Separation of Labeled Inositol Phosphate Isomers by High-Pressure Liquid Chromatography (HPLC)

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 ( 1 , 2 ). Phosphodiesteric cleavage of phosphatidylinositol-4,5-bis-phosphate by phospholipase C produces Ins(1,4,5)P ₃ and sn-1,2-diacylglycerol, which release Ca ²⁺ from intracellular stores and activate protein kinase(s) C, respectively ( 1 – 4 ). Metabolism of Ins(1,4,5)P ₃ is rapid and can follow two possible pathways (Fig. 1 .; for review, see ref. 4 ). The simple dephosphory lation route proceeds via a 5-phosphatase to Ins(1,4)P ₂ , and Ins(1,3,4)P ₃ /Ins(1,4)P ₂ 1-phosphatase to Ins(4)P ₁ . Alternatively, phosphorylation of Ins(1,4,5)P ₃ by a 3-kinase generates the putative second messenger Ins(1,3,4,5)P ₄ , which then undergoes sequential dephosphorylation via a 5-phosphatase to Ins(1,3,4)P ₃ and either a 4-phosphatase, an Ins(1,3,4)P ₃ /Ins(1,4)P ₂ 1-phosphatase, or a 6-kinase route to produce Ins(1,3)P ₂ , Ins(3,4)P ₂ , or Ins(1,3,4,6)P ₄ , respectively. Subsequent dephosphorylation of Ins(1,3)P ₂ and Ins(3,4)P ₂ produces Ins(l)P ₁ and Ins(3)P ₁ , which, along with Ins(4)P ₁ , are metabolized by the inositol monophosphatase to release myo-inositol ( 4 ). This complex pathway of Ins(1,4,5)P ₃ , 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.

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