Assessment of agonism at G-protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells.

Several different molecular species of phosphatidic acid (PA) bind to a G-protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from G(i/o)). The de-acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an approximately 100 - 1000 fold lower potency compared with the different molecular species of PA. RT - PCR using gene-specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.

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

We have investigated the extracellular and intracellular actions of sphingosine 1-phosphate (S1P) by using cultured airway smooth muscle cells. We have demonstrated that exogenous S1P elicited an activation of mitogen-activated protein kinase (p42/p44 MAPK) that was abolished by pertussis toxin (0.1 microg/mL, 24 h), which was used to inactivate Gi. The effect of exogenous S1P might therefore be attributed to an action at a putative Gi-coupled receptor. The regulation of the p42/p44 MAPK cascade by S1P was also shown to include a protein kinase C (PKC)-dependent intermediate step. Platelet-derived growth factor (PDGF) stimulates intracellular S1P formation and was therefore used to evaluate the intracellular action of S1P. This has previously 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 MAPK. However, both are also PKC inhibitors, which might account for their effect because PDGF utilises PKC as an intermediate in the regulation of the p42/p44 MAPK cascade. Significantly, sphingosine, which is the substrate of sphingosine kinase and a PKC inhibitor, blocked the activation of p42/p44 MAPK by PDGF with an almost identical concentration dependence compared with D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. Therefore, the use of so-called sphingosine kinase inhibitors might lead to misleading interpretations because of their additional effect on PKC. Other approaches, such as oligodeoxynucleotide anti-sense against sphingosine kinase, are required to address the intracellular role of S1P.

PDGF-stimulated cyclic AMP formation in airway smooth muscle: assessment of the roles of MAP kinase, cytosolic phospholipase A2, and arachidonate metabolites.

Platelet-derived growth factor (PDGF) stimulates cyclic AMP (cAMP) synthesis in cultured guinea-pig airway smooth muscle (ASM) cells. However, this stimulation is normally countered by the action of cAMP phosphodiesterases. Thus, cAMP synthesis was observed only in cells pre-treated with either 3-isobutyl-1-methylxanthine (IBMX) or with cholera toxin. cAMP synthesis was inhibited by pre-treating cells with well-defined inhibitors of arachidonate metabolite synthesis, such as AACOCF3 [a cytosolic phospholipase A2 (cPLA2) inhibitor] and indomethacin (a cyclooxygenase inhibitor). This suggests that arachidonate metabolites (e.g., prostaglandins) released in response to PDGF stimulate cAMP synthesis. The presence of functional prostaglandin (PG) receptors was confirmed by experiments that showed that exogenous PGE2 stimulated cAMP formation. cPLA2 is regulated by mitogen-activated protein kinase (MAPK) in a number of cell types. The presence of this pathway in ASM cells and its role in regulating arachidonate metabolism were supported by the finding that pre-treatment of cells with PD098059 (an inhibitor of mitogen-activated protein kinase kinase-1 activation) reduced PDGF-stimulated cAMP synthesis. The cAMP formed in response to the arachidonate metabolites subsequently reduced the PDGF-dependent activation of c-Raf, MAPK, and DNA synthesis, suggesting the presence of a negative feedback pathway.