Extracellular Na+ dependency of free cytosolic Ca2+ regulation in aortic vascular smooth muscle cells

Abstract

This study examined contribution of Na(+)-dependent processes to the regulation of free cytosolic calcium (Ca2+i) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca2+i that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca2+i increase was followed by partial recovery to a new steady state that was still significantly higher than that seen before removal of external Na+ (Na+o). In ouabain-pretreated cells lowering of Na+o caused progressive increases in Ca2+i. Addition of NiCl2, a Na(+)-Ca2+ exchange inhibitor, completely blocked the increase in Ca2+i produced by removal of Na+o, indicating that the Na(+)-Ca2+ antiporter was responsible for observed Ca2+i changes. Ca2+i increase produced by reduction of Na+o was also seen after depletion of inositol trisphosphate-sensitive Ca2+ stores with repeated pulses of angiotensin II or after blockade of sarcoplasmatic reticulum Ca2+ release with TMB-8 but was not observed in the absence of external Ca2+. These observations indicate that the source of Ca2+i increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca2+i surge by ouabain suggests Ca2+ influx via the Na(+)-Ca2+ exchanger operating in the reverse mode. The relative contribution of a Na(+)-dependent and -independent component of Ca2+i recovery was investigated by superfusing cells with ionomycin in a Na(+)-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca2+i to a peak, and this was followed by a rapid but partial recovery to a new steady state. Readdition of varying amounts of Na+ to the superfusate, in the continued presence of ionomycin, resulted in concentration-related decline in Ca2+i, thereby uncovering a substantial contribution of a Na(+)-dependent mechanism of Ca2+i regulation. Decline of Ca2+i produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca2+i regulation in VSMC via a Na(+)-dependent mechanism, consistent with a Na(+)-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca2+i is elevated. Na(+)-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.