EXCESS MEMBRANE CHOLESTEROL ALTERS CALCIUM MOVEMENTS, CYTOSOLIC CALCIUM LEVELS, AND MEMBRANE FLUIDITY IN ARTERIAL SMOOTH-MUSCLE CELLS

The relations between membrane cholesterol content, basal (unstimulated) transmembrane Ca-45(2+) movements, cytosolic calcium levels, and membrane fluidity were investigated in cultured rabbit aortic smooth muscle cells (SMCs) and isolated SMC plasma membrane microsomes. SMCs were enriched with unesterified (free) cholesterol (FC) for 18-24 hours with medium containing human low density lipoprotein and FC-rich phospholipid (PL) liposomes. This procedure increased cholesterol mass without affecting PL mass, resulting in an increase in the FC/PL molar ratio compared with controls in cells (67% FC increase, p < 0.001; 43% FC/PL ratio increase, p < 0.01) and in SMC microsomes (52% FC increase, p < 0.05; 43% FC/PL ratio increase, p < 0.05). Cholesterol enrichment also increased unstimulated Ca-45(2+) influx (p < 0.001) and efflux (p < 0.05). Cellular cholesterol content correlated in a linear fashion with these changes (influx: r = 0.722, p < 0.01; efflux: r = 0.951, p < 0.05). In addition, cytosolic calcium levels increased approximately 34% (p < 0.01) with cholesterol enrichment. The cholesterol-induced increase in Ca-45(2+) influx was reversible with time and demonstrated sensitivity to the channel blockers. Fluorescence anisotropy measured from 5-degrees-C to 40-degrees-C using the fluorophore diphenylhexatriene showed decreased membrane fluidity in microsomal membranes obtained from cholesterol-enriched SMCs compared with controls (p < 0.02). These results suggest that the SMC plasma membrane is very sensitive to cholesterol enrichment with liposomes or human low density lipoprotein and that increases in membrane cholesterol content increase cytosolic calcium levels in SMCs, are associated with a decrease in membrane fluidity, and unmask a new, or otherwise silent, dihydropyridine-sensitive calcium channel that may be involved in altered arterial wall properties with serum hypercholesterolemia.