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

Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca2+ concentration ([Ca2+](i)) in human SH-SY5Y neuroblastoma cells. The 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 magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca2+ from intracellular stores, as assessed by inhibition 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 contrast, 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 antagonist of InsP(3)Rs, blocked Ca2+-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P-3, but failed to inhibit Ca2+-release induced by LPA. Elevation of [Ca2+](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca2+], responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca2+, N,N-dimethylsphingosine (DMS, 30 mu M), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca2+ responses by almost 90%. In addition, MCh-induced Ca2+ responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca2+-stores 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 sphingosine kinase activation and the potential generation of the putative second messenger sphingosine I-phosphate.