RESUMO
Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Lisofosfolipídeos/farmacologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Cafeína/farmacologia , Canais de Cálcio/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Neuroblastoma , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Ácidos Lisofosfatídicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingosina/metabolismo , Esfingosina/farmacologia , Células Tumorais CultivadasRESUMO
The sphingosine kinase inhibitor, dimethylsphingosine, is an important tool for investigating intracellular effects of the putative second messenger compound, sphingosine 1-phosphate. However, the specificity of action of dimethylsphingosine has not been fully investigated. In human SH-SY5Y neuroblastoma cells, dimethylsphingosine (30 microM), produced a 25-fold increase in the EC(50) for methacholine-induced Ca(2+) mobilisation, and reduced the maximum response by 57+/-5%, suggesting the involvement of sphingosine 1-phosphate production in the Ca(2+) signal. However, dimethylsphingosine also inhibited [3H]N-methylscopolamine binding to whole SH-SY5Y cells and reduced methacholine-induced phosphoinositide turnover. Thus, this compound must be used with caution when investigating the role of sphingosine kinase in G-protein coupled receptor-mediated Ca(2+) mobilisation responses.