The important discrepancy between the apparent affinity observed in Ca2+ mobilization studies and the Kd measured in binding studies is a consequence of the quantal process by which inositol 1,4,5-trisphosphate releases Ca2+ from bovine adrenal cortex microsomes.
Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from a non-mitochondrial intracellular store. An important discrepancy has been observed between the affinity measured in binding studies (Kd) and the apparent affinity obtained in Ca2+ mobilization studies (EC50). It has been proposed that this discrepancy could be due to different experimental conditions used for Ca2+ mobilization studies and for InsP3 binding studies. With the fluorescent indicator Fura-2, we studied InsP3-induced Ca2+ release activity at 7 degrees C and at 37 degrees C, in bovine adrenal cortex microsomes. Under both conditions, the Ca2+ releasing effect of InsP3 (1 microM) was completed within about 2 s, as a result of the quantal process of InsP3 receptor action. The apparent affinity (EC50) observed for InsP3-induced Ca2+ release at 7 degrees C and at 37 degrees C were 0.64 +/- 0.2 microM and 0.9 +/- 0.2 microM respectively. InsP3 degradation studies, at 37 degrees C, indicated that less than 10% of [3H]-InsP3 was degraded within the first 10 s of incubation. InsP3 association rates were evaluated, at low temperature, with increasing concentrations of [3H]-InsP3. These kinetic studies revealed a direct relationship between the initial rate of association (Vi) and InsP3 concentration. From this relationship, we evaluated that the concentration of InsP3 needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy between Kd and EC50 is related to a kinetic constraint dictated by the quantal process by which InsP3 releases Ca2+.
Ribeiro-do-Valle
R M
RM
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Québec, Canada.
Poitras
M
M
Boulay
G
G
Guillemette
G
G
D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium