RESOLUTION OF THE BASAL PLASMA-MEMBRANE CALCIUM FLUX IN VASCULAR SMOOTH-MUSCLE CELLS

Steady-state cytosolic calcium (Ca-i(2+)) concentration in a vascular smooth muscle cell is determined by Ca2+ influx and Ca2+ extrusion acr oss the plasma membrane, yet no means for determining the absolute mag nitude of these transmembrane Ca2+ fluxes in the basal state of the re sting cell has been devised. We now report a method that combines fluo rescence measurement of Ca-i(2+), Ca-45 kinetics, and computer modelin g to yield the basal plasma membrane Ca2+ flux in A7r5 vascular smooth muscle cells. Kinetic analysis of basal Ca-i(2+) and Ca-i(2+) transie nts following chelation of extracellular Ca2+ yields a unique value fo r the ratio of the rate constant governing Ca2+ pumping into the sarco plasmic reticulum (SR) to that for plasma membrane Ca2+ extrusion (1.1 2 +/- 0.06). When this ratio was used to constrain the least-squares f itting of Ca-45 efflux data from A7r5 cells, it was possible to determ ine unique values for the unidirectional, steady-state Ca2+ duxes acro ss both SR and plasma membranes. The basal unidirectional plasma membr ane Ca2+ flux was 0.062 +/- 0.018 fmol . min(-1). cell(-1), and the ba sal SR Ca2+ flux was 0.069 +/- 0.019 fmol . min(-1). cell(-1). These r esults demonstrate, within the limitations of measuring the absolute v alue of Ca-i(2+), the feasibility of measuring previously unresolvable subpicoamp basal Ca2+ fluxes in intact cells under normal physiologic al conditions.