Separation of labeled inositol phosphate isomers by high-pressure liquid chromatography (HPLC).

Abstract

It has now been established that agonist stimulation of a large variety of cell-surface receptors promotes hydrolysis of inositol phospholipids through activation of a phosphoinositide-specific phospholipase C (, ). Phosphodiesteric cleavage of phosphatidylinositol-4,5-bis-phosphate by phospholipase C produces Ins(1,4,5)P3 and sn-1,2-diacylglycerol, which release Ca2+ from intracellular stores and activate protein kinase(s) C, respectively (, , , ). Metabolism of Ins(1,4,5)P3 is rapid and can follow two possible pathways (Fig. 1.; for review, see ref. ). The simple dephosphory lation route proceeds via a 5-phosphatase to Ins(1,4)P2, and Ins(1,3,4)P3/Ins(1,4)P2 1-phosphatase to Ins(4)P1. Alternatively, phosphorylation of Ins(1,4,5)P3 by a 3-kinase generates the putative second messenger Ins(1,3,4,5)P4, which then undergoes sequential dephosphorylation via a 5-phosphatase to Ins(1,3,4)P3 and either a 4-phosphatase, an Ins(1,3,4)P3/Ins(1,4)P2 1-phosphatase, or a 6-kinase route to produce Ins(1,3)P2, Ins(3,4)P2, or Ins(1,3,4,6)P4, respectively. Subsequent dephosphorylation of Ins(1,3)P2 and Ins(3,4)P2 produces Ins(l)P1 and Ins(3)P1, which, along with Ins(4)P1, are metabolized by the inositol monophosphatase to release myo-inositol (). This complex pathway of Ins(1,4,5)P3, serves to terminate second-messenger action and to conserve efficiently the cellular myo-inositol pool for the resynthesis of inositol phospholipids. Fig. 1. The phosphoinositide cycle. Open image in new window Fig. 1. The phosphoinositide cycle.