Regulation of Ca(2+) Release by InsP(3) in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca(2+)] ([Ca(2+)](i)) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP(3)-evoked Ca(2+) release. The kinetics of both processes were studied with flash photolytic release of InsP(3) and time resolved measurements of [Ca(2+)](i) in single cells. InsP(3) evoked Ca(2+) flux into the cytosol was measured as d[Ca(2+)](i)/dt, and the kinetics of Ca(2+) release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP(3) concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP(3) suggests that metabolism of InsP(3) is important in determining the minimal concentration needed to produce Ca(2+) release. A distinct latency or delay of several hundred milliseconds after release of low InsP(3) concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca(2+)](i), suggesting a cooperative action of Ca(2+) in InsP(3) receptor activation. InsP(3)-evoked flux and peak [Ca(2+)](i) increased with InsP(3) concentration up to 5–10 μM, with large variation from cell to cell at each InsP(3) concentration. The duration of InsP(3)-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca(2+)](i)/dt and much less cell to cell variation than the dependence of flux on InsP(3) concentration, suggesting that the rate of termination of the Ca(2+) flux depends on the free Ca(2+) flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca(2+) flux, up to 50 μM · s(−1) and in Purkinje neurons at high flux up to 1,400 μM · s(−1). Experiments in which [Ca(2+)](i) was controlled at resting or elevated levels support a mechanism in which InsP(3)-evoked Ca(2+) flux is inhibited by Ca(2+) inactivation of closed receptor/channels due to Ca(2+) accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP(3)-evoked Ca(2+) flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP(3) receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP(3) receptor density promotes fast rising, rapidly inactivating InsP(3)-evoked [Ca(2+)](i) transients.