Role of kinins in anaphylactic-induced bronchoconstriction mediated by tachykinins in guinea-pigs.

1. In the present study, we have investigated the role of kinins in allergen-induced bronchoconstriction. 2. Anaesthetized guinea-pigs were sensitized to ovalbumin, ventilated artificially, pretreated with atropine (1.4 mumol kg-1, i.v.) and total pulmonary resistance (RL) measured. In preliminary studies in the presence of the neutral endopeptidase inhibitor, phosphoramidon (4.5 mumol kg-1, i.v.), the bradykinin B2 receptor antagonist Hoe 140 (0.1 mumol kg-1, i.v.) completely abolished the increase in RL following aerosolized bradykinin (1 mM, 40 breaths), but had no effect on the increase in RL following aerosolized neurokinin A (NKA, 10 microM, 40 breaths). On the other hand, a combination of the NK1 (CP-96,345, 2 mumol kg-1, i.v.) and NK2 (SR 48968, 0.3 mumol kg-1, i.v.) tachykinin receptor antagonists abolished completely the increase in RL produced by NKA and partially inhibited the increase in RL produced by bradykinin. These results confirm previous studies that suggest that bradykinin induces the release of tachykinins from sensory nerves in guinea-pig airways. 3. Aerosolized ovalbumin (0.5%, 5 breaths) increased RL in sensitized guinea-pigs pretreated with atropine (1.4 mmol kg-1, i.v.), an effect that began within 2 min and reached a maximum within 5 min; RL remained above baseline at 20 min. Pretreatment with the bradykinin B2 receptor antagonist, Hoe 140, decreased the bronchoconstrictor effect of ovalbumin markedly at 10 to 20 min. In the presence of phosphoramidon (4.5 mumol kg-1, i.v.) the inhibition induced by Hoe 140 was apparent earlier and remained over the 20 min period of study. 4. Pretreatment with a combination of NK1 (CP-96,345) and NK2 (SR 48968) tachykinin receptor antagonists also markedly inhibited ovalbumin-induced bronchoconstriction; addition of the bradykinin B2 receptor antagonist to the NK1 and NK2 tachykinin receptor antagonists had no additional inhibitory effect on antigen-induced bronchoconstriction.5. These findings confirm that activation of sensory nerves to release tachykinins in guinea-pig airways contribute to antigen-induced bronchoconstriction, and provide evidence that tachykinin release is due to kinins generated during the allergic response.

Involvement of neurogenic inflammation in antigen-induced bronchoconstriction in guinea pigs.

The role of tachykinins released from sensory nerves in bronchoconstriction induced by antigen was studied in sensitized guinea pigs anesthetized with pentobarbital sodium and pretreated with atropine. The combination of NK2 (SR-48968) and NK1 (CP-96,345) tachykinin-receptor antagonists abolished the increase in total pulmonary resistance (RL) evoked by intravenous capsaicin but did not affect the response evoked by intravenous histamine. A small dose of aerosolized ovalbumin (OVA, 0.1%) produced a small increase in RL that was further increased and markedly prolonged by the neutral endopeptidase (NEP) inhibitor phosphoramidon; this bronchoconstrictor effect of OVA was markedly reduced by the NK2-receptor antagonist and was abolished by the combination of the NK1 and NK2-receptor antagonists together. When a larger dose of OVA (0.5%) was used, a maximal bronchoconstrictor response was obtained. Phosphoramidon did not potentiate this response significantly. The combination of NK1- and NK2-receptor antagonists blunted the response at 5 min only slightly but markedly attenuated the later (10-20 min) response. These results show that tachykinins released from sensory nerves play a significant role in antigen-induced bronchoconstriction in guinea pigs. This effect is exaggerated when the normal modulation of neuropeptides by NEP is inhibited and is mediated predominantly by NK2-receptor activation, with a smaller contribution by NK1 receptors.

Capsaicin increases airflow resistance in guinea pigs in vivo by activating both NK2 and NK1 tachykinin receptors.

The role of NK1 tachykinin receptors in the increase in total pulmonary resistance (RL) produced by release of endogenous tachykinins was investigated in anesthetized guinea pigs pretreated intravenously with atropine (1 mumol/kg) by using the novel nonpeptide antagonists of NK1 (CP-96,345) and NK2 (SR 48968) tachykinin receptors. SR 48968 (0.3 mumol/kg) and CP-96,345 (2 mumol/kg) given intravenously completely blocked the response to the selective NK2 receptor agonist [beta Ala8]neurokinin A(4-10) and NK1 receptor agonist [Sar9,Met(O2)11]substance P, respectively. The response to neurokinin A was reduced dose-dependently, but not abolished, by SR 48968, and it was completely prevented by a combination of SR 48968 and CP-96,345. The response to capsaicin was reduced, but not blocked, by SR 48968, and it was completely abolished by the combination of the two antagonists. The combination of SR 48968 and CP-96,345 did not affect the increase in RL evoked by histamine. Thus, NK1, as well as NK2, receptor activation contributes to the noncholinergic increase in RL evoked by capsaicin and ascribed to release of endogenous tachykinins from sensory nerves.

Interleukin-8 induces neutrophil accumulation but not protease secretion in the canine trachea.

The neutrophil enzyme elastase is a potent secretagogue of airway secretory cells, and elastase is present in high concentrations in sputum of patients with hypersecretion (e.g., cystic fibrosis, bronchiectasis). Interleukin-8 (IL-8), a recently discovered cytokine with potent neutrophil chemotactic properties in vitro, is also found in the sputum of these patients. We used an isolated tracheal segment in dogs in vivo to study the effect of IL-8 in causing neutrophil accumulation, elastase release, and secretion (by measuring lysozyme concentrations) in the luminal superfusate. IL-8 caused a potent time-dependent neutrophil accumulation at between 3 and 6 h. The effect was significant at 10(-9) and maximum at 10(-8) M. No increase in free elastase, cathepsin G, or lysozyme was detected in the superfusate. Thus, in contrast to previous studies showing that ragweed antigen causes the accumulation of neutrophil elastase which in turn causes lysozyme secretion, IL-8 causes neutrophil accumulation without granule secretion (or subsequent secretagogue activity). The findings were confirmed with dog and human neutrophils in vitro.

Bronchial smooth muscle responses evoked by toluene diisocyanate are inhibited by ruthenium red and by indomethacin.

We have investigated the ability of ruthenium red, an inorganic dye with Ca2+ entry-blocking properties and a selective antagonist of capsaicin, and of indomethacin, a cyclooxygenase inhibitor, to inhibit bronchial smooth muscle responses evoked by toluene diisocyanate in guinea pigs. Previous exposure of isolated guinea pig bronchi to ruthenium red significantly decreased the response produced by toluene diisocyanate. Further, the response to toluene diisocyanate was significantly decreased by pretreatment with indomethacin. These findings provide evidence that toluene diisocyanate-induced contractions of guinea pig bronchi are produced indirectly by generation of a prostanoid that activates capsaicin-sensitive afferents via a ruthenium red-sensitive mechanism.

Toluene diisocyanate contracts guinea pig bronchial smooth muscle by activating capsaicin-sensitive sensory nerves.

This study was designed to evaluate the mechanism of action of toluene diisocyanate (TDI) and the role of endogenous neutral endopeptidase in modulating in vitro contractile responses to TDI in guinea pigs. TDI (0.01-1 mM) produced a concentration-dependent contraction of the guinea pig main bronchi. Sensory nerve desensitization with capsaicin greatly reduced and in some cases almost abolished TDI-induced contractions. The neutral endopeptidase inhibitor phosphoramidon significantly increased the contractile response to TDI. Pretreatment with the substance P antagonist (D-Arg1,D-Pro2,D-Trp7,9,Leu11)-substance P greatly reduced TDI-induced contractions. These results suggest that TDI activates the "efferent" function of capsaicin-sensitive sensory nerves and that neutral endopeptidase may play a role in modulating the response in guinea pigs.

Mast cell chymase potentiates histamine-induced wheal formation in the skin of ragweed-allergic dogs.

Skin mast cells release the neutral protease chymase along with histamine during degranulation. To test the hypothesis that chymase modulates histamine-induced plasma extravasation, we measured wheal formation following intradermal injection of purified mast cell chymase and histamine into the skin of ragweed-allergic dogs. We found that chymase greatly augments histamine-induced wheal formation. The magnitude of the potentiating effect increases with increasing doses of chymase and becomes maximal approximately 30 min after administration. Injection of chymase without histamine does not evoke wheal formation. The chymase potentiation of histamine-induced skin responses is prevented completely by pretreatment with the H1-receptor antagonist pyrilamine, and is prevented by inactivation of chymase with soybean trypsin inhibitor, suggesting that both histamine and preserved catalytic activity are required for the effects of chymase. To examine the effects of histamine and chymase released in situ in further experiments, we measured wheal size following local degranulation of mast cells by intradermal injection of ragweed antigen or compound 48/80. We found that pretreatment with either soybean trypsin inhibitor or pyrilamine markedly reduces ragweed antigen- or 48/80-induced wheal formation, supporting the results obtained by injection of exogenous chymase and histamine. These findings suggest a novel and important proinflammatory role for chymase in modulating the effects of histamine on vascular permeability during mast cell activation.

Somatostatin potentiates cholinergic neurotransmission in ferret trachea.

We studied the effect of somatostatin on contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. Somatostatin (up to 10(-5) M) did not change resting tension, but it potentiated the contractile response to EFS dose dependently, with a maximum effect at 10(-6) M. Thus, at a concentration of 10(-6) M, somatostatin significantly decreased the mean log of EFS frequency producing 50% of maximum contraction from a control value of 0.52 +/- 0.07 to 0.24 +/- 0.06 (SE) Hz (P less than 0.01). The potentiating effect of somatostatin (10(-6) M) was not inhibited by hexamethonium, indomethacin, BW755C, pyrilamine, methysergide, or D,Pro2,D,Trp7,9-SP, but it was inhibited by atropine or by the somatostatin antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)]. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by somatostatin at a concentration of 10(-6) M. These results suggest that somatostatin potentiates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific somatostatin receptor.

Prostaglandin D2 causes accumulation of eosinophils in the lumen of the dog trachea.

Prostaglandin D2 (PGD2), the major product of arachidonic acid metabolism via the cyclooxygenase pathway in most mast cells, is present in the airways of atopic asthmatic patients after antigen challenge. Because eosinophilia is characteristic of asthma, we asked whether PGD2 causes eosinophils to accumulate in the airways in vivo. Using an endotracheal tube with two inflatable balloons we isolated a segment of trachea in four anesthetized mechanically ventilated dogs, and we superfused this segment with either a control solution (Hanks' balanced salt solution and antibiotics) or solution containing PGD2 (10(-6) M). Total and differential cell counts were determined at base line and every hour for 4 h during the study. PGD2 caused eosinophil accumulation in the trachea [7.0 +/- 3.4, 28.7 +/- 17.8, 33.7 +/- 13.6, and 35.4 +/- 10.7 (SD) cells/cm2 trachea after 1, 2, 3, and 4 h, respectively, P less than 0.05 vs. controls] but had no significant effect on neutrophil accumulation. The effect of PGD2 on eosinophil accumulation was significantly inhibited by the prostaglandin receptor antagonist SKF 88046 (5 mg/kg iv). We conclude that PGD2 is a selective stimulus that causes accumulation of eosinophils in the tracheal lumen of dogs in vivo.

Mast cell tryptase and chymase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret.

Recent evidence suggests that nonadrenergic airway relaxation may be controlled by vasoactive intestinal peptide (VIP). The magnitude and duration of smooth muscle relaxation in response to VIP may be influenced by rates of peptide degradation after release from efferent peptidergic neurons. To explore the potential role of mast cell mediators in modulating neural control of airway tone, we studied the effect of the mast cell proteases tryptase and chymase on airway smooth muscle relaxation induced by VIP in ferret airway. Tracheal rings precontracted by serotonin (10(-6) M) in a muscle bath were relaxed by VIP (10(-7) M). We found that protease-rich supernatant obtained by degranulation of dog mastocytoma cells reversed VIP-induced relaxation, as did highly purified tryptase and chymase incubated with the tracheal rings. Either enzyme completely reversed the effect of VIP, but tryptase was more potent than chymase, paralleling previous test tube observations on the relative rates of VIP cleavage by the two enzymes. Inhibitors of mast cell tryptase and chymase preincubated with the supernatant or with the purified proteases prevented reversal of VIP-induced relaxation. Mast cell proteases did not reverse the tracheal relaxation caused by the nonpeptide adrenergic agonist isoproterenol. These findings show that mast cell proteases tryptase and chymase counteract the smooth muscle relaxant effects of VIP in ferret trachea and suggest a potential role for the mast cell proteases in the modulation of nonadrenergic neural control of airway tone by VIP.

Inhibitors of neutral endopeptidase potentiate electrically and capsaicin-induced noncholinergic contraction in guinea pig bronchi.

To evaluate the role of airway neutral endopeptidase (NEP) in the regulation of contraction of airway smooth muscle in response to endogenous tachykinins, we studied the effects of the NEP inhibitor phosphoramidon on contractions of guinea pig bronchial smooth muscle strips induced by either electrical field stimulation (EFS) or by capsaicin. In the presence of atropine (10(-6) M), propranolol (10(-6) M), phentolamine (10(-5) M), indomethacin (10(-6) M) and pyrilamine (5 x 10(-6) M) EFS (biphasic; pulse width, 1.0 msec; frequency 0.5-5 Hz for 30 sec; intensity, 20 V) produced noncholinergic, nonadrenergic muscle contraction in a frequency-dependent fashion (P less than .001). Phosphoramidon potentiated the contractile responses to EFS (P less than .01). Leucine-thiorphan (10(-5) M), another NEP inhibitor, potentiated EFS-induced contraction in a similar fashion as phosphoramidon (186 and 182% of control, respectively; each comparison, P less than .025). Captopril, bestatin, leupeptin and physostigmine (each drug, 10(-5) M) were without effect (P greater than .5, N = 5). Capsaicin (1.5 x 10(-8) M) produced long-lasting atropine-resistant smooth muscle contraction, an effect potentiated by phosphoramidon (10(-5) M (P less than .001). Removal of the epithelium slightly but significantly (P less than .05) increased the contractile responses to capsaicin and to EFS at impulse frequencies of 2 and 5 Hz, and phosphoramidon substantially increased contractions in tissues without epithelium. The trachea, bronchi and lungs each contained significant NEP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway neutral endopeptidase-like enzyme modulates tachykinin-induced bronchoconstriction in vivo.

To determine whether neutral endopeptidase (NEP), also called enkephalinase (EC 3.4.24.11), modulates the effects of exogenous and endogenous tachykinins in vivo, we studied the effects of aerosolized phosphoramidon, a specific NEP inhibitor, on the responses to aerosolized substance P (SP) and on the atropine-resistant response to vagus nerve stimulation (10 V, 5 ms for 20 s) in guinea pigs. SP alone (10(-7) to 10(-4) M; each concentration, 7 breaths) caused no change in total pulmonary resistance (RL, P greater than 0.5). Phosphoramidon (10(-4) M, 90 breaths) caused no change either in base-line RL (P greater than 0.5) or in the response to aerosolized acetylcholine (P greater than 0.5). However, in the presence of phosphoramidon, SP (7 breaths) produced a concentration-dependent increase in RL at concentrations greater than or equal to 10(-5) M (P less than 0.001). Phosphoramidon (10(-7) to 10(-4) M; each concentration, 90 breaths) induced a concentration-dependent potentiation of SP-induced bronchoconstriction (10(-4) M, 7 breaths; P less than 0.01). Vagus nerve stimulation (0.5-3 Hz), in the presence of atropine, induced a frequency-dependent increase in RL (P less than 0.001). Phosphoramidon potentiated the atropine-resistant responses to vagus nerve stimulation (P less than 0.001) at frequencies greater than 0.5 Hz. The tachykinin antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-substance P abolished the effects of phosphoramidon on the atropine-resistant response to vagus nerve stimulation (2 Hz, P less than 0.005). NEP-like activity in tracheal homogenates of guinea pig was inhibited by phosphoramidon with a concentration producing 50% inhibition of 5.3 +/- 0.8 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutral endopeptidase inhibitors potentiate substance P- and capsaicin-induced cough in awake guinea pigs.

To study the roles of substance P and endogenous neutral endopeptidase in mediating cough, we measured cough responses in awake guinea pigs in response to exogenous substance P and capsaicin aerosols in the presence and absence of the neutral endopeptidase inhibitors leucine-thiorphan and phosphoramidon. Substance P stimulated cough in very low concentrations (10(-17)-10(-16) M). In a second study where the investigator did not know whether substance P or diluent alone was aerosolized, substance P (10(-16) M) caused cough. Leucine-thiorphan (10(-5) M) and phosphoramidon (10(-5) M) potentiated substance P-induced cough; NEP inhibitors also potentiated capsaicin-induced cough significantly. These findings suggest that substance P is a potent stimulator of cough responses, that capsaicin-induced cough is mediated by substance P or another similar neuropeptide, and that cough responses are modulated by endogenous neutral endopeptidase.

Modulation of cholinergic neurotransmission by vasoactive intestinal peptide in ferret trachea.

We studied the effect of vasoactive intestinal peptide (VIP) on the contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. VIP did not change resting tension up to 2 X 10(-7) M, but it showed a biphasic effect on the responses to EFS. In concentrations up to 10(-9) M, VIP potentiated the response; at higher concentrations VIP reduced responses. Thus, at a concentration of 10(-9) M, VIP decreased the mean (+/- SE) log EFS frequency, producing 50% of maximum contraction significantly from a control value of 0.476 +/- 0.062 to 0.214 +/- 0.057 Hz (P less than 0.01); at a concentration of 2 X 10(-7) M VIP increased the half-maximal frequency from a control value of 0.513 +/- 0.086 to 0.752 +/- 0.053 Hz (P less than 0.05). The potentiating effect of VIP (10(-9) M) was not inhibited by hexamethonium, indomethacin, pyrilamine, methysergide, or [D-Pro2,D-Trp7,9] substance P. The inhibitory effect of VIP (2 X 10(-7) M) was also not inhibited by hexamethonium, indomethacin, or naloxone. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by VIP at concentrations of 10(-9) and 2 X 10(-7) M. These results suggest that VIP modulates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific VIP receptor.

An in vivo chemotaxis assay in the dog trachea: evidence for chemotactic activity of 8,15-diHETE.

We describe a new in vivo chemotaxis assay in the dog trachea using a double-balloon endotracheal catheter. When inflated, the two balloons isolate a segment of trachea, which is perfused through Silastic tubes using a peristaltic pump. After instilling a chemotactic agent, the perfusate is sampled periodically to permit characterization of the chemotactic response. We anesthetized four mongrel dogs and ventilated them mechanically through the double-balloon catheter. Two mediators, leukotriene B4 (LTB4) and 8S,15S-dihydroxyeicosatetraenoic acid (8,15-diHETE) were tested in each dog by perfusing the trachea with each mediator in Hanks' balanced salt solution (HBSS) containing ethanol and antibiotics. Aliquots were removed for differential cell counts at fixed time intervals over a 4-h period. Control experiments performed in each dog with the identical concentrations of ethanol and antibiotics in HBSS showed no cellular response before 180 min. At 240 min, the cell counts were 86 +/- 28 (SE) granulocytes/microliter (n = 4). In contrast, both LTB4 and 8,15-diHETE gave a significant cellular response at 120 min (309 +/- 125 and 141 +/- 41 granulocytes/microliter, respectively; P less than 0.05) but did not differ significantly from each other. These results suggest that both LTB4 and 8,15-diHETE can incite inflammatory responses in the dog trachea in vivo. Furthermore, the double-balloon catheter technique promises to be a useful in vivo chemotaxis assay.

Neutral endopeptidase and angiotensin converting enzyme inhibitors potentiate kinin-induced contraction of ferret trachea.

Neutral endopeptidase (NEP) (enkephalinase, EC 3.4.24.11) and angiotensin converting enzyme (ACE) are two peptidases that can cleave the C-terminal dipeptide bradykinin(8-9) from bradykinin. To determine whether these peptidases play roles in modulating kinin-induced contractions in the airways, we studied the effects of captopril, an ACE inhibitor, and of leucine-thiorphan and phosphoramidon, two NEP inhibitors, on the contractile responses to bradykinin and lysyl-bradykinin in isolated segments of ferret trachea. Bradykinin and lysyl-bradykinin-induced contractions in a concentration-dependent fashion (P less than .001), with a threshold of 10(-7) M and 5 x 10(-7) M, respectively. In contrast, the bradykinin(8-9) and the N-terminal heptapeptide bradykinin(1-7), the major fragments of hydrolysis of bradykinin by NEP and ACE, had a very weak or no effect on tracheal contraction in concentrations as great as 10(-5) M. Captopril, leucine-thiorphan and phosphoramidon (each inhibitor at 10(-5) M, 15 min) shifted the concentration-response curves to lower concentrations by approximately 1 to 1.5 log U (P less than .05). Both NEP inhibitors and the ACE inhibitor potentiated the response to bradykinin in a concentration-dependent fashion (P = .0001), and the combination of phosphoramidon and captopril resulted in an additive potentiation of bradykinin-induced contraction (P less than .02). [D-Pro2-D-Trp7,9]-substance P, a substance P antagonist, did not modify the potentiation of bradykinin-induced contraction by NEP inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea.

To determine the roles of endogenous enkephalinase (EC.3.4.24.11) in regulating tachykinin-induced contraction of airway smooth muscle, the authors studied the effects of the enkephalinase inhibitor leucine-thiorphan on the contractile responses to substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) in isolated ferret tracheal smooth muscle segments. Leucine-thiorphan shifted, in concentration-dependent fashions, the dose-response curves to all tachykinins to lower concentrations. Leucine-thiorphan changed the rank order of tachykinin potency from NKA greater than SP greater than NKB to NKA = NKB greater than SP. Removal of the epithelium slightly enhanced the contractile responses to SP and NKA but not to NKB. Atropine shifted the dose-response curves of all tachykinins to higher concentrations. Each tachykinin increased the contractile response to electrical field stimulation (5 Hz, 20 sec of duration, 20 V) in a dose-dependent fashion. This effect was not altered by hexamethonium, indomethacin, BW755C or naloxone but was potentiated by leucine-thiorphan and inhibited by the tachykinin receptor antagonist (D-Pro2, D-Trp7,9)-SP and by atropine. Because tachykinins did not affect contractile responses to acetylcholine significantly, their effects were probably on presynaptic postganglionic nerves. Captopril, bestatin and leupeptin did not alter contractile responses, suggesting that angiotensin converting enzyme, aminopeptidases and serine proteinases did not modulate tachykinin-induced effects. Enkephalinase immunofluorescence was found in the smooth muscle and epithelium and confirmed the authors' finding of enkephalinase-like activity in the muscle. The results suggest that tracheal enkephalinase is an important modulator of tachykinin-induced effects.

Effect of gamma-aminobutyric acid on neurally mediated contraction of guinea pig trachealis smooth muscle.

To determine whether gamma-aminobutyric acid (GABA) affects the contractile properties of airway smooth muscle and, if so, what the mechanism of action is, the authors studied guinea pig tracheal rings under isometric conditions in vitro. GABA and related substances, baclofen and muscimol, had no effect on the resting tension but reversibly depressed contractions induced by electrical field stimulation in a dose-dependent fashion, IC50 values (mean +/- S.E.) being 5.6 +/- 1.4 X 10(-6) M, 6.8 +/- 0.9 X 10(-6) M and 8.5 +/- 1.5 X 10(-5) M, respectively. In contrast, GABA did not alter the response to exogenous acetylcholine or the nonadrenergic noncholinergic inhibitory component. Pretreatment of tissues with bicuculline antagonized the inhibitory effect of GABA as well as that of baclofen. This inhibitory effect was not modified by propranolol, phentolamine, hemicholinium-3 or naloxone, but it was blocked by the Cl channel blocker furosemide and by the substitution of external Cl. These results suggest that GABA decreases the contractile response of airway smooth muscle to cholinergic nerve stimulation by inhibiting the evoked release of acetylcholine and that this effect is exerted by activating Cl-dependent, bicuculline-sensitive GABA receptors.

Cholinergic neuromodulation by prostaglandin D2 in canine airway smooth muscle.

To determine whether prostaglandin D2 (PGD2) modulates cholinergic neurotransmission in airway smooth muscle and, if so, what the mechanism of action is, we studied bronchial segments from dogs under isometric conditions in vitro. PGD2 (10(-8)-10(-5) M) elicited dose-dependent muscle contraction, which was reduced after blockade of muscarinic receptors, so that 50% effective dose (ED50) increased from 1.3 +/- 0.3 X 10(-6) to 3.9 +/- 1.0 X 10(-6) M by atropine (10(-6) M) (mean +/- SE, P less than 0.05). Physostigmine, at a concentration insufficient to alter base-line tension (10(-8) M), enhanced the PGD2-induced contraction and decreased ED50 to 6.4 +/- 0.5 X 10(-7) M (P less than 0.05). When added at the highest doses that did not cause spontaneous contraction (1.9 +/- 0.5 X 10(-7) M), PGD2 increased the contractile response to electrical field stimulation (1-50 Hz) by 21.9 +/- 6.6% (P less than 0.001). In contrast to this effect, the response to administered acetylcholine was not affected by PGD2. On the other hand, PGD2-induced augmentation of the response to electrical field stimulation (5 Hz) was further increased from 23.6 +/- 3.0 to 70.4 +/- 8.8% in the presence of physostigmine (10(-8) M) and was abolished by atropine but not affected by the alpha-adrenergic antagonist phentolamine or the histamine H1-blocker pyrilamine. These results suggest that the contraction of airway smooth muscle induced by PGD2 is in in part mediated by a cholinergic action and that PGD2 prejunctionally augments the parasympathetic contractile response, likely involving the accelerated release of acetylcholine at the neuromuscular junction.