Zagari M, Stephens M, Earp H S, Herman B
Department of Cell Biology and Anatomy, University of North Carolina School of Medicine, Chapel Hill 27599.
J Cell Physiol. 1989 Apr;139(1):167-74. doi: 10.1002/jcp.1041390123.
Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.
血小板衍生生长因子（PDGF）和其他激活蛋白激酶C（PKC）的因子可迅速改变BALB/c - 3T3成纤维细胞的胞质pH值（pHi）和细胞内游离钙浓度（[Ca++]i）。为了确定pHi或[Ca++]i的变化是否与PDGF刺激的细胞增殖有关，我们在对照和成PKC缺失的成纤维细胞中评估了这些参数。向BALB/c - 3T3成纤维细胞中添加PDGF会导致细胞质短暂酸化，随后是长时间的胞质碱化。将细胞暴露于12 - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA），一种激活PKC的佛波酯，会导致胞质碱化而无先前的酸化。用600 nM TPA过夜孵育可使BALB/c - 3T3成纤维细胞中的总细胞PKC组蛋白磷酸化活性降低超过90%。在PKC缺陷的成纤维细胞中，TPA和PDGF诱导的碱化被消除。此外，在用PDGF处理的对照细胞中最初观察到的pHi短暂下降会持续到pHi比对照细胞值低0.6 - 0.7个pH单位长达30分钟。PDGF使[Ca++]i增加了三倍；这种[Ca++]i的短暂升高仅受到极小影响（小于15%），这是通过用乙二醇双（β - 氨基乙醚） - N，N，N' - 四乙酸（EGTA）降低细胞外钙水平或用CoCl2阻断钙内流实现的。相反，8 - （二乙胺） - 辛基 - 3,4,5 - 三甲氧基苯甲酸酯（TMB - 8），一种被认为可抑制细胞内钙库释放钙的试剂，显著抑制了PDGF引起的[Ca++]i升高。TPA和1 - 油酰 - 2 - 乙酰甘油（OAG）增加了[Ca++]i，但与PDGF不同的是，这种作用被用EGTA或CoCl2预处理细胞所阻断。在PKC缺陷的成纤维细胞中，PDGF仍然像在对照细胞中一样有效地增加[Ca++]i并刺激DNA合成。然而，TPA和OAG不再增加[Ca++]i。在持续酸化且缺乏PKC活性的情况下，PDGF仍具有刺激DNA合成的能力，这表明胞质碱化和PKC激活对于BALB/c - 3T3成纤维细胞中PDGF诱导的细胞增殖能力并非必不可少。
