Evidence for the involvement of multiple mechanisms in the excitatory action of bradykinin in the circular muscle of guinea-pig colon.

We have investigated, by using the sucrose gap technique, the mechanisms of the excitatory action of bradykinin in the circular muscle of the guinea-pig proximal colon. In the presence of atropine (1 microM) and S-ketoprofen (3 microM), the application of bradykinin (1 microM for 20 s) produced complex changes in membrane potential and muscle tension. The prevailing response was a small hyperpolarization followed by a slowly developing depolarization and a tonic contraction. The selective B2 receptor antagonist, HOE 140 (0.3 microM) blocked the responses to bradykinin (1 microM) while tetrodotoxin (0.3 microM) had no affect. The selective B1 receptor agonist, [des-Arg9]bradykinin (1 microM) did not affect the electrical or mechanical activities of the circular muscle. Apamin (0.1 microM) blocked the transient hyperpolarization and potentiated the bradykinin-induced depolarization and contraction. In the presence of apamin, nifedipine (1 microM) blocked spikes (when present) and the phasic contraction while leaving the tonic contraction unaffected. The excitatory action of bradykinin was further investigated in the presence of atropine (1 microM), S-ketoprofen (3 microM), apamin (0.1 microM) and nifedipine (1 microM). The depolarization but not the contraction induced by bradykinin was reduced by about 30% in low-Na+ (25 mM) but not in low Cl- (9.7 mM) Krebs solution. The depolarization and contraction evoked by bradykinin were reduced (by about 30 and 75%, respectively) in Ca2+-free (2 min) Krebs solution. The blocker of the sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (CPA, 10 microM) reduced the nifedipine-resistant depolarization and contraction induced by bradykinin by about 40 and 60%, respectively. The inhibitor of receptor-operated cation channels, SKF 96365 (50 microM) reduced the nifedipine-resistant bradykinin-induced depolarization and contraction by about 40 and 30%, respectively, whereas the inhibitor of Ca2+-dependent chloride channels, niflumic acid (100 microM) was without effect. The inhibitory effect of SKF 96365 (50 microM) and CPA (10 microM) was additive: in the presence of both drugs the bradykinin-induced depolarization and contraction were reduced by about 70-80%. The protein kinase C inhibitor, GF 109203x (10 microM) did not affect the nifedipine-resistant bradykinin-induced depolarization and contraction. At a concentration of 30 microM, GF 109203x reduced the bradykinin-induced contraction by about 50% while leaving the bradykinin-induced depolarization unaffected. The KCl (40 mM)-induced contraction was significantly reduced (by about 30%) by GF 109203x (30 microM). The present findings indicate that, in the presence of apamin and nifedipine, the bradykinin-induced contraction of circular muscle of the guinea-pig colon is due to the influx of extracellular Ca2+ via non-selective cation channels and, in part, to the release of Ca2+ from a loosely bound internal store. Intracellular Ca2+ facilitates the bradykinin-induced depolarization, a response which does not involve a protein kinase C-dependent mechanism.