Inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate binding sites in smooth muscle

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Abstract
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 [3H]-InsP3 and [3H]-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 [3H]-InsP3 binding sites was 335.3 ± 19.3 fmol mg−1 protein which was approximately 2.5 fold greater than that of [3H]-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 [3H]-InsP3 binding was maximal, whereas [3H]-InsP4 binding was only 10% that of [3H]-InsP3. Conversely, at pH 5.0, [3H]-InsP4 binding was maximal, while [3H]-InsP3 binding was reduced to 15% of its binding at pH 9.0. 4 InsP3 was about 20 fold less potent (K1 = 50.7 ± 8.3 nm) than InsP4 in competing for [3H]-InsP4 binding sites and could compete for only 60% of [3H]-InsP4 specific binding. InsP4 was also capable of high affinity competition with [3H]-InsP3 binding (K1 = 103.5 ± 1.5 nm), and could compete for 100% of [3H]-InsP3 specific binding. 5 [3H]-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 [3H]-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.

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