Influence of epithelial removal on the antihistaminic activity of azelastine in the guinea-pig airway smooth muscles

The influence of epithelial removal on the antihistaminic activity of azelastine in the guinea-pig isolated cervical trachea and main bronchus was examined. In both preparations with an intact epithelium, histamine produced a concentration-dependent contraction, but its potency (pD₂) and contractiliy (Emax) were significantly higher in the main bronchus than in the cervical trachea. Epithelial removal from these preparations significantly increased the pD₂ values in both tracheal and bronchial preparation, while the Emax value was significantly increased only in the cervical trachea. When these preparations were pretreated with azelastine (20 or 200 nM), concentration-response curves for histamine shifted downward, but were not influenced by the presence of an intact epithelium. Neither concentration of azelastine significantly modified the contractile responsiveness of tracheal and bronchial preparations to carbachol or neurokinin A. These results indicate that azelastine selectively antagonizes the histamine-induced contraction of the guinea-pig airway smooth muscles with or without an intact epithelium.     

Substance P and capsaicin-induced contraction of human bronchi

Substance P induced a dose-dependent contraction of human segmental bronchi in vitro with a threshold dose of about 10(-6) M. These preparations were obtained from patients undergoing lung tumor surgery. The substance P-induced contractions were resistant to mepyramine and atropine, suggesting a direct effect on the bronchial smooth muscle. Capsaicin (10(-5) M) also induced a slowly developing strong atropine-resistant contraction of human bronchi in vitro. a rapid tachyphylaxis developed for the response to capsaicin. Both substance P and capsaicin were less potent than acetylcholine and histamine in inducing contractions of human bronchi. This finding may however be partly due to the experimental conditions and both substance P and capsaicin were comparatively much more potent in guinea-pig preparations. Transmural field stimulation of the bronchial preparations in man resulted in contractions that were largely sensitive to atropine. The presence of capsaicin-induced bronchial contractions however indicates the existence of a local non-cholinergic axon-reflex control of bronchial smooth muscle tone by substance P in man.     

Pharmacologic characteristics of non-prostanoid, non-nitric oxide mediated and endothelium-dependent relaxation of guinea-pig aorta in response to substance P.

The pharmacologic characteristics of the non-prostanoid (prostacyclin, PGI2), non-nitric oxide (NO) mediated endothelium-dependent relaxation in response to substance P were examined in the guinea-pig aorta. Substance P, in a concentration-dependent manner, relaxed the ring preparations of guinea-pig thoracic aorta preconstricted with norepinephrine (NE) in an endothelium-dependent manner. Substance P-induced endothelium-dependent relaxation was not affected by indomethacin (3 x 10(-6) M) as in the case of acetylcholine (ACh)-induced endothelium-dependent relaxation. Although N(G)-nitro-L-arginine (L-NNA, 3 x 10(-5) M), an inhibitor of nitric oxide (NO) synthase, significantly inhibited substance P-induced endothelium-dependent relaxation in the presence of indomethacin, about 50% of the vasorelaxant response to substance P remained in the combined presence of L-NNA and indomethacin. By comparison, indomethacin-resistant component of endothelium-dependent relaxation to ACh was mostly suppressed by the treatment with L-NNA plus indomethacin. Substance P-induced non-PGI2, non-NO mediated vascular relaxation was attenuated markedly in high (40 mM) KCl solution or by tetraethylammonium (TEA, 5 x 10(-3) M). Furthermore, substance P-induced non- PGI2, non-NO mediated vascular relaxation was not appreciably affected by glibenclamide (10(-6) M), apamin (10(-7) M), iberiotoxin (1(-7) M), but was greatly attenuated by the combined treatment with charybdotoxin (10(-7) M) plus apamin (10(-7) M), which suggesting that endothelium-derived hyperpolarizing factor(s) (EDHF(s)) mediates the response. Interestingly, after applied repetitively, the substance P-induced vasorelaxant component remaining in the combined presence of indomethacin and L-NNA was decreased more profoundly than the response to substance P in the presence of indomethacin alone. Possible contribution of non-PGI2, non-NO vasorelaxant(s) (EDHF(s)) from the endothelium to the total relaxation response to substance P was greater in thoracic aorta isolated from adult guinea-pigs than that from neonatal ones. These findings suggest that 1) endothelium-dependent vascular relaxation of guinea-pig thoracic aorta in response to substance P is attributable to the release of both NO and EDHF(s); 2) possible release of EDHF(s) from the endothelium of guinea-pig thoracic aorta decreases after repetitive stimulation with substance P; and 3) contribution of EDHF(s) to substance P-induced functional relaxation of the thoracic aorta is greater in adult guinea-pigs than neonatal ones.     

A neuroactive steroid, allotetrahydrocorticosterone inhibits sensory nerves activation in guinea-pig airways

We examined the effects of a neuroactive steroid, allotetrahydrocorticosterone on the activation of capsaicin-sensitive afferent sensory nerves (C-fibers). Allotetrahydrocorticosterone (0.0001-1.0 microg/ml) dose-dependently inhibited electrical field stimulation-induced guinea-pig bronchial smooth muscle contraction, but not the substance P-induced contraction at 1.0 microg/ml. Allotetrahydrocorticosterone (0.01-1.0 microg/ml) also reduced the capsaicin-induced release of substance P-like immunoreactivity from guinea-pig airway tissues in a dose-dependent manner. The inhibitory effect of allotetrahydrocorticosterone on electrical field stimulation-induced bronchial contraction were reduced by the pretreatment of voltage-dependent K+ channel blockers, tetraethylammonium (1 mM). This evidence suggests that allotetrahydrocorticosterone negatively modulate the activation of C-fibers and substance P release from their endings in airway tissues via the opening of voltage-dependent K+ channels.     

A Neutrophil Elastase Inhibitor,Sivelestat, Reduces Lung Injury Following Endotoxin-Induced Shock in Rats by Inhibiting HMGB1

Neutrophil elastase (NE) plays an important role in the progression of acute lung injury (ALI). Sivelestat sodium hydrate (Sivelestat) is a highly specific synthetic inhibitor of NE. High mobility group box 1 (HMGB1) is one of the key mediators in the development of sepsis. The aim of this study was to evaluate the effect of sivelestat and to determine whether it can reduce lipopolysaccharide (LPS)-induced acute lung injury in rats. Rats were randomly divided into a negative control group, an LPS-induced sepsis group, and a group treated with sivelestat prior to LPS administration. Animals in the sivelestat group received a bolus of 10 mg/kg of sivelestat injected into the intraperitoneal cavity before the LPS treatment. Furthermore, rats were administered sivelestat at 0, 1, 3, and 6 h following LPS treatment. We measured cytokine and HMGB1 levels in the serum after the induction of sepsis. In addition, we observed histopathology, wet/dry weight ratio, inducible nitric oxide synthase and HMGB1 expression in the lung tissue. Lung histopathology was significantly improved in the sivelestat group compared to the LPS group. Serum and pulmonary HMGB1 levels were lower over time among sivelestat-treated animals. Furthermore, inhibition of NF-kappaB activity was observed with the administration of sivelestat. These results suggest that sivelestat reduces LPS-induced lung injury at least partially by inhibiting inflammation and NF-kappaB activity.     

Inhibitory effect of bronchodilators on cholinergic and peptidergic contractions of guinea-pig isolated bronchial muscle

The effect of bronchodilators on neurogenic contractions was studied using isolated bronchial strip-chain preparations from guinea-pigs. Electrical field stimulation (8 Hz, 0.5 msec, 30 V, 40 pulses) evoked a biphasic contraction of the bronchial muscle, consisting of an initial phasic component followed by a sustained one which was mediated by cholinergic and peptidergic nerve stimulations, respectively. All the beta-stimulants and xanthines tested caused a concentration-dependent inhibition in the height of the electrically-induced biphasic contraction, with the following order of potency: procaterol greater than fenoterol greater than salbutamol greater than isoproterenol greater than adrenaline greater than enprofylline greater than aminophylline greater than ephedrine. Ipratropium bromide selectively inhibited the electrically-induced cholinergic contraction. Submaximal contractions of the bronchial muscle evoked by exogenous substance P (2 X 10(-7)M) were less potently inhibited by these drugs than those evoked by exogenous acetylcholine (2 X 10(-6)M). These results indicate that in isolated guinea-pig bronchial muscle, bronchodilators may inhibit cholinergic and peptidergic neurotransmissions mainly by postsynaptic depression of the responsiveness and partly by presynaptic reduction of the transmitter release.     

Combined effects of a neutrophil elastase inhibitor (sivelestat sodium) and a free radical scavenger (edaravone) on lipopolysaccharide-induced acute lung injury in rats

Objective and design

The present study aimed to investigate the combined effects of a neutrophil elastase inhibitor, sivelestat sodium, with a free radical scavenger, edaravone, on lipolysaccharide (LPS)-induced acute lung injury (ALI).

Materials and methods

Adult male Sprague–Dawley rats were anesthetized and instilled intratracheally with 2?mg/kg LPS. Sivelestat sodium (10?mg/kg, i.p.) and/or edaravone (8?mg/kg, i.p.) were administered 1?h after LPS instillation. The severity of pulmonary injuries was evaluated 12?h after inducing acute lung injury.

Results

In lung tissues, either sivelestat or edaravone treatment alone showed significant protective effects against neutrophil infiltration and tissue injury, as demonstrated by myeloperoxidase activity and histopathological analysis. Sivelestat or edaravone treatment also attenuated the LPS-induced production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in rat lungs. However, the LPS-induced elevation of malondialdehyde levels in rat lungs was reduced only by edaravone, but not by sivelestat. In addition, combined treatment with both sivelestat and edaravone demonstrated additive protective effects on LPS-induced lung injury, compared with single treatments.

Conclusions

Combination of sivelestat and edaravone shows promise as a new treatment option for ALI/acute respiratory distress syndrome patients.     

Neutrophil elastase inhibitor (sivelestat) reduces the Levels of inflammatory mediators by inhibiting NF-kB

Objective:  Sivelestat sodium hydrate (sivelestat) is a specific synthetic inhibitor of neutrophil elastase (NE). Various studies suggest that sivelestat treatment reduces inflammation. In this study, we tested the hypothesis that sivelestat acts as an inhibitor of inflammatory mediators and prevents nuclear factor-kB (NF-kB) activation. Methods:  In the presence and absence of sivelestat, the mouse macrophage cell line RAW 264.7 was stimulated with lipopolysaccharide (LPS) and the levels of inflammatory mediators (TNF-α, IL-6 and high mobility group box 1 (HMGB1)) and nitrite in the cell supernatant were measured, along with inducible nitric oxide synthase (iNOS) expression. Results:  While LPS administration increased the secretion of inflammatory mediators and nitric oxide (NO), sivelestat decreased the secretion of these mediators. Cell signaling studies demonstrated that sivelestat decreased NF-kB activation by inhibiting IkB phosphorylation. Conclusion:  Sivelestat may inhibit the various inflammatory mediators through NF-kB inhibition. Received 14 June 2008; returned for revision 11 July 2008; received from final revision 4 September 2008; accepted by H. Katori 9 September 2008     

Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term

The aim of this study was firstly to elucidate whether the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)-regulated contractility of myometrium obtained from near-term pregnant women, and secondly to investigate the receptor subtype(s) responsible. In the presence of peptidase inhibitors, i.e. thiorphan (3 micromol/l; endopeptidase 24.11 inhibitor), captopril (10 micromol/l; angiotensin converting enzyme inhibitor) and bestatin (10 micromol/l; aminopeptidase inhibitor); all three mammalian tachykinins elicited concentration-related contractions of isolated myometrial preparations. The rank order of agonist potency of the mammalian tachykinins in the presence of the peptidase inhibitors was NKA > SP = NKB, indicating that the contractile effects were mediated by activation of an NK(2) receptor. The NK(2) receptor-selective agonist, [Lys(5), MeLeu(9), Nle(10)]NKA(4-10), produced concentration-related contractile responses, while the respective NK(1) and NK(3) receptor-selective agonists, [Sar(9), Met(O(2))(11)]SP and [N-MePhe(7)]NKB, had no effect either in the absence or presence of the peptidase inhibitors. The NK(2) receptor-selective antagonist, SR48968, produced concentration-related rightward shift in the log concentration curve to [Lys(5), MeLeu(9), Nle(10)]NKA(4-10). This study shows that tachykinins elicit contractile effects on human myometrium obtained from pregnant women near term, and that these effects are mediated by an NK(2) receptor. An excitatory effect of the tachykinins on these preparations could indicate a physiological role for these peptides in enhancing contractility of the uterus in women at term.     

Capsaicin induced release of multiple tachykinins (substance P, neurokinin A and eledoisin-like material) from guinea-pig spinal cord and ureter

The release of tachykinins from isolated slice preparations of the guinea-pig spinal cord and ureter was studied in vitro. Capsaicin (10 microM) caused release of substance P, neurokinin A and an eledoisin-like component from both the spinal cord and ureter. The release of tachykinins induced by capsaicin or potassium (60 mM) was calcium dependent. No detectable release of neurokinin B or neuropeptide K, an N-terminally extended form of neurokinin A, was induced by capsaicin. No detectable release of tachykinins could be demonstrated after exposure to agents which are known to activate C-fibre afferents, such as histamine, bradykinin, serotonin, prostaglandins E1, E2 or acetylcholine. Protein extravasation in the ureter, as determined by the Evans Blue extravasation technique was used as a functional correlate to the tachykinin release. Protein extravasation was induced in vivo by local intraluminal injections of capsaicin at several hundred-fold lower concentrations than those required to induce a detectable release of tachykinins in vitro. The difference may, however, partly depend on the experimental conditions and the detection limit of the tachykinin assay used. The protein extravasation response to capsaicin was absent after systemic capsaicin pretreatment, which causes a marked depletion of tachykinins in the ureter. In conclusion, capsaicin evokes release of several tachykinins from both central and peripheral endings of primary afferent neurons. The peptides released from sensory nerves in the periphery may induce effects such as protein extravasation and smooth muscle contraction.     

Substance P and calcitonin gene-related peptide in the ureter of chicken and guinea-pig: distribution, binding sites and possible functions.

To elucidate the possible functional significance of sensory neuropeptides in visceral organs of mammals and birds the distribution, binding sites and the effects on ureteric peristalsis of substance P and calcitonin gene-related peptide (CGRP) were investigated in the ureter of guinea-pigs and chickens. In the guinea-pig numerous substance P and CGRP-immunoreactive fibres were located in the adventitia, smooth muscle layer, submucosa and occasionally in the epithelium. Varicose peptidergic fibres were often found on blood vessels. Binding sites for substance P were associated with blood vessels and epithelium in the following density order: venules greater than epithelium greater than arterioles. The highest density of CGRP binding sites was detected on the smooth muscle; venules and arterioles expressed moderate binding. The peristalsis frequency of the isolated ureter of the guinea-pig was increased by neurokinin A and substance P, whereas CGRP inhibited ureteric motility. In the chicken the immunoreactivity to substance P and CGRP was less pronounced. Immunoreactive fibres were found in the submucosa close to the epithelium and around ureteric ganglion cells. Correspondingly, substance P binding sites were located in the epithelium and in ureteric ganglia; however, specific CGRP binding was restricted to large blood vessels. In the chicken none of the sensory neuropeptides affected ureteric motility. Only high doses of the sensory neurotoxin capsaicin (greater than 10 microM) repeatedly produced a non-specific inhibitory effect, similar to that found in a capsaicin-desensitized guinea-pig ureter preparation. The data suggest that in the guinea-pig ureter sensory neuropeptides play a modulatory role in the regulation of ureteric motility and might have vascular and epithelial functions. In the chicken, substance P might be involved in the regulation of epithelial function and modulation of ganglionic transmission. The physiological or pathophysiological role of sensory neuropeptides and the efferent functions of afferent fibres appears to be much better developed in the guinea-pig than in the chicken.     

Effects of capsaicin on the circular muscle motility of the isolated guinea-pig ileum

Effects of capsaicin on the circular muscle motility of the isolated guinea-pig ileum were investigated. Capsaicin produced a contraction followed by a relaxation of the circular muscle. Both responses were easily desensitized. As the late relaxation response was not sufficiently intense to be analyzed, the inhibitory effect of capsaicin on substance P-induced contractions was explored. Capsaicin abolished the substance P-induced contractions. This inhibitory effect was not affected by tetrodotoxin, and the effect was desensitized. Therefore, all effects of capsaicin on circular muscle motility seem to be due to the release of sensory neuropeptides, similarly to those elicited in the longitudinal muscle.     

Effect of baclofen on different models of bronchial hyperreactivity in the guinea-pig

In this paper we report an inhibitory effect of (–)-baclofen on many models of bronchial hyperreactivity bothin vivo andin vitro. (–)-Baclofen protects guinea-pigs from the anaphylactic bronchospasm induced in sensitized animals by an ovalbumin aerosol and from that induced by aerosols of histamine and PGF₂. Moreover (–)-baclofen reduces the TXA₂ and TXB₂ output induced by ovalbumin from isolated sensitized guinea-pig lungs. On the other hand (–)-baclofen does not show antihistaminic, anticholinergic or antiprostaglandinic action on isolated tracheal preparations. It is concluded that baclofen can provide protection from bronchial hyperreactivity possibly through a modulation of autonomic nervous system activity.     

血小板活化因子的支气管收缩作用及其机理研究

本文研究了血小板活化因子对豚鼠在体和离体支气管平滑肌的收缩作用，同时对ＰＡＦ的这一作用机制进行了讨论。结果：１．ＰＡＦ静脉和雾化吸入均迅速引起支气管收缩，并伴有外周循环血小板数减少。２．单纯ＰＡＦ对离体支气管平滑肌条的收缩作用微弱，当加入洗涤血小板后其收缩作用明显增强。３．血栓素Ａ２受体拮抗剂（ＡＡ－２４１４）明显抑制了ＰＡＦ加血小板而引起的支气管平滑肌收缩。提示ＰＡＦ对支气管平滑肌可有直接作用，     

Capsaicin can abolish spontaneous tone in guinea-pig trachealis

The properties of spontaneous tone in isolated preparations of guinea-pig tracheal smooth muscle were examined. Experiments with control preparations revealed that 5–15 min after stretching the muscle with 0.15 mN, the spontaneous tone assumed a plateau value from which it declined gradually during the following hour. During the plateau, the force amounted to ～35% and 1 h later to ～20% of a maximum KC1 contraction. The tone was independent of tetrodotoxin, atropine and propranolol. Indomethacin quickly and completely relaxed the tone in 15 of 21 preparations. However, four preparations retained some tone even after 1 h of treatment. Exposure to the C-fibre influencing drug capsaicin resulted in a dose-dependent, reversible suppression of spontaneous tone, normally preceded by a transient increase in force. No spontaneous tone at all remained after 1 h of 10 μM capsaicin. This effect was also found in preparations pretreated with tetrodotoxin, atropine and propranolol. Preparations, deprived of spontaneous tone by capsaicin-treatment, contracted distinctly when exposed to 10 μM arachidonic acid. This contraction was almost completely abolished by indomethacin, which indicates that the prostaglandin synthesis is functioning after capsaicin treatment and, thus, that inhibition of this synthesis is not responsible for the capsaicin effect. Exposure to phosphoramidon increased the spontaneous tone almost threefold. Addition of 3 nM neurokinin A in the permanent presence of capsaicin gave weaker contractions in preparations where prostaglandin synthesis had been abolished by indomethacin, as compared to contractions in preparations with intact prostaglandin synthesis. The data indicate that a continuous release of tachykinins from sensory C-fibres is essential for the generation of spontaneous tone and that a combination of tachykinins and prostaglandins determine the size of the tone in this preparation.     

Histopathology of the airway epithelium in an experimental dual-phase model of bronchial asthma

BACKGROUND: Lesions of trachea cuticles are a pathological histological characteristic of bronchial asthma. Furthermore, collected tracheal cuticles desquamated from the respiratory tract are found in patients' sputum when asthma attacks occur or after the induction of allergen inhalation. From these facts, it is assumed that desquamation of trachea cuticle cells is a pathological symptom of bronchial asthma. However, there has not been any chronological report of desquamation of trachea cuticles through the process of bronchial asthma attacks. OBJECTIVE: For this report, we made an experimental bronchial asthma model using guinea pigs, and conducted chronological examinations of trachea cuticle lesions related to pathological symptoms of bronchial asthma using a transmission electron microscope and a scanning electron microscope. METHODS: The experimental asthma models were made by injection of ovalbumin into the abdominal cavity of guinea pigs. Then the airway responses to inhaled aerosolized ovalbumin were induced. The trachea were enucleated and examined under an optical microscope, a transmission electron microscope (hereafter abbreviated as TEM), and a scanning electron microscope (hereafter abbreviated as SEM) after 1, 2, 4, 8, 12, 24 h and 7 days after the immediate airway responses. RESULTS: Intercellular oedema of ciliated epithelium was observed in the sensitization groups immediately after the immediate airway response. SEM observation revealed increased mucus secretion and shortening of cilium. A slight case of desquamation or deciduation of ciliated epithelium was also beginning to appear. TEM observation revealed a dilation of ciliated epithelium intervals. Infiltration of eosinophilic leucocytes was already detectable beneath the ciliated epithelium. The degree of ciliated epithelium desquamation and infiltration of eosinophilic leucocytes progressed with time. When the late airway response occurred 4 hours later, eosinophilic leucocytes had increased drastically, and ciliate epithelium had desquamated to the extent that basal cells were exposed. Seven days after the immediate airway response, epithelium intercellular oedema had improved, and cilium had been reproduced. CONCLUSION: These results suggest that desquamation of epithelium caused by trachea cuticle lesions appears at an early stage of an asthma attack, owing to the contraction of the trachea, and that the damage is intensified by the infiltration of eosinophilic leucocytes.     

Thymoquinone-induced relaxation of guinea-pig isolated trachea

The effect of thymoquinone (TQ), the main constituent of the volatile oil of black seed (Nigella sativa), on the guinea-pig isolated tracheal zig-zag preparation was investigated. TQ caused a concentration-dependent decrease in the tension of the tracheal smooth muscle precontracted by carbachol. The effects of TQ were significantly potentiated by pretreatment of the tracheal preparations with quinacrine, a phospholipase A2 inhibitor, nordihydroguiaretic acid, a lipoxygenase inhibitor and by pretreatment with methylene blue, an inhibitor of soluble guanylyl cyclase. On the other hand, the effects of TQ were not influenced by pretreatment of the tracheal preparations with indomethacin, a cyclooxygenase inhibitor, propranolol, a non-selective beta-adrenoceptor blocker or by the pretreatment with theophylline, an adenosine receptors antagonist TQ totally abolished the pressor effects of histamine and serotonin on the guinea-pig isolated tracheal and ileum smooth muscles. The results of the present study suggest that TQ induced relaxation of precontracted tracheal preparation is probably mediated, at least in part, by inhibition of lipoxygenase products of arachidonic acid metabolism and possibly by non-selective blocking of the histamine and serotonin receptors. This relaxant effect of TQ, further support the traditional use of black seeds either alone or in combination with honey to treat bronchial asthma.     

Tachykinin receptors are involved in the "local efferent" motor response to capsaicin in the guinea-pig small intestine and oesophagus

The sensory neuron stimulant drug capsaicin stimulates primary afferent nerve endings in the guinea-pig small intestine, which in turn activate myenteric cholinergic neurons by an unknown mechanism. The tachykinins substance P and neurokinin A are present in primary afferent neurons. This study was performed to assess the possible involvement of endogenous tachykinins acting via neurokinin-1, neurokinin-2 and neurokinin-3 receptors in the contractile effect of capsaicin in the isolated guinea-pig ileum and oesophagus by using the receptor-specific antagonists GR 82334 (3 microM) for neurokinin-1 receptors, MEN 10627 (3 microM; ileum) or MEN 11420 (1 microM; oesophagus) for neurokinin-2 receptors and SR 142801 (0.1 microM) for neurokinin-3 receptors. In the ileum, the peak contraction evoked by capsaicin (2 microM) was not reduced when tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors were blocked separately, whereas an inhibition of neurokinin-3 receptors diminished the area under the curve of the capsaicin response. A combined blockade of neurokinin-1 and neurokinin-3 receptors significantly depressed the effect of capsaicin; the amplitude of the contractile response was 53.3+/-3.7% of the maximal longitudinal spasm in control preparations, whereas in the presence of GR 82334 plus SR 142801 it reached only 27.6+/-5% (P<0.001, Kruskal-Wallis test; n=9 and 10, respectively). Also, the area under the curve of the contractile response to capsaicin was more than 85% lower in the group of preparations treated with GR 82334 plus SR 142801 than in the control group (P<0.001). Including a neurokinin-2 blocker in the combination did not produce any further inhibition. A concomitant tachyphylaxis to substance P (natural neurokinin-1 receptor stimulant) and the neurokinin-3 receptor agonist senktide (5 and 1 microM, respectively) also reduced the contractile effect of capsaicin. In the oesophagus, capsaicin (1 microM) induced biphasic contractions which were strongly inhibited by atropine (1 microM) or capsaicin pretreatment (1 microM for 10 min). Here again, a blockade of tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors separately failed to inhibit the response to capsaicin, whereas a combined blockade of any two tachykinin receptors caused a partial inhibition. The reduction of the contractile effect of capsaicin was strongest when all three tachykinin receptors were blocked. In seven control preparations, peaks for the first and second phases of contraction reached 35.3+/-3.7% and 20+/-3.2% of maximal longitudinal spasm; the corresponding values in the presence of a combination of GR 82334, MEN 11420 and SR 142801 were 7.5+/-0.8% and 9.1+/-2.2%, respectively (n=6, P<0.001 and 0.05, respectively). Tetrodotoxin (0.5 microM) practically abolished the contractile effect of capsaicin in both tissues studied. It is concluded that an interplay of neuronal tachykinin neurokinin-1 and neurokinin-3 receptors (ileum) and neurokinin-1, neurokinin-2 and neurokinin-3 receptors (oesophagus) is involved in the contractile action of capsaicin, probably in mediating excitation of myenteric neurons by tachykinins released from primary afferents. In both tissues, there also seems to be a non-tachykininergic component of the capsaicin-induced contraction.     

Detection of nitric oxide release induced by bradykinin in guinea pig trachea and main bronchi using a porphyrinic microsensor

Indirect evidence using nitric oxide (NO) synthase (NOS) inhibitors suggests that in guinea-pig airways bradykinin releases bronchoprotective NO. In this study, using a recently developed electrochemical method of NO measurement based on a porphyrinic microsensor, we investigated whether bradykinin releases NO from guinea-pig airways and whether the epithelium is the main source of NO. Further, the Ca(2+)-dependence of bradykinin-induced NO release was assessed stimulating airway preparations with bradykinin in Ca(2+)-free conditions. We also studied the immunohistochemical distribution of the Ca(2+)- dependent constitutive isoforms of NOS (constitutive NOS [cNOS]: neuronal and endothelial [ecNOS]) in our preparations. The porphyrinic microsensor was placed in the bathing fluid onto the mucosal surface of tracheal or main bronchial segments. Addition of bradykinin vehicle (0.9% saline) did not cause any detectable change of the baseline signal. Addition of bradykinin caused an upward shift of the baseline that reached a maximum within 1 to 2 s. The amplitude of the response to bradykinin was concentration-dependent between the range 1 nM to 10 microM, with a maximum effect at 10 microM. Bradykinin-induced NO release was higher in tracheal than in main bronchial segments. The selective bradykinin B(2) receptor antagonist D-Arg(0)-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]bradykinin (1 microM) inhibited NO release induced by a submaximum concentration of bradykinin (1 microM). The ability of bradykinin to release NO was markedly reduced in epithelium-denuded segments, and abolished in Ca(2+)-free conditions and after pretreatment with N(G)-monomethyl-L-arginine (100 microM), but not with N(G)-monomethyl-D-arginine. Both cNOS isoforms were present in trachea and main bronchi, ecNOS being the predominant isoform in the epithelium. The study shows that bradykinin via B(2) receptor activation caused a rapid and Ca(2+)-dependent release of NO, mainly, but not exclusively, derived from the epithelium. It also shows that both cNOS isoforms may be involved in bradykinin-evoked NO release.