Lysophosphatidic acid-mediated Ca2+ mobilization in human SH-SY5Y neuroblastoma cells is independent of phosphoinositide signalling, but dependent onsphingosine kinase activation

Extracellular application of lysophosphatidic acid (LPA) elevated intracell ular Ca2+ concentration ([Ca2+](i)) in human SH-SY5Y neuroblastoma cells. T he maximal response to LPA occurred between 0.1 and 1 mu M, at which point [Ca2+](i) was increased by approx. 500 nM. This increase was of similar mag nitude to that caused by the muscarinic acetylcholine receptor agonist meth acholine (MCh), although the initial rate of release by LPA was slower. Bot h LPA and MCh released Ca2+ from intracellular stores, as assessed by inhib ition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca2+ uptake, and by the persistence of their action in nominally Ca2+-free extracellular medium. Similarly, both agonists appeared to stimulate store- refilling Ca2+ entry. MCh produced a marked elevation in cellular Ins(1,4,5 )P-3 and stimulated [H-3]InsP accumulation in the presence of Li+. In contr ast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P-3 receptor (InsP(3)R) proteins with MCh did not affect Ca2+ responses to LPA. In addition, heparin, a competitive antag onist of InsP(3)Rs, blocked Ca2+-mobilization in permeabilized SH-SY5Y cell s in response to MCh or exogenously added Ins(1,4,5)P-3, but failed to inhi bit Ca2+-release induced by LPA. Elevation of [Ca2+](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]diphosphate, indicating that this agoni st acts via a G-protein-coupled receptor. However, pertussis toxin was with out effect on LPA-evoked [Ca2+], responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca2+, N,N-dimethylsphin gosine (DMS, 30 mu M), a competitive inhibitor of sphingosine kinase, block ed LPA-induced Ca2+ responses by almost 90%. In addition, MCh-induced Ca2responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca2+-st ores in SH-SY5Y cells independently of the generation and action of Ins(1,4 ,5)P-3. Furthermore, the Ca2+-response to LPA appears to be dependent on sp hingosine kinase activation and the potential generation of the putative se cond messenger sphingosine I-phosphate.