Inositol bis-, tris-, and tetrakis(phosphate)s: analysis in tissues by HPLC

Abstract

The concentration of isomers of inositol tris(phosphate) (InsP3) was measured in tissues of intact animals. The method employed involved anion-exchange HPLC with on-line enzymatic hydrolysis of the phosphate esters and detection of the inorganic phosphate formed. All seven organs tested from rats killed by decapitation contained Ins(1,4,5)P3 in concentrations of 13-40 nmol/g; a distribution that bears no resemblance to that reported for its precursor [phosphatidylinositol bis(phosphate)]. A second InsP3 isomer [probably Ins(1,3,4)P3] was also detectable in brain and salivary gland. The content of Ins(1,4,5)P3 in brain and salivary gland from rats killed by decapitation was 10-60 times greater than that from rats killed by focused microwave irradiation to block postmortem metabolism. Inositol bis(phosphate) concentrations also changed dramatically postmortem. A much smaller postmortem change was seen in the content of Ins(1,3,4)P3. Receptor stimulation by muscarinic cholinergic agonists increased the content not only of Ins(1,3,4)P3, but also its recently discovered probable precursor, inositol tetrakis-(phosphate).