title: Characterization of specific binding sites for alpha-trinositol (D-myo-inositol 1,2,6-trisphosphate) in rat tissues.
abstract: Alfa-trinositol (or D-myo-inositol 1,2,6-trisphosphate) was recently found to, e.g., inhibit agonist-induced vasoconstriction and display antiinflammatory properties. However, its mechanism of action is unknown, although effects on Ca2+ fluxes, perhaps by interfering with endogenous inositol phosphate(s), have been suggested. Here we describe the existence of specific [3H]alpha-trinositol binding sites and compare these with binding sites for naturally occurring inositol phosphates. For this purpose we developed a tritiated analog of alpha-trinositol and used it in a centrifugation binding assay on extensively washed membranes from rat tissues. The degree of specific [3H] alpha-trinositol binding was markedly increased as a result of the many wash steps, indicating the existence of endogenous binding inhibitor(s). A single population of [3H] alpha-trinositol binding sites, displaying a KD of 159 nM and a Bmax of 71 pmol/mg protein, was present in cardiac membranes assayed at pH 7.4. Similar binding site densities were detected also in liver > lung > brain. The relative density of [3H] alpha-trinositol sites in cardiac membranes was 8-fold higher than [3H]Ins(1,4,5)P3 but 2-fold and 4-fold lower than [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding sites, respectively. Competition binding studies indicated the ability of Ins(1,3,4,5)P4 and InsP6, but not Ins(1,4,5)P3, to potently displace [3H] alpha-trinositol binding. Conversely, unlabelled alpha-trinositol showed relatively low potency vs. [3H]InsP6, but the novel inositol phosphate was virtually equipotent with Ins(1,3,4,5)P4 in inhibiting [3H]Ins(1,3,4,5)P4 binding. Finally, analyses of binding at different pH and ionic conditions revealed differences between alpha-trinositol and the three other previously studied inositol phosphates, although distinct similarities between alpha-trinositol and Ins(1,3,4,5)P4 were again observed.
authors.affiliation: Department of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021.
authors.forename: H
authors.initials: H
authors.lastname: Yoo
authors.forename: B
authors.initials: B
authors.lastname: Fallgren
authors.forename: A
authors.initials: A
authors.lastname: Lindahl
authors.forename: C
authors.initials: C
authors.lastname: Wahlestedt
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D006321:Heart / Q000187:drug effects; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats
chemical_list: D002412:Cations; D007295:Inositol Phosphates; D006493:Heparin; C059368:atrinositol