Characterisation of stereospecific binding sites for inositol 1,4,5‐trisphosphate in airway smooth muscle

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Abstract
A ‘P2’ membrane fraction of bovine tracheal smooth muscle displays high affinity (KD 3.8 ± 0.2 nm), saturable (Bmax 1003 ± 170 fmol mg−1 protein) and reversible binding of d‐myo[3H]‐inositol 1,4,5‐trisphosphate ([3H]‐Ins(1,4,5)P3). This binding site shows strict stereo‐ and positional specificity for the d‐Ins(1,4,5)P3 isomer with l‐Ins(1,4,5)P3, dl‐Ins(1,3,4,5)P4 and d‐Ins(1,3,4)P3 displacing [3H]‐Ins(1,4,5)P3 with Ki values of 20 μm, 0.35 μm and 2.4 μm, respectively. Specific binding of [3H]‐Ins(1,4,5)P3 is enhanced at alkaline pH values (maximal at pH 7.75) and, in distinct contrast to [3H]‐Ins(1,4,5)P3 binding in rat cerebellum membranes, is not inhibited by Ca2+ (5–500 μm). Heparin displaces [3H]‐Ins(1,4,5)P3 specific binding with an IC50 of 7.6 ± 1.0 μg ml−1. Comparative studies demonstrated specific [3H]‐Ins(1,4,5)P3 binding in bovine cardiac atrial preparations (Bmax 75 ± 5 fmol mg−1 protein) and very low specific [3H]‐Ins(1,4,5)P3 binding in bovine cardiac ventricle and skeletal muscle membranes (≤ 25 fmol mg−1 protein).

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