Study of mechanisms of ionic transport and intracellular signaling pathways of shortening in cochlear outer hairce

使用蛋白激酶和蛋白磷酸酶的抑制剂和/或激活剂研究了离体豚鼠耳蜗外毛细胞缓慢缩短的细胞内机制。强电场刺激可使其缓慢缩短。使用氯敏感荧光染料显微镜下监测细胞长度、体积和细胞内CI^-浓度的变化。钙调素抑制剂抑制了缓慢缩短。W-7，以及钙钙调素依赖性蛋白激酶II （CaMKII）抑制剂。kn - 62。W-7或KN-62的抑制作用被补充万霉素的K^+电导所消除。在蛋白磷酸酶抑制剂中检测了1型和2A型蛋白磷酸酶抑制剂。calyculin a .抑制缓慢缩短。calyculin A的抑制作用被CI^-通透性的增加所消除。但与valinomycin和A23187均不增加K^+电导和Ca^<2+>电导。一种蛋白质丝氨酸/苏氨酸磷酸酶激活剂，n -乙酰鞘氨酸抑制了被缬霉素或2A型蛋白磷酸酶抑制剂所消除的缩短。这些发现提示外毛细胞缓慢缩短的信号机制如下：K^+通道的打开是通过CaMKII的蛋白磷酸化促进的，并通过冈田酸敏感的去磷酸化抑制，C1^-通道的打开依赖于calyculin a敏感的蛋白磷酸酶活性。

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation.and changes in the cell length volume and intracellular CI^- concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor. W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor. KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K^+ with valinomycin. Among the protein phosphatase inhibitors tested a type 1 and 2A protein phosphatase inhibitor. calyculin A.inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased CI^- permeability. but neither by the increased K^+ conductance with valinomycin nor by the increased Ca^<2+> conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine. inhibited the shortening which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor. okadaic acid but not by calyculin A.These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells : the K^+ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the C1^- channel opening depends on calyculin A-sensitive protein phosphatase activity.B.V.