Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes

Abstract

The binding of [3H]Ins(1,4,5)P3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites (KD = 6.82+/-2.3 nM, Bmax = 370+/-38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e. Ins(2,4,5)P3 was able to compete with [3H]Ins(1,4,5)P3 whereas Ins(1,3,4)P3 was not. Ins(1,3,4,5)P4 showed a similar affinity for the receptor as Ins(2,4,5)P3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [3H]Ins(1,4,5)P3-binding was independent of free Ca2+ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determine the basal and vasopressin-stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P3 were calculated to be 0.22+/-0.15 microM basal and 2.53+/-1.8 microM at peak stimulation. This assay provides a simple, specific and quantitative method for the measurement of Ins(1,4,5)P3 concentrations in the picomolar range.