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Objective: Bakumondo-to (Maimendong tang) is a traditional oriental herbal medicine that has been used as an antitussive agent. We previously demonstrated that Bakumondo-to attenuates airway hyperresponsiveness induced by ozone. However, the mechanism(s) responsible for this effect remains unclear. In the present study, we examined the mechanism whereby Bakumondo-to inhibits ozone-induced airway hyperresponsiveness. First, we examined the effect of Bakumondo-to on prostanoids production, which are key mediators to airway hyperresponsiveness after ozone exposure. Second, we studied its effects on the vagal neuroeffector transmission, because vagal nerve is likely to play an important role in airway hyperresponsiveness after ozone., Methods: We measured the effects of Bakumondo-to on the concentrations of prostanoids in bronchoalveolar lavage fluid before and after ozone. We evaluated the effects of Bakumondo-to on the contraction of guinea pig tracheal smooth muscle evoked by electrical field stimulation (EFS) or the exogenous application of acetylcholine (ACh). Isometric tension of tracheal strips was measured in the presence of indomethacin (10(-6) M) and of guanethidine (10(-6) M)., Results: Ozone caused significant increase in prostaglandin E2 (PGE2) and thromboxane B2 (TXB2); however, Bakumondo-to did not affect the increase in these prostanoids. Bakumondo-to (0.01 mg/mL-1 mg/mL) significantly suppressed the contraction evoked by EFS, but did not affect the ACh-evoked contraction, indicating that Bakumondo-to suppressed tracheal smooth muscle contraction pre-junctionally., Conclusion: These results suggest that the mechanism by which Bakumondo-to inhibits airway hyperresponsiveness depends on inhibiting the release of acetylcholine from vagal nerve terminals.

Goblet cell metaplasia is an important morphological feature in the airways of patients with chronic airway diseases; however, the precise mechanisms that cause this feature are unknown. We investigated the role of endogenous platelet-activating factor (PAF) in airway goblet cell metaplasia induced by cigarette smoke in vivo. Guinea pigs were exposed repeatedly to cigarette smoke for 14 consecutive days. The number of goblet cells in each trachea was determined with Alcian blue-periodic acid-Schiff staining. Differential cell counts and PAF levels in bronchoalveolar lavage fluid were also evaluated. Cigarette smoke exposure significantly increased the number of goblet cells. Eosinophils, neutrophils, and PAF levels in bronchoalveolar lavage fluid were also significantly increased after cigarette smoke. Treatment with a specific PAF receptor antagonist, E-6123, significantly attenuated the increases in the number of airway goblet cells, eosinophils, and neutrophils observed after cigarette smoke exposure. These results suggest that endogenous PAF may play a key role in goblet cell metaplasia induced by cigarette smoke and that potential roles exist for inhibitors of PAF receptor in the treatment of hypersecretory airway diseases.

A non-phorbol ester-type tumor promoter, thapsigargin has been reported to deplete Ca(2+) stores in endothelial cells by inhibiting Ca(2+)-ATPase, which in turn increases intracellular Ca(2+) by mobilization of extracellular Ca(2+), leading to activation of constitutive nitric oxide synthase (cNOS) and resultant generation of nitric oxide (NO). In the present study, to evaluate the role of Ca(2+) in the release of epithelium-dependent relaxing factor (EpDRF), we determined the effect of thapsigargin (10(-6) M) on the contraction evoked by exogenous Ca(2+) or acetylcholine (10(-5) M) in epithelium-denuded or epithelium-intact smooth muscle from guinea pig trachea. The following results were obtained: (1) In epithelium-denuded smooth muscle, the contraction evoked by exogenous Ca(2+) in Ca(2+)-free solution or by acetylcholine (10(-5) M) in Ca(2+)-containing solution did not change within 20 min after thapsigargin application, but the contraction evoked by exogenous Ca(2+) increased markedly after 120 min, indicating that thapsigargin had no effect on smooth muscle itself within 20 min of application. The following experiments were performed within 20 min of thapsigargin application. (2) In epithelium-intact smooth muscle, thapsigargin significantly suppressed the contraction evoked by acetylcholine, suggesting that thapsigargin stimulate the epithelium to produce EpDRF. N(G)-nitro-L-arginine methylester (L-NAME) partly, but significantly, attenuated this inhibitory effect of thapsigargin. (3) In epithelium-denuded smooth muscle, atropine (10(-6) M) and L-NAME (10(-5) M) did not change the contraction evoked by exogenous Ca(2+) after application of thapsigargin, suggesting that thapsigargin did not stimulate acetylcholine and NO release from nerve terminals. These results suggest that thapsigargin (10(-6) M) may stimulate EpDRF, including NO and other factor(s) by Ca(2+)-dependent mechanisms.

We investigated the contribution of pituitary adenylate cyclase activating peptide (PACAP) to inhibitory nonadrenergic noncholinergic (inhibitory-NANC) relaxation of tracheal smooth muscle in cats. We also investigated the roles of vasoactive intestinal peptide (VIP) and nitric oxide (NO) on this function. Smooth muscle strips prepared from feline trachea were precontracted with 1 microM serotonin, and inhibitory-NANC relaxation was induced by electrical-field stimulation in the presence of atropine and propranolol. PACAP-(6-38) (a selective antagonist of PACAP; 1, 3 and 10 microM), VIP-(10-28) (a selective antagonist of VIP; 1, 3 and 10 microM) and N(omega)-nitro-L-arginine methyl ester (L-NAME, a selective NO synthase inhibitor; 3, 10 and 30 microM) each partially but significantly attenuated the amplitude of inhibitory-NANC relaxation. The effects of PACAP-(6-38) and VIP-(10-28) were additive. Addition of PACAP-(6-38) and/or VIP-(10-28) further attenuated relaxation in the presence of L-NAME. These results suggest that PACAP, VIP and NO contribute to the relaxation induced by inhibitory-NANC in tracheal smooth muscle in cats, and that they mediate this relaxation via different pathways.

We determined the effects of epinastine hydrochloride, an anti-asthmatic drug, on cholinergic neuro-effector transmission in canine trachea. Isometric tension of tracheal strips was measured in the presence of indomethacin and propranolol. Epinastine (10(-6) M) significantly suppressed the contraction evoked by electrical field stimulation, but had no effect on the acetylcholine-evoked contraction. An L-type Ca2+ channel blocker, nicardipine, did not suppress the electrical field stimulation-induced smooth muscle contraction and did not alter the inhibitory effect of epinastine. An N-type Ca2+ channel blocker, omega-conotoxin, suppressed the electrical field stimulation-induced contraction in a dose-dependent manner, and in a subthreshold/intermediate concentration abolished the inhibitory effect of epinastine. These findings indicate that epinastine exerts prejunctional inhibitory effects on airway smooth muscle of dogs, presumably by inhibiting acetylcholine release from vagal nerve terminals, and suggest that this effect is mediated by N-type Ca2+ channels.

The neurotransmitters responsible for neurogenic airway relaxation are still unknown. We investigated the effects of N omega-nitro-L-arginine methylester (L-NAME) on nonadrenergic and noncholinergic (NANC) relaxation evoked by electrical stimulation of vagus nerves in vivo and in vitro in cat. To that end, we measured pulmonary resistance during vagal nerve stimulation (VS) in vivo, and isometric tension of small bronchi (1-3 mm outer diameter) during electrical field stimulation (EFS) in vitro. During infusion of 5-hydroxytryptamine (5-HT), VS transiently decreased total pulmonary resistance in the presence of atropine and propranolol, with peak relaxation at several seconds after the VS and a gradual return to baseline within 2-3 min. L-NAME abolished the initial peak relaxation and reduced the peak amplitude, but did not affect the duration of the NANC relaxation. In small bronchi obtained from control cats, EFS evoked a biphasic NANC relaxation, comprising an initial fast component followed by a second slow component, and L-NAME (10(-5) M) selectively abolished the first component without affecting the second. Whilst in the small bronchi obtained from L-NAME pretreated cats, EFS elicited only the slow component of NANC relaxation, which was insensitive to L-NAME but sensitive to tetrodotoxin. These results indicate that nonadrenergic noncholinergic relaxation induced by vagal nerve stimulation during infusion of 5-hydroxytryptamine can be classified into two components, and that at least two neurotransmitters, including nitric oxide, are involved in the relaxation.

To determine the role of VIP-like peptides on neurotransmission of vagus nerve, we evaluated the effects of helodermin, helospectin, and vasoactive intestinal peptide (VIP) on the contraction of guinea pig tracheal smooth muscle evoked by electrical field stimulation (EFS) or the exogenous application of actylcholine (ACh). Isometric tension of tracheal strips was measured in the presence of indomethacin (10(-6) M) and of guanethidine (10(-6) M). VIP (10(-9) M to 10(-7) M) significantly suppressed the contraction evoked by EFS. VIP, at concentrations of 10(-9) M and 10(-8) M, did not affect the ACh-evoked contraction, but a concentration of 10(-7) M suppressed ACh-evoked contraction. Helospectin and helodermin (10(-8) M and 10(-7) M) significantly suppressed the EFS-evoked contraction, but 10(-9) M showed no effect. Helospectin and helodermin had no effect on the ACh sensitivity of smooth muscle up to 10(-8) M, but a concentration of 10(-7) M suppressed the ACh-evoked contraction. These results indicate that helodermin, helospectin, and VIP exert both pre- and post-junctional inhibitory effects on the airway smooth muscle of guinea pigs. These peptides, thus, inhibited tracheal smooth muscle contraction prejunctionally at low concentrations, and acted postjunctionally at higher concentrations.

Although the bronchoconstriction induced by leukotriene D4 (LTD4) has been reported to be partly mediated by thromboxane A2 (TXA2) in the guinea-pig airway, it is not known which part of the airway is susceptible to TXA2. In order to determine the role of TXA2 in the central and peripheral airways, we compared the effect of a TXA2 antagonist on tracheal strips to its effect on parenchymal strips of guinea-pigs. Tracheal and parenchymal strips were mounted in a 3.5 ml organ bath filled with Krebs-Henseleit solution aerated with 95% O2, 5% CO2 and kept at 37 degrees C. After equilibration for 60 min in Krebs solution, the strip was contracted by exposure to 10(-5) M of acetylcholine (ACh). Sixty minutes after ACh was eliminated, the concentration-response curve to LTD4 (10(-9) M-10(-7) M) was obtained, and the LTD4-induced contractions were expressed as the percent of the contraction evoked by 10(-5) M of ACh. We measured the contractile response to LTD4 in the presence or absence of the TXA2 antagonist, BAY u3405 (10(-8) M-10(-6) M). In the tracheal strips, BAY u3405 had no effect on the LTD4-induced contraction. However, in parenchymal strips, BAY u3405 significantly suppressed the contractile response to LTD4. These results suggest that in the central airway LTD4 contracts smooth muscle directly, but that in the peripheral airway LTD4 induces smooth muscle contraction both directly and indirectly, via TXA2.

It has been reported that cholinergic agonists induce bronchoconstriction by directly stimulating M3 muscarinic receptors on the surfaces of smooth muscle cells. Although thromboxane A2 (TXA2) has been demonstrated to induce airway hyperresponsiveness to cholinergic agonists in vivo, it does not affect the contractile response of smooth muscle to cholinergic agonists in vitro. To investigate the causes for the discrepancy between the in vivo and in vitro data, we compared the effects exerted by a TXA2 mimetic, U-46619, on the smooth muscle of canine trachea and bronchiole. We measured the contractile response to exogenously applied acetylcholine (ACh) before and after the application of a subthreshold dose of U-46619. The subthreshold dose was determined as that dose which did not induce smooth muscle contraction, this being 10(-9) M in the present study. The contractile responses of tracheal strips to ACh were not affected by the subthreshold dose of U-46619. By contrast, the responses of bronchiolar rings were significantly enhanced by this subthreshold dose. The excitatory effect of U-46619 on the ACh-induced contraction was completely prevented by treatment with a TXA2 antagonist, BAY u3405. These results indicate that TXA2 directly increases the responsiveness of smooth muscle in the bronchiole, and suggest that increases in the responsiveness of small airways may play an important role in the development of the airway hyperresponsiveness induced by TXA2.

We observed the effects of supernatants from cultured epithelial cells on the contraction of tracheal smooth muscle evoked by acetylcholine (ACh) or by electrical field stimulation (EFS). Cultured canine tracheal epithelial cells were incubated in Krebs solution with or without indomethacin (10(-5)M) for 30 and 120 min. The amplitude of the tracheal smooth muscle contractions evoked by EFS or exogenously applied ACh were measured before and after the application of each supernatant in the combined presence of both indomethacin (10(-5)M) and propranolol (10(-6)M). The control supernatant incubated without indomethacin markedly suppressed the amplitude of the contraction evoked by EFS, but not by ACh. The supernatant incubated with indomethacin for 30 min did not show any effects on the contractile responses evoked by EFS or ACh. However, the supernatants from the cultured epithelial cells incubated for a longer period (120 min) in the presence of indomethacin significantly suppressed the contraction evoked by EFS, but not by ACh. The prostaglandin E2 (PGE2) concentration was markedly higher in the supernatants incubated without indomethacin (1.39 +/- 0.51 ng/ml, 30 min incubation) than in those with indomethacin (0.02 +/- 0.01 ng/ml, 30 min incubation, and 0.06 +/- 0.01 ng/ml, 120 min incubation). To determine whether PGE2 is responsible for the inhibitory effect of the supernatants, we evaluated the effects of PGE2 on the resting tone, and EFS- or ACh-evoked contraction. 10(-12) to 10(-6) M of PGE2 showed no significant effect on the resting tone. 10(-9) M of PGE2, corresponding to the concentration of the supernatants incubated without indomethacin, and 10(-11) M of PGE2, to that of the supernatants incubated with indomethacin, showed a similar extent of inhibitory effects to the corresponding supernatants on the EFS-evoked contraction, and no effect on the ACh-evoked contraction. These results suggest that cultured airway epithelial cells release at least two factors spontaneously even without stimulation. One of these factors may be prostanoid (PGE2), which acts prejunctionally to inhibit the contractile response. The other factor is distinct from prostanoid and inhibits smooth muscle contraction, presumably by suppressing ACh release from vagus nerve termini.

Thromboxane A2 (TXA2) is reported to potentiate vagal nerve neuro-effector transmission in airway smooth muscle tissue. We investigated the effects of BAY u3405 (3(R)-[[4-fluorophenyl)sulfonyl]amino]-1,2,3,4,-tetrahydro-9H-carbazole - 9-propanoic acid), a potent and selective TXA2 receptor antagonist, on the increase in vagal nerve neuro-effector transmission induced by a TXA2 mimetic, U-46619, in the canine trachea. We measured the contractions of canine tracheal smooth muscle evoked by electrical field stimulation (EFS) and by acetylcholine (ACh) in the presence and absence of a subthreshold dose of U-46619 (the highest dose that did not induce any smooth muscle contraction). We then examined whether BAY u3405 inhibited the effect of U-46619 on tracheal smooth muscle. The following results were obtained: (i) subthreshold doses of U-46619 (10(-10) M and 10(-9) M) significantly increased the amplitude of the contractions evoked by EFS; (ii) by contrast, U-46619 had no effect on the contractile response of smooth muscle to exogenously applied ACh; (iii) the contraction evoked by EFS was completely abolished by the application of atropine (10(-6) M) or tetrodotoxin (10(-7) M), indicating that EFS caused the smooth muscle contraction through the release of ACh from vagal nerve terminals; and (iv) pretreatment with BAY u3405 (10(-6) M) abolished the excitatory action of U-46619 on the amplitude of twitch contraction evoked by EFS in the trachea. These results indicate that U-46619, at low concentrations, has a prejunctional action stimulating neuro-effector transmission, presumably increasing ACh release from vagal nerve terminals through TXA2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of fetal bovine serum (FBS) on the resting membrane potential and muscle tone of canine airway smooth muscles using tension recording and microelectrode methods. At concentrations > 0.1%, FBS induced a sustained increase in muscle tone. At concentrations > 1%, FBS depolarized the resting membrane potential of dog trachea in a dose-dependent manner. When FBS was dialyzed, it failed to induce a sustained increase in muscle tone or to depolarize the membrane, indicating that FBS contained factor(s) that induced contraction of dog airway smooth muscles. When FBS was dialyzed against distilled water, the outer solution which was freeze dried and then reconstituted (> 1% in original vol) induced a sustained increase in muscle tone, indicating that FBS contained dialyzable factor(s) that increased muscle tone and depolarized the resting membrane potential of dog airway smooth muscles. Methysergide (10(-6) M) or cyproheptadine (10(-6) M), nonspecific antagonists to serotonin receptors, 5-hydroxytryptamines (5-HT1 and 5-HT2), markedly reduced the FBS-induced increase in the muscle tone to approximately 20% of the original value but did not abolish the response. On the other hand, methysergide (10(-6) M) completely suppressed the increase in the muscle tone evoked by peak fractions obtained by application of the dialysate of FBS or 5-HT to a C18 reverse-phase column, indicating the peak fractions contained only 5-HT. These observations indicate that FBS contained 5-HT and an unknown factor(s) responsible for increase in the muscle tone of the airway smooth muscle induced by FBS.

We investigated the effects of cultured epithelial cells and supernatants on resting membrane potential and excitatory neuroeffector transmission in smooth muscle cells of dog trachea and bronchioles. The mean resting membrane potential of the mucosa-free tracheal smooth muscle cells was -59.5 +/- 1.5 mV (+/- SD). Application of cultured epithelial cells (> 2.5 x 10(5) cells/ml) hyperpolarized the membrane, resulting in a potential of -64.5 +/- 1.7 mV. The supernatant of the cultured epithelial cells also increased the resting membrane potential of the mucosa-free tracheal smooth muscle cells by 4 to 9 mV. These hyperpolarizing actions were not modified by indomethacin (10(-5) M), l-NG-nitroarginine (10(-5) M), or oxyhemoglobin (10(-5) M), but were inhibited by glibenclamide (10(-6) M). The supernatants of the cultured epithelial cells completely or partially suppressed the contractile response of epithelium-denuded bronchioles to electrical field stimulations and suppressed the amplitude of excitatory junction potentials of the trachealis evoked by electrical field stimulations. Indomethacin prevented the inhibitory effect of supernatants on the amplitude of twitch contractions and excitatory junction potentials and markedly suppressed supernatant-associated inhibition of the excitatory neuroeffector transmission. Furthermore, indomethacin with AA861, a lipoxygenase inhibitor, completely suppressed this effect. Our findings suggest that cultured airway epithelial cells spontaneously release at least two factors. One factor selectively modulates the resting membrane potential, and the other inhibits the excitatory neuroeffector transmission.

We investigated the effects of epithelial cells on excitatory cholinergic neurotransmission in dog trachea, to shed more light on the role of airway epithelial cells in regulating airway responsiveness. Airway epithelial cells were prepared by an enzymatic dissociation of the tracheal mucosa using protease-free collagenase. Tracheal smooth muscle contractions evoked by electrical field stimulation (EFS) or acetylcholine (ACh) were measured before and after the application of epithelial cells. Isolated and dispersed epithelial cells (3 x 10(5) cells/ml) suppressed the amplitude of the twitch-like contractions evoked by EFS in the combined presence of guanethidine sulfate (10(-6) M) and indomethacin (10(-5) M). In contrast, epithelial cells did not affect the contraction evoked by exogenously applied ACh. Atropine (10(-6) M) or tetrodotoxin (10(-7) M) abolished the contraction evoked by electrical field stimulation. These findings indicate that airway epithelial cells inhibit the excitatory neurotransmission of the vagus nerve, presumably by suppressing the release of ACh. Airway epithelial cells may therefore play an important role in regulating the response of smooth muscle.

To elucidate the role of the axon reflex in the airways, we studied the effects of atropine (10(-6) M) and tetrodotoxin (10(-7) M) on the bradykinin-induced contraction of guinea pig tracheal smooth muscle, with or without pretreatment of the animals with capsaicin. The concentration-response curves to bradykinin (10(-9) to 10(-5) M) were measured in the presence of both indomethacin and propranolol. In the guinea pigs not given capsaicin pretreatment, baseline tension values did not differ before versus after the application of atropine or tetrodotoxin. Tetrodotoxin reduced the bradykinin-induced contraction significantly, but atropine did not change the contraction induced by bradykinin. These observations indicate that bradykinin-induced contraction is potentiated by a neurally mediated action, but that is not mediated by acetylcholine released from the efferent vagal nerve terminals. The contractile response to bradykinin was significantly reduced in the animals treated with capsaicin as compared with those administered vehicle only. Furthermore, in the animals treated with capsaicin, tetrodotoxin did not affect the response to bradykinin. These observations indicate that bradykinin-induced airway smooth muscle contraction is mediated in part by tachykinins released from C-fiber endings, presumably via an axon reflex.

It has been reported that tracheal smooth muscle of guinea pig is innervated by both the adrenergic and nonadrenergic noncholinergic (NANC) inhibitory nervous system. However, NANC inhibitory nerve supply to the lower airway of guinea pig has not yet been demonstrated in in vivo experiments. We performed the present study to evaluate the physiological role of NANC inhibitory nerves in tracheal smooth muscle of guinea pig in vitro, and in anesthetized guinea pigs in vivo. Innervation of NANC inhibitory nerves to tracheal smooth muscle was much greater than that of adrenergic nerves (77.8 +/- 3.8%, 22.2 +/- 3.8%, respectively, p less than 0.01). Neither decrease in RL nor increase in CL, however, was observed with vagal stimulation during serotonin infusion after the administration of propranolol. The role of NANC inhibitory nerves in histamine-induced bronchoconstriction (HIB) was investigated. HIB was enhanced by vagotomy in guinea pig pretreated with propranolol (p less than 0.01). These results suggest the role of the NANC inhibitory nervous system in the attenuation of HIB in vivo in the guinea pig.

It has been reported that low concentrations of vasoactive intestinal peptide (VIP) suppress the release of acetylcholine (ACh) from vagal nerve terminals. However, there is little documentation of the neuro-effector transmission of VIP in human airways. In the present study, the effects of VIP on excitatory neuro-effector transmission in the human bronchus were investigated, in comparison with feline trachea. In feline trachea, VIP (10(-10) to 10(-7) M) reduced the amplitude of contractions evoked by field stimulation in the presence of indomethacin (10(-6) M). By contrast, VIP (10(-10) to 10(-9) M) had no effect on ACh-sensitivity of smooth muscle cells. These results indicate that VIP in low concentrations has a pre-junctional action inhibiting excitatory neuro-effector transmission, in addition to a post-synaptic action, presumably suppressing transmitter release from the vagal nerve terminals. On the other hand, in human bronchi, VIP (10(-9) to 10(-7) M) did not affect either contractions evoked by field stimulation or those evoked by ACh. These results indicate that VIP does not exhibit any pre-junctional effects in the human bronchus at these concentrations.

To examine the possible role of vasoactive intestinal polypeptide (VIP) in excitatory and inhibitory neurotransmission and physiological function of coexistence VIP and acetylcholine at vagus nerve terminals in the cat trachea, we immunized five cats each against conjugate of VIP-bovine serum albumin (BSA) and BSA, respectively. A booster injection of VIP-BSA given 3 wk after the primary one elevated antibodies against VIP, as detected by enzyme-linked immunosorbent assay, but not in the control cats identically injected with BSA. After immunization, in vitro, summation phenomena observed in the amplitude of contractions and excitatory junction potentials of the trachea evoked by repetitive field stimulation were markedly enhanced, whereas the amplitude of phasic relaxation evoked by repetitive field stimulation in the presence of serotonin, atropine, and guanethidine was markedly reduced. In contrast, in vivo, the change in pulmonary resistance evoked by vagal stimulation was not affected in the immunized cats. We conclude that antibodies against neurotransmitter VIP are a potential modulatory factor in excitatory and inhibitory neurotransmission in airway smooth muscle at tissue or cellular levels. The lack of correlation between in vivo and in vitro conditions was discussed in relation to VIP autoantibodies in normal and asthmatic humans.

Azelastine is a newly developed antiallergic drug that is reported to antagonize histamine and leukotrienes in addition to its inhibitory action on release of chemical mediators. In the present study, the effects of azelastine on neuroeffector transmission in the airway smooth muscles with double sucrose gap and isometric tension-recording methods were evaluated. Azelastine (10(-8) to 10(-6) mol/L) markedly decreased the contractile response of human bronchial and dog tracheal muscle strips to electrical field stimulation (10 pulses at 20 V, 20 Hz, 800 microseconds) in a dose-dependent manner. In parallel with actions on twitch contractions, azelastine suppressed the amplitude of excitatory junction potentials of dog trachea without changing the resting membrane potential and input resistance of smooth muscle cells. However, azelastine did not alter acetylcholine sensitivity of the smooth muscle cells. These results indicate that azelastine possesses an inhibitory action on the release of acetylcholine by vagal nerve terminals. This inhibitory effect of azelastine may contribute to the treatment of asthma in addition to its antiallergic actions.

To elucidate the effect of endogenous tachykinins on neuro-effector transmission of vagal nerves, we performed in vitro experiments using guinea-pig tracheal smooth muscle. The subthreshold dose (the highest dose which did not induce any smooth muscle contraction) of capsaicin (10(-8) to 10(-7) M) increased the amplitudes of contractions evoked by electrical field stimulation (EFS) significantly, but not those by acetylcholine (ACh). The inhibitor of neutral endopeptidase, phosphoramidon (10(-7) to 10(-6) M), increased the contractions evoked by EFS significantly. The inhibitor of cholinesterase, physostigmine (10(-6) to 10(-5) M), induced smooth muscle contractions, but such contractions were inhibited by atropine, suggesting the spontaneous release of ACh from the vagal nerve terminals. The subthreshold dose of substance P or capsaicin increased the contractions evoked by physostigmine. These results indicated that endogenous tachykinins increase the spontaneous ACh release as well as the ACh release in response to vagal stimulation from the nerve terminals. Furthermore, it is suggested that the excitatory effects of the tachykinins on the vagal neuro-effector transmission may be modulated by neutral endopeptidase in the guinea pig.

To be able to study the release of mediators from the canine trachea noninvasively in vivo, we modified an endotracheal tube to allow superfusion of the lumen of an isolated segment of trachea. We tested this apparatus by measuring prostaglandin E2 (PGE2) output in the presence and absence of a stimulant (bradykinin) and a blocker (indomethacin) and comparing the results with results of in vitro studies. We found that PGE2 output increased with time over the 180 min of the study. Bradykinin (10(-5) M) applied to the luminal surface increased PGE2 output markedly. Indomethacin (10(-5) M) decreased PGE2 and prevented the usual increase in PGE2 caused by bradykinin. These findings are qualitatively similar to those for in vitro experiments. We conclude that this apparatus can be used to measure the release of mediators from the trachea of dogs noninvasively in vivo.

The incubation of suspensions of canine tracheal epithelial cells of greater than 95% purity with arachidonic acid (25-200 micrograms/ml) for 60-120 min resulted in the generation of a maximum of 36.2 +/- 9.1 picomoles of leukotriene B4/10(6) cells, less than 2.0 picomoles of leukotrienes C4, D4, and E4/10(6) cells, and 1030 +/- 463, 767 +/- 500, and 324 +/- 100 picomoles/10(6) cells of 15-, 12-, and 5-hydroxy-eicosatetraenoic acids, respectively (mean +/- SEM, n = 8). The identity of leukotriene B4 was established by chromatographic and spectral properties, by reactivity with mono-specific anti-plasma, and by the chemotactic activity for neutrophils. Thus, the epithelium may be an important source of mediators of inflammation and hypersensitivity of pulmonary airways.