Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: Relationship to diacylglycerol and phosphatidic acid accumulation

Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P₂ cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [³H]choline administration, resulted in an enhanced release of [³H]phosphocholine (PCho) into the external medium; enhanced [³H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [³H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [³H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [³H]phosphatidylethanol in cells prelabelled with [³H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [³H]myristic acid and [¹⁴]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P₂₊ whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.