Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8-site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.

Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) is a G protein-coupled receptor that is required for humoral immune responses and polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1-3. The natural ligand for EBI2 has been unknown. Here we describe identification of 7α, 25-dihydroxycholesterol (5-cholesten-3β, 7α, 25-triol; 7α, 25-OHC) as a potent and selective agonist of EBI2. Functional activation of EBI2 by 7α, 25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high affinity radioligand binding. Furthermore we find that 7α, 25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A key enzyme required for the generation of 7α, 25-OHC is cholesterol 25-hydroxylase (Ch25h)4. Similar to EBI2 receptor knockout mice, mice deficient in Ch25h fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that Ch25h generates EBI2 bioactivity in vivo and suggests that the EBI2 − oxysterol signaling pathway plays an important role in the adaptive immune response.

BACKGROUND AND PURPOSEBAF312 is a next-generation sphingosine 1-phosphate (S1P) receptor modulator, selective for S1P1 and S1P5 receptors. S1P1 receptors are essential for lymphocyte egress from lymph nodes and a drug target in immune-mediated diseases. Here, we have characterized the immunomodulatory potential of BAF312 and the S1P receptor-mediated effects on heart rate using preclinical and human data.EXPERIMENTAL APPROACHBAF312 was tested in a rat experimental autoimmune encephalomyelitis (EAE) model. Electrophysiological recordings of G-protein-coupled inwardly rectifying potassium (GIRK) channels were carried out in human atrial myocytes. A Phase I multiple-dose trial studied the pharmacokinetics, pharmacodynamics and safety of BAF312 in 48 healthy subjects.KEY RESULTSBAF312 effectively suppressed EAE in rats by internalizing S1P1 receptors, rendering them insensitive to the egress signal from lymph nodes. In healthy volunteers, BAF312 caused preferential decreases in CD4+ T cells, Tnaïve, Tcentral memory and B cells within 4–6 h. Cell counts returned to normal ranges within a week after stopping treatment, in line with the elimination half-life of BAF312. Despite sparing S1P3 receptors (associated with bradycardia in mice), BAF312 induced rapid, transient (day 1 only) bradycardia in humans. BAF312-mediated activation of GIRK channels in human atrial myocytes can fully explain the bradycardia.CONCLUSION AND IMPLICATIONSThis study illustrates species-specific differences in S1P receptor specificity for first-dose cardiac effects. Based on its profound but rapidly reversible inhibition of lymphocyte trafficking, BAF312 may have potential as a treatment for immune-mediated diseases.

Targeting sphingosine-1-phosphate receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-phosphate (FTY720P) are thought to be mediated via S1P1 receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a "functional antagonist" by promoting efficient internalization of S1P1 receptors. We demonstrate here that S1P1 receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.

Antibodies against the four isoforms of the human plasma membrane Ca(2+)-ATPase (PMCA) were raised using an N-terminal sequence of the pump as epitope. The antibodies against PMCA isoforms 1, 2, and 3 were not species-specific, e.g. they also recognized the corresponding proteins in rat, whereas that against the human PMCA isoform 4 failed to do so. The tissue distribution of the four isoforms was estimated by Western blot analysis. Two, PMCA1 and PMCA4, were expressed in all tissues tested (with the exception of the choroid plexus, where the former was not detected). In most tissues the signal from the PMCA1 protein exceeded that of PMCA4, the exception being the erythrocyte. The PMCA2 and PMCA3 proteins were only found in neuronal tissues; the PMCA2 protein was present in high concentrations in the cerebellum and in the cerebral cortex. At variance with previous results on mRNA (e.g. the kidney) no other tissues contained the PMCA2 protein. PMCA3 was the other tissue-specific isoform; in agreement with results in the rat, the protein was found in human neuronal tissues, particularly in the choroid plexus, but was practically absent in all other tissues tested.