Assessment of the extracellular and intracellular actions of sphingosine 1-phosphate by using the p42/p44 mitogen-activated protein kinase cascade asa model

We have investigated the extracellular and intracellular actions of sphingo sine 1-phosphate (S1P) by using cultured airway smooth muscle cells. We hav e demonstrated that exogenous S1P elicited an activation of mitogen activat ed protein kinase (p42/p44 MAPK) that was abolished by pertussis toxin (0.1 mu g/mL, 24 h), which was used to inactivate G(i). The effect of exogenous S1P might therefore be attributed to an action at a putative Gi-coupled re ceptor. The regulation of the p42/p44 MAPK cascade by S1P was also shown to include a protein kinase C (PKC)-dependent intermediate step. Platelet-der ived growth factor (PDGF) stimulates intracellular S1P formation and was th erefore used to evaluate the intracellular action of S1P. This has previous ly been investigated by others using the sphingosine kinase inhibitors D,L- threo-dihydrosphingosine and N,N-dimethylsphingosine. We have demonstrated here that both inhibitors block the PDGF-dependent activation of p42/p44 MA PK. However, both are also PKC inhibitors, which might account for their ef fect. because PDGF utilises PKC as an intermediate in the regulation of the p42/p44 MAPK cascade. Significantly, sphingosine, which is the substrate o f sphingosine kinase and a PKC inhibitor, blocked the activation of p42/p44 MAPK by PDGF with an almost identical concentration dependence compared wi th D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. Therefore, the use of so-called sphingosine kinase inhibitors might lead to misleading in terpretations because of their additional effect on PKC. Other approaches, such as oligodeoxynucleotide anti-sense against sphingosine kinase, are req uired to address the intracellular role of S1P. CELL SIGNAL 11;5:349-354, 1 999. (C) 1999 Elsevier Science Inc.