Abstract
We developed a unidirectional 45Ca2+ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP3), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP3 action, which was around 32 nM InsP3 under control conditions. The InsP3-induced Ca2+ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP3 concentrations. Stimulatory concentrations of thimerosal (10 microM) shifted the threshold to 4.5 nM InsP3 and increased both the cooperativity and the maximal normalized rate of Ca2+ release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 microM) but then the effects on the cooperativity and on the normalized rate of Ca2+ release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP3 concentrations. Luminal Ca2+ did not affect the threshold for InsP3 action but stimulated the normalized release at each InsP3 concentration. The inhibitory effect of 10 microM free cytosolic Ca2+ was associated with a shift in threshold to higher InsP3 concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP3 action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP3 receptor.
Highlights
Many hormones, neurotransmitters, and growth factors induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby produce inositol 1,4,5-trisphosphate (InsP3)1 as an intracellular messenger (1)
InsP3 acts on the threshold value. This novel technique of measuring thresholds for InsP3 action allowed a refined analysis of the kinetic parameters controlling the mechanisms of action of several regulators of the InsP3R
Concluding Remarks on the Kinetics of InsP3-induced Ca2ϩ Release—The response to a long-lasting stimulation with InsP3 is characterized by an initial fast release, which progressively slows down
Summary
Neurotransmitters, and growth factors induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby produce inositol 1,4,5-trisphosphate (InsP3) as an intracellular messenger (1). We have developed a 45Ca2ϩ efflux technique in which cumulative doses of InsP3 are subsequently added. This technique, which mimics the gradual accumulation of InsP3 at the onset of a Ca2ϩ response, allows an accurate measurement of the threshold [InsP3] for InsP3-induced Ca2ϩ release. With this technique, we have investigated how the. This novel technique of measuring thresholds for InsP3 action allowed a refined analysis of the kinetic parameters controlling the mechanisms of action of several regulators of the InsP3R
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