Pezzi V, Clark B J, Ando S, Stocco D M, Rainey W E
Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas 75235, U.S.A.
J Steroid Biochem Mol Biol. 1996 Jul;58(4):417-24. doi: 10.1016/0960-0760(96)00052-0.
Acute aldosterone production in adrenocortical cells is highly dependent on calcium (Ca2+) and calmodulin (CaM) activation. To determine the role of calmodulin-dependent protein kinase II (CaM kinase II) in human adrenal aldosterone production, the action of KN93 (a specific CaM kinase II inhibitor) on human adrenocortical H295R cells was examined. The stimulation of aldosterone, production by angiotensin II (Ang II) and potassium (K+) were inhibited by KN93 in a concentration-dependent manner with an IC50 of approximately 0.9 and approximately 0.5 microM, respectively. Aldosterone production was also stimulated by treatment with the calcium channel activator Bay K 8644 (Bay K) (1 microM). This production was inhibited in a concentration-dependent manner by KN93 with an IC50 of between 1 and 3 microM. No inhibition by KN93 (0.3-3 microM) or by the calmodulin inhibitor calmidazolium (0.03-0.3 microM) was observed for 22R-hydroxycholesterol (22R-OHChol) stimulation of aldosterone production. Because 22R-OHChol is a substrate for the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and does not require active transport to the mitochondria, these results indicate that KN93 does not directly inhibit P450scc or later steps leading to aldosterone synthesis. To investigate the site of KN93 action further we examined its effect on agonists induction of steroidogenic acute regulatory (StAR) protein, which was recently shown to regulate the movement of cholesterol from the outer to the inner mitochondrial membranes. Induction of StAR protein in H295R cells by Ang II, or Bay K was not affected by co-treatment with KN93 at concentration which blocked steroidogenesis by 60-80%. These results indicate a direct role of CaM kinase II in Ang II and K+ simulation of aldosterone production and support the hypothesis that CaM kinase II may be involved in the process of cholesterol mobilization to the mitochondria.
肾上腺皮质细胞中醛固酮的急性产生高度依赖于钙（Ca2+）和钙调蛋白（CaM）的激活。为了确定钙调蛋白依赖性蛋白激酶II（CaM激酶II）在人肾上腺醛固酮产生中的作用，研究了KN93（一种特异性CaM激酶II抑制剂）对人肾上腺皮质H295R细胞的作用。KN93以浓度依赖性方式抑制血管紧张素II（Ang II）和钾（K+）刺激的醛固酮产生，IC50分别约为0.9和0.5 microM。用钙通道激活剂Bay K 8644（Bay K）（1 microM）处理也刺激了醛固酮产生。KN93以浓度依赖性方式抑制这种产生，IC50在1至3 microM之间。未观察到KN93（0.3 - 3 microM）或钙调蛋白抑制剂氯咪达唑（0.03 - 0.3 microM）对22R-羟基胆固醇（22R-OHChol）刺激醛固酮产生的抑制作用。由于22R-OHChol是细胞色素P450胆固醇侧链裂解酶（P450scc）的底物，且不需要主动转运到线粒体，这些结果表明KN93不会直接抑制P450scc或导致醛固酮合成的后续步骤。为了进一步研究KN93的作用位点，我们研究了其对类固醇生成急性调节（StAR）蛋白激动剂诱导的影响，最近发现StAR蛋白可调节胆固醇从线粒体外膜向内膜的转运。Ang II或Bay K诱导H295R细胞中StAR蛋白的过程不受与KN93共同处理的影响，此时KN93的浓度可使类固醇生成受阻60 - 80%。这些结果表明CaM激酶II在Ang II和K+刺激醛固酮产生过程中起直接作用，并支持CaM激酶II可能参与胆固醇向线粒体转运过程的假说。
