Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca²⁺ system in FRTL-5 thyroid cells: Possible involvement of guanosine triphosphate-binding proteins in the lipid signaling

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H₂O₂) generation in association with an increase in intracellular Ca²⁺ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N⁶-(L-2-phenylisopropyl)adenosine, an A₁-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPγS), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTPγS-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross- desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca²⁺ mobilization, no detectable cross- desensitization of S1P and bradykinin was observed. This suggests that the primary action of SIP in its activation of the PLC-Ca²⁺ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross- desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H₂O₂ generation through a PLC-Ca²⁺ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.