Prostaglandin I2 Production and cAMP Accumulation in Response to Acidic Extracellular pH through OGR1 in Human Aortic Smooth Muscle Cells

Abstract

Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4 have recently been identified as proton-sensing or extracellular pHresponsive G-protein-coupled receptors stimulating inositol phosphate production and cAMP accumulation, respectively. In the present study, we found that OGR1 and GPR4 mRNAs were expressed in human aortic smooth muscle cells (AoSMCs). Acidic extracellular pH induced inositol phosphate production, a transient increase in intracellular Ca 2؉ concentration ([Ca 2؉ ] i ), and cAMP accumulation in these cells. When small interfering RNAs (siRNAs) targeted for OGR1 and GPR4 were transfected to the cells, the acidinduced inositol phosphate production and [Ca 2؉ ] i increase were markedly inhibited by the OGR1 siRNA but not by the GPR4 siRNA. Unexpectedly, the acid-induced cAMP accumulation was also largely inhibited by OGR1 siRNA but only slightly by GPR4 siRNA. Acidic extracellular pH also stimulated prostaglandin I 2 (PGI 2 ) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI 2 production and cAMP accumulation without changes in the inositol phosphate production. A specific inhibitor of phospholipase C also inhibited the acid-induced cAMP accumulation. In conclusion, OGR1 is a major receptor involved in the extracellular acid-induced stimulation of PGI 2 production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI 2 pathway.Ludwig et al. (1) have recently shown that ovarian cancer G-proteincoupled receptor 1 (OGR1) 2 and GPR4, which were previously described as receptors for lysolipids, such as SPC (2) and LPC (3), sense extracellular protons and are coupled to G-proteins to stimulate intracellular signaling pathways. Thus, OGR1 stimulation causes inositol phosphate production and the subsequent increase in [Ca 2ϩ ] i and GPR4stimulation induces cAMP accumulation, probably reflecting the activation of adenylyl cyclase, in response to extracellular pH change (1, 4). Later, G2A (5) and TDAG8 (4, 6, 7), sharing homology with OGR1 and GPR4, were also shown to sense extracellular proton concentration, resulting in stimulation of the early intracellular signaling pathways, such as phospholipase C and adenylyl cyclase. Both G2A and TDAG8 had been reported to be receptors for lysolipids, LPC for G2A (5, 8 -11) and psychosine for TDAG8 (12). Thus, OGR1/GPR4/TDAG8/G2A are unique GPCRs that recognize both lipids and protons as ligands, although the agonistic actions of lipid molecules have not been always confirmed (1,5,6,13). Acidification of extracellular or interstitial space has been proposed to occur under many physiological and pathophysiological circumstances, such as ischemia, tumor, inflammation, and exercise. Acidosis has been shown to induce a variety of responses at whole animal, tissues, and cellular levels. In vascular systems, for example, acidosis causes vasodilation of sy...

Expression of and Role for Ovarian Cancer G-protein-coupled Receptor 1 (OGR1) during Osteoclastogenesis

“…Both proton-and lipid-induced signaling through OGR1 have been studied in a variety of cell systems in which OGR1 was transfected and overexpressed. The lone exception to date was a study done in primary aortic smooth muscle cells (19), in which OGR1 mediated cAMP and prostaglandin I 2 accumulation in response to lowering pH. Transfected overexpression systems are valuable but also carry significant risk of artifactual G-protein associations due to abnormal stoichiometry of receptors to other cellular constituents (20).…”

Involvement of Proton-Sensing TDAG8 in Extracellular Acidification-Induced Inhibition of Proinflammatory Cytokine Production in Peritoneal Macrophages

“…The mouse G2A (Mm00490809)-, OGR1 (Mm01335272)-, TDAG8 (Mm00433695)-, GPR4 (Mm00558777)-, TLR4 (Mm00445274)-, TNF-␣ (Mm00443258)-, IL-6 (Mm00446190)-, and GAPDH (4352932E)-specific probes for realtime PCR were obtained from TaqMan gene expression assays (Applied Biosystems). The sources of all other reagents were described previously (13,15,18,22,23).…”

“…To evaluate the expression level of the OGR1, TDAG8, G2A, GPR4, TLR4, TNF-␣, and IL-6 mRNAs, quantitative real-time PCR was performed using real-time TaqMan technology with a Sequence Detection System model 7700 (Applied Biosystems). Other experimental conditions were described previously (22). The expression level of the target mRNA was normalized to the relative ratio to the expression of GAPDH mRNA.…”

Emerging roles for the pH-sensing G protein-coupled receptors in response to acidotic stress

“…44,47 increases expression of cardiomyogenic and pro-survival genes in the heart; as well as mediates gene expression in aortic smooth muscle cells. [50][51][52] Renal N/A Regulates renal acid excretion in mice and H + -K + -ATPase expression. 59,60 Regulates proton transporter activity and proton extrusion in the kidney.…”

“…50 In human aortic smooth muscle cells, GPR68 is the major receptor responsible for extracellular acidic pH-induced production of inositol phosphate, PGI 2 , and cAMP. 51 Additionally, acidic pH-induced vascular actions of aortic smooth muscle cells can be divided into GPR68-dependent effects such as COX-2 expression, PGI 2 production, and MAPK phosphatase-1 expression and GPR68-independent effects such as PAI-1 expression and cell proliferation. 52 Taken together, both GPR4 and GPR68 play roles in regulating the function of the cardiovascular system.…”