Autoradiographic localization and characterization of [3H]alpha-trinositol (1D-myo-inositol 1,2,6-trisphosphate) binding sites in human and mammalian tissues

Abstract

alpha-Trinositol (1D-myo-inositol 1,2,6-trisphosphate, PP56) selectively and potently inhibits the vasoconstrictor effects of neuropeptide Y (NPY). The authors used quantitative in vitro receptor autoradiography to localize and characterize [3H]alpha-trinositol binding sites in human and mammalian tissues. [3H]alpha-trinositol bound specifically to vascular and nonvascular smooth muscle in human, porcine and rat tissues. Binding was time dependent, reversible, saturable and specific for alpha-trinositol compared with inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and inositol hexakisphosphate (Ins-P6). Binding to each structure gave Kd values of 5 to 20 nM and was consistent with a homogeneous population of sites. Binding was optimal at pH 5 and at low calcium concentrations. Comparison with [125I]Bolton Hunter-labeled NPY ([125I]BH-NPY) binding in porcine tissues revealed 1) a partial colocalization but Bmax values for [3H]alpha-trinositol binding some two orders of magnitude higher than for [125I]BH-NPY and 2) failure of each of the two ligands to inhibit binding of the other. Comparison of [3H]alpha-trinositol with [3H]Ins-1,3,4,5-P4 binding in human umbilical cord revealed that both ligands bound specifically to vascular smooth muscle but that only [3H]Ins-1,3,4,5-P4 bound to arterial endothelium. Both ligands bound to sites with rank orders of affinity Ins-1,3,4,5-P4 > Ins-P6 > inositol 1,4,5-trisphosphate. alpha-Trinositol had, however, three orders of magnitude higher affinity for [3H]alpha-trinositol than [3H]Ins-1,3,4,5-P4 binding sites; Ins-1,3,4,5-P4 and Ins-P6 had higher affinity for [3H]Ins-1,3,4,5-P4 binding sites. Specific [3H]alpha-trinositol binding sites may represent receptors by which alpha-trinositol inhibits NPY effects on vascular tone.(ABSTRACT TRUNCATED AT 250 WORDS)