Wright L C, Chen S, Roufogalis B D
Department of Pharmacy, University of Sydney, NSW, Australia.
Arch Biochem Biophys. 1995 Aug 20;321(2):459-66. doi: 10.1006/abbi.1995.1418.
We have previously shown in intact human erythrocytes that both the plasma membrane Ca2+ pump activity and its phosphorylation can be increased by phorbol-12-myristate 13-acetate (PMA), a known stimulator of protein kinase C. These effects were inhibited by high doses of adriamycin (L. C. Wright et al., 1993, Arch. Biochem. Biophys. 306, 277-284). We now show that low doses of adriamycin (ADR) (maximum effect at 10 microM for 1-6 min) decrease the amplitude of the intracellular calcium ([Ca2+]i) transient induced by 2.5 microM CaCl2 and 10 microM A23187 in intact human erythrocytes. This is reflected by a parallel increase in Ca(2+)-ATPase activity in plasma membranes isolated from pretreated intact cells. When 10 microM ADR and 1 microM PMA were combined the effects were additive, with a maximum decrease in the Ca2+ transient amplitude of 50%. A similar effect was seen on the Ca(2+)-ATPase activities in isolated membranes. In erythrocytes labeled with [32P]orthophosphate 10 microM ADR induced a 1.5-fold increase in the phosphorylation of the Ca2+ pump and when combined with 1 microM PMA the phosphorylation was greatly enhanced (2.3 times that induced by PMA alone). ADR alone and in combination with PMA was found to decrease both 32P labeling and lipid phosphate content of phosphatidylinositol 4,5-bisphosphate (PIP2). This was accompanied by an increase in the amount of 1,2-diacylglycerol formed in response to 10 microM ADR. We conclude that low doses of ADR are able to stimulate the breakdown of 6-13% of erythrocyte PIP2 by phospholipase C at an intracellular calcium concentration of 2.5 microM, normally regarded as below threshold for phospholipase C activation in erythrocytes. The diacylglycerol formed appears to stimulate protein kinase C to activate the Ca2+ pump and enhance its phosphorylation and Ca2+ efflux in intact human erythrocytes.
我们之前在完整的人体红细胞中发现，佛波醇-12-肉豆蔻酸酯13-乙酸酯（PMA），一种已知的蛋白激酶C刺激剂，可增加质膜Ca2+泵活性及其磷酸化水平。高剂量阿霉素可抑制这些效应（L.C. Wright等人，1993年，《生物化学与生物物理学档案》306卷，277 - 284页）。我们现在发现，低剂量阿霉素（ADR）（1 - 6分钟内10 microM时效果最佳）可降低完整人体红细胞中由2.5 microM CaCl2和10 microM A23187诱导产生的细胞内钙（[Ca2+]i）瞬变幅度。这表现为从预处理的完整细胞中分离出的质膜中Ca(2+)-ATP酶活性的平行增加。当10 microM ADR与1 microM PMA联合使用时，效果具有加和性，Ca2+瞬变幅度最大降低50%。在分离的膜中对Ca(2+)-ATP酶活性也观察到类似效果。在用[32P]正磷酸盐标记的红细胞中，10 microM ADR使Ca2+泵的磷酸化增加1.5倍，当与1 microM PMA联合使用时，磷酸化大大增强（是单独PMA诱导的2.3倍）。单独的ADR以及与PMA联合使用均被发现可降低磷脂酰肌醇4,5-二磷酸（PIP2）的32P标记和脂质磷酸盐含量。这伴随着响应10 microM ADR形成的1,2-二酰基甘油量的增加。我们得出结论，低剂量ADR能够在细胞内钙浓度为2.5 microM时，通过磷脂酶C刺激红细胞中6 - 13%的PIP2分解，而该钙浓度通常被认为低于红细胞中磷脂酶C激活的阈值。形成的二酰基甘油似乎刺激蛋白激酶C激活Ca2+泵，并增强其在完整人体红细胞中的磷酸化和Ca2+外流。
