Suzuki A, Shinoda J, Oiso Y, Kozawa O
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
J Endocrinol. 1995 Apr;145(1):81-6. doi: 10.1677/joe.0.1450081.
We have previously reported that extracellular ATP stimulates Ca2+ influx from extracellular space, resulting in the production of prostaglandin E2 which mediates, at least in part, its proliferative effect on osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) stimulates phospholipase D in these cells. In the present study, we examined the effect of extracellular ATP on phosphatidylcholine-hydrolysing phospholipase D activity in MC3T3-E1 cells. ATP stimulated the formation of both choline and inositol phosphates dose-dependently in the range between 0.1 and 1 mM. The formation of choline by a combination of ATP and NaF, an activator of GTP-binding protein, was synergistic, whereas that of inositol phosphates was not. A combination of ATP and 12-O-tetradecanoylphorbol-13-acetate, a PKC activating phorbol ester, additively stimulated the formation of choline. Staurosporine, an inhibitor of PKC, had little effect on ATP-stimulated formation of choline. Choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA, while being inhibited by W-7, an antagonist of calmodulin. These results suggest that extracellular ATP stimulates phospholipase D in a Ca2+/calmodulin-dependent manner in osteoblast-like cells, and that neither PKC activation nor GTP-binding protein is involved in this mechanism.
我们之前曾报道，细胞外ATP可刺激细胞外空间的Ca2+内流，导致前列腺素E2生成，而前列腺素E2至少部分介导其对成骨样MC3T3-E1细胞的增殖作用，并且蛋白激酶C（PKC）的激活可刺激这些细胞中的磷脂酶D。在本研究中，我们检测了细胞外ATP对MC3T3-E1细胞中水解磷脂酰胆碱的磷脂酶D活性的影响。ATP在0.1至1 mM范围内剂量依赖性地刺激胆碱和肌醇磷酸的生成。ATP与GTP结合蛋白激活剂NaF联合使用时，胆碱的生成具有协同作用，而肌醇磷酸的生成则不然。ATP与PKC激活佛波酯12-O-十四酰佛波醇-13-乙酸酯联合使用时，可加成性刺激胆碱的生成。PKC抑制剂星形孢菌素对ATP刺激的胆碱生成影响很小。用EGTA螯合细胞外Ca2+可显著降低胆碱生成，同时被钙调蛋白拮抗剂W-7抑制。这些结果表明，细胞外ATP以Ca2+/钙调蛋白依赖性方式刺激成骨样细胞中的磷脂酶D，且该机制不涉及PKC激活或GTP结合蛋白。
