Characterisation 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 (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). 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 Ki values of 20 microM, 0.35 microM and 2.4 microM, 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 microM). 4. Heparin displaces [3H]-Ins(1,4,5)P3 specific binding with an IC50 of 7.6 +/- 1.0 micrograms ml-1. 5. 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 (less than or equal to 25 fmol mg-1 protein).
Chilvers
E R
ER
Department of Pharmacology and Therapeutics, University of Leicester.
Challiss
R A
RA
Willcocks
A L
AL
Potter
B V
BV
Barnes
P J
PJ
Nahorski
S R
SR
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels; D002417:Cattle; D002531:Cerebellum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D013237:Stereoisomerism; D014132:Trachea / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium