POLYPHOSPHOINOSITIDES ARE THE MAJOR SOURCE OF INOSITOL PHOSPHATES IN CARBAMOYLCHOLINE-STIMULATED SK-N-SH NEUROBLASTOMA-CELLS

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Abstract
The contribution of polyphosphoinositides to muscarinic receptor-stimulated phosphoinositide turnover has been evaluated for intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of carbamoylcholine to [3H]inositol-prelabeled intact cells resulted in a rapid (5-10 sec) loss of phosphatidylinositol-4,5-bisphosphate and the concomitant appearance of radiolabeled inositol-1,4,5-trisphosphate, inositol-1,3,4-trisphosphate, and inositol tetrakisphosphate. In the presence of the agonist, production of these inositol polyphosphates remained enhanced for up to 45 min. Inositol mono- and biphosphates steadily accumulated in response to receptor activation and in the presence of Li+ comprised > 95% of agonist-stimulated inositol phosphate formation at incubation times > 5 min. The major inositol bisphosphate isomer was the 1,4-species. Of the two inositol monophosphates produced radioactivity recovered in inositol-4-monophosphate increased continuously, whereas that in the inositol-1-monophosphate/inositol-3-monophosphate fraction was delayed in appearance but thereafter progressively accumulated. Omission of Ca2+ reduced carbamoylcholine-stimulated inositol phosphate release by > 50% but did not significantly influence the ratio of inositol monophosphates formed. Upon addition of atropine to agonist-pretreated cells, radiaoctivity was lost from inositol phosphates in the following order: inositol-1,4,5-trisphosphate > inositol-1,3,4-trisphosphate > inositol-1,4-bisphosphate = inositol-4-monophosphate > inositol-1-monophosphate/inositol-3-monophosphate. Although carbamoylcholine addition to digitonin-permeabilized cells also resulted in a sustained release of inositol monophosphates, relatively more inositol-4-monophosphate was produced in these preparations. Omission of ATP from permeabilized cell incubations inhibited carbamoylcholine-stimulated inositol phosphate formation by > 70%. Whole homogenates of SK-N-SH cells metabolized added inositol-1,4,5-trisphosphate and inositol-1,4-bisphosphate exclusively to inositol-4-monophosphate, whereas inositol-1,3,4,5-tetrakisphosphate was degraded to inositol-1 or 3-monophosphate. Measurement of inositol trisphosphate 3''-kinase and 5''-phosphatase activities revealed that, following permeabilization, 3''-kinase activity was diminished, whereas that of 5''-phosphatase was enhanced. The results indicate that occupancy of muscarinic cholinergic receptors in SK-N-SH cells elicits a continuous Ca2+-dependent breakdown of the polyphosphoinositides rather than of phosphatidylinositol. In intact cells, inositol-1,4,5-trisphosphate produced upon receptor activation is metabolized both to inositol bisphosphate and inositol tetrakisphosphate via the 5''-phosphatase and 3''-kinase pathways, respectively. The latter pathway may account for much of the radioactivity present in the inositol-1-monophosphate/inositol-3-monophosphate fraction recovered from intact cell stimulated with carbamoylcholine. In contrast, the 5''-phosphatase route of degradation predominates in the permeabilized cell.