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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 μM), a calcium channel blocker, or dantrolene (10 μM), 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 μg/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+]iat 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+]ivs 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.
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© 1997 Academic Press. Published by Elsevier Inc. All rights reserved.