Relation between mechanisms of intracellular signaling pathways and auditory transmission system of shortening in cochlear hair cells

用蛋白激酶和蛋白磷酸酶的抑制剂和/或激活剂研究了豚鼠耳蜗外毛细胞缓慢缩短的细胞内机制。用强直电场刺激诱导细胞的缓慢缩短，用氯离子敏感的荧光染料显微镜监测细胞长度、体积和细胞内氯离子浓度的变化。钙调素抑制剂W-7和钙调素依赖的蛋白激酶11(CaMKII)抑制剂可抑制细胞的缓慢缩短。KN-62W-7或KN-62的抑制作用可被K~+与呋喃霉素的电导所抵消。在所测试的蛋白磷酸酶抑制剂中，1和2A型蛋白磷酸酶抑制剂Calyculin A可抑制缓慢缩短。Calyculin A的抑制作用可被增加的氯离子通透性所消除，但既不能被呋喃霉素增加的K+电导所消除，也不能被A…的钙电导增加所消除超过23187。蛋白丝氨酸/苏氨酸磷酸酶激动剂N-乙酰鞘氨醇可抑制外毛细胞的缩短，这种缩短可被瓦林霉素或2A型蛋白磷酸酶抑制剂冈田酸所阻断，但不能被花盏蛋白A所阻断。这些结果提示，外毛细胞缓慢缩短的信号机制如下：K~+通道开放通过CaMKII蛋白磷酸化促进，而K~+通道开放通过冈田克酸敏感的去磷酸化而抑制，而Cl~+通道开放依赖于Calyculin A敏感蛋白磷酸酶活性。这种收缩在刺激后30-40秒出现，持续约1分钟。用数字成像显微镜和对氯离子敏感的荧光染料N-(6-甲氧基喹啉)乙酰乙酯，细胞体积和细胞内氯离子浓度随细胞浓度的增加而减小。细胞松弛素B抑制了这一事件，表明收缩是由肌动蛋白介导的。较少

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 Cl^- 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 11 (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 Cl^- permeability, but neither by the increased K^+ conductance with valinomycin nor by the increased Ca^<2+> conductance with A … More 23187. 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 Cl^- channel opening depends on calyculin A-sensitive protein phosphatase activity.Tetanic electrical field stimulation elicited a prominent contraction of isolated guinea pig cochlear inner hair cells. This contraction appeared 30-40 sec after field stimulation and lasted for about 1 min. Using a digital imaging microscope and the Cl^- sensitive fluorescence dye, N-(6-methoxyquinolyl) acetoethyl ester, the decrease in cell volume and intracellular Cl^- concentration were concomitant with the cell concentration. Cytochalasin B inhibited this event, suggesting that the contraction is mediated by actin. Less