Roles of c-kit in functional development of Ca^<2+> release mechanism in smooth muscle cells

为了阐明受体酪氨酸激酶c-kit在平滑肌细胞功能发育中的作用，我们研究了中和抗c-kit单抗(ACK2)处理BALB/c小鼠新鲜分离的胃肠道平滑肌细胞时，由Ca~(2+)诱导的Ca~(2+)释放特性。在制霉菌素穿孔膜片钳技术的电压钳制条件下，c-kit表达(c-kit^+)细胞产生节律性钙激活的氯离子电流，对照组的平滑肌细胞产生自发外向电流和咖啡因和氨基甲胆碱诱导的瞬时外向电流。在ACK2处理的小鼠的平滑肌细胞中，当用咖啡因诱导的Ca^&lt；2+&gt；激活的K^+电流和Fura-2荧光测量细胞内Ca^&lt；2+&gt；浓度时，表明胞浆中兰诺定敏感的Ca^&lt；2+&gt；存储功能障碍。对照组大鼠血管平滑肌细胞钙离子释放和咖啡因激活的钾电流对兰诺定敏感，并依赖L型电压门控性钙通道的活动。在等长张力的测量中，咖啡因引起的松弛在高K~+去极化或卡巴胆碱预收缩的平滑肌上显著加速。综上所述，Ryanodine敏感的Ca~(2+)释放是胞浆内Ca~(2+)动态平衡和小鼠胃肠平滑肌收缩的重要调节剂，c-kit^+细胞通过胞浆内的节律性兴奋和Ca~(2+)波动在平滑肌发育中发挥重要作用。

To clarify the roles of c-kit, a receptor-type tyrosine kinase, in the functional development of smooth muscle cells, we investigated the property of the Ca^<2+>-induced Ca^<2+> release in smooth muscle cells freshly dissociated from gastrointestinal tract of BALB/c mice treated with neutralizing anti-c-kit-monoclonal antibody (ACK2). Under voltage-clamped conditions with the nystatin-perforated patch-clamp technique, c-kit-expressing (c-kit^+) cells developed rhythmic Ca^<2+>-activated Cl^- currents and smooth muscle cells from the control animals developed spontaneous outward currents and caffeine-and carbachol-induced transient outward currents. In smooth muscle cells from the ACK2-treated mice, dysfunction in the ryanodine-sensitive Ca^<2+> storage in the cytoplasm was suggested when tested with caffeine-induced Ca^<2+>-activated K^+ currents and fura-2 fluorometric measurements of intracellular Ca^<2+> concentration. The Ca^<2+> release and caffeine-induced Ca^<2+>-activated K^+ currents in smooth muscle cells from the control animals were ryanodine-sensitive and had great dependence upon the activity of L-type voltage-gated Ca^<2+> channels. In measurements of isometric tension, caffeine-induced relaxation was significantly accelerated in smooth muscles precontracted with high K^+ depolarization or carbachol. Altogether, it is suggested that ryanodine-sensitive Ca^<2+> release is an important modifier of Ca^<2+> homeostasis in the cytoplasm and the contractility of gastrointestinal smooth muscle of mice and that the c-kit^+ cells play important roles in the development of smooth muscle through the rhythmic excitation and Ca^<2+>-fluctuation in the cytoplasm.