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Background and purpose: Sensory nerves regulate central and local reflexes such as airway plasma protein leakage, bronchoconstriction and cough. Sensory nerve activity may be enhanced during inflammation such that these protective effects become exacerbated and deleterious. Cannabinoids are known to inhibit airway sensory nerve function. However, there is still controversy surrounding which receptor is involved in eliciting these effects. Experimental approach: We have adopted a pharmacological approach, including using a novel, more selective CB2 receptor agonist, GW 833972A (1000-fold selective CB2/CB1), and receptor selective antagonists to investigate the inhibitory activity of cannabinoids on sensory nerve activity in vitro and in vivo in guinea-pig models of cough and plasma extravasation. Key results: GW 833972A inhibited capsaicin-induced depolarization of the human and guinea-pig and prostaglandin E2 (PGE2) and hypertonic saline-induced depolarization of the guinea-pig isolated vagus nerve in vitro. GW 833972A also inhibited citric acid-induced cough but not plasma extravasation in the guinea-pig and this effect was blocked by a CB2 receptor antagonist. Conclusions and implications: This confirms and extends previous studies highlighting the role of CB2 receptors in the modulation of sensory nerve activity elicited both by the exogenous ligands capsaicin and hypertonic saline but also by endogenous modulators such as PGE2 and low pH stimuli. These data establish the CB2 receptor as an interesting target for the treatment of chronic cough. British Journal of Pharmacology (2008) 155, 547–557; doi:10.1038/bjp.2008.298; published online 11 August 2008

In the present study, we examined the effect of E-ring 8-isoprostanes on cholinergic neurotransmission in guinea-pig trachea and identified the receptor(s) involved. As isoprostanes are isomeric with prostaglandins, PGE2 and sulprostone (a selective EP3-receptor agonist) were examined in parallel. 8-Iso-PGE1, 8-iso-PGE2 (0.1 nM–1 μM), sulprostone (1 nM–1 μM) and PGE2 (1 μM) suppressed EFS-evoked [3H]ACh release from guinea-pig trachea in a concentration-dependent manner, producing 39.5, 53.9, 61.2 and 59.9% inhibition, respectively, at 1 μM. It should be noted that an established maximum effective concentration was not determined. Neither SQ 29,548 (1 μM; a TP-receptor antagonist) nor AH 6809 (10 μM; an EP1-/EP2-/DP-receptor antagonist) reversed the inhibitory effect of these compounds. L-798,106, a novel and highly selective EP3-receptor antagonist, produced a parallel shift to the right of the concentration–response curves that described the inhibitory action of sulprostone on EFS-evoked contractile responses in guinea-pig vas deferens (an established EP3-receptor-expressing tissue), from which a mean pA2 of 7.48 was derived. On guinea-pig trachea, L-798,106 also antagonised sulprostone-induced inhibition of EFS-induced twitch responses, with similar potency (mean pA2=7.82). The inhibitory effects of 8-iso-PGE1, 8-iso-PGE2, sulprostone and PGE2 on EFS-induced [3H]ACh release was blocked by L-798,106 at a concentration (10 μM) that binds only weakly to human recombinant EP1-, EP2- and EP4-receptor subtypes expressed in HEK 293 cells. These data suggest that E-ring 8-isoprostanes, PGE2 and sulprostone inhibit EFS-evoked [3H]ACh release from cholinergic nerves innervating guinea-pig trachea, by interacting with prejunctional prostanoid receptors of the EP3-subtype. British Journal of Pharmacology (2004) 141, 600–609. doi:10.1038/sj.bjp.0705648

1. There is considerable interest in novel therapies for cough, since currently used agents such as codeine have limited beneficial value due to the associated side effects. Sensory nerves in the airways mediate the cough reflex via activation of C-fibres and RARs. Evidence suggests that cannabinoids may inhibit sensory nerve-mediated responses. 2. We have investigated the inhibitory actions of cannabinoids on sensory nerve depolarisation mediated by capsaicin, hypertonic saline and PGE2 on isolated guinea-pig and human vagus nerve preparations, and the cough reflex in conscious guinea-pigs. 3. The non-selective cannabinoid (CB) receptor agonist, CP 55940, and the selective CB2 agonist, JWH 133 inhibited sensory nerve depolarisations of the guinea-pig vagus nerve induced by hypertonic saline, capsaicin and PGE2. These responses were abolished by the CB2 receptor antagonist SR144528, and unaffected by the CB1 antagonist SR141716A. Similarly, JWH 133 inhibited capsaicin-evoked nerve depolarisations in the human vagus nerve, and was prevented by SR144528. 4. Using a guinea-pig in vivo model of cough, JWH 133 (10 mg kg-1, i.p., 20 min) significantly reduced citric acid-induced cough in conscious guinea pigs compared to those treated with the vehicle control. 5. These data show that activation of the CB2 receptor subtype inhibits sensory nerve activation of guinea-pig and human vagus nerve, and the cough reflex in guinea-pigs, suggesting that the development of CB2 agonists, devoid of CB1-mediated central effects, will provide a new and safe antitussive treatment for chronic cough.

Sensory nerves regulate central and local reflexes such as airway plasma leakage, and cough and their function may be enhanced during inflammation. Evidence suggests that dopamine receptor agonists may inhibit sensory nerve-mediated responses. In this study dopamine inhibited vagal sensory nerve induced microvascular leakage in the rat. In order to characterize the receptor involved rat vagus preparations were utilized. Quinagolide (D2/3 agonist), ropinirole (D2/3/4 agonist), SKF 38393 (D1/5 agonist), AR-C68397AA (Viozan™) (dual D2/B2 agonist) and dopamine inhibited hypertonic saline induced depolarization by approximately 50%. Data suggests that AR-C68397AA and quinagolide also inhibited depolarization of the human vagus. The quinagolide response was blocked by sulpiride (D2/3 antagonist) but not SCH 23390 (D1/5 antagonist); ropinirole was partially blocked by sulpiride, totally blocked by spiperone (at a concentration that blocks all dopamine receptors) but not by SCH 23390. The response to SKF 38393 was not blocked by sulpiride but was by SCH 23390. The inhibition evoked by AR-C68397AA was only partially blocked by SCH 23390 but not by sulpiride or spiperone whereas dopamine was blocked by spiperone. The effect of dopamine was not stimulus-specific as it inhibited capsaicin-induced depolarization of the rat vagus in a spiperone sensitive manner. In conclusion, dopamine receptor ligands inhibit depolarization of the rat and human vagus. These data suggest that dopamine receptor agonists may be of therapeutic benefit in the treatment of symptoms such as cough and mucus secretion which are evident in respiratory diseases such as asthma and chronic obstructive pulmonary disease. British Journal of Pharmacology (2002) 136, 620–628; doi:10.1038/sj.bjp.0704758

In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

Elevated levels of nerve growth factor (NGF) have been detected in the bronchoalveolar lavage fluid of patients with asthma. However, the source of this enhanced mediator production is not known. Here, we investigate the production of NGF from a human airway epithelial cell line (A549). Under basal conditions, A549 cells generated NGF in a time-dependent fashion. However, basal release was significantly augmented in a concentration-dependent manner in cells treated with interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) and inhibited by dexamethasone. These data suggest that NGF released from structural cells may be an important target for the anti-inflammatory effects of steroids in asthma therapy.

The anti-spasmogenic potential of SK&F 94120 (PDE3-selective), rolipram (PDE4-selective), zaprinast (PDE5-selective), zardaverine (dual PDE3/4 inhibitor) and theophylline (non-selective) was evaluated in guinea-pig trachealis. SK&F 94120 or rolipram (10 and 100 μM) antagonized histamine-induced tension generation in a concentration-dependent and non-competitive manner whereas ACh-induced contractions were unaffected. Similarly, SK&F 94120 and rolipram in combination were anti-spasmogenic with respect to both contractile agonists to an extent that was greater than the effect of either drug alone. Identical results were obtained with zardaverine (1, 10 and 100 μM) and theophylline (100 μM and 1 mM). Zaprinast protected guinea-pig trachealis against histamine-, but not ACh-induced contractile responses in a manner that was indistinguishable from the results obtained with SK&F 94120. However, in contrast to the interaction between SK&F 94120 and rolipram, no further antagonism was seen when zaprinast and rolipram were used in combination. Pre-treatment of tissues with SNP (10 and 100 μM) antagonized histamine-induced tension generation in a concentration-dependent and non-competitive manner. However, no further antagonism was produced when SNP and rolipram were used concurrently. Likewise, the protection afforded by a combination SNP and SK&F 94120 was no greater than that produced by SNP alone. These results demonstrate that an inhibitor of PDE3 enhances the anti-spasmogenic activity of rolipram but not drugs that elevate cyclic GMP mass. Moreover, the ability of SNP and zaprinast to protect guinea-pig trachealis against histamine-induced contractions apparently is not due to the inhibition of PDE3. British Journal of Pharmacology (1999) 128, 327–336; doi:10.1038/sj.bjp.0702779

We have assessed the effect of sensitization to allergen on airway smooth muscle responsiveness and acetylcholine (ACh) release from cholinergic nerves in tracheal preparations from rabbits immunized at birth to Alternaria tenuis and littermate control rabbits injected with saline. ACh release induced by EFS was significantly greater in tracheal preparations obtained from immunized rabbits compared with littermate controls. The ability of the muscarinic-receptor agonist, oxotremorine, to inhibit ACh release to EFS (4 Hz) was not altered by immunization. The contractile response evoked by electrical field stimulation (EFS), ACh and 5-hydroxytryptamine (5-HT) was not significantly altered in tracheal preparations from antigen immunized rabbits compared with littermate controls. Antigen challenge of immunized rabbits did not affect the release of ACh from isolated trachea following EFS, or the ability of oxotremorine to inhibit ACh release. Furthermore, antigen challenge of immunized rabbits failed to alter the contractile response to EFS or ACh, but reduced the contractile potency of 5-HT. These results demonstrate increased ACh release in tracheal preparations following immunization which had no functional consequence on airway smooth muscle responsiveness. Moreover, the increased release in ACh was not associated with an alteration in M(2)-receptor function. Thus, antigen-induced bronchial hyperresponsiveness in the rabbit does not appear to depend upon M(2)-receptor dysfunction.

Eotaxin is a novel C-C chemokine with selective chemoattractant activity for eosinophils. We determined whether eotaxin could be produced by human airway smooth muscle (HASM) cells in culture and examined its regulation by interleukin-10 (IL-10) and the corticosteroid, dexamethasone. Stimulation of the cells with interleukin-1beta (IL-1beta) or tumour necrosis factor (TNFalpha) each at 10 ng ml(-1) induced the release of eotaxin protein with maximal accumulation by 24 h. Interferon-gamma (IFNgamma) alone at 10 ng ml(-1) had no effect and there was no synergy between these cytokines on the release of eotaxin. Reverse phase high performance liquid chromatographic (HPLC) analysis of supernatents from cells treated with TNFalpha (10 ng ml(-1) for 96 h showed immunoreactivity to eotaxin which eluted with the expected retention time of 34.5-35 min. Both IL-1beta and TNFalpha-induced release of eotaxin was not inhibited by dexamethasone (1 microM), however IL-10 (10 ng ml(-1)) had a significant inhibitory effect. Dexamethasone and IL-10 did not inhibit the induction of eotaxin mRNA induced by IL-1beta or TNFalpha. Thus, human airway smooth muscle cells can release eotaxin and could be an important source of chemokine production during airway inflammatory events.

In this study we have evaluated the pharmacological profile of the muscarinic antagonist glycopyrrolate in guinea-pig and human airways in comparison with the commonly used antagonist ipratropium bromide. Glycopyrrolate and ipratropium bromide inhibited EFS-induced contraction of guinea-pig trachea and human airways in a concentration-dependent manner. Glycopyrrolate was more potent than ipratropium bromide. The onset of action (time to attainment of 50% of maximum response) of glycopyrrolate was similar to that obtained with ipratropium bromide in both preparations. In guinea-pig trachea, the offset of action (time taken for response to return to 50% recovery after wash out of the test antagonist) for glycopyrrolate (t1/2 [offset]=26.4±0.5 min) was less than that obtained with ipratropium bromide (81.2±3.7 min). In human airways, however, the duration of action of glycopyrrolate (t1/2 [offset]>96 min) was significantly more prolonged compared to ipratropium bromide (t1/2 [offset]=59.2±17.8 min). In competition studies, glycopyrrolate and ipratropium bromide bind human peripheral lung and human airway smooth muscle (HASM) muscarinic receptors with affinities in the nanomolar range (Ki values 0.5–3.6 nM). Similar to ipratropium bromide, glycopyrrolate showed no selectivity in its binding to the M1–M3 receptors. Kinetics studies, however, showed that glycopyrrolate dissociates slowly from HASM muscarinic receptors (60% protection against [3H]-NMS binding at 30 nM) compared to ipratropium bromide. These results suggest that glycopyrrolate bind human and guinea-pig airway muscarinic receptors with high affinity. Furthermore, we suggest that the slow dissociation profile of glycopyrrolate might be the underlying mechanism by which this drug accomplishes its long duration of action. British Journal of Pharmacology (1999) 127, 413–420; doi:10.1038/sj.bjp.0702573

1. Previous studies have provided evidence that activation of beta-adrenoceptors on cholinergic nerve terminals can inhibit neurotransmission in the airways. However, in most cases, this conclusion has been based on indirect evidence obtained from mechanical experiments where changes in airways smooth muscle tone were measured. 2. We have assessed whether modulation of cholinergic neurotransmission by beta-adrenoceptor agonists is due to a pre- or post-junctional action by investigating the effect of isoprenaline on contractile responses evoked by exogenous acetylcholine (ACh) and electrical field stimulation (EFS; 4 Hz, 40 V, 0.5 ms pulse width every 15 s), and on EFS-induced ACh release from cholinergic nerves innervating guinea-pig and human trachea. Furthermore, the subtype of beta-adrenoceptor which modulates neurotransmission and the potential role of cyclic AMP in this response were evaluated. 3. In guinea-pig trachea, isoprenaline (1 nM-1 microM) inhibited the contractile response evoked by exogenous ACh (1 microM) to a similar extent to that evoked by EFS (EC50 = 19.9 and 23 nM, respectively). 4. In epithelium-denuded guinea-pig strips treated with indomethacin (10 microM), isoprenaline significantly enhanced EFS-induced ACh release from cholinergic nerve terminals (by 36% at 0.3 microM). This effect was blocked by propranolol and ICI 118, 551 (each 0.1 microM). In contrast, isoprenaline failed to affect EFS-induced ACh release from parasympathetic nerves innervating human trachea. 5. To evaluate the role of cyclic AMP in the beta-adrenoceptor-induced facilitation of cholinergic neurotransmission, the effects of various cyclic AMP elevating drugs on ACh release were studied. Forskolin (10 microM) significantly augmented (by 17%) EFS-induced ACh release, an effect which was not reproduced by 1,9-dideoxyforskolin (10 microM) which does not activate adenylyl cyclase. Similarly, the cyclic AMP analogue, 8-bromo-cyclic AMP (1 mM) and cholera toxin (1 microgram ml-1) facilitated ACh output by 22 and 47% respectively, whereas prostaglandin E2 (PGE2, 0.1 nM-1 microM) inhibited this response (by 67% at 1 microM). 6. Zardaverine (10 microM), a dual inhibitor of the phosphodiesterase (PDE)3 and PDE4 isoenzyme families, did not affect EFS-induced ACh release and failed to facilitate the actions of either isoprenaline or PGE2. Similarly, neither SK&F 94120 (10 microM) nor rolipram (10 microM), selective inhibitors of PDE3 and PDE4 respectively, significantly affected the release of ACh in response to EFS. 7. The result of this study suggests that isoprenaline facilitates cholinergic neurotransmission in guinea-pig, but not human, trachea by activation of pre-junctional beta 2-adrenoceptors, an effect that may be mediated via activation of the cyclic AMP/cyclic AMP-dependent protein kinase cascade. Furthermore, the data presented herein illustrate the need to undertake direct measurements of neurotransmitter release when examining the effect of agents purported to act pre-junctionally.

1. In the present study, we examined the effect of E-ring 8-isoprostanes on cholinergic neurotransmission in guinea-pig trachea and identified the receptor(s) involved. As isoprostanes are isomeric with prostaglandins, PGE(2) and sulprostone (a selective EP(3)-receptor agonist) were examined in parallel. 2. 8-Iso-PGE(1), 8-iso-PGE(2) (0.1 nm-1 microM), sulprostone (1 nm-1 microM) and PGE(2) (1 microM) suppressed EFS-evoked [(3)H]ACh release from guinea-pig trachea in a concentration-dependent manner, producing 39.5, 53.9, 61.2 and 59.9% inhibition, respectively, at 1 microM. It should be noted that an established maximum effective concentration was not determined. 3. Neither SQ 29,548 (1 microm; a TP-receptor antagonist) nor AH 6809 (10 microM; an EP(1)-/EP(2)-/DP-receptor antagonist) reversed the inhibitory effect of these compounds. 4. L-798,106, a novel and highly selective EP(3)-receptor antagonist, produced a parallel shift to the right of the concentration-response curves that described the inhibitory action of sulprostone on EFS-evoked contractile responses in guinea-pig vas deferens (an established EP(3)-receptor-expressing tissue), from which a mean pA(2) of 7.48 was derived. On guinea-pig trachea, L-798,106 also antagonised sulprostone-induced inhibition of EFS-induced twitch responses, with similar potency (mean pA(2)=7.82). 5. The inhibitory effects of 8-iso-PGE(1), 8-iso-PGE(2), sulprostone and PGE(2) on EFS-induced [(3)H]ACh release was blocked by L-798,106 at a concentration (10 microM) that binds only weakly to human recombinant EP(1)-, EP(2)- and EP(4)-receptor subtypes expressed in HEK 293 cells. 6. These data suggest that E-ring 8-isoprostanes, PGE(2) and sulprostone inhibit EFS-evoked [(3)H]ACh release from cholinergic nerves innervating guinea-pig trachea, by interacting with prejunctional prostanoid receptors of the EP(3)-subtype.

Airway smooth muscle is actively involved in the inflammatory process in diseases such as chronic obstructive pulmonary disease and asthma by 1) contributing to airway narrowing through hyperplasia and hypertrophy and 2) the release of GM-CSF and G-CSF, which promotes the survival and activation of infiltrating leukocytes. Thus, the identification of novel anti-inflammatory pathways in airway smooth muscle will have important implications for the treatment of inflammatory airway disease. This study identifies such a pathway in the activation of peroxisome proliferator-activated receptors (PPARs). PPAR ligands are known therapeutic agents in the treatment of diabetes; however, their role in human airway disease is unknown. We demonstrate, for the first time, that human airway smooth muscle cells express PPARα and -γ subtypes. Activation of PPARγ by natural and synthetic ligands inhibits serum-induced cell growth more effectively than does the steroid dexamethasone, and induces apoptosis. Moreover, PPARγ activation, like dexamethasone, inhibits the release of GM-CSF. However, PPARγ ligands, but not dexamethasone, similarly inhibits G-CSF release. These results reveal a novel anti-inflammatory pathway in human airway smooth muscle, where PPARγ activation has additional anti-inflammatory effects to those of steroids. Hence, PPAR ligands might act as potential treatments in human respiratory diseases.

Sensory nerves regulate central and local reflexes such as airway plasma leakage, and cough and their function may be enhanced during inflammation. Evidence suggests that dopamine receptor agonists may inhibit sensory nerve-mediated responses. In this study dopamine inhibited vagal sensory nerve induced microvascular leakage in the rat. In order to characterize the receptor involved rat vagus preparations were utilized. Quinagolide (D(2/3) agonist), ropinirole (D(2/3/4) agonist), SKF 38393 (D(1/5) agonist), AR-C68397AA (Viozan) (dual D(2)/B(2) agonist) and dopamine inhibited hypertonic saline induced depolarization by approximately 50%. Data suggests that AR-C68397AA and quinagolide also inhibited depolarization of the human vagus. The quinagolide response was blocked by sulpiride (D(2/3) antagonist) but not SCH 23390 (D(1/5) antagonist); ropinirole was partially blocked by sulpiride, totally blocked by spiperone (at a concentration that blocks all dopamine receptors) but not by SCH 23390. The response to SKF 38393 was not blocked by sulpiride but was by SCH 23390. The inhibition evoked by AR-C68397AA was only partially blocked by SCH 23390 but not by sulpiride or spiperone whereas dopamine was blocked by spiperone. The effect of dopamine was not stimulus-specific as it inhibited capsaicin-induced depolarization of the rat vagus in a spiperone sensitive manner. In conclusion, dopamine receptor ligands inhibit depolarization of the rat and human vagus. These data suggest that dopamine receptor agonists may be of therapeutic benefit in the treatment of symptoms such as cough and mucus secretion which are evident in respiratory diseases such as asthma and chronic obstructive pulmonary disease.

Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

Endomorphin-1 and -2 (mu-opioid receptor agonists) produced a concentration-dependent and naloxone-sensitive inhibition of cholinergic contractile responses in guinea-pig trachea (at 10 microM, 46.1 +/- 8.0% and 33.8 +/- 8.6%, respectively). Endomorphin-1 and -2 also inhibited electrically-evoked acetylcholine release from cholinergic nerves innervating guinea-pig (at 0.1 microM, 41.8 +/- 10.9%; at 1 microM 60.1 +/- 6.3%, respectively) and human trachea (at 10 microM, 76.2 +/- 18.1%, and 77.7 +/- 14.3%, respectively). Naloxone prevented the inhibition by endomorphin-1 and -2 in both guinea-pig and human trachea, suggesting that these peptides can inhibit cholinergic, parasympathetic neurotransmission to the airways via the activation of classical opioid receptors.

The novel peptide, nociceptin and the mu-opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) produced a concentration-dependent inhibition of electrical field stimulation (EFS)-evoked release of acetylcholine (ACh) from cholinergic nerves innervating guinea-pig trachea. The non-selective opioid receptor antagonist, naloxone, did not antagonize the inhibitory action of nociceptin under conditions where the inhibition of ACh release evoked by DAMGO was completely reversed. It is suggested that DAMGO and nociceptin can inhibit cholinergic, parasympathetic neurotransmission to the airways via the activation of classical (naloxone-sensitive) and novel (naloxone-insensitive) opioid receptors, respectively.

Functional studies suggest the presence of prejunctional muscarinic autoreceptors on cholinergic nerves in human airways. However, these studies are an indirect method of evaluating changes in neurally evoked acetylcholine (ACh) release. We have investigated the presence of muscarinic autoreceptors in human and guinea pig trachea by comparing the effects of the muscarinic receptor antagonists pirenzepine (M1), methoctramine (M2), 4-DAMP (M3), and rispenzepine (M1/M3) on cholinergic neural contractile responses evoked by electrical field stimulation (EFS) and [3H]ACh release. The M1, M1/M3, or M3 antagonists inhibited the EFS-evoked cholinergic contractile response in a concentration-dependent manner (4-DAMPrispenzepinepirenzepine), whereas methoctramine facilitated this response at low concentrations (3 microM). In ACh release studies, the M3 antagonist had no significant effect, whereas pirenzepine, methoctramine, and rispenzepine significantly increased ACh release in guinea pig trachea. In contrast, ACh release was significantly inhibited by the muscarinic agonist oxotremorine M. Methoctramine and the nonselective antagonist ipratropium bromide, but not the M1, M1/M3, or M3 antagonists, significantly increased ACh release in human trachea. These data suggest the presence of an autoinhibitory receptor on cholinergic nerve terminals in human and guinea pig trachea. In addition, the action of ipratropium bromide at the autoinhibitory receptor may limit its use in the treatment of obstructive airways disease.