Ca2+ signaling induced by sphingosylphosphorylcholine and sphingosine 1-phosphate via distinct mechanisms in rat glomerular mesangial cells

Abstract

To elucidate the molecular mechanism underlying sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediated signaling, we compared their effects with those of adenosine triphosphate (ATP) and angiotensin II (Ang II) on the cytosolic free Ca2+ concentration ([Ca2+]i), inositol 1,4, 5-trisphosphate (IP3) generation and arachidonic acid release in rat glomerular mesangial cells. The fluorescent Ca2+ indicator, Fura-2, was used to measure the [Ca2+]i changes in cultured rat glomerular mesangial cells either in suspension or attached to the coverslips. SPC 5 microM, S1P 5 microM, ATP 100 microM and Ang II 90 nM all induced increases in the [Ca2+]i, and the effect showed marked homologous desensitization, while heterologous desensitization was less. After the initial exposure of the cells to SPC, the increase in [Ca2+]i induced by subsequent addition of ATP or Ang II was only reduced by about 14.3% and 4.8%, respectively. After the initial exposure to S1P, a greater reduction was seen (42. 1% and 47.7%, respectively). Both arachidonic acid release and IP3 generation were activated by all four agonists with an identical rank order of effectiveness of SPC >> S1P > ATP = Ang II; both were pertussis toxin-sensitive and cholera toxin-resistant. The arachidonic acid release induced by all four agonists showed identical susceptibility to removal of extracellular Ca2+, whereas IP3 generation displayed differential extracellular Ca2+ dependence. Only SPC-induced IP3 generation was highly sensitive to extracellular Ca2+ level, and this Ca2+ dependence was abolished after pretreatment of cells with arachidonyl trifluoromethyl ketone (AACOCF3), a phospholipase A2 inhibitor. Furthermore, the Mn2+ influx was markedly greater in SPC-stimulated cells than in either control or other agonist-stimulated cells, and was decreased by prior exposure of cells to AACOCF3. After phospholipase A2 was inhibited or in the absence of extracellular Ca2+, SPC displayed identical effectiveness as S1P on desensitizing the action of ATP or Ang II on the increase in [Ca2+]i. Conclusions. Our results indicate that all four agents primarily activate phospholipase C through their receptor occupancies, but that SPC alone also induces further significant Mn2+ influx and IP3 generation attributable to its primary stimulatory effect on arachidonic acid release. Thus, the heterologous desensitization to ATP or Ang II induced by SPC was less profound than that induced by S1P, since SPC induced a Ca2+ influx.