Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA1–6, showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18∶1) being 10-fold more potent than acyl-LPA(18∶1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA2, LPA5 and LPA6 receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA5 receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA5 as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

Background and purpose: Recently, some ligands targeting the sphingosine-1-phosphate receptor subtype 3 (S1P3) have become available. The characterization of these compounds was mainly based on one functional read-out system, although S1P3 receptors are known to activate different signal transduction pathways. Therefore, this study pharmacologically characterizes these compounds using different assays. Experimental approach: Using CHO-FlpIn cells expressing the human S1P3 receptor the potencies and maximal effects of S1P, FTY720-P, VPC23019, VPC23153 and VPC24191 were determined in three different assays [inhibition of cAMP accumulation, elevation of intracellular calcium concentrations ([Ca 2+ ]i) and S1P3 receptor internalization].Key results: All compounds tested inhibited forskolin-induced cAMP accumulation, increased [Ca 2+ ]i and induced S1P3 receptor internalization but with different potencies and maximal effects. S1P was the most potent compound in all assays followed by FTY720-P. The VPC compounds were generally less potent than S1P and FTY720-P. Regarding the maximal effects, all compounds except VPC23153, behaved as full agonists in the cAMP accumulation assay. In the calcium assay, FTY720-P, VPC23019 and VPC24191 displayed partial and VPC23153 weak partial agonist activity, relative to S1P. Interestingly, treatment with the Gi inactivator Pertussis toxin, did not affect S1P-induced [Ca 2+ ]i elevations but inhibited those in response to the other compounds, by about 50%. Conclusions and implications:This study demonstrated differential response patterns at the S1P3 receptor for a range of ligands. These differences could indicate the presence of functional selectivity at this receptor as FTY720-P and the VPC compounds seemed to signal predominantly via Gi -whereas S1P activated Gi and Gq-coupled pathways.

Background and purpose: The thiazolidine carboxylic acid, BML-241, has been proposed as a lead compound in development of selective antagonists at the sphingosine-1-phosphate receptor (S1P 3 ), based on its inhibition of the rise in intracellular calcium concentrations ([Ca 2 þ ] i ) in HeLa cells overexpressing S1P receptors. We have studied the antagonistic properties of BML-241 for the S1P 3 receptor in more detail. Experimental approach: Chinese hamster ovary (CHO) cells stably transfected with the S1P 3 , S1P 2 or a 1A -adrenoceptors were used to investigate the effect of BML-241 on increases in [Ca 2 þ ] i mediated via different receptors. CHO-K1 cells were used to study ATP-induced [Ca 2 þ ] i elevations. Effects on S1P 3 -mediated inhibition of forskolin-induced cAMP accumulation and on binding to a 1A -adrenoceptors were also investigated. In addition, the effect of BML-241 on contractions of rat mesenteric artery induced by phenylephrine was studied in an organ bath. Key results: High concentrations of BML-241 (10 mM) inhibited the rise in [Ca 2 þ ] i induced by S1P 3 and S1P 2 receptor stimulation; lower concentrations were ineffective. This high concentration of BML-241 also inhibited [Ca 2 þ ] i increases via P2 (nucleotide) receptor or a 1A -adrenoceptor stimulation. Moreover, BML-241 (10 mM) inhibited a 1 -adrenoceptor-mediated contraction of rat mesenteric artery but did not displace [3 H]-prazosin from a 1A -adrenoceptors in concentrations up to 100 mM. BML-241 (10 mM) did not affect the S1P 3 -mediated decrease of forskolin-induced cAMP accumulation. Conclusions and Implications: We conclude that BML-241 is a low potency, non-selective inhibitor of increases in [Ca 2 þ ] i , rather than a specific antagonist at the S1P 3 receptor.

We conclude that sphingomyelin metabolites are important endogenous modulators of vascular function, which may contribute to the pathophysiology of some diseases and be targets for therapeutic interventions.