LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.

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 LPA(1-6), 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 LPA(2), LPA(5) and LPA(6) receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5) receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5) 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.

Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors.

Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P(1) receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands.

Vascular effects of sphingolipids.

UNLABELLED: The sphingomyelin metabolites ceramide, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) are emerging modulators of vascular tone. While ceramide appears to act primarily intracellularly, S1P and SPC appear to mainly work via specific receptors, although those for SPC have not yet been defined unequivocally. Each of the sphingomyelin metabolites can induce both vasoconstriction and vasodilatation and, in some cases--ceramide on the one hand, and S1P and SPC on the other hand--have opposite effects on vascular tone. The differences in effects between vessels may relate to the relative roles of endothelial and smooth muscle cells in mediating them, as well as to the distinct expression patterns of S1P receptors among vascular beds and among endothelial and smooth muscle cells. Recent evidence suggests that vascular tone is not only modulated by sphingomyelin metabolites which are exogenously added or reach the vessel wall via the bloodstream but also by those formed locally by cells in the vessel wall. Such local formation can be induced by known vasoactive agents such as angiotensin II and may serve a signalling function. CONCLUSION: 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.