Characteristics of inositol 1,4,5-trisphosphate binding to rat cerebellar and bovine adrenal cortical membranes: evidence for the heterogeneity of binding sites

Abstract

The equilibrium and kinetic binding characteristics of D-inositol 1,4,5-trisphosphate (Ins(1.4.5)P 3) have been studied in membrane preparations of rat cerebellum and bovine adrenal cortex. Saturation analysis of isotopic dilution binding data demonstrated apparent K D values for Ins(1,4,5)P 3 binding of 23 ± 5 nM and 3.0 ± 1.3 nM for cerebellar and adrenal cortical preparations, respectively, with approximately 20-fold greater receptor density present in the cerebellar preparation (B max: 10.2 ± 2.5 pmol/mg protein). Kinetic analysis confirmed the equilibrium binding-derived K D value for cerebellum (K D: 39.9 nM), but revealed a second, very high affinity site (K D: 0.06 nM) to be present in adrenal cortex. The affinity differences between the investigated preparations was also observed with respect to the IC 50 values obtained for inhibition of specific [ 3H]Ins(1,4,5)P 3 binding by a number of inositol polyphosphate analogues including D-inositol 2,4,5-trisphosphate, DL-inositol 1,4,5-triphosphorothioate and L-Ins(1,4,5)P 3. In contrast, the Ins(1,4,5)P 3-receptor antagonist heparin displayed greater potency for the cerebellar (IC 50: 16.5 ± 6.2 μg · ml −1) compared to the adrenal cortical preparation (IC 50: 51.0 ± 6.1 μg · ml −1). The apparent differences between the Ins(1,4,5)P 3 receptors characterized in the two tissue preparations are discussed.