Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.
Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.
复制标题
蛋白激酶 C 可能不参与胰岛素对 cAMP 磷酸二酯酶作用的证据：对对胰岛素高度敏感的电穿孔大鼠脂肪细胞进行的研究。
DOI：
10.1016/0003-9861(91)90333-e
复制
发表时间：
1991
影响因子：
3.9
通讯作者：
Kono,T
中科院分区：
文献类型：
--
作者：
Shibata,H;Robinson,FW;Benzing,CF;Kono,T
用电穿孔法制备了对胰岛素具有高反应性的部分通透的大鼠脂肪细胞，并研究了1-（5-异喹啉磺酰基）-2-甲基哌嗪（H-7）对脂肪细胞胰岛素作用的影响。H-7是几种蛋白激酶的抑制剂，包括蛋白激酶C;然而，它不能迅速进入受完整质膜保护的脂肪细胞。将细胞悬浮于缓冲液X [4.74 mmNaCl、118.0 mmKCl、0.38 mmCaCl 2、1.00 mmEGTA、1.19 mmMg 2SO 4、1.19 mmKH 2 PO 4、25.0 mmHepes/K、20 mg/ml牛血清白蛋白和3 mm丙酮酸/Na，pH 7.4]中，并用设定为25 μF和2 kV/cm的Gene-Pulser（来自Bio-Rad）电穿孔6次。在如上电穿孔的细胞中，胰岛素刺激（a）当激素浓度为10 nm时膜结合cAMP磷酸二酯酶约2.6倍，和（B）当激素浓度升高至100 nm时葡萄糖转运活性约4.5倍。H-7强烈抑制胰岛素对电穿孔脂肪细胞葡萄糖转运（表观Ki = 0.3 mM）和cAMP磷酸二酯酶（表观Ki = 1.2 mM）的作用。H-7也抑制脂肪细胞中的脂解;在完整细胞中的反应的appartentKi值为0.45 mm，在电穿孔的细胞中为0.075 mm。这表明，某些蛋白激酶或激酶，是显着敏感的H-7可能参与胰岛素依赖的刺激葡萄糖转运和磷酸二酯酶。然而，蛋白激酶C（或Ca ~（2+）/磷脂依赖性蛋白激酶）可能不参与，至少，在激素作用磷酸二酯酶，因为反应的H-7的appartKi值太高。
Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mmNaCl, 118.0 mmKCl, 0.38 mmCaCl2, 1.00 mmEGTA, 1.19 mmMg2SO4, 1.19 mmKH2PO4, 25.0 mmHepes/K, 20 mg/ml bovine serum albumin, and 3 mmpyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 μF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nmand (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nm. H-7 strongly inhibited the actions of insulin on both glucose transport (apparentKi= 0.3 mM) and cAMP phosphodiesterase (apparentKi= 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparentKivalue for the reaction in intact cells was 0.45 mm, and that in electroporated cells was 0.075 mm. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparentKivalue of H-7 for the reaction is too high.