Novel kinetics of single cell Ca2+ transients in stimulated hepatocytes and A10 cells measured using fura-2 and fluorescent videomicroscopy.
The kinetics of agonist-induced increases in cytosolic free Ca2+ have been measured in single A10 vascular smooth muscle cells and rat hepatocytes using fluorescent videomicroscopy with fura-2 as a Ca2+ indicator. At high agonist concentrations there was no difference in the kinetics of the Ca2+ transient measured in vasopressin-stimulated single A10 cells or in cell populations. However, stimulation of single A10 cells with concentrations of vasopressin below 0.5 nM produced characteristic Ca2+ transients composed of two distinct peaks. The two peaks appeared to represent a temporal separation between release of intracellular Ca2+ and influx of extracellular Ca2+. The double transient was not observed in single rat hepatocytes stimulated with low concentrations of vasopressin or phenylephrine. In both A10 cells and hepatocytes, the initial rate of increase in Ca2+ concentrations in response to submaximal agonist concentrations was faster in single cells than in cell populations. This difference was due to asynchrony of the cellular response, where there was a latent period of variable length before onset of a rapid increase in Ca2+ concentration. The duration of the latent period was dependent on the agonist concentration, higher concentrations of agonist giving a reduced latent period. The hormone-stimulated Ca2+ transient measured in single hepatocytes with fura-2 was different from the series of transient spikes as previously reported using aequorin as the Ca2+ indicator, suggesting that fura-2 and aequorin may report different aspects of the Ca2+ response in stimulated cells. Collectively, these results demonstrate that measurement of Ca2+ transients in single cells provide novel information concerning the nature of the Ca2+ transient that is not apparent from studies with cell populations.
Monck
J R
JR
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Reynolds
E E
EE
Thomas
A P
AP
Williamson
J R
JR
D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D005456:Fluorescent Dyes; D002118:Calcium; D016257:Fura-2