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

A ‘P 2 ’ membrane fraction of bovine tracheal smooth muscle displays high affinity ( K D 3.8 ± 0.2 n m ), saturable ( B max 1003 ± 170 fmol mg −1 protein) and reversible binding of d ‐ myo [ 3 H]‐inositol 1,4,5‐trisphosphate ([ 3 H]‐Ins(1,4,5)P 3 ). This binding site shows strict stereo‐ and positional specificity for the d ‐Ins(1,4,5)P 3 isomer with l ‐Ins(1,4,5)P 3 , dl ‐Ins(1,3,4,5)P 4 and d ‐Ins(1,3,4)P3 displacing [ 3 H]‐Ins(1,4,5)P 3 with K i values of 20 μ m , 0.35 μ m and 2.4 μ m , respectively. Specific binding of [ 3 H]‐Ins(1,4,5)P 3 is enhanced at alkaline pH values (maximal at pH 7.75) and, in distinct contrast to [ 3 H]‐Ins(1,4,5)P 3 binding in rat cerebellum membranes, is not inhibited by Ca 2+ (5–500 μ m ). Heparin displaces [ 3 H]‐Ins(1,4,5)P 3 specific binding with an IC 50 of 7.6 ± 1.0 μg ml −1 . Comparative studies demonstrated specific [ 3 H]‐Ins(1,4,5)P 3 binding in bovine cardiac atrial preparations ( B max 75 ± 5 fmol mg −1 protein) and very low specific [ 3 H]‐Ins(1,4,5)P 3 binding in bovine cardiac ventricle and skeletal muscle membranes (≤ 25 fmol mg −1 protein).