RAPID FILTRATION STUDIES OF THE EFFECT OF CYTOSOLIC CA2-TRISPHOSPHATE-INDUCED CA-45(2+) RELEASE FROM CEREBELLAR MICROSOMES( ON INOSITOL 1,4,5)

Using microsomal membrane vesicles derived from sheep cerebellum, we m easured the rate of inositol 1,4,5-trisphosphate (InsP(3))-dependent C a-45(2+) afflux from Ca-45(2+)-loaded compartments during rapid perfus ion with a medium containing InsP(3) and various concentrations of fre e Ca-40(2+) on the cytosolic side (pH 7.1, 5 mM Mg2+, in the absence o f ATP at 20 degrees C). At 0.15 mu M InsP(3) and pCa 6.5, half-Ca-45(2 +) release was attained within less than 200 ms. At low Ca2+ concentra tions, the initial rate of Ca-45(2+) release depended smoothly on InsP (3) concentration, and InsP(3) activated release with moderate positiv e cooperativity. Preliminary experiments performed at various free Ca- 40(2+) concentrations were consistent with a bell-shaped Ca-40(2+) dep endence of Ca-45(2+) release. In the range of micromolar or higher fre e Ca-40(2+) concentrations, the apparent inhibition of Ca-45(2+) relea se was dependent on InsP(3) concentration, and Ca-45(2+) release for i ntermediate InsP(3) concentrations was transient; under selected condi tions, a second perfusion period, identical to the first one but separ ated from it by a short recovery period, was found to allow renewed Ca -45(2+) efflux. At high Ca2+ concentration, fast reversible Ca2+-depen dent desensitization of the channel, and not heterogeneity, was theref ore responsible for the termination of InsP(3)-triggered Ca-45(2+) aff lux at submaximal concentrations of InsP(3). At lower Ca2+ concentrati ons, a large fraction of the apparent activating effect of submicromol ar Ca-40(2+) concentrations on Ca-45(2+) efflux that we had observed i n the preliminary experiments proved to be the artifactual consequence of an inhibitory effect exerted by metal-free Ca2+ chelators on InsP( 3)-dependent afflux at nanomolar Ca-40(2+) concentrations. ,2-Bis(2-am inophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3 were al l effective inhibitors. When this inhibition was taken into account, a rise in free Ca-40(2+) concentration from 30 to 300 nM only weakly en hanced Ca-45(2+) fluxes in the presence of a low concentration of InsP (3+). As a result, submicromolar free Ca-40(2+) appears to be only a p oor activator of InsP(3)-induced Ca2+ release under these experimental conditions.