Abstract
Inositol 1,4,5-trisphosphate (InsP3) is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. The properties of InsP3 receptor sites in the rat pancreas were evaluated by binding studies with InsP3 labeled with 3H. Specific binding was very sensitive to pH variations between 7 and 9. Kinetic studies showed that specific binding of InsP3 at 0 degrees C was half-maximal in about 10 min and reached a plateau within 60 min with a K+1 = 3.37 x 10(9) mol-1min-1. Binding was reversible as addition of 10(-6) M unlabeled InsP3 was followed by dissociation of the bound ligand with a K-1 = 0.016 min-1. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with KD of 9.9 +/- 2.47 nM and a maximal binding capacity of 210 +/- 55 fmol/mg of protein (n = 7). The specificity of [3H]InsP3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1,3,4,5-tetrakisphosphate, phytic acid, fructose 1,6-bisphosphate, and heparin. To assess the functional relevance of the InsP3 binding sites, the Ca(2+)-releasing activity of InsP3 was measured in permeabilized acinar cell preparations. In the presence of oligomycin (10 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 4 mM ATP caused rapid uptake of Ca2+ by the vesicular component of the acinar cells. Addition of InsP3 (0.1-15 microM) caused a dose-dependent release of Ca2+ with half-maximal effect at 3.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
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