A LINGERING ELEVATION OF CA(I) ACCOMPANIES INHIBITION OF INOSITOL 1,4,5 TRISPHOSPHATE-INDUCED CA RELEASE IN LIMULUS VENTRAL PHOTORECEPTORS

Injection of inositol 1,4,5 trisphosphate (InsP3) into Limulus ventral photoreceptors causes an elevation of intracellular free Ca concentration (Ca(i)) and depolarizes the photoreceptors. When measured with the photoprotein aequorin, the InsP3-induced Ca(i) increase follows the time course of depolarization and declines within 1-2 s. However, sensitivity to further injections of InsP3 remains suppressed for several tens of seconds. The possibility that the suppression of Ca release (feedback inhibition) is due to a small lingering elevation of Ca(i), below the existing detection limit of aequorin, was investigated by measuring Ca(i) with Ca-sensitive electrodes. Double-barreled, Ca-selective microelectrodes were used to pressure inject InsP3 and measure Ca(i) at the same point. Light or InsP3 injections into the light-sensitive compartment depolarized the photoreceptors and induced an elevation of Ca(i) that persisted for tens of seconds. Injections of InsP3 during the decay of Ca(i) showed that sensitivity to InsP3 recovered as resting Ca(i) approached the prestimulus level. The relationship between elevated Ca(i) and feedback inhibition was very steep. An elevation of Ca(i) of 1 muM or more was associated with inhibitions of 79 +/- 12.4% (SEM; n = 7) for the InsP3-induced Ca(i) increase and of 76 +/- 8% for depolarizations. With a residual Ca(i) elevation of 0.01 muM or less, the mean inhibition was 10 +/- 7.4% for InsP3-induced Ca(i) increase and 6.6 +/- 4% for InsP3-induced depolarization. Injections of InsP3 into a light-insensitive compartment within the cell induced elevations of Ca(i) with no associated depolarizations or feedback inhibition. To verify that a sustained elevation of Ca(i) is necessary for. inhibition of InsP3-induced Ca(i) increase and depolarization, we injected ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) between two injections of InsP3. Injection of 1 mM EGTA or the related Ca chelator BAPTA, delivered 750 ms after the first injection of InsP3, restored the peak depolarization caused by the second injection of InsP3 to > 80 +/- 3% of control, compared with 13 +/- 8% without an intervening injection of EGTA. Measurement of Ca(i) with aequorin showed that an intervening injection of EGTA partially restored the InsP3-induced Ca(i) increase. The results suggest that feedback inhibition of InsP3-induced Ca(i) increase and depolarization is mediated by a lingering elevation of Ca(i) and not by depletion of intracellular Ca stores.