Jamieson G A, Etscheid B G, Muldoon L L, Villereal M L
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
J Cell Physiol. 1988 Feb;134(2):220-8. doi: 10.1002/jcp.1041340207.
Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)
用血清或生长因子混合物（由血管加压素、缓激肽、表皮生长因子和胰岛素组成）对静止的人成纤维细胞（HSWP）进行促有丝分裂刺激，会刺激肌醇磷酸的释放、细胞内钙的动员、钠/氢交换的激活以及随后[3H] - 胸腺嘧啶的掺入。我们之前已经确定，用促肿瘤的佛波酯12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）预处理可抑制HSWP细胞中有丝分裂原刺激的钠内流。我们在此报告，TPA预处理也能显著抑制HSWP细胞中有丝分裂原刺激的肌醇磷酸释放。在1 - 2 nM TPA时，对有丝分裂原刺激的肌醇磷酸释放的半数最大抑制作用出现。单独用TPA处理细胞对肌醇磷酸释放没有影响。还确定了TPA预处理对单个生长因子诱导的肌醇磷酸释放的影响。据Cassel等人（1984年）报道，原钒酸盐可增加A431细胞中的钠/氢交换，已证明它能刺激HSWP细胞中的钠内流和肌醇磷酸释放。TPA预处理也能抑制原钒酸盐刺激的肌醇磷酸释放和钠内流。此外，用荧光细胞内钙探针fura - 2测定发现，原钒酸盐通过动员细胞内钙储存来增加细胞内钙活性。TPA预处理可阻断原钒酸盐刺激的细胞内钙储存的动员。很明显，在HSWP细胞中，用佛波酯预处理细胞能够通过在肌醇磷酸释放之前的某个点阻断相关信号转导机制，人为地使细胞对有丝分裂刺激（生长因子、原钒酸盐）的早期反应脱敏。我们假设，在HSWP细胞中，肌醇磷酸释放和钠/氢交换的正常脱敏是通过刺激介导的磷脂酶C激活和蛋白激酶C激活剂二酰甘油释放后蛋白激酶C的激活来介导的。然而，有趣的是要注意到，TPA介导的对HSWP细胞中这些早期反应的抑制并不抑制它们被刺激掺入[3H] - 胸腺嘧啶的能力。（摘要截断于400字）
