EXTRACELLULAR NA+ DEPENDENCY OF FREE CYTOSOLIC CA2+ REGULATION IN AORTIC VASCULAR SMOOTH-MUSCLE CELLS

This study examined contribution of Na+-dependent processes to the regulation of free cytosolic calcium (Ca(i)2+) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca(i)2+ that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca(i)2+ 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 Ca(i)2+. Addition of NiCl2, a Na+-Ca2+ exchange inhibitor, completely blocked the increase in Ca(i)2+ produced by removal of Na(o)+, indicating that the Na+-Ca2+ antiporter was responsible for observed Ca(i)2+ changes. Ca(i)2+ 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 Ca(i)2+ increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca(i)2+ 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 Ca(i)2+ recovery was investigated by superfusing cells with ionomycin in a Na+-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca(i)2+ 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 Ca(i)2+, thereby uncovering a substantial contribution of a Na+-dependent mechanism of Ca(i)2+ regulation. Decline of Ca(i)2+ produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca(i)2+ regulation in VSMC via a Na+-dependent mechanism, consistent with a Na+-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca(i)2+ is elevated. Na+-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.