Antitussive drugs--past, present, and future.

Cough remains a serious unmet clinical problem, both as a symptom of a range of other conditions such as asthma, chronic obstructive pulmonary disease, gastroesophageal reflux, and as a problem in its own right in patients with chronic cough of unknown origin. This article reviews our current understanding of the pathogenesis of cough and the hypertussive state characterizing a number of diseases as well as reviewing the evidence for the different classes of antitussive drug currently in clinical use. For completeness, the review also discusses a number of major drug classes often clinically used to treat cough but that are not generally classified as antitussive drugs. We also reviewed a number of drug classes in various stages of development as antitussive drugs. Perhaps surprising for drugs used to treat such a common symptom, there is a paucity of well-controlled clinical studies documenting evidence for the use of many of the drug classes in use today, particularly those available over the counter. Nonetheless, there has been a considerable increase in our understanding of the cough reflex over the last decade that has led to a number of promising new targets for antitussive drugs being identified and thus giving some hope of new drugs being available in the not too distant future for the treatment of this often debilitating symptom.

Sensory nerves and airway irritability.

The lung, like many other organs, is innervated by a variety of sensory nerves and by nerves of the parasympathetic and sympathetic nervous systems that regulate the function of cells within the respiratory tract. Activation of sensory nerves by both mechanical and chemical stimuli elicits a number of defensive reflexes, including cough, altered breathing pattern, and altered autonomic drive, which are important for normal lung homeostasis. However, diseases that afflict the lung are associated with altered reflexes, resulting in a variety of symptoms, including increased cough, dyspnea, airways obstruction, and bronchial hyperresponsiveness. This review summarizes the current knowledge concerning the physiological role of different sensory nerve subtypes that innervate the lung, the factors which lead to their activation, and pharmacological approaches that have been used to interrogate the function of these nerves. This information may potentially facilitate the identification of novel drug targets for the treatment of respiratory disorders such as cough, asthma, and chronic obstructive pulmonary disease.

Cough sensors. I. Physiological and pharmacological properties of the afferent nerves regulating cough.

The afferent nerves regulating cough have been reasonably well defined. The selective effects of general anesthesia on C-fiber-dependent cough and the opposing effects of C-fiber subtypes in cough have led to some uncertainty about their regulation of this defensive reflex. But a role for C-fibers in cough seems almost certain, given the unique pharmacological properties of these unmyelinated vagal afferent nerves and the ability of many C-fiber-selective stimulants to evoke cough. The role of myelinated laryngeal, tracheal, and bronchial afferent nerve subtypes that can be activated by punctate mechanical stimuli, inhaled particulates, accumulated secretions, and acid has also been demonstrated. These "cough receptors" are distinct from the slowly and rapidly adapting intrapulmonary stretch receptors responding to lung inflation. Indeed, intrapulmonary rapidly and slowly adapting receptors and pulmonary C-fibers may play no role or a nonessential role in cough, or might even actively inhibit cough upon activation. A critical review of the studies of the afferent nerve subtypes most often implicated in cough is provided.

Evidence for an esophageal origin of VIP-IR and NO synthase-IR nerves innervating the guinea pig trachealis: a retrograde neuronal tracing and immunohistochemical analysis.

Nonadrenergic noncholinergic (NANC) relaxations of the guinea pig trachea are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Physiological studies have indicated that the parasympathetic ganglion neurons mediating NANC relaxations of the guinea pig trachea but not the ganglion neurons mediating cholinergic contractions are in some way associated with the adjacent esophagus. In the present study, we attempted to locate precisely the noncholinergic parasympathetic ganglia innervating the trachealis. Two days after injection of the retrograde neuronal tracer DiI into the trachealis of organotypic cultures of the guinea pig trachea and esophagus, neurons within the myenteric plexus of the esophagus or closely associated with the outer striated longitudinal muscle layers of the esophagus were labeled. Subsequent immunohistochemical analyses revealed that a majority of the retrogradely labeled neurons possessed VIP immunoreactivity (IR) or NO synthase (NOS)-IR or had VIP-IR nerve fibers associated with their cell bodies. By contrast, no labeling of esophageal neurons was seen when the tissue between the trachea and esophagus had been disrupted by blunt dissection prior to tracer injection or when the cultures were treated with the axonal transport inhibitor colchicine. The results of these experiments provide the first direct evidence that VIP-IR and NOS-IR neurons intrinsic to the guinea pig esophagus project axons to the adjacent trachealis. Based on their location and phenotype and the results of our previous studies, it is likely that these neurons are the postganglionic parasympathetic neurons mediating NANC relaxations of the trachealis.

Role of nerves in asthmatic inflammation and potential influence of gastroesophageal reflux disease.

Inflammation of the lower airways is a defining characteristic of asthma. Microaspiration of refluxate may initiate an inflammatory response in the airways of patients with gastroesophageal reflux disease (GERD), thereby precipitating asthma. Airway nerves are likely to play a role in the pathogenesis of asthma and could potentially mediate airway inflammation initiated by GERD through axonal reflexes. Alternatively, refluxate may initiate airway reflexes from the esophagus that are markedly exaggerated by inflammation-induced enhancement of airway neuronal excitability. The characteristic features of inflammation in asthma are defined, and the potential role of nerves in inflammation is discussed. Mechanisms by which GERD may initiate airway inflammation or act synergistically with airway inflammation to produce asthma also are discussed.

Adenosine receptor-mediated contraction and relaxation of guinea-pig isolated tracheal smooth muscle: effects of adenosine antagonists.

1. The effects of several adenosine analogues and antagonists on guinea-pig isolated trachea have been examined. 2. 5'-N-ethylcarboxamidoadenosine (NECA), 5'-N-methylcarboxamidoadenosine (MECA) and adenosine (in the presence and absence of dipyridamole) elicited concentration-dependent tracheal relaxation. 3. The R(-)- and S(+)-enantiomers of N6-(2-phenylisopropyl)adenosine (R-PIA and S-PIA respectively), N6-cyclohexyladenosine (CHA) and 2-chloroadenosine (CADO) caused contractions at low concentrations (0.05-2.0 microM), whereas at higher concentrations, relaxation resulted. 4. For tracheal relaxation, the adenosine analogues exhibited the following rank order of potency: NECA greater than CADO greater than R-PIA = MECA greater than S-PIA greater than adenosine. The rank order of potency for inducing contractions was R-PIA greater than CHA greater than CADO greater than S-PIA. These data suggest that relaxation is mediated by adenosine A2-receptors, whereas contraction is the result of activation of A1-receptors. 5. 8-Phenyltheophylline (8-PT), aminophylline, the triazoloquinazoline CGS 15943A and NPC205 (1,3-di-n-propyl-8-(4-hydroxyphenyl)xanthine) each inhibited the R-PIA-induced contractile response, whereas enprofylline was without effect. NPC205, aminophylline and 8-PT were competitive antagonists, but CGS15943A was non-competitive. 6. That the most potent antagonist was the A1-selective agent, NPC205 (pA2 = 7.80), further suggests that the contraction is mediated by A1-receptors. Moreover, NPC205 was 13 times more potent as an antagonist of R-PIA-induced contractions (A1) than of NECA-induced relaxations (A2). 7. The antagonists were also found to relax the trachea by an unknown mechanism. That enprofylline did not antagonize the R-PIA-induced contractions, but was 3-4 times more potent a tracheal relaxant than aminophylline, further suggests that a direct effect on airway smooth muscle, rather than antagonism of endogenous adenosine, is more relevant to the bronchodilator effect of alkylxanthines in the treatment of asthma.

The effects of ozone on immune function.

A review of the literature reveals that ozone (O3) exposure can either suppress or enhance immune responsiveness. These disparate effects elicited by O3 exposure depend, in large part, on the experimental design used, the immune parameters examined as well as the animal species studied. Despite the apparent contradictions, a general pattern of response to O3 exposure can be recognized. Most studies indicate that continuous O3 exposure leads to an early (days 0-3) impairment of immune responsiveness followed, with continued exposures, by a form of adaptation to O3 that results in a re-establishment of the immune response. The effects of O3 exposure on the response to antigenic stimulation also depend on the time at which O3 exposure occurred. Whereas O3 exposure prior to immunization is without effect on the response to antigen, O3 exposure subsequent to immunization suppresses the response to antigen. Although most studies have focused on immune responses in the lung, numerous investigators have provided functional and anatomical evidence to support the hypothesis that O3 exposure can have profound effects on systemic immunity.

Correlation of vasoactive intestinal peptide and nitric oxide synthase with choline acetyltransferase in the airway innervation.

Vasoactive intestinal peptide (VIP) and nitric oxide (NO) are potent mediators of neural airway smooth muscle relaxation. The major contractile mediator released by airway nerves under physiological conditions is acetylcholine (ACh). In the present study, we have correlated the immunohistochemical distribution of the relaxant mediators using antisera to VIP, to the marker enzyme of catecholamine synthesis tyrosine hydroxylase (TH) and to the NO-generating enzyme NO-synthase (NOS) with the distribution of the ACh-synthesizing enzyme, choline acetyltransferase (ChAT), and of substance P (SP), a neuropeptide present in sensory nerve fibers. In guinea-pig airways, VIP- and NOS-immunoreactivity (IR) were present in numerous nerve fibers in the airway smooth muscle and around submucosal glands; some fibers were also seen in the lamina propria and around blood vessels. The neuronal cell bodies in the intrinsic ganglia were devoid of both VIP- and NOS-IR. In contrast, all neuronal cell bodies in the intrinsic ganglia were immunoreactive for ChAT. In human airways, immunoreactivity for VIP, NOS, and ChAT was found in airway intrinsic neuronal perikarya. Whereas ChAT-IR appeared to be most frequent in the cell bodies, VIP-IR was seen in the largest number of nerve fibers in the airways. Therefore, in guinea pigs, a clear neuroanatomical and neurochemical separation of relaxant and of constrictor pathways is seen, whereas in human airways, both separate pathways as well as coexpression of VIP-/NOS- and of ChAT-IR are found.

Multiple mechanisms of reflex bronchospasm in guinea pigs.

The mechanisms of histamine- and bradykinin-induced reflex bronchospasm were determined in anesthetized guinea pigs. With intravenous administration, both autacoids evoked dose-dependent increases in tracheal cholinergic tone. Vagotomy or atropine prevented these tracheal reflexes. When delivered as an aerosol, bradykinin readily increased tracheal cholinergic tone, whereas histamine aerosols were much less effective at inducing tracheal reflexes. Also, unlike histamine, bradykinin could evoke profound increases in cholinergic tone without directly or indirectly (e.g., prostanoid dependent) inducing measurable airway smooth muscle contraction resulting in bronchospasm. Neither autacoid required de novo synthesis of prostanoids or nitric oxide to induce reflex tracheal contractions. Combined cyclooxygenase inhibition and tachykinin-receptor antagonism did, however, abolish all effects of bradykinin in the airways, whereas responses to histamine were unaffected by these pretreatments. The data indicate that histamine and bradykinin initiate reflex bronchospasm by differential activation of vagal afferent nerve subtypes. We speculate that selective activation of either airway C fibers or airway rapid adapting receptors can initiate reflex bronchospasm.

The role of trigeminal nasal TRPM8-expressing afferent neurons in the antitussive effects of menthol.

The cold-sensitive cation channel TRPM8 is a target for menthol, which is used routinely as a cough suppressant and as an additive to tobacco and food products. Given that cold temperatures and menthol activate neurons through gating of TRPM8, it is unclear how menthol actively suppresses cough. In this study we describe the antitussive effects of (-)-menthol in conscious and anesthetized guinea pigs. In anesthetized guinea pigs, cough evoked by citric acid applied topically to the tracheal mucosa was suppressed by menthol only when it was selectively administered as vapors to the upper airways. Menthol applied topically to the tracheal mucosa prior to and during citric acid application or administered continuously as vapors or as an aerosol to the lower airways was without effect on cough. These actions of upper airway menthol treatment were mimicked by cold air delivered to the upper airways but not by (+)-menthol, the inactive isomer of menthol, or by the TRPM8/TRPA1 agonist icilin administered directly to the trachea. Subsequent molecular analyses confirmed the expression of TRPM8 in a subset of nasal trigeminal afferent neurons that do not coincidently express TRPA1 or TRPV1. We conclude that menthol suppresses cough evoked in the lower airways primarily through a reflex initiated from the nose.

Cholinergic brush cells in the trachea mediate respiratory responses to quorum sensing molecules.

AIM: The airway epithelial surface is constantly exposed to inhaled environmental factors and pathogens. Bitter "tasting" bacterial products such as quorum sensing molecules (QSM) can be detected by solitary chemosensory cells of the upper respiratory tract. Recently, we have shown that tracheal brush cells are cholinergic chemosensory cells affecting the respiration upon stimulation with bitter substances. Here, we explore the hypothesis that tracheal brush cells are capable of detection of bacterial products such as QSM resulting in changes in respiration and in induction of local effects, e.g. regulation of mucociliary clearance. MAIN METHODS: Functional analyses of respiration were performed in the trachea using a newly established model for investigation of respiration in spontaneously breathing anesthetized mice upon isolated tracheal stimulation. Influence of N-3-oxododecanoyl-homoserine lactone (3-OxoC(12)-HSL) on cilia-driven particle transport speed (PTS) in the airways was investigated in acutely excised and submerged mouse tracheae. KEY FINDINGS: 3-OxoC(12)-HSL, a Pseudomonas aeruginosa quorum sensing autoinducer, caused a drop in the respiratory rate 2 min after the application at the mucosal surface. The 3-OxoC(12)-HSL-induced effect on respiration was abolished by inhibition of nicotinic receptors with mecamylamine and by removal of the respiratory epithelium. At the same concentration, 3-OxoC(12)-HSL enhanced significantly PTS on the mucosal surface. SIGNIFICANCE: We conclude that cholinergic airway epithelial cells sense bacterial QSM in the airway lining fluid and communicate this to the CNS via ACh release and nicotinic stimulation of sensory neurons. In addition, QSM enhance PTS.

Nicotinic acetylcholine receptor assays.

Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves and the motor end plates of striated musculature. Pharmacologic analyses of nicotinic receptor antagonist potencies can be conducted by assessing the ability of these compounds to inhibit responses elicited by preganglionic autonomic nerve stimulation or stimulation of the motor nerves innervating striated muscle in isolated tissue preparations. In addition, in some isolated tissues innervated by autonomic nerves, nicotinic receptor mediated responses can be elicited by exogenously administered agonists, and the effects of antagonists on these responses can be assessed using pharmacologic analyses. This unit describes the guinea pig trachea/esophagus preparation, in which nicotinic receptor pharmacology can be studied at synapses of the parasympathetic and sympathetic nervous system and the striated musculature of the esophagus. In addition, a preparation whereby the nicotinic receptors of the striated musculature of the diaphragm can be studied is described as are techniques for studying exogenous nicotinic agonist mediated effects in two smooth muscle preparations.Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves.

Regulation of baseline cholinergic tone in guinea-pig airway smooth muscle.

1. We quantified baseline cholinergic tone in the trachealis of mechanically ventilated guinea-pigs and determined the influence of vagal afferent nerve activity on this parasympathetic tone. 2. There was a substantial amount of baseline cholinergic tone in the guinea-pig trachea, eliciting contractions of the trachealis that averaged 24.6 +/- 3.5 % (mean +/- s.e.m.) of the maximum attainable contraction. This tone was essentially abolished by vagotomy or ganglionic blockade, suggesting that it was dependent upon on-going pre-ganglionic input arising from the central nervous system. 3. Cholinergic tone in the trachealis could be markedly and rapidly altered (either increased or decreased) by changes in ventilation (e. g. cessation of ventilation; hyperpnoea; slow, deep breathing) and by lung distention (via positive end-expiratory pressure). These effects were not accompanied by marked alterations in blood gases and were abolished by vagotomy or atropine. By contrast, tachykinin receptor antagonists, which abolished capsaicin-induced bronchospasm, were without effect on baseline cholinergic tone. This and other evidence suggests that capsaicin-sensitive nerves have little if any influence on baseline parasympathetic tone. Likewise, while activation of afferent nerves innervating the larynx can alter airway parasympathetic nerve activity, transection of the superior laryngeal nerves was without effect on baseline cholinergic tone. 4. Cutting the vagus nerves caudal to the recurrent laryngeal nerves, thus leaving the preganglionic parasympathetic innervation of the trachealis intact but disrupting all afferent nerves innervating the lungs and intrapulmonary airways, abolished baseline cholinergic tone in the trachea. Sham vagotomy or cutting the vagi caudal to the lungs did not reduce baseline cholinergic tone. 5. The results indicate that baseline airway cholinergic nerve activity is necessarily dependent upon afferent nerve activity arising from the intrapulmonary airways and lungs. More specifically, the data are consistent with the hypothesis that on-going activity arising from the nerve terminals of intrapulmonary rapidly adapting receptors determines the level of baseline airway cholinergic tone.

Neural regulation of airway smooth muscle tone.

Airway smooth muscle is innervated by sympathetic and parasympathetic nerves. When activated, airway nerves can markedly constrict bronchi either in vivo or in vitro, or can completely dilate a precontracted airway. The nervous system therefore plays a primary role in regulating airway caliber and its dysfunction is likely to contribute to the pathogenesis of airways diseases. The predominant contractile innervation of airway smooth muscle is parasympathetic and cholinergic in nature, while the primary relaxant innervation of the airways is comprised of noncholinergic (nitric oxide synthase- and vasoactive intestinal peptide-containing) parasympathetic nerves. These parasympathetic nerves are anatomically and physiologically distinct from one another and differentially regulated by reflexes. Sympathetic-adrenergic nerves play little if any role in directly regulating smooth muscle tone in the human airways. Activation of airway afferent nerves (rapidly adapting receptors, C-fibers) can evoke increases in airway smooth muscle parasympathetic nerve activity, or decreases in parasympathetic nerve activity (through activation of slowly adapting receptors). Extrapulmonary afferents can also modulate nerve mediated regulation of airway smooth muscle tone. In guinea pigs and rats, peripheral activation of tachykinin-containing airway afferent nerves evokes bronchospasm via release of substance P and neurokinin A. This effect of airway afferent nerve activation appears to be unique to guinea pigs and rats. The actions and interactions between the components of airway innervation are discussed.

Localization of cholinergic nerves in lower airways of guinea pigs using antisera to choline acetyltransferase.

Primary antiserum to choline acetyltransferase (ChAT), a specific marker for cholinergic nerves, was used to characterize the distribution of cholinergic nerve fibers and nerve cell bodies in guinea pig airways. ChAT immunoreactive nerve fibers were localized to the smooth muscle throughout the conducting airways and in the lamina propria of the trachea and large bronchi. Likewise, all nerve cell bodies in the ganglia intrinsic to the trachea and bronchi displayed a cholinergic phenotype. By contrast, ChAT immunoreactive nerve fibers were infrequently seen in the lamina propria of the peripheral airways and were absent in the airway epithelium. No evidence for colocalization of ChAT and the enzyme synthesizing the putative relaxant neurotransmitter nitric oxide was observed. These results provide further evidence for the key role played by cholinergic nerves in regulating airway smooth muscle tone and bronchial blood flow and provide further evidence that acetylcholine is not coreleased with the neurotransmitter(s) mediating relaxations of airway smooth muscle.

Evidence that antidromically stimulated vagal afferents activate inhibitory neurones innervating guinea-pig trachealis.

1. We recently described a capsaicin-sensitive vagal pathway mediating non-adrenergic, non-cholinergic (NANC) relaxations of an isolated, innervated rostral guinea-pig tracheal preparation. These afferent fibres are carried by the superior laryngeal nerves and relaxations elicited by their activation are insensitive to autonomic ganglion blockers such as hexamethonium. In the present study this vagal relaxant pathway was further characterized. 2. Relaxations of the trachealis elicited by electrical stimulation of capsaicin-sensitive vagal afferents were mimicked by bath application of capsaicin. Relaxations elicited by both methods were abolished when the tissue between the trachea and the adjacent oesophagus was disrupted. Indeed, separating the trachea from the oesophagus uncovered a contractile effect of capsaicin administration on the trachealis. 3. Capsaicin-induced, oesophagus-dependent relaxations of the trachealis were blocked by pretreatment with the fast sodium channel blocker tetrodotoxin (TTX). By contrast, capsaicin-induced contractions of the trachealis (obtained in the absence of the oesophagus) were unaffected by tetrodotoxin. 4. Substance P, neurokinin A (NKA) and neurokinin B (NKB) also elicited NANC relaxations of precontracted trachealis that were abolished by separating the trachea from the oesophagus or by TTX pretreatment. Like capsaicin, the tachykinins elicited only contractions of the trachealis following TTX pretreatment or separation of the trachea from the adjacent oesophagus. 5. Relaxations elicited by stimulation of the capsaicin-sensitive nerves were unaffected by a concentration of the tachykinin NK2 receptor-selective antagonist, SR 48968, that is selective for NK2 receptor blockade and were not mimicked by the NK2 receptor-selective agonist [beta-Ala8]-NKA(4-10). This suggests that NK2 receptors are not responsible for these relaxations. By contrast, the NK3 receptor-selective agonist, senktide analogue, and the NK1 receptor-selective agonist, acetyl-[Arg6, Sar9, Met (O2)11]-SP(6-11), elicited oesophagus-dependent relaxations of the trachealis that were abolished by oesophagus removal. Furthermore, pretreatment with the NK1-selective antagonists, CP 96345 and CP 99994, or pretreatment with a concentration of SR 48968 that also blocks NK3 receptors, markedly attenuated relaxations elicited by stimulation of the capsaicin-sensitive vagal pathways. 6. The data are consistent with the hypothesis that relaxations elicited by stimulation of capsaicin-sensitive vagal afferents involve tachykinin-mediated activation of peripheral NANC inhibitory neurones that are in some way associated with the oesophagus. The data also indicate that airway smooth muscle tone might be regulated by peripheral reflexes initiated by activation of capsaicin-sensitive afferent fibres.

Relaxant innervation of the guinea-pig trachealis: demonstration of capsaicin-sensitive and -insensitive vagal pathways.

1. The guinea-pig trachea was isolated with its extrinsic innervation intact and placed in a water-jacketed dissecting dish containing warmed, oxygenated Krebs solution. The trachea was not separated from the oesophagus. Two adjacent cartilage rings of the rostral portion of the trachea were cut open opposite the trachealis and prepared for isometric tension measurements. 2. Following the addition of atropine and contraction of the trachealis with prostaglandin F2 alpha (PGF2 alpha), stimulation of the cervical sympathetic trunks elicited relaxations that were abolished by propranolol or hexamethonium. Stimulation of the vagus nerves caudal to the nodose ganglia also elicited relaxations. These vagally mediated relaxations were unaffected by propranolol but were abolished by hexamethonium or by cutting the recurrent laryngeal nerves. 3. After cutting the vagi caudal to the nodose ganglia, stimulation of the vagi rostral to the nodose ganglia elicited relaxations of the trachealis that were not significantly affected by either propranolol or hexamethonium but were abolished by cutting the superior laryngeal nerves. Stimulation of right vagi which had undergone supranodose vagotomy 14 days prior to experimentation was without effect on the smooth muscle of the guinea-pig trachea while the response to stimulation of the left vagus was unchanged. 4. Acute capsaicin desensitization abolished relaxations of the guinea-pig trachealis elicited by stimulation of the vagal fibres carried by the superior laryngeal nerves. In contrast, capsaicin desensitization only modestly inhibited relaxations elicited by stimulation of the preganglionic parasympathetic fibres carried by the recurrent laryngeal nerves and had no effect on sympathetic nerve-induced relaxations. 5. Removing the oesophagus selectively abolished relaxations elicited by stimulation of both vagal pathways of non-adrenergic relaxant innervation. Non-adrenergic relaxations of the trachealis elicited by electrical field stimulation were unaffected by removing the oesophagus. Oesophagus removal also had no effect on the parasympathetic-cholinergic contractile innervation or the sympathetic relaxant innervation of the trachealis. 6. The results indicate that the guinea-pig trachealis receives non-adrenergic relaxant innervation from both parasympathetic and capsaicin-sensitive vagal pathways. The results also suggest that the neurones mediating non-adrenergic relaxations of the trachea are sensitive to oesophagus removal. The observation that oesophagus removal abolishes parasympathetic relaxations of the trachealis while having no effect on parasympathetic contractions supports the hypothesis that the guinea-pig trachealis receives excitatory and inhibitory innervation from distinct vagal parasympathetic pathways.

Localization of heme oxygenase-2 immunoreactivity to parasympathetic ganglia of human and guinea-pig airways.

Carbon monoxide (CO), an activator of soluble guanylate cyclase and generated enzymatically by heme oxygenase-2 (HO-2), is thought to function as an intra- and intercellular neurotransmitter in the central and peripheral nervous system. In the present study, the distribution of HO-2 in airway nerves from both humans and guinea pigs was assessed. HO-2 was found in all neuronal perikarya of the intrinsic ganglia of guinea-pig airways and in all ganglion nerve cell bodies localized to the trachea and bronchi of humans. By contrast, nerve fibers innervating the smooth muscle, lamina propria, and epithelium of the airways in both species were devoid of HO-2 immunoreactivity. HO-1, the inducible isoform of heme oxygenase, was not found in airway nerves. The pattern of distribution of HO-2 observed suggests that CO might serve as a modulator of synaptic neurotransmission in the lung and airways rather than as a bona fide neurotransmitter in the smooth muscle, vasculature, or glands. Consistent with this hypothesis, 8-bromo-cyclic guanosine monophosphate (cGMP) (30 microM), a stable, pharmacologically active analog of cGMP, markedly inhibited vagally-mediated cholinergic contractions of the isolated guinea-pig trachea. In subsequent studies, however, neither inhibiting heme oxygenase with zinc protoporphyrin-IX (30 microM) nor inhibiting the soluble isoform of guanylate cyclase with ODQ (3 microM) had measurable effects on vagally-mediated cholinergic contractions of the trachea. These results indicate that CO could play a modulatory role in efferent (parasympathetic) synaptic neurotransmission in the airways, but under normal conditions may not be activated to an appreciable extent during periods of elevated vagal activity.

Evidence that distinct neural pathways mediate parasympathetic contractions and relaxations of guinea-pig trachealis.

1. The guinea-pig trachea was isolated with its extrinsic innervation intact and pinned to the bottom of a water-jacketed dissecting dish filled with warmed, oxygenated Krebs solution. The trachea was not separated from the oesophagus. Isometric tension was measured in a segment of the rostral portion of the trachea. 2. Stimulation of the vagus nerves caudal to the nodose ganglia elicited contractions of the trachealis that were blocked by the muscarinic receptor antagonist atropine. Following addition of atropine and contraction of the trachealis with prostaglandin F2 alpha (PGF2 alpha), vagus nerve stimulation elicited non-adrenergic, non-cholinergic relaxations. Both responses elicited by stimulation of the vagi were abolished by cutting the recurrent laryngeal nerves and were considered parasympathetic in nature as they were sensitive to the autonomic ganglion blockers trimetaphan and hexamethonium. 3. Experiments were designed in which ganglionic blockers were added to the buffer bathing the entire preparation or, alternatively, added only to the buffer perfusing the tracheal lumen. When given equal access to the trachea and oesophagus, hexamethonium was 56-fold more potent an inhibitor of vagally mediated relaxations of the trachealis than vagally mediated contractions. Selective administration of hexamethonium to the buffer perfusing the tracheal lumen did not decrease the potency of the ganglionic blocker versus vagally mediated contractions. By contrast, even at a concentration of 1 mM, intratracheally administered hexamethonium failed to inhibit vagally mediated relaxations by 50%. Comparable results were obtained using trimetaphan. 4. Consistent with previous observations, removing the portion of the oesophagus contiguous with the region of the trachea at which isometric tension was measured abolished parasympathetic relaxations of the trachealis. Oesophagus removal was without effect on parasympathetic nerve-induced contractions. Removing the dorsal half of the oesophagus or the mucosa and submucosa of the oesophagus did not affect the parasympathetic relaxant innervation. 5. The compound action potential of guinea-pig recurrent laryngeal nerves evoked by vagus nerve stimulation consisted of three distinct peaks representing populations of axons with fast, intermediate and slow conduction velocities. The voltage-response characteristics of vagally mediated contractions were identical to those of the compound action potential peak representing fibres with intermediate (10 m/s) conduction velocities. By contrast, the voltage-response characteristics of the vagally mediated relaxations were best correlated with the compound action potential peak representing fibres with slow (0.4-3 m/s) conduction velocities.(ABSTRACT TRUNCATED AT 400 WORDS)