Resolution of the basal plasma membrane calcium flux in vascular smooth muscle cells. Fayazi, Amir H., Smadar A. Lapidot, Bill K. Huang, Robert W. Tucker, Robert D. Phair. Department of Biomedical Engineering, Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
APStracts 2:0480H, 1995.
Steady state cytosolic calcium concentration, Cai, in a vascular smooth muscle cell is determined by Ca influx and Ca extrusion across the plasma membrane yet no means for determining the absolute magnitude of these trans-membrane Ca fluxes in the basal state of the resting cell has been devised. We now report a method that combines fluorescence measurement of Cai, 45Ca kinetics, and computer modeling to yield the basal plasma membrane Ca flux in A7r5 vascular smooth muscle cells. Kinetic analysis of basal Cai and Cai transients following chelation of extracellular Ca yields a unique value for the ratio of the rate constant governing Ca pumping into the sarcoplasmic reticulum (SR) to that for plasma membrane Ca extrusion (1.12+/ -0.06). When this ratio was used to constrain the least-squares fitting of 45Ca efflux data from A7r5 cells, it was possible to determine unique values for the unidirectional, steady state Ca fluxes across both SR and plasma membranes. The basal unidirectional plasma membrane calcium flux was 0.062+/-0.018 fmol min-1 cell-1 and the basal SR Ca flux was 0.069+/-0.019 fmol min-1 cell-1. These results demonstrate, within the limitations of measuring the absolute value of Cai, the feasibility of measuring previously unresolvable sub-picoamp basal Ca fluxes in intact cells under normal physiological conditions. 

Received 26 May 1994; accepted in final form 5 October 1995.
APS Manuscript Number H463-4.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 November 95