TRANSMEMBRANE CALCIUM FLUX REGULATES LDL OXIDATION BY ARTERIAL SMOOTH-MUSCLE CELLS

Although clinical and experimental studies have supported a role of ca lcium in atherogenesis, the nature of this relationship has not been d etermined. The following investigation was performed to examine the ef fect of transmembrane calcium flux on the oxidative modification of lo w-density lipoprotein (LDL) by arterial smooth muscle cells (SMC). Con fluent SMC were incubated in basal medium alone or in medium containin g either nifedipine (0.25 mu M), a calcium channel blocker, or dantrol ene (10 mu M), a blocker of calcium release from the endoplasmic retic ulum. Cells were then suspended in medium with either physiologic (1.2 5 mM) or supraphysiologic (2.5 mM) calcium concentrations ([Ca2+](e)) and were exposed to oxidized LDL (20 mu g/protein/ml; 4.96 +/- 0.76 nm ole malondialdehyde/mg LDL protein). Changes in cytosolic calcium ([Ca 2+](i)) were measured by spectrofluorometric analysis using a Fura 2-A M indicator. In similar studies, the cellular oxidation of native LDL was determined by fluorometric measurement of thiobarbituric acid-reac tive substances in the media. Nifedipine and, to a lesser extent, dant rolene 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 syner gism. These results indicate that the increase in cytosolic calcium in SMC exposed to Ox-LDL may occur through both membrane channels and re ticular release. Inhibition of transmembrane calcium flux severely lim its the cellular oxidation of low-density lipoprotein. These alternate means of calcium signal generation may allow a more varied response o f the SMC to atherogenic stimuli and provide an opportunity for specif ic therapeutic intervention. (C) 1997 Academic Press.