Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line

Abstract

Prolonged exposure to oxidized low density lipoprotein (oxLDL) can alter various aspects of cell biology, including modification of vasomotor responses and downregulation of calcium channel proteins in aortic smooth muscle cells. However, the components of oxLDL responsible for these effects have not been fully elucidated. The study reported here aimed at examining the consequences of extended exposure to oxysterols, cholesterol oxidation products whose levels are elevated in oxLDL as compared to unmodified LDL, on calcium signalling mechanisms in A7r5 cells, a model aortic smooth muscle cell-line. Within 24h of exposure, all three oxysterol congeners tested caused an elevation in the resting cytoplasmic Ca(2+) concentration. These oxysterols also inhibited Ca(2+) transients in response to arginine vasopressin and bradykinin, and some but not all congeners ablated Ca(2+) signals triggered by platelet activating factor, the ryanodine receptor calcium channel agonist 4-choloro-meta-cresol, or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) uptake. The effects of long-term exposure to the oxysterol congener 7β-hydroxycholesterol on arginine vasopressin stimulated Ca(2+) signals were mainly at the level of Ca(2+) release from intracellular stores rather than on Ca(2+) influx mechanisms. Of the calcium signalling proteins tested, only the type 1 ryanodine receptor and the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) were significantly downregulated by 24h exposure to oxysterols. Decreases in IP(3)R1 protein triggered by 7β-hydroxycholesterol were both time and concentration dependent, occurring over a concentration range encountered within atherosclerotic lesions. IP(3)R1 downregulation by certain oxysterols is mediated by proteasomal proteolysis, since it can be abolished by co-incubation with epoxomicin. Overall, these data demonstrate that major oxysterol components of oxLDL cause long-term alterations in Ca(2+) signalling in a model aortic smooth muscle cell. Such effects could contribute to the pathology of atherosclerotic disease.