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Inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate binding sites in smooth muscle
Author(s) -
Zhang Lubo,
Bradley Michael E.,
Khoyi Mohammad,
Westfall David P.,
Buxton Iain L.O.
Publication year - 1993
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1993.tb13706.x
Subject(s) - inositol , inositol phosphate , binding site , inositol trisphosphate , receptor , radioligand , biochemistry , chemistry , biophysics , radioligand assay , affinities , biology
1 We have previously demonstrated that activation of M 3 muscarinic receptors increases inositol 1,4,5‐trisphosphate (InsP 3 ) and inositol 1,3,4,5‐tetrakisphosphate (InsP 4 ) accumulation in colonic smooth muscle. 2 In the present study, we demonstrate the existence of InsP 3 and InsP 4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [ 3 H]‐InsP 3 and [ 3 H]‐InsP 4 bound rapidly and reversibly to a single class of saturable sites in detergent‐solubilized colonic membranes with affinities of 5.04 ± 1.03 n m and 3.41 ± 0.78 n m , respectively. The density of [ 3 H]‐InsP 3 binding sites was 335.3 ± 19.3 fmol mg −1 protein which was approximately 2.5 fold greater than that of [ 3 H]‐InsP 4 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 [ 3 H]‐InsP 3 binding was maximal, whereas [ 3 H]‐InsP 4 binding was only 10% that of [ 3 H]‐InsP 3 . Conversely, at pH 5.0, [ 3 H]‐InsP 4 binding was maximal, while [ 3 H]‐InsP 3 binding was reduced to 15% of its binding at pH 9.0. 4 InsP 3 was about 20 fold less potent ( K 1 = 50.7 ± 8.3 n m ) than InsP 4 in competing for [ 3 H]‐InsP 4 binding sites and could compete for only 60% of [ 3 H]‐InsP 4 specific binding. InsP 4 was also capable of high affinity competition with [ 3 H]‐InsP 3 binding ( K 1 = 103.5 ± 1.5 n m ), and could compete for 100% of [ 3 H]‐InsP 3 specific binding. 5 [ 3 H]‐InsP 3 binding in subcellular fractions separated by discontinuous sucrose density gradients followed NADPH‐cytochrome c reductase activity, suggesting an intracellular localization for the majority of InsP 3 receptors in this tissue, whereas [ 3 H]‐InsP 4 binding appeared to be equally distributed between plasma membrane and intracellular membrane populations. 6 These results suggest the existence of distinct and specific InsP 3 and InsP 4 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.