Polyphosphoinositides are the major source of inositol phosphates in carbamoylcholine-stimulated SK-N-SH neuroblastoma cells

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 bisphosphates steadily accumulated in response to receptor activation and in the presence of Li+ comprised greater than 95% of agonist-stimulated inositol phosphate formation at incubation times greater than 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 greater than 50% but did not significantly influence the ratio of inositol monophosphates formed. Upon addition of atropine to agonist-pretreated cells, radioactivity was lost from inositol phosphates in the following order: inositol-1,4,5-trisphosphate greater than inositol-1,3,4-trisphosphate greater than inositol-1,4-bisphosphate = inositol-4-monophosphate greater than 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 greater than 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.(ABSTRACT TRUNCATED AT 400 WORDS)