Clyne C D, Nguyen A, Rainey W E
Dept of Obstetrics & Gynecology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Endocr Res. 1995 Feb-May;21(1-2):259-65. doi: 10.3109/07435809509030441.
The effects of KN62 on aldosterone secretion have been studied using an angiotensin II (AII)- and K(+)-responsive human adrenocortical tumor cell line (H295R). Basal aldosterone secretion (measured by RIA) was 0.57 +/- 0.22 pmol/mg protein.h. The physiologicial agonists AII (10 nM) and K+ (14 mM) increased aldosterone secretion by 6.9- and 5.0-fold, respectively. Aldosterone secretion was also stimulated by dibutyryl cyclic AMP (dbcAMP, 1 mM, 10.3-fold over basal). Nifedipine dose-dependently inhibited K(+)- and AII-stimulated aldosterone secretion. In contrast, dbcAMP-stimulated secretion was relatively insensitive to this agent (26.8% inhibition at 1 microM nifedipine). K(+)- and AII-stimulated aldosterone production was also dose-dependently inhibited by KN62, which produced 93.9% and 82.3% inhibition at 10 microM KN62 (both p < 0.01). In order to test the specificity of KN62 in H295R cells, its effects on various other steroidogenic agonists were assessed. KN62 dose-dependently inhibited aldosterone secretion stimulated by dbcAMP, 22-hydroxycholesterol and pregnenolone. In addition, KNO4, a derivative of KN62 which is not a potent inhibitor of CaM Kinase II, exhibited a similar pattern of inhibition. These data confirm the requirement for extracellular Ca2+ in the stimulation of human adrenocortical cell aldosterone secretion by AII and K+. However, the non-specific inhibitory effects of KN62 in H295R cells limit the usefulness of this agent as a tool for investigations of the involvement of CaM kinase II in adrenocortical steroidogenesis.
已使用对血管紧张素II（AII）和K⁺有反应的人肾上腺皮质肿瘤细胞系（H295R）研究了KN62对醛固酮分泌的影响。基础醛固酮分泌（通过放射免疫分析法测定）为0.57±0.22 pmol/mg蛋白质·小时。生理激动剂AII（10 nM）和K⁺（14 mM）分别使醛固酮分泌增加6.9倍和5.0倍。二丁酰环磷腺苷（dbcAMP，1 mM，比基础值高10.3倍）也刺激了醛固酮分泌。硝苯地平剂量依赖性地抑制K⁺和AII刺激的醛固酮分泌。相比之下，dbcAMP刺激的分泌对该药物相对不敏感（在1 μM硝苯地平下抑制26.8%）。K⁺和AII刺激的醛固酮生成也被KN62剂量依赖性地抑制，在10 μM KN62时分别产生93.9%和82.3%的抑制（两者p<0.01）。为了测试KN62在H295R细胞中的特异性，评估了其对各种其他类固醇生成激动剂的影响。KN62剂量依赖性地抑制dbcAMP、22-羟胆固醇和孕烯醇酮刺激的醛固酮分泌。此外，KN62的衍生物KNO4不是钙调蛋白激酶II的有效抑制剂，也表现出类似的抑制模式。这些数据证实了细胞外Ca²⁺在AII和K⁺刺激人肾上腺皮质细胞醛固酮分泌中的必要性。然而，KN62在H295R细胞中的非特异性抑制作用限制了该药物作为研究钙调蛋白激酶II参与肾上腺皮质类固醇生成工具的实用性。
