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

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。通过强直电场刺激诱导缓慢缩短，并使用氯化物敏感的荧光染料显微镜下监测细胞长度体积和细胞内Cl ^-浓度的变化。钙调素抑制剂可抑制缓慢缩短。W-7和钙钙调蛋白依赖性蛋白激酶II（CaMKII）抑制剂。KN-62 W-7或KN-62的抑制作用可被缬氨霉素补充的K ^+电导所消除。在测试的蛋白磷酸酶抑制剂中，有1型和2A型蛋白磷酸酶抑制剂。calyculin A抑制缓慢缩短。C1-β-渗透性的增加消除了calyculin A的抑制作用。但这既不是由于缬氨霉素增加了K ^+电导，也不是由于A23187增加了Ca ^2+电导。一种蛋白质丝氨酸/苏氨酸磷酸酶激活剂，N-乙酰鞘氨醇。抑制被缬氨霉素或2A型蛋白磷酸酶抑制剂消除的缩短。这些发现表明，在外毛细胞缓慢缩短过程中存在以下信号机制：CaMKII通过蛋白磷酸化促进K ^+通道开放，而冈田酸敏感性去磷酸化抑制K ^+通道开放，C1 ^-通道开放依赖于calyculin A敏感性蛋白磷酸酶活性。

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.