CHARACTERIZATION OF STEREOSPECIFIC BINDING-SITES FOR INOSITOL 1,4,5-TRISPHOSPHATE IN AIRWAY SMOOTH-MUSCLE

1. A 'P2' membrane fraction of bovine tracheal smooth muscle displays high affinity (K(D) 3.8±0.2 nM), saturable (B(max) 1003±170 fmol mg-1 protein) and reversible binding of D-myo[3H]-inositol 1,4,5-trisphosphate ([3H]-Ins(1,4,5)P3). 2. 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 K(i) values of 20 μM, 0.35 μM and 2.4 μM, respectively. 3. 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). 4. Heparin displaces [3H]-Ins(1,4,5)P3 specific binding with an IC50 of 7.6±1.0 μg ml-1. 5. Comparative studies demonstrated specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac atrial preparations (B(max) 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).