ACTIVATION AND COORDINATION OF INSP(3)-MEDIATED ELEMENTARY CA2+ EVENTS DURING GLOBAL CA2+ SIGNALS IN XENOPUS OOCYTES

1. The activation of elementary calcium release events ('puff') and th eir co-ordination to generate calcium waves was studied in Xenopus ooc ytes by confocal linescan imaging together with photorelease of inosit ol. 1,4,5-trisphosphate (InsP(3)) from a caged precursor. 2. Weak phot olysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occur ring in flurries as abortive waves, and then a near-simultaneous calci um liberation originating at multiple sites. The numbers of sites acti vated increased initially as about the fourth power of photoreleased [ InsP(3)]. 3. Following repeated, identical photolysis flashes, puffs a rose after stochastically varying latencies of a few hundred milliseco nds to several seconds. The cumulative number of events initially incr eased as about the third power of time. No rise in free [Ca2+] was det ected preceding the puffs, suggesting that this co-operativity arises through binding of multiple InsP(3) molecules, rather than through cal cium feedback. 4, The mean latency to onset of calcium liberation shor tened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly. 5. Weak stimuli oft en evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non-constant velocit y. During waves, [Ca2+] rose slowly between puff sites, but more abrup tly at active sites following an initial diffusive rise in calcium. 6. Initial rates of rise of local [Ca2+] at release sites were similar d uring puffs and release induced by much (> 10-fold) greater [InsP(3)]. In contrast, macroscopic calcium measurements averaged over the scan line shamed a graded dependence of rate of calcium liberation upon [In sP(3)], due to recruitment of additional sites and decreasing dispersi on in activation latencies. 7. We conclude that the initiation of calc ium liberation depends co-operatively upon [InsP(3)] whereas the subse quent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP(3)]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which i s primed by InsP(3).