Kinetic Analysis of A23187-Mediated Polyphosphoinositide Breakdown in Rat Cortical Synaptosomes Suggests that Inositol Bisphosphate Does Not Arise Primarily by Degradation of Inositol Trisphosphate

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Abstract
The kinetics of polyphosphoinositide breakdown and inositol phosphate formation have been studied in rat cortical synaptomes labelled in vitro with myo-[2-3H]inositol. Intrasynaptosomal Ca2+ concentrations have been varied by the use of Ca-EGTA buffers or by adding the ionophore A23187 in the presence and absence of 1 mM Ca2+. The former studies have revealed that, at very low (20 nM) intrasynaptosomal free Ca2+ levels, inositol bisphosphate, but not inositol monophosphate levels are reduced. Addition of A23187 in the absence of added Ca2+ gives rise to greatly enhanced inositol bisphosphate accumulation, which if further enhanced if 1 mM Ca2+ is present in the extrasynaptosomal medium. At all time points examined (down to 2 s after adding ionophore), the ratio of inositol trisphosphate/inositol bisphosphate accumulation does not exceed 0.2, and calculations based on inositol bis- and tris-phosphate breakdown rates in synaptosomal lysates suggest that only a minority of the inositol bisphosphate arises from degradation of inositol trisphosphate. Addition of ionophore in the presence (but not in the absence) of 1 mM Ca2+ leads to rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and ATP and slower breakdown of phosphatidylinositol 4-phosphate (PtdInsP). The rates of loss of PtdinP2 and ATP are very highly correlated, suggesting that polyphosphoinositide resynthesis may be limited by ATP availability at high Ca2+ levels. Analysis of 32P-labelled synaptosomes also also reveals that A23187 produces Ca2+-dependent losses of PtdInsP2, PtdInsP, ATP, and GTP radioactivity and a marked increase in the radioactivity of a compound distinct from nucleotides or any of the lipid breakdown products testes. This compound may be a hexose bisphosphate arising from stimulation of glycolysis by A23187.