Wells K E, Miguel R, Alexander J J
Case Western Reserve University, MetroHealth Medical Center, Cleveland, Ohio 44109, USA.
J Surg Res. 1997 Feb 1;67(2):126-31. doi: 10.1006/jsre.1996.4991.
Although clinical and experimental studies have supported a role of calcium in atherogenesis, the nature of this relationship has not been determined. The following investigation was performed to examine the effect of transmembrane calcium flux on the oxidative modification of low-density lipoprotein (LDL) by arterial smooth muscle cells (SMC). Confluent SMC were incubated in basal medium alone or in medium containing either nifedipine (0.25 microM), a calcium channel blocker, or dantrolene (10 microM), a blocker of calcium release from the endoplasmic reticulum. Cells were then suspended in medium with either physiologic (1.25 mM) or supraphysiologic (2.5 mM) calcium concentrations ([Ca2+]e) and were exposed to oxidized LDL (20 micrograms/protein/ml; 4.96 +/- 0.76 nmole malondialdehyde/mg LDL protein). Changes in cytosolic calcium ([Ca2+]i) were measured by spectrofluorometric analysis using a Fura 2-AM indicator. In similar studies, the cellular oxidation of native LDL was determined by fluorometric measurement of thiobarbituric acid-reactive substances in the media. Nifedipine and, to a lesser extent, dantrolene lowered steady-state [Ca2+]i at supraphysiologic [Ca2+]e (2.5 mM; P < 0.0002). Exposure of SMC to Ox-LDL increased [Ca2+]i (P < 10(7), which was further augmented by increasing [Ca2+]e (P < 10(-7). Nifedipine significantly reduced the calcium response to Ox-LDL proportionate to [Ca2+]e (P < 0.0002). A similar reduction in [Ca2+]i vs control was seen with dantrolene, but was independent of [Ca2+]e. TBARS assays revealed a significantly greater degree of cellular oxidation of native LDL following preincubation of SMC with Ox-LDL (P < 10(8). This effect was markedly inhibited by both nifedipine (P < 10(-8) and dantrolene (P < 10(-8), which showed some degree of synergism. These results indicate that the increase in cytosolic calcium in SMC exposed to Ox-LDL may occur through both membrane channels and reticular release. Inhibition of transmembrane calcium flux severely limits the cellular oxidation of low-density lipoprotein. These alternate means of calcium signal generation may allow a more varied response of the SMC to atherogenic stimuli and provide an opportunity for specific therapeutic intervention.
尽管临床和实验研究都支持钙在动脉粥样硬化形成过程中发挥作用，但这种关系的本质尚未确定。进行以下研究以检验跨膜钙通量对动脉平滑肌细胞（SMC）氧化修饰低密度脂蛋白（LDL）的影响。将汇合的SMC单独培养在基础培养基中，或培养在含有钙通道阻滞剂硝苯地平（0.25微摩尔）或内质网钙释放阻滞剂丹曲林（10微摩尔）的培养基中。然后将细胞悬浮于生理钙浓度（1.25毫摩尔）或超生理钙浓度（2.5毫摩尔）（[Ca2+]e）的培养基中，并暴露于氧化型LDL（20微克/蛋白/毫升；4.96±0.76纳摩尔丙二醛/毫克LDL蛋白）。使用Fura 2-AM指示剂通过荧光分光光度分析测量胞质钙（[Ca2+]i）的变化。在类似研究中，通过荧光法测量培养基中硫代巴比妥酸反应性物质来确定天然LDL的细胞氧化情况。硝苯地平以及程度稍轻的丹曲林在超生理[Ca2+]e（2.5毫摩尔）时降低了稳态[Ca2+]i（P<0.0002）。将SMC暴露于氧化型LDL会增加[Ca2+]i（P<10^-7），而增加[Ca2+]e会进一步增强这种增加（P<10^-7）。硝苯地平显著降低了与[Ca2+]e成比例的对氧化型LDL的钙反应（P<0.0002）。丹曲林也观察到[Ca2+]i相对于对照有类似降低，但与[Ca2+]e无关。硫代巴比妥酸反应物（TBARS）分析显示，SMC与氧化型LDL预孵育后，天然LDL的细胞氧化程度显著更高（P<10^-8）。这种效应被硝苯地平（P<10^-8）和丹曲林（P<10^-8）均显著抑制，且二者表现出一定程度的协同作用。这些结果表明，暴露于氧化型LDL的SMC中胞质钙的增加可能通过膜通道和内质网释放两种途径发生。抑制跨膜钙通量会严重限制低密度脂蛋白的细胞氧化。这些钙信号产生的替代方式可能使SMC对动脉粥样硬化刺激产生更多样化的反应，并为特异性治疗干预提供机会。
