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

为了阐明受体型酪氨酸激酶c-kit在平滑肌细胞功能发育中的作用，我们研究了Ca^<2+>诱导的BALB/c小鼠胃肠道平滑肌细胞Ca^<2+>释放的特性。在使用制霉菌素穿孔膜片钳技术的电压钳制条件下，表达c-kit^+的细胞（c-kit^+）产生了节律性的Ca^2+激活的Cl^-电流，而对照动物的平滑肌细胞产生了自发外向电流以及咖啡因和卡巴胆碱诱导的瞬时外向电流。用咖啡因诱导的Ca^2+激活的K^+电流和fura-2荧光法测量细胞内Ca^2+浓度，结果表明，在接受ACK 2处理的小鼠的平滑肌细胞中，细胞质中对ryanodine敏感的Ca^2+储存功能障碍。对照组平滑肌细胞的Ca^2+释放和咖啡因诱导的Ca^2+激活的K^+电流均对ryanodine敏感，并且对L型电压门控Ca^2+通道的活性有很大的依赖性。在测量等长张力时，在高K^+去极化或卡巴胆碱预收缩的平滑肌中，咖啡因诱导的舒张显著加速。以上结果提示，ryanodine敏感性Ca^2+释放是调节细胞质Ca^2+稳态和胃肠平滑肌收缩力的重要因素，c-kit^+细胞通过节律性兴奋和胞质Ca^2+波动在平滑肌发育中起重要作用。

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.