Importance of inositol (1,4,5)-trisphosphate, intracellular Ca2+ release and myofilament Ca2+ sensitization in 5-hydroxytryptamine-evoked contraction of rabbit mesenteric artery.

1. Small strips from third-order branches of rabbit mesenteric artery (approximately 150-200 microM wide) contracted in response to noradrenaline (10 microM) or 5-hydroxytryptamine (5-HT; 10 microM) in oxygenated Krebs solution containing 2.5 mM Ca2+. In a Ca(2+)-free mock intracellular solution (0 Ca2+ plus 0.2 mM EGTA), noradrenaline (10 microM) and caffeine (10 mM) induced only a single, transient contraction in artery strips, while 5-HT (10 microM) failed to induce any response. 2. In strips of mesenteric artery which had been permeabilized with Staphylococcus alpha-toxin and bathed in Ca(2+)-free mock intracellular solution, noradrenaline (10 microM), caffeine (10 mM) and D-myo-inositol (1,4,5)-trisphosphate (IP3, 100 microM), but not 5-HT (10 or 100 microM) induced a transient contraction. In contrast to the non-permeabilized strips, contractions to noradrenaline, caffeine and IP3 were restored by prior incubation (10 min) in solution containing 0.08 microM Ca2+. The contractions to noradrenaline and IP3 in permeabilized muscle strips required the presence of 100 microM guanosine 5'-triphosphate (GTP), although in the absence of Ca2+. GTP alone did not induce contraction. 3. Exposure of permeabilized mesenteric artery strips to IP3 significantly reduced the subsequent contractile responses to caffeine. Contractile responses to caffeine and IP3 were abolished by the Ca(2+)-ATPase inhibitor, thapsigargin (1 microM). 4. Ca2+ (0.1-10 microM) induced concentration-dependent contraction in permeabilized artery strips. In strips which were submaximally contracted with 0.5 microM Ca2+/100 microM GTP, the subsequent addition of 5-HT (10 microM) stimulated further contraction. The protein kinase C inhibitor, H-7 (1 microM) abolished the 5-HT/GTP-induced contraction, but did not alter the contraction to Ca2+. 5. In non-permeabilized, endothelium-denuded segments of rabbit mesenteric artery bathed in Ca2+-replete Krebs solution, noradrenaline (10 microM) stimulated a rapid, transient accumulation of IP3. 5-HT(100 microM) failed to stimulate IP3 accumulation during exposure periods of up to 5 min. 5-HT (100 microM)did stimulate IP3 accumulation if the external K+ concentration was raised (to around 25 mM). This concentration of K+ alone did not stimulate IP3 production and the 5-HT-stimulated IP3 accumulation in the presence of elevated extracellular [K+] was abolished by the alpha l-adrenoceptor antagonist, prazosin(O.1 microM).6. These results suggest that intracellular Ca2+ release does not play an important role in 5-HT-induced smooth muscle contraction in the rabbit mesenteric artery. This is despite the fact that a significant intracellular Ca2+ pool is present in these cells, which can be discharged by either noradrenaline or IP3.However, 5-HT did stimulate smooth muscle contraction in the presence of raised intracellular calcium,suggesting that a component of the contraction to 5-HT will reflect an increase in myofilament Ca2+sensitivity, possibly due to the activation of protein kinase C.