Cyclic ADP-ribose-gated Ca2+ Release in Sea Urchin Eggs Requires an Elevated [Ca2+]

Abstract

Cyclic ADP-ribose (cADPr) 1 has been found to be a potent mobilizer of intracellular Ca 2ϩ in sea urchin eggs and in numerous vertebrate cell types (for review see Ref. 1). Over the last few years, the accumulating evidence has strengthened the proposal that cADPr is a true calcium-mobilizing second messenger. For example, both cADPr and its metabolic enzymes have been found to be widely distributed in mammalian tissue (2-4). Further, cADPr production and Ca 2ϩ release are linked to activation of the cGMP signal transduction pathway in sea urchin eggs and in cultured neurosecretory cells (5-7). Thus, cADPr appears to meet the criteria to be considered a second messenger of Ca 2ϩ signaling. The mechanism by which cADPr gates Ca 2ϩ release remains unclear. cADPr-mediated Ca 2ϩ release has been found to be enhanced by Ca 2ϩ and sensitive to classical pharmacologic agonists and antagonists of ryanodine receptor/channels (RyRCs) (8 -11 ]. We found that the delay before cADPr-induced Ca 2ϩ release was eliminated when the [Ca 2ϩ ] was step-elevated coincident with the photoliberation of cADPr and greatly prolonged in the presence of exogenous Ca 2ϩ buffers. Thus, the slow onset of Ca 2ϩ release was not due to an intrinsically slow rate by which cADPr (or Ca 2ϩ ) gated release channels. Rather, our results show that cADPr is relatively ineffective at releasing Ca 2ϩ at normal resting [Ca 2ϩ ] levels and that a positive Ca 2ϩ feedback is required to achieve full Ca 2ϩ release. Further, these findings underscore the importance of controlling the [Ca 2ϩ ] in investigations of cADPr-mediated Ca 2ϩ release. EXPERIMENTAL PROCEDURESEgg Preparation-Eggs were released from female Lytechinus pictus sea urchins by intracoelomic injection of 0.5 M KCl and washed twice in artificial sea water containing (in mM): 460 NaCl, 27 MgCl 2 , 28 MgSO 4 , 10 CaCl 2 , 10 KCl, 2.5 NaHCO 3 ; pH adjusted to 8.0 with NaOH. The jelly was removed by multiple filtrations through a 100-m pore nylon filter. The eggs were transferred onto a poly-L-lysine-treated quartz coverslip that formed the bottom of a cell chamber filled with artificial sea water for microinjection and study. The microinjection solution contained a Ca 2ϩ indicator dye (either 250 M fluo-3 or 1-4 mM spectrally similar Calcium Green 5N (CG-5N)) and various concentrations of either caged cADPr, caged IP 3 , caged calcium, and/or heparin dissolved in a microinjection buffer (0.5 M KCl, 50 M EGTA, 10 mM MOPS, pH 6.7, with KOH). The injected volume was estimated from the Ca 2ϩ dye fluorescence calibrated against fluorescence measurements made of droplets of dye solution created in oil as described previously (21) and was typically 2-3% of the egg volume. Unless otherwise noted, caged IP 3 and caged cADPr were loaded to intracellular concentrations greater than 10 M. All experiments were performed at room temperature. All values reported in the text are the means Ϯ S.E.Detection of Ca 2ϩ Dye Fluorescence-Whole egg Ca 2ϩ dye fluorescence was monitored with a high time resolution microf...

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