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

Abstract

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 [14C]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.

Effect of n-6 polyunsaturated fatty acids on growth and lipid composition of neoplastic and non-neoplastic canine prostate epithelial cell cultures

“…The tumor cell line contained a putative ether lipid (1-alkyl-2,3-diacylglycerol) with high levels of triacylglycerol and a lower phospholipid content. High phospholipase C activity has been found in some tumorous tissues [21,22] and this would also account for low phospholipid content of the prostate neoplastic cells. Phospholipase C activity would also generate the diacylglycerol, which on further acylation could yield the high levels of triacylglycerol found in the cells.…”

Negative regulation of diacylglycerol kinase θ mediates adenosine-dependent hepatocyte preconditioning

“…Biochemical evidence both in neuroblastoma 17 and in C. elegans motoneurons 14 shows that the enzymatic activity of DGKy and of its orthologue DGK-1 is strongly inhibited on direct interaction with the small GTPase RhoA. 18 Thus, we set to investigate the interaction between active RhoA and DGKy in PC12 cells,, a rat cell line expressing A2aR 19,20 transiently co-transfected with FLAGtagged DGKy and myc-tagged RhoA.…”

Choline-containing phospholipids: relevance to brain functional pathways

“…The reason for these inconsistencies is unclear. In cells of neural origin, muscarinic hydrolysis of PC has been described in human LA-N-2 neuroblastoma cells [ 50 ] and in SK-N human neuroblastoma cells [ 51 ]. In the latter case, PtdCho hydrolyses may have been due to a PtdCho-PLC activation.…”