INOSITOL 1,4,5-TRISPHOSPHATE AND INOSITOL 1,3,4,5-TETRAKISPHOSPHATE BINDING-SITES IN SMOOTH-MUSCLE

1 We have previously demonstrated that activation of M3 muscarinic receptors increases inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) accumulation in colonic smooth muscle. 2 In the present study, we demonstrate the existence of InSP3 and InsP4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [H-3]-InSP3 and [H-3]-InSP4 bound rapidly and reversibly to a single class of saturable sites in detergent-solubilized colonic membranes with affinities of 5.04 +/- 1.03 nm and 3.41 +/- 0.78 nm, respectively. The density of [H-3]-InSP3 binding sites was 335.3 +/- 19.3 fmol mg-1 protein which was approximately 2.5 fold greater than that of [H-3]-InSP4 sites (127.3 +/- 9.1 fmol mg-1 protein). 3 The two high affinity inositol phosphate binding sites exhibited markedly different pH optima for binding of each radioligand. At pH 9.0, specific [H-3]-InSP3 binding was maximal, whereas [H-3]-InSP4 binding was only 10% that of [H-3]-InsP3. Conversely, at pH 5.0, [H-3]-InSP4 binding was maximal, while [H-3]-InSP3 binding was reduced to 15% of its binding at pH 9.0. 4 InsP3 was about 20 fold less potent (K(I) = 50.7 +/- 8.3 nm) than InSP4 in competing for [H-3]-InsP4 binding sites and could compete for only 60% of [H-3]-InSP4 specific binding. InsP4 was also capable of high affinity competition with [H-3]-InsP3 binding (K(I) = 103.5 +/- 1.5 nm), and could compete for 100% of [H-3]-InSP3 Specific binding. 5 [H-3]-InSP3 binding in subcellular fractions separated by discontinuous sucrose density gradients followed NADPH-cytochrome c reductase activity, suggesting an intracellular localization for the majority of InsP3 receptors in this tissue, whereas [H-3]-InsP4 binding appeared to be equally distributed between plasma membrane and intracellular membrane populations. 6 These results suggest the existence of distinct and specific InSP3 and InsP4 binding sites which may represent the physiological receptors for these second messengers; differences in the subcellular distribution of these receptors may contribute to differences in their putative physiological roles.