AJP - Heart and Circulatory Physiology, Vol 270, Issue 6 1972-H1978, Copyright © 1996 by American Physiological Society
Resolution of the basal plasma membrane calcium flux in vascular smooth muscle cells
A. H. Fayazi, S. A. Lapidot, B. K. Huang, R. W. Tucker and R. D. Phair
Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Steady-state cytosolic calcium (Ca2+i) concentration in a vascular smooth
muscle cell is determined by Ca2+ influx and Ca2+ extrusion across the
plasma membrane, yet no means for determining the absolute magnitude of
these transmembrane Ca2+ fluxes in the basal state of the resting cell has
been devised. We now report a method that combines fluorescence measurement
of Ca2+i, 45Ca kinetics, and computer modeling to yield the basal plasma
membrane Ca2+ flux in A7r5 vascular smooth muscle cells. Kinetic analysis
of basal Ca2+i and Ca2+i transients following chelation of extracellular
Ca2+ yields a unique value for the ratio of the rate constant governing
Ca2+ pumping into the sarcoplasmic reticulum (SR) to that for plasma
membrane Ca2+ 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 Ca2+ fluxes across both SR and plasma membranes. The basal
unidirectional plasma membrane Ca2+ flux was 0.062 +/- 0.018 fmol . min-1 .
cell, and the basal SR Ca2+ flux was 0.069 +/- 0.019 fmol . min-1 . cell-1.
These results demonstrate, within the limitations of measuring the absolute
value of Ca2+i, the feasibility of measuring previously unresolvable
subpicoamp basal Ca2+ fluxes in intact cells under normal physiological
conditions.
