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 Ca
2+ fluxes, perhaps by interfering with endogenous inositol phosphate(s), have been suggested. Here we describe the existence of specific [
3H]α-trinositol binding sites and compare these with binding sites for naturally occurring inositol phosphates. For this purpose we developed a tritiated analog of α-trinositol and used it in a centrifugation binding assay on extensively washed membranes from rat tissues. The degree of specific [
3H]α-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]α-trinositol binding sites, displaying a
K
D of 159 nM and a
B
max 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]α-trinositol sites in cardiac membranes was 8-fold higher than
[
3
H]
Ins(1,4,5)P
3
but 2-fold and 4-fold lower than
[
3
H]
Ins(1,3,4,5)P
4
and
[
3
H]
InsP
6
binding sites, respectively. Competition binding studies indicated the ability of Ins(1,3,4,5)
P
4 and Ins
P
6, but not Ins(1,4,5)
P
3, to potently displace [
3H]α-trinositol binding. Conversely, unlabelled α-trinositol showed relatively low potency vs.
[
3
H]
InsP
6
, but the novel inositol phosphate was virtually equipotent with Ins(1,3,4,5)
P
4 in inhibiting
[
3
H]
Ins(1,3,4,5)P
4
binding. Finally, analyses of binding at different pH and ionic conditions revealed differences between α-trinositol and the three other previously studied inositol phosphates, although distinct similarities between α-trinositol and Ins(1,3,4,5)
P
4 were again observed.
