Is Inositol Bisphosphate the Product of A23187 and Carbachol‐Mediated Polyphosphoinositide Breakdown in Synaptosomes?

Synaptosomes have been isolated from rat cerebral cortex and labelled in vitro with [ 32 P]orthophosphate and myo ‐[2‐ 3 H]inositol. Subsequent addition of the Ca 2+ ionophore A23187 in the presence of 2 m M extrasynaptosomal Ca 2+ raised intrasynaptosomal free [Ca 2+ ] to >2 μ M from a resting level of 200 n M and led to rapid breakdown of polyphosphoinositides. This was accompanied by a small increase in the level of inositol monophosphate, greatly enhanced accumulation of inositol bisphosphate, but no detectable increase in inositol trisphosphate. Depolarising (25 m M ) extrasynaptosomal K + produced a smaller increase in intrasynaptosomal free [Ca 2+ ] (to around 400 n M ) and a proportional increase in inositol bisphosphate radioactivity. Carbachol (1 m M ) alone elicited only limited polyphosphoinositide breakdown and inositol mono‐ and bisphosphate formation, but this was greatly increased in the presence of 25 m M K + . The effect of carbachol in the presence of depolarising K + was time‐ and dose‐dependent and was antagonised by atropine (10 μ M ). There was no detectable accumulation of inositol trisphosphate in the presence of carbachol, K + , or carbachol plus K + , even after short (30 s.) incubations. The lack of inositol trisphosphate accumulation does not appear to result from rapid formation of inositol tetrakisphosphate or from enhanced breakdown of the trisphosphate in synaptosomes.