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 concentration 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⁺ concentration 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.