title: Characterisation of inositol 1,4,5-trisphosphate binding sites in rabbit aortic smooth muscle.
abstract: The present study investigated the characteristics of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites in crude membrane preparations of rabbit aortic smooth muscle. A particular aim was to demonstrate if increases in cytoplasmic cyclic guanosine 3':5' monophosphate (cGMP), which mediates the effect of nitrovasodilators, may cause smooth muscle relaxation in part by the displacement of Ins(1,4,5)P3 binding. Negligible Ins(1,4,5)P3 binding was observed at pH < 7, while maximum binding occurred over the pH range 8-9. Saturation analysis of isotopic dilution binding data revealed an apparently homogenous population of Ins(1,4,5)P3 binding sites with a KD of 4.02 +/- 0.53 nM and a Bmax of 27.7 +/- 4.6 fmol/mg protein. Heparin, an Ins(1,4,5)P3 receptor antagonist, inhibited binding with an IC50 of 11.43 +/- 2.81 micrograms/ml. The ability of other polyphosphate compounds to inhibit Ins(1,4,5)P3 binding in this preparation was also examined. D-myo-Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP) inhibited Ins(1,4,5)P3 binding, although each was significantly less potent that Ins(1,4,5)P3. In contrast, cyclic guanosine 3':5' monophosphate (cGMP) did not significantly alter Ins(1,4,5)P3 binding in rabbit aortic smooth muscle. This observation suggests that competitive inhibition of Ins(1,4,5)P3 receptor binding is not an important consideration in cGMP-mediated vascular smooth muscle cell relaxation.
authors.affiliation: Department of Pharmacology, School of Medical Sciences, University of Bristol, UK.
authors.forename: T V
authors.initials: TV
authors.lastname: Murphy
authors.forename: L M
authors.initials: LM
authors.lastname: Broad
authors.forename: C J
authors.initials: CJ
authors.lastname: Garland
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D006152:Cyclic GMP / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011817:Rabbits; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*
chemical_list: D000227:Adenine Nucleotides; D015220:Calcium Channels; D006150:Guanine Nucleotides; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D006152:Cyclic GMP