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Muscarinic-receptor-mediated phosphoinositide hydrolysis in rat cerebral cortex was investigated by analysis of the kinetics of [3H]inositol phosphate formation and degradation in myo-[2-3H]inositol-labelled tissue slices. Carbachol stimulated rapid (5 s) increases in the concentrations of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,4)P2. Stimulated accumulation of [3H]Ins(1,3,4)P3, [3H]Ins(1,3)P2 and [3H]Ins(3,4)P2 and [3H]Ins(1/3)P or of [3H]Ins(4)P occurred only subsequently and with a sequence indicating formation by successive dephosphorylation of [3H]Ins(1,3,4,5)P4 or of Ins(1,4)P2 respectively. A similar sequence was inferred from the order of rapidity with which the accumulations of [3H]inositol polyphosphates, resulting from sustained (5 min) carbachol stimulation in the presence of LiCl, were reversed when muscarinic receptors were subsequently blocked with atropine. During this latter period of receptor blockade, radiolabel lost from [3H]inositol polyphosphates was quantitively recovered as [3H]inositol monophosphates owing to effective inhibition of monophosphatase by Li+, and the rate of poly- into mono-phosphate conversion was similar to agonist-stimulated rates of monophosphate accumulation. This implies that, even during persistent stimulation, polyphosphoinositide, not PtdIns, is the substrate for phosphoinositidase C. Quantitative comparison of the degradation of [3H]inositol poly- to mono-phosphates after receptor blockade unexpectedly suggests the dual hydrolysis of [3H]Ins(1,4)P2 to [3H]Ins(1)P and [3H]Ins(4)P. This result advises cautious interpretation of the origin of [3H]Ins(1)P in stimulated tissue, but, with other data presented, allows calculation from the observed ratio of [3H]Ins(1/3)P:[3H]Ins(4)P that a minimum of approx. 50% of the [3H]Ins(1,4,5)P3 produced during persistent muscarinic-receptor stimulation is metabolized by Ins(1,4,5)P3 3-kinase.

Analysis of [3H]inositol phosphate formation and metabolism in cerebral-cortical slices. Evidence for a dual metabolism of inositol 1,4-bisphosphate