Resolution of the basal plasma membrane calcium flux in vascular smooth muscle cells.
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.
Fayazi
A H
AH
Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Lapidot
S A
SA
Huang
B K
BK
Tucker
R W
RW
Phair
R D
RD
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D001330:Electronic Data Processing; D005456:Fluorescent Dyes; D016257:Fura-2; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008955:Models, Cardiovascular; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*
D005456:Fluorescent Dyes; D002118:Calcium; D016257:Fura-2