Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-x beta in Xenopus oocytes

Abstract

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory CI-currents by acting through membrane-bound receptors. External application of 50 mu M S1P elicited a longlasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-mu M LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18)as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent CI-currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because CI-currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-x beta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-x beta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein or subunits that were identified in Xenopus laevis; G(q) alpha, G(11)alpha, G(0) alpha, and G(11)alpha. Among AS-ODNs against the Gas tested, AS-G(q) alpha and AS-G(l1)alpha to S1P and AS-G(q) alpha and AS-G(11)alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. (C) 1998 Wiley-Liss, Inc.