Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

BACKGROUND: Extracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca(2+)](i )signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways. METHODS: Isometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca(2+)](i )was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student's t tests for quantitative variables and χ(2 )tests for qualitative variables. Results were considered significant at P < 0.05. RESULTS: In rat airways, extracellular ATP (10(-6)–10(-3 )M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca(2+)](i )response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca(2+), external Na(+ )and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways. CONCLUSION: Extracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca(2+ )influx in addition with, in IPB, P2Y receptors stimulation and Ca(2+ )release from intracellular Ca(2+ )stores. Extracellular Ca(2+ )influx occurs through L-type voltage-dependent channels activated by external Na(+ )entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation

Expression and function of human hemokinin-1 in human and guinea pig airways

<p>Abstract</p> <p>Background</p> <p>Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the <it>TAC4 </it>gene. <it>TAC4 </it>and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study.</p> <p>Methods</p> <p>RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages.</p> <p>Results</p> <p>In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK₁-and NK₂-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK₂-receptors, which blockade unmasked a NK₁-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK₁-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages.</p> <p>Conclusions</p> <p>We demonstrate endogenous expression of <it>TAC4 </it>in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.</p

Relaxant effect of the H2-receptor antagonist oxmetidine on guinea-pig and human airways.

The effects of three different H2-receptor antagonists (cimetidine, ranitidine and oxmetidine) were tested on isolated preparations of guinea-pig trachea and human bronchus against contractions induced by acetylcholine, histamine and potassium chloride (KCl). In addition, their influence on calcium concentration-response curves in guinea-pig tracheal spirals was examined in a potassium-rich solution (30 mM). Finally, their effects were studied in vivo against acetylcholine and histamine-induced bronchoconstriction in anaesthetized guinea-pigs. In guinea-pig isolated trachea, oxmetidine--in contrast to cimetidine and ranitidine, which were completely inactive--induced a concentration-dependent relaxation regardless of the excitatory stimulus: its--log EC50 values (i.e. the negative log concentration that caused a 50% relaxation) were 3.46 +/- 0.11, 4.61 +/- 0.09 and 4.20 +/- 0.12 against acetylcholine, histamine and KCl, respectively. In Ca2+-free, K+-enriched solution, the compound was able to inhibit Ca2+-induced contractions at concentrations close to those needed to counteract the spasmogenic effect of histamine in normal Krebs solution. Results obtained in the human bronchus preparation were similar to those observed in guinea-pig tracheal spirals. When tested against acetylcholine or histamine-induced bronchoconstriction in vivo, oxmetidine (10 and 30 mg Kg-1 intravenously) significantly reduced the increase in pulmonary airway resistance (Raw) induced by both agents. Once again, cimetidine and ranitidine were completely ineffective. In summary, oxmetidine displayed non-specific antispasmogenic activity on guinea-pig and human airways. This effect, which is independent of H2-receptor blockade, represents a side-effect of the drug which may be connected to its interference with Ca2+ influx and the action or release of intracellular Ca2+

Relaxant effect of the H2-receptor antagonist oxmetidine on guinea-pig and human airways.

The effects of three different H2-receptor antagonists (cimetidine, ranitidine and oxmetidine) were tested on isolated preparations of guinea-pig trachea and human bronchus against contractions induced by acetylcholine, histamine and potassium chloride (KCl). In addition, their influence on calcium concentration-response curves in guinea-pig tracheal spirals was examined in a potassium-rich solution (30 mM). Finally, their effects were studied in vivo against acetylcholine and histamine-induced bronchoconstriction in anaesthetized guinea-pigs. In guinea-pig isolated trachea, oxmetidine--in contrast to cimetidine and ranitidine, which were completely inactive--induced a concentration-dependent relaxation regardless of the excitatory stimulus: its--log EC50 values (i.e. the negative log concentration that caused a 50% relaxation) were 3.46 +/- 0.11, 4.61 +/- 0.09 and 4.20 +/- 0.12 against acetylcholine, histamine and KCl, respectively. In Ca2+-free, K+-enriched solution, the compound was able to inhibit Ca2+-induced contractions at concentrations close to those needed to counteract the spasmogenic effect of histamine in normal Krebs solution. Results obtained in the human bronchus preparation were similar to those observed in guinea-pig tracheal spirals. When tested against acetylcholine or histamine-induced bronchoconstriction in vivo, oxmetidine (10 and 30 mg Kg-1 intravenously) significantly reduced the increase in pulmonary airway resistance (Raw) induced by both agents. Once again, cimetidine and ranitidine were completely ineffective. In summary, oxmetidine displayed non-specific antispasmogenic activity on guinea-pig and human airways. This effect, which is independent of H2-receptor blockade, represents a side-effect of the drug which may be connected to its interference with Ca2+ influx and the action or release of intracellular Ca2+

Role of bradykinin and tachykinins in the potentiation by enalapril of coughing induced by citric acid in pigs.

Angiotensin-converting enzyme (ACE) inhibitors are among the first-choice drugs for treating hypertension and congestive heart disease. It has been reported, however, that these drugs could induce chronic cough and airway hyperresponsiveness. The aim of this work was to assess in pigs the effects of bradykinin and tachykinins on citric-acid-induced coughing after ACE inhibitor pretreatment. Coughing was induced by challenging pigs with an aerosol of 0.8 M citric acid over 15 min. Coughs were counted by a trained observer for 30 min. The animals underwent two cough induction tests two days apart (days 1 and 3), the first being taken as a control. All drugs were injected intravenously 30 min before the second challenge. In the control group, no difference was observed between days 1 and 3. The ACE inhibitor enalapril (7.5 and 15 microg/kg) caused the cough frequency to increase significantly. In contrast, a dose-related decrease was observed with Hoe140 (icatibant), a bradykinin B2 receptor antagonist (0.5 and 1 mg/kg). When both drugs were administered simultaneously (15 microg/kg for enalapril and 1 mg/kg for Hoe140), a significant increase was observed as compared with the control value obtained on day 1. When enalapril was combined with the three tachykinin receptor antagonists SR 140333 (NK1 receptor antagonist), SR 48968 (NK2 receptor antagonist) and SR 142801 (NK3 receptor antagonist), a significant decrease was observed as compared with control value obtained on day 1; the percentage of variation was also significantly different as compared with those observed in enalapril groups at both doses. These data suggest that ACE-inhibitor-induced enhancement of the cough reflex is mainly due to tachykinins and not to bradykinin in our pig model. Bradykinin, however, plays a major role in coughing induced by citric acid alone

Effects of Age and Indomethacin on Response and Sensitivity of Pulmonary Artery to Phenylephrine and to Histamine in Pigs

The vasoconstrictor effects of phenylephrine and histamine were investigated in isolated strips of pulmonary arteries in pigs during ageing. Interactions between phenylephrine-induced responses and arachidonic acid derivatives were also studied by incubating the blood-vessels with indomethacin. Potency (pD2 values) and maximal effects (Emaxx) recorded in 5-week-old piglets (group I, n= 5) with phenylephrine [5.71 ± 0.17 and 0.76 ± 0.22 g/mg of dry tissue respectively (mean ± SEM)] were similar to values found in 12-week-old animals (group 2, n = 5) (5.49 ± 0.30 and 1.06 ± 0.27 g/mg of dry tissue respectively). The sensitivity and responsiveness of tissues to this agonist were significantly reduced in 26-week-old mature pigs (group 3, n = 6) as indicated by the decrease in pD2 (3.91 ± 0.23; P < 0.01) and Emax (0.27 ± 0.13 g/mg of dry tissue; P < 0.05) values observed in this group. Histamine (10_3M)-induced maximal responses (Emax) were significantly higher in group 2 (2.23 ± 0.49 g/mg) than in group 1 (0.85 ± 0.11 g/mg; P < 0.05) and in group 3 (0.48 ± 0.10 g/mg; P < 0.01). In 5-week-old animals, indomethacin (3.10˜5M) significantly (P < 0.05) shifted the concentration-response curve to phenylephrine to the right (0.28 log. units) and depressed contractions to this drug as shown by the significant decrease of 39.5% (P < 0.05) in Emax. This cyclo-oxygenase inhibitor had no effect in other groups. These data indicate that phenylephrine is a potent and effective vasoconstrictor agent for the main pulmonary arteries in 5-week-old piglets and that alpha-1-adrenergic-induced contractions are enhanced by cyclo-oxygenase products. These findings can be related with the high reactivity of pulmonary vascular smooth muscles in these animal