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

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。用强电刺激诱导细胞缓慢缩短，并用氯敏荧光染料显微镜观察细胞长度、体积和细胞内Cl^-浓度的变化。钙调素抑制剂W-7和钙调素依赖性蛋白激酶11 （CaMKII）抑制剂抑制了缓慢缩短。kn - 62。W-7或KN-62的抑制作用被补充了valinomycin的K^+电导所消除。在所测试的蛋白磷酸酶抑制剂中，1型和2A型蛋白磷酸酶抑制剂calyculin a抑制了缓慢缩短。calyculin A的抑制作用被Cl^-通透性的增加所消除，但既不能被valinomycin增加的K^+电导所消除，也不能被A增加的Ca^<2+>电导所消除。More 23187。蛋白丝氨酸/苏氨酸磷酸酶激活剂n -乙酰鞘氨醇能抑制毛细胞的缩短，而这一作用可被缬霉素或2A型蛋白磷酸酶抑制剂冈田酸所消除，但不能被calyculin A所消除。这些发现提示外毛细胞缓慢缩短的信号机制如下：K^+通道的打开通过CaMKII的蛋白磷酸化促进，并通过冈田酸敏感的去磷酸化抑制，而Cl^-通道的打开依赖于calyculin a敏感的蛋白磷酸酶活性。强电刺激引起离体豚鼠耳蜗内毛细胞明显收缩。这种收缩在现场刺激后30-40秒出现，持续约1分钟。利用数码成像显微镜和对Cl^-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Cl^-浓度随细胞浓度的降低而降低。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。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