Carvallo P, Aguilera G
Section of Endocrine Physiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Mol Endocrinol. 1989 Dec;3(12):1935-43. doi: 10.1210/mend-3-12-1935.
The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 nM TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKC-depleted cells, cAMP responses to stimulation with 100 nM CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC- depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 +/- 0.47 and 1.93 +/- 0.11 pmol 32P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [3H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP- and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [125I]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
通过用佛波酯长时间孵育细胞抑制内源性蛋白激酶C（PKC）以及直接测量垂体细胞中的PKC活性，研究了蛋白激酶C（PKC）在加压素（VP）作用中的角色。在37℃下用100 nM佛波醇-12-十四酸酯-13-乙酸酯（TPA）预孵育细胞6小时，导致总PKC活性降低90%。在PKC耗竭的细胞中，对100 nM促肾上腺皮质激素释放因子（CRF）刺激30分钟的cAMP反应正常，但VP和佛波酯（PMA）对CRF刺激的cAMP产生的增强作用被消除。在PKC耗竭的细胞中，单独由VP和PMA刺激促肾上腺皮质激素（ACTH）分泌的作用也被消除。通过酶促测定和免疫印迹技术直接测量了通过离心淘析获得的富集垂体促肾上腺皮质细胞的胞质和去污剂增溶膜组分中的PKC活性。基础PKC活性在胞质中高于膜中（分别为8.43±0.47和1.93±0.11 pmol 32P掺入/10分钟）。在用VP孵育细胞15分钟或用[3H]佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）孵育30分钟后，胞质中的PKC活性分别降低了40%和89%，而膜中的活性分别增加了138%和405%。当使用特异性抗PKC抗体和[125I]蛋白A通过免疫印迹测量胞质和膜中的酶含量时，也观察到了这种由VP和PMA诱导的PKC易位。免疫印迹的放射自显影分析显示了一条80千道尔顿的PKC特征带，其光密度在胞质组分中高于膜组分。（摘要截短于250字）
