the ARL was enhanced significantly at 4, 8, and 16 Hz. Gallamine also augmented the changes in C at 4, 8, and 12 dyn Hz. However, I was still unable to demonstrate M2 inhibitory receptors in vivo in the cervical trachea of the cat with increasing stimulation frequencies following gallamine injection. Using the techniques described for this study, I was able to demonstrate augmented changes in RL and Cdyn following gallamine injection at greater frequencies of stimulation. A plateau in intra- thoracic airway constriction occurred at the highest frequencies. However, 123 although vagal stimulation significantly decreased tracheal diameter with increasing stimulation frequencies, gallamine injection had no effect on the amount of tracheal constriction. An increase in tracheal constriction following gallamine injection still could not be demonstrated even with stimulation frequencies greater than 4 Hz Therefore, I was unable to demonstrate a physiological role for M2 inhibitory receptors in limiting tracheal constriction in cat trachea. A low density of M2 autoreceptors in the cervical trachea of the cat could be one explanation for my data. Although direct receptor binding techniques suggest a high density of muscarinic receptors in tracheal smooth muscle (Cheng and Townley 1982, Barnes et al. 1983), to my knowledge, studies with selective radiolabelled M2 receptors antagonists in cat cervical trachea have not been done. Non-selective radioligand binding of whole lung homogenates do not differentiate M1, M2, and M3 receptors associated with smooth muscle, neuronal tissue, and tracheal gland cells (Culp and Marin 1986). These nonspecific ligands indicate a high density of muscarinic receptors but do not identify specific receptor subtypes. My physiological findings could be explained by a preponderance of M1 and/or M3 receptors with a low number or no detectable neuronal M2 receptors in cat cervical trachea. The cartilaginous rings could be important structures for limiting tracheal constriction, whereas M2 inhibitory receptors could play an important role in limiting intrathoracic airway constriction during neural stimulation where cartilaginous rings do not exist. Studies describing differential distribution of muscarinic receptor subtypes in human brain and rat cerebellum have been published (Palacios et 124 al. 1985, Lapchak et 31.1989). Selective receptor binding studies in rat cerebellum demonstrated positive feedback control of acetylcholine release by M1 facilitatory receptors but failed to demonstrate muscarinic autoreceptors using the M2 selective antagonist AF-DX 116 (Lapchak et al.1989). Failure to show M2 autoreceptors in rat cerebellum is comparable to my inability to demonstrate a functional role for M2 receptors in cat trachea. Another possible explanation for the apparent absence of M2 receptors in cat trachea is that my in vivo physiological studies may not be sensitive enough to detect the presence of presynaptic inhibitory muscarinic receptors. Only with recent techniques designed to measure newly synthesized [3H]acetylcholine release have presynaptic autoreceptors, including inhibitory muscarinic receptors, been clearly demonstrated in vitro associated with autonomic and motor nerves (Szerb 1975, Wessler and Kilbinger 1986, Wessler 1989). The question of the correlation between in vitro findings and the significance of M2 autoreceptors in tracheal caliber regulation in vivo remains unanswered. Although M2 receptors might be demonstrated with highly sensitive in vitro techniques, a functional role for M2 autoreceptors in cat trachea was not demonstrated in my studies. Simultaneous activation of both inhibitory and facilitatory muscarinic receptors during nerve stimulation by accumulated endogenous ACh might prevent demonstration of M2 inhibitory receptor modulation (Wessler et 31.1987, Wessler et al. 1988, Wessler 1989). Studies of rat phrenic nerve have demonstrated facilitatory and inhibitory muscarinic receptors in the motor nerve (Wessler et al.1988). In addition, muscarinic receptor inhibition has been reported during short periods of stimulation of the phrenic nerve, whereas 125 during long periods of continuous nerve stimulation the positive muscarinic feedback mechanism was activated predominantly (Wessler et al.1987, Wessler et al. 1988). Therefore, inhibitory autoreceptors may be present in the cat cervical trachea but their function may have been masked by primary activation of facilitatory muscarinic receptors during vagal stimulation in this study. As described in the previous study, I wanted to compare the effects of gallamine on the tracheal response during presynaptic vagal stimulation and postjunctional muscarinic receptors activation with locally injected ACh. If M2 autoreceptors are located at a presynaptic site and could be demonstrated at stimulation frequencies greater than 4 Hz, then gallamine would enhance smooth muscle contraction induced by vagal stimulation but have no effect on contraction caused by ACh. However, similar to the study described in Chapter 4, gallamine had no effect on the tracheal response to either vagal stimulation or exogenous ACh. As expected, local administration of ACh contracted the trachea but had a variable effect on RL and CM because ACh is rapidly hydrolyzed and needs to reach pulmonary circulation in order to produce intrathoracic airway smooth muscle contraction (Colebatch et al. 1966). These data suggest that M2 receptors play an important role in limiting intrathoracic airway constriction during vagal stimulation. I have not demonstrated inhibitory autoreceptors in the cervical trachea with either increasing doses of iv gallamine or with increasing frequencies of stimulation between 05 and 16 Hz. Therefore, a functional role of M2 receptors to limit smooth muscle constriction was not demonstrated in the cervical trachea 126 regardless of the amount of tracheal constriction that was quantitated during vagal stimulation. CHAPTER 6 Summary and Conclusions Viral respiratory infections can exacerbate bronchial asthma and enhance airway reactivity to smooth muscle contractile substances. Empey and coworkers showed that histamine aerosols produced a greater degree of bronchoconstriction in subjects with colds than in a control group (Empey et al. 1976). This exaggerated response returned to normal during several weeks. Similar findings were reported after inoculation of live, attenuated influenza virus in humans (Laitinen et al. 1976). Because atropine sulfate prevented the exaggerated bronchomotor responses, these investigators concluded that the effect was not simply an increased accessibility of the histamine aerosol to the airway smooth muscle, but implicated the parasympathetic nervous system in the exaggerated responses (Laitinen et al. 1976). Although cats do not contract the same upper respiratory viruses as humans, cats are quite susceptible to naturally occurring viral infections that bear many similarities to upper respiratory infections in persons. Feline rhinotracheitis, designated herpesvirus-I, causes naturally occurring upper respiratory infection, and is of a single antigenic type specific to the cat (Hoover et al. 1970, Povey 1979). Because the mechanisms of virus-provoked 127 128 airway hyperreactivity are unknown, I have used an animal model to determine how respiratory viral infections affect cholinergic responses. Early in my studies, a device with matched microfoil strain gages was designed to quantify airway smooth muscle shortening. The instrument measures changes in the outer diameter of the airway and is unaffected by airway wall edema or mucus accumulation. Depending on the configuration of the device, it can measure either isometric or isotonic muscle contractions. In these studies, the device was attached in situ to the external surface of the fourth tracheal ring with minimal surgery, allowing the airway to retain its normal innervation and perfusion. This device is sensitive and maintains its calibration for long periods of time. The instrument is easy to attach to the airways and interferes minimally with air flow, lining epithelium, and the blood and nerve supply of the study segment. Rather than measuring isometric force developed by the tissue, the device can be calibrated in units of distance to measure isotonic smooth muscle shortening, which parallels in vivo airway constriction. The next studies were designed to determine the effect of acute infection with feline herpesvirus-I on airway responsiveness in the cranial cervical trachea (tracheal ring 4) and in the intrathoracic airways (RL and Cdyn)' Although herpesvirus-I infects primarily the upper respiratory tract in cats, upper airway viral infection can cause heightened lower airway reactivity in humans (National Heart, Lung Blood Institutes Workshop 1988). These protocols were designed to explore the pathophysiologic interrelationship between the upper and lower respiratory tracts in the cat following acute viral infection. 129 Bilateral vagal nerve stimulation and intra-arterial ACh were used to separate the responses of airway smooth muscle resulting from pre- and post junctional muscarinic receptor stimulation. Increased tracheal constriction during vagal stimulation detected in infected cats studied at PID3 was absent by PID6 and in control cats. Tracheal hyperreactivity at PID3 was completely eliminated by injection of the muscarinic receptor antagonist atropine. Postjunctional muscarinic receptor activation with locally injected ACh produced no difference in tracheal constriction between control and virus- infected cats. Intrathoracic airway hyperresponsiveness during vagal stimulation was not detected at either PID3 or PID6. Virus quantitation studies at PID3 revealed herpesvirus associated with the tracheal study segment and mainstem bronchi, but virus was not found in swabs taken distal to the mainstem bronchi. By PID6, virus was no longer isolated from the trachea and bronchi but was still present at the sites of inoculation. The absence of herpesvirus distal to the mainstem bronchi may account for the lack of differences in RL and Cd),n in PID3-infected and control cats. Hyperresponsiveness to vagal stimulation was detected in the trachea but not in the pulmonary airways. Furthermore, at PID6 the trachea was no longer hyperresponsive and virus could not be isolated. Therefore, these physiologic studies at PID3 and PID6 largely parallel the virus isolation findings. Tracheal hyperresponsiveness to vagal stimulation appears to be mediated by a presynaptic mechanism, and this hyperreactivity correlates with presence of virus at PID3. Obtaining cats that were truly "disease-free" for investigation as control or herpesvirus-infected cats proved to be quite difficult. While studying 130 random source cats obtained from the University Laboratory Animal Resources facility at Michigan State University, I found a group of adult, mixed-breed cats of either gender with tracheal hyperresponsiveness to vagal stimulation independent of viral infection. Currently, the etiology of their tracheal hyperresponsiveness is unknown. These cats have been used to investigate the role of presynaptic muscarinic inhibitory receptors in limiting airway constriction. The subsequent studies were designed to investigate whether dysfunction of the prejunctional inhibitory (M2) receptors is responsible for the exaggerated tracheal response to vagal stimulation detected in some cats (tracheal hyperresponsive cats) and whether it is accompanied by hyperresponsiveness in intrathoracic airways. Airway constriction was produced by intra-arterial acetylcholine injection and bilateral vagal stimulation at 24 V, 05 msec duration, and 4 Hz. The changes in tracheal ring 4 diameter, RL, and Cdyn were quantitated before and after cumulative iv doses of the M2 receptor antagonist gallamine. Gallamine is an antagonist for the neuronal muscarinic receptor at doses that have little postsynaptic action (Blaber et al. 1985) There was no significant difference pre- or post-gallamine administration in the response of tracheal ring 4, Ru or Cdyn in any of the cats following activation of postsynaptic muscarinic receptors with ia ACh. Vagal stimulation caused more tracheal ring 4 narrowing in the hyperresponsive cats than in control animals at every gallamine dose, but there was no modulatory role of M2 receptors; increasing doses of gallamine did not alter the amount of constriction in either group. These data did not demonstrate a functional role for M2 inhibitory receptors in limiting tracheal 131 constriction at any dose of gallamine. The change in RL and C(in1 during vagal stimulation was particularly enhanced by iv gallamine in hyperresponsive cats demonstrating the presence and physiological significance of M2 inhibitory receptors in limiting intrathoracic airway constriction. Because receptor activation is frequency dependent during nerve stimulation (Blaber et al. 1985, Wessler 1989), the next protocols were designed to determine if M2 inhibitory receptors might be demonstrated in the cervical tracheal at stimulation frequencies greater than 4 Hz. Vagal stimulation caused tracheal ring 4 narrowing in a frequency- dependent manner, but constriction was unchanged by gallamine administration either in cats with control responses or those with tracheal hyperresponsiveness to nerve stimulation. 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