Prostaglandin I2 Production and cAMP Accumulation in Response to ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 41, pp. 34458 –34464, October 14, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Prostaglandin I2 Production and cAMP Accumulation in Response to Acidic Extracellular pH through OGR1 in Human Aortic Smooth Muscle Cells* Received for publication, May 13, 2005, and in revised form, August 4, 2005 Published, JBC Papers in Press, August 8, 2005, DOI 10.1074/jbc.M505287200

Hideaki Tomura‡1, Ju-Qiang Wang‡, Mayumi Komachi‡, Alatangaole Damirin‡, Chihiro Mogi‡, Masayuki Tobo‡, Junko Kon‡, Norihiko Misawa§, Koichi Sato‡, and Fumikazu Okajima‡ From the ‡Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan and the §Central Laboratories for Key Technology, Kirin Brewery Co., LTD, 1-13-5, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan 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 Ca2ⴙ concentration ([Ca2ⴙ]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 [Ca2ⴙ]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 I2 (PGI2) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI2 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 PGI2 production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI2 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

* This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science, a grant of the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and grants from The Nakatomi Foundation and The Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 3718512, Japan. Tel.: 81-27-220-8851; Fax: 81-27-220-8895; E-mail: tomurah@ showa.gunma-u.ac.jp. 2 The abbreviations used are: OGR1, ovarian G-protein-coupled receptor 1; SPC, sphingosylphosphorylcholine; LPC, lysophosphatidylcholine; G-protein, GTP-binding regulatory protein; TDAG8, T cell death-associated gene 8; psychosine, galactosylsphingosine; GPCR, G-protein-coupled receptor; AoSMC, human aortic smooth muscle cell; PG, prostaglandin; 6-keto-PGF1␣, 6-keto-prostaglandin F1␣; PMA, phorbol 12-myristate 13-acetate; Fura2/AM, Fura2/acetoxymethyl ester; BSA, bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine; S1P, sphingosine 1-phosphate; ERK, extracellular signal-regulated kinase; COX, cyclooxygenase; CASMC, human coronary artery smooth muscle cell; siRNA, small interfering RNA; D-SPC, D-erythro-SPC; L-SPC, L-threo-

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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 [Ca2⫹]i and GPR4 stimulation 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 systemic circulation at whole animal levels (14), relaxation in isolated vessels (15–19), and alteration of a variety of cellular activities at the cell levels, including cAMP accumulation (20) and changes in intracellular Ca2⫹ concentration ([Ca2⫹]i) (21, 22). Cyclic AMP accumulation and changes in [Ca2⫹]i have been shown to regulate a variety of cellular responses. Therefore, acid-induced cAMP accumulation and changes in [Ca2⫹]i may play an important role in the acidinduced variety of physiological and pathophysiological responses that were previously reported (see above). However, the mechanism of how acidification modulates the cellular levels of cAMP and [Ca2⫹]i has not yet been characterized. In our search for model systems in which acidification actually induces change in cellular cAMP and [Ca2⫹]i levels, we found that AoSMCs respond to acidic pH leading to substantial cAMP accumulation and [Ca2⫹]i increase. Moreover, we found that acidification causes PGI2 production in AoSMCs. PGI2 has been known to regulate the cellular activities of SMC, for example, the inhibition of DNA synthesis and induction of relaxation (23). In the present study, we showed that these responses are mediated mainly by OGR1 and only slightly, if at all, by GPR4. This is the first indication that OGR1 is actually involved in the regulation of the cellular activities of vascular SMCs.

SPC; IPR, PGI2 receptor; EPPS, N⬘-(2-hydroxyethyl)piperazine-N⬘-3-propanesulphonic acid; MES, N-morpholino)ethanesulfonic acid.

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Acid-induced PGI2 and cAMP Accumulation through OGR1 EXPERIMENTAL PROCEDURES Materials—AoSMCs were purchased from CAMBREX (East Rutherford, NJ); PGD2, PGI2, PGE2, PMA, indomethacin, isoproterenol, and psychosine (galactosylsphingosine) were from Sigma-Aldrich. L-␣-Lysophosphatidylcholine palmitoyl (LPC, C16:0), and sphingosine 1-phosphate (S1P) were from Cayman Chemical Co. (Ann Arbor, MI). Fatty acid-free BSA was from Calbiochem-Novabiochem Co. (San Diego, CA). Fura2/AM and A23187 were from Dojindo (Tokyo, Japan). U73122 and U73343 were generously provided by Upjohn Co. (Kalamazoo, MI). The D-erythro and L-threo forms of sphingosylphosphorylcholine (SPC) were prepared by acid methanolysis of bovine brain sphingomyelin (Sigma-Aldrich) and the following chromatographic separation was described previously (24). Cyclic AMP radioimmunoassay kit was from Yamasa (Chosi, Japan). [myo-2-3H]Inositol (23.0 Ci/mmol) was from American Radiolabeled Chemicals, Inc.(St Louis, MO). The sources of all other reagents were the same as described previously (25, 26) Cell Culture—AoSMCs (passages 7–10) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (Invitrogen), human epidermal growth factor (0.5 ng/ml), human fibroblast growth factor-2 (2 ng/ml), and insulin (5 ␮g/ml) in a humidified air/CO2 (19:1) atmosphere. For the cAMP assay and inositol phosphate assay, the cells were plated on 12- or 24-multiplates. Twenty-four hours before the experiments, the medium was changed to fresh Dulbecco’s modified Eagle’s medium (without serum) containing 0.1% (w/v) BSA (fraction V) for cAMP response and was changed to TCM199 (without serum) containing 2 ␮Ci of [myo-3H]inositol (in 1 ml) and 0.1% BSA for the inositol phosphate assay. cAMP Accumulation—AoSMCs were washed once and preincubated for 20 min (with PD98059 or indomethacin) or 5 min (with U73122 or U73343) at 37 °C in HEPES-buffered medium. The HEPESbuffered medium was composed of 20 mM HEPES (pH 7.6), 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 2.5 mM NaHCO3, 5 mM glucose, and 0.1% (w/v) BSA (fraction V). The cells were then incubated for 30 min under the indicated pH in the presence of 0.5 mM IBMX and these inhibitors in a final volume of 0.6 ml. To cover a wider pH range, the incubation medium was buffered with the mixture of HEPES/EPPS/MES (8 mM each). Where indicated, appropriate test agents were supplemented with the incubation medium. All data in this report are referenced to pH at room temperature. The reaction was terminated by adding 100 ␮l of 1 N HCl. The cAMP in the acid extract was measured using the cAMP radioimmunoassay kit (Yamasa, Chosi, Japan). Measurement of [Ca2⫹]i—The cells were harvested from 10-cm dishes with trypsin and labeled with Fura2/AM. Intracellular Ca2⫹ concentration ([Ca2⫹]i) was measured based on the change in Fura2 fluorescence as described previously (25). In the case of U73122 or U73343 treatment, these agents were added to the HEPES-buffered medium 2 min before the measurement of [Ca2⫹]i. Inositol Phosphate Production—[myo-2-3H]Inositol-labeled AoSMCs were washed once and preincubated in a similar way as that for cAMP assay as described above. The cells were then further incubated for 30 min at 37 °C in HEPES/EPPS/MES-buffered medium with an appropriate pH in the presence of 10 mM LiCl and test inhibitors. Total inositol phosphates including inositol mono-, di-, and tri-phosphate were measured (25). PGI2 Measurement—PGI2 levels were determined using an enzyme immunoassay kit, according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). AoSMCs were washed once and preincubated for 20 min (PD98059 or indomethacin) at 37 °C in HEPESbuffered medium. The cells were then incubated for 10 min at 37 °C in

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HEPES/EPPS/MES-buffered medium (0.5 ml) with an appropriate pH and inhibitors. The supernatant (0.3 ml) was immediately taken up after the incubation and transferred to microtubes (1.5 ml) on ice. The amounts of PGI2 released in the supernatant were estimated as the levels of its stable metabolite 6-keto-PGF1␣ (27). Quantitative RT-PCR using Real-time TaqMan Technology—Total RNA was isolated using TRI Reagent (Sigma-Aldrich) according to the instructions from the manufacturer. After DNase I (Promega, Madison, WI) treatment to remove possible traces of genomic DNA contaminating in the RNA preparations, 5 ␮g of the total RNA was reverse-transcribed using random priming and Multiscribe reverse transcriptase according to the instructions from the manufacturer (Applied Biosystems, Foster City, CA). To evaluate the expression level of the OGR1, TDAG8, G2A, GPR4, and PGI2 receptor (IPR) mRNAs, quantitative RT-PCR was performed using real-time TaqMan technology with a Sequence Detection System model 7700 (Applied Biosystems). The human OGR1, GPR4, G2A, TDAG8, and IPR-specific probes were obtained from TaqMan gene expression assays (Applied Biosystems). The ID number of each specific probe is Hs00203431 for G2A, Hs00268858 for OGR1, Hs00269247 for TDAG8, Hs00270999 for GPR4, Hs00168765 for IPR and Hs99999905 for glyceraldehyde-3phosphate dehydrogenase, respectively. Other experimental conditions were described previously (28). The expression level of the target mRNA was normalized to the relative ratio of the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ⫾ S.E. unless otherwise indicated. Transfection of siRNA—AoSMCs were harvested from 10-cm dishes. Each siRNA (60 pmol) was introduced into AoSMCs (about 1 ⫻ 106 cells) using Human AoSMC Nucleofector kit (Amaxa Inc, Gaithersburg, MD) according to the manufacturer’s instructions. The cells were further cultured for 4 days. The mRNA level was measured using real-time TaqMan technology as described above. The siRNA targeted for OGR1, GPR4, IPR, and non-silencing (NS) was obtained from Dharmacon Inc (Lafayette, CO). The ID number is M-005591-01 for OGR1, M-005570-01 for GPR4, M-005716-00 for IPR, and D-001206-13-05 for NS. Data Presentation—All experiments were performed in duplicate or triplicate. The results of multiple observations are presented as the mean ⫾ S.E. or as a representative result from more than three different batches of cells unless otherwise stated. Statistical significance was assessed by the Student’s t test; values were considered significant at p ⬍ 0.05 (*).

RESULTS AoSMC Senses Extracellular pH, Resulting in Inositol Phosphate Production, [Ca2⫹]i Increase, and cAMP Accumulation—We first examined whether acidification of an extracellular medium modulates the early signaling pathways in AoSMCs. Acidification of the extracellular medium induced a marked inositol phosphate production (Fig. 1A), which was accompanied by a transient [Ca2⫹]i increase (Fig. 1B). The [Ca2⫹]i increase was inhibited by a phospholipase C inhibitor U73122 but not by U77343, an inactive form of U73122, suggesting an involvement of phospholipase C (Fig. 1C). Cyclic AMP was also accumulated in response to the acidification of extracellular medium, as shown in Fig. 1D. In this experiment, a phosphodiesterase inhibitor, IBMX, was included in the assay medium, even though the cAMP response was also observed in its absence (data not shown). Thus, extracellular acidification activates phospholipase C and adenylyl cyclase, although ED50 (a half-maximal concentration required for the response) was slightly lower for adenylyl cyclase (cAMP response) than for phospholipase C

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Acid-induced PGI2 and cAMP Accumulation through OGR1

FIGURE 1. Effect of extracellular proton on inositol phosphate production, [Ca2ⴙ]i increase, and cAMP accumulation in AoSMCs. A, AoSMCs prelabeled with [myo-23 H]inositol were incubated at the indicated pH for 30 min in the presence of 10 mM LiCl to measure inositol phosphate production. Results are expressed as the sum of mono-, di-, tri-inositol phosphates (dpm) per 105 dpm of total counts incorporated into the cells. A representative result is shown. The data are means ⫾ S.D. of three values. The other two independent experiments gave similar results. B, AoSMCs harvested from 10-cm dishes were prelabeled with Fura2/AM. The cells were then further incubated at the indicated pH to monitor [Ca2⫹]i. A representative trace is shown. The other two independent experiments gave similar results. C, AoSMCs prelabeled with Fura2/AM were pretreated with Me2SO, 5 ␮M U73122, or 5 ␮M U73343 for 2 min. The cells were then further incubated at pH 6.7 to monitor [Ca2⫹]i. The net [Ca2⫹]i change (peak value-basal value) at around 15 s was calculated. Data are means ⫾ S.E. from four to five determinations of at least two separate experiments. D, cAMP accumulation for 30 min at the indicated pH in the presence of 0.5 mM IBMX was measured. Results are expressed as cAMP accumulation (pmol) per mg of cell proteins. A representative result is shown. The data are means ⫾ S.D. of three values. The other two experiments gave similar results. E, cAMP accumulation at the indicated pH or in the presence of 100 nM PGI2 at pH 7.6 was measured with (black column) or without (open column) psychosine (10 ␮M). Results are expressed as cAMP accumulation (pmol) per mg of cell proteins. A representative result is shown. The data are means ⫾ S.D. of three values. The other two experiments gave similar results.

(inositol phosphate response) (see Fig. 1, A and D). To clarify the involvement of proton-sensing GPCRs in acid-induced actions, we examined the effect of psychosine, which was shown to selectively antagonize the proton-sensing GPCR-mediated actions (6). Psychosine significantly inhibited the cAMP accumulation and inositol phosphate production (data not shown) at pH 6.8, whereas the lipid did not affect the increased acid (pH 6.5)-induced cAMP accumulation and PGI2induced action at pH 7.6 (Fig. 1E). Thus, psychosine specifically inhibited the acid-induced action in a manner dependent on extracellular pH, as previously shown (6). These results suggest that proton-sensing GPCRs mediate the acid-induced early signaling events in AoSMCs. OGR1 Is Involved in Proton-induced Actions—Fig. 2A shows the mRNA expression patterns of OGR1, GPR4, TDAG8, and G2A, which


FIGURE 2. Effects of siRNAs targeted for OGR1 and GPR4 on the proton-induced inositol phosphate production, [Ca2ⴙ]i increase, and cAMP accumulation in AoSMCs. A, mRNA expression of proton-sensing GPCRs was assessed by real-time TaqMan PCR. Results are expressed as the relative ratios to glyceraldehyde-3-phosphate dehydrogenase mRNA expression. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ⫾ S.E. B, AoSMCs were transfected with non-silencing (NS; open column), OGR1 siRNA (OGR1; closed column), or GPR4 siRNA (GPR4; hatched column). OGR1 and GPR4 mRNA expressions were measured as described in A. The mRNA expression is expressed as percentages of the value of NS treatment. The expression of OGR1 and GPR4 was not significantly changed by NS treatment. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ⫾ S.E. C–E, AoSMCs were transfected with NS (open column), OGR1 siRNA (closed column), or GPR4 siRNA (hatched column). Inositol phosphate production (C), [Ca2⫹]i increase (D), and cAMP accumulation (E) at the indicated pH were measured as described in the legend to Fig. 1. Where indicated, 1 ␮M S1P was added to the incubation medium. Each experiment was performed in triplicate. Data are means ⫾ S.E. from three independent experiments.

have been reported to be proton-sensing GPCRs so far, measured by the real-time TaqMan PCR method in AoSMCs. Among them, the expression of OGR1 and GPR4 was clearly detected. In order to examine the possible roles of OGR1 and GPR4 in the proton-induced actions, we performed siRNA experiments. As shown in Fig. 2B, the siRNA specific to OGR1 decreased the OGR1 mRNA expression to about 25% of the initial level without any significant effect on GPR4 mRNA expression. Similarly, the siRNA specific to GPR4 decreased the GPR4 mRNA expression to about 40% of the initial expression without any significant effect on OGR1 expression. As shown in Fig. 2C, the proton-induced inositol phosphate production was markedly inhibited by OGR1 siRNA but not by GPR4 siRNA.



FIGURE 3. Effects of various inhibitors for cAMP accumulation and inositol phosphate production in AoSMCs. A and B, AoSMCs were pretreated with 10 ␮M indomethacin (INDO) or PD98059 (PD) for 20 min. The cells were then incubated for 30 min at the indicated pH to measure cAMP accumulation (A) or inositol phosphate production (B) as described in Fig. 1. C and D, AoSMCs were pretreated with 10 ␮M U73122 or vehicle for 5 min. The cells were then incubated for 30 min at the indicated pH to measure cAMP accumulation (C) or inositol phosphate production (D). In some experiments, 1 ␮M isoproterenol (ISP) was added to the incubation medium at pH 7.6. E, AoSMCs were incubated with 100 ng/ml of PMA or 10 ␮M A23187 for 30 min to measure cAMP accumulation as described in the legend to Fig. 1. Each experiment was performed in triplicate. Data are means ⫾ S.E. from three independent experiments.

As expected, the proton-induced [Ca2⫹]i increase was also significantly inhibited by OGR1 siRNA but not by GPR4 siRNA. Neither OGR1 siRNA nor GPR4 siRNA attenuated the S1P-induced [Ca2⫹]i increase at all (Fig. 2D), indicating that the inhibitory effect of OGR1 siRNA is specific to the proton actions. These results are quite consistent with the reported role of OGR1 in the phospholipase C/Ca2⫹ system (1). On the other hand, a previous report showed that GPR4 is coupled to adenylyl cyclase (1). We, therefore, expected that the proton-induced cAMP accumulation might be inhibited by GPR4 siRNA. As shown in Fig. 2E, however, acidic pH-induced cAMP accumulation was inhibited about 70% by OGR1 siRNA but only about 20% by GPR4 siRNA. The effect of siRNAs was specific, as evidenced by the lack of any significant effect on S1P-induced cAMP accumulation by any siRNA treatment. This result suggests that acidic pH-induced cAMP accumulation is also mediated mainly through OGR1 and only slightly through GPR4. Intracellular Signaling Pathways of the Acid-induced cAMP Accumulation—The unexpected observation might be in part explained by the involvement of COX/prostaglandin (PG) pathways. Our recent study in CASMC showed that cAMP accumulation is associated with the production of PGs, especially PGI2, which was produced through the phospholipase C/ERK/COX pathway (27). As shown in Fig. 3A, the protoninduced cAMP accumulation was almost completely inhibited by


indomethacin, a COX inhibitor. In a previous CASMC study, ERK was shown to work upstream of PG production (27). Consistent with this scheme, PD98059, an ERK kinase inhibitor, also inhibited the protoninduced cAMP accumulation (Fig. 3A). These inhibitors did not affect the proton-induced inositol phosphate production, indicating the specificity of these inhibitors (Fig. 3B). These results suggest that ERK-regulated and COX-catalyzed PG production is responsible for cAMP accumulation. We next examined the possible role of phospholipase C in the acidinduced activation of the PG/cAMP system. The proton-induced cAMP accumulation was inhibited by the phospholipase C inhibitor U73122 (Fig. 3C) in association with the inhibition of inositol phosphate production (Fig. 3D), whereas the cAMP response to the ␤-adrenergic receptor agonist isoproterenol was hardly affected by the phospholipase C inhibitor (Fig. 3C). Furthermore, we found that either PMA, a protein kinase C activator, or A23187, Ca2⫹ ionophore, caused cAMP accumulation. These results suggest that phospholipase C activation and the subsequent [Ca2⫹]i increase and protein kinase C activation may lead to the stimulation of the PG/cAMP system. OGR1 Is Involved in the Proton-induced PGI2 Production Leading to cAMP Accumulation—Among the PGs employed, PGI2 was the most potent stimulator of cAMP accumulation in AoSMCs (Fig. 4A). As shown in Fig. 4B, PGI2 was actually produced from the cells, depending on the extracellular pH. The PGI2 production at pH 6.5 corresponded to about 200 nM, which is a high enough concentration to increase cAMP accumulation, as shown in Fig. 4A (100 nM PGI2). The PGI2 production was almost completely inhibited by indomethacin and PD98059 (Fig. 4C), suggesting an involvement of the ERK/COX pathway (Fig. 3A). We further examined the role of OGR1 and GPR4. As expected, siRNA targeted for OGR1 but not for GPR4 specifically reduced the PGI2 production at pH 6.8, indicating that the PGI2 production is also mediated by OGR1. The involvement of PGI2 was further supported by a finding of an inhibition of acidification-induced cAMP accumulation by siRNA specific to PGI2 receptor (IPR). Thus, the IPR-specific siRNA decreased its mRNA expression to about 10% of the initial level without any significant effect on OGR1 mRNA expression (Fig. 4E). Under these conditions, the cAMP accumulation induced by acidification (pH 6.8 and pH 6.5), but not by isoproterenol, was significantly attenuated (Fig. 4F). OGR1 Does Not Seem to Mediate Ca2⫹ Response to SPC and LPC in AoSMCs—OGR1 and GPR4 have been reported as receptors for SPC (2) and LPC (3). We finally examined whether or not these lysolipids modulate the Ca2⫹ metabolism in AoSMCs. As shown in Fig. 5, D-SPC but not L-SPC induced a [Ca2⫹]i increase, as reported previously (2). The [Ca2⫹]i increase, however, was not attenuated by siRNA targeted for OGR1, whereas the proton-induced [Ca2⫹]i increase was markedly inhibited under the conditions. The [Ca2⫹]i increase by S1P was not attenuated by the siRNA treatment, indicating the specificity of the OGR1 siRNA (Fig. 5). LPC at 10 ␮M, which is a saturated concentration for the [Ca2⫹]i increase in the previous report (3), never induced any [Ca2⫹]i increase in AoSMCs (Fig. 5).

DISCUSSION Proton-sensing GPCRs, including OGR1, GPR4, G2A, and TDAG8, have all been shown to be receptors for lysolipids and are known to be expressed in various tissues and cell types (3, 29 –31). The roles of lysolipid ligands have been discussed, especially in relation to disorders, such as inherited sphingolipidoses, cancers, vascular diseases, and immune diseases (32–36). However, little is known about the physiological role of another feature of these receptors, the “proton sensing.”


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FIGURE 5. Effect of siRNA targeted for OGR1 on lysolipid-induced [Ca2ⴙ]i increase. AoSMCs were transfected with siRNA targeted for OGR (black column) or non-silencing (NS, open column). The cells harvested from 10-cm dishes were prelabeled with Fura2/ AM. The cells were then further incubated at the indicated pH or in the presence of 10 ␮M 2⫹ D-SPC, 10 ␮M L-SPC, 10 ␮M LPC (16:0), or 100 nM S1P at pH 7.6 to monitor [Ca ]i. The net [Ca2⫹]i change (peak value-basal value) at around 15 s was calculated. Data are means ⫾ S.E. from four to five determinations of two to three separate experiments.

FIGURE 4. Effects of inhibitors for intracellular signaling enzymes, siRNAs against OGR1 or GPR4 on proton-induced PGI2 production and siRNA against PGI2 receptor on proton-induced cAMP production. A, AoSMCs were incubated with 100 nM each of PGD2, PGE2, or PGI2 for 30 min in the presence of 0.5 mM IBMX to measure cAMP accumulation. Results are expressed as the net change of cAMP accumulation (pmol) per mg of cell proteins. The basal value of cAMP is 4.2 ⫾ 2.3 pmol/mg. A representative result is shown. The data are means ⫾ S.D. of three values. Other two independent experiments gave similar results. B, AoSMCs were incubated at the indicated pH for 10 min to measure 6-keto-PGF1␣ production. Each experiment was performed in triplicate. Data are means ⫾ S.E. from three independent experiments. C, AoSMCs were pretreated with 10 ␮M indomethacin (INDO) or 10 ␮M PD98059 (PD) for 20 min and then further incubated at the indicated pH for 10 min to measure 6-keto-PGF1␣ production. Each experiment was performed in triplicate. Data are means ⫾ S.E. from three independent experiments. D, AoSMCs were transfected with siRNA targeted for OGR1 or GPR4. The cells were incubated at the indicated pH for 10 min to measure 6-keto-PGF1␣ production. The results are expressed as 6-keto-PGF1␣ production (ng) per mg cell proteins. Each experiment was performed in triplicate. Data are means ⫾ S.E. from three independent experiments E, AoSMCs were transfected with non-silencing (NS; open column), or PGI2 receptor siRNA (IPR; closed column). PGI2 receptor and OGR1 mRNA expressions were measured as described in A. Each RT-PCR assay was performed at least three times, and the results are expressed as percentages ⫾ S.E. of the value of NS treatment. The 100% value of IPR is 0.0052 ⫾ 0.0013. The values of OGR1 (refer to Fig. 2A) and IPR were not significantly changed by NS treatment. F, AoSMCs were transfected with NS (open column) or PGI2 receptor (IPR) siRNA (closed column). The cAMP accumulation at the indicated pH was


The proton-sensing mechanism has been demonstrated by using cells that overexpressed these GPCRs. Among native cells, TDAG8 has been suggested to be involved in extracellular acidification-induced cAMP accumulation only in thymocytes so far (4, 6). In the present study, we demonstrated that OGR1 and GPR4 are expressed in AoSMCs and that OGR1 is responsible for extracellular proton-induced PGI2 production and cAMP accumulation in cells. This finding suggests that the protonsensing mechanism actually plays a role in a wide area of biological systems in native cells. OGR1 and GPR4 have also been shown to be receptors for SPC (2) and LPC (3). We detected a [Ca2⫹]i increase by D-SPC, as described previously (2), whereas LPC was ineffective (Fig. 5). The [Ca2⫹]i increase by D-SPC, however, was not inhibited by siRNA targeted for OGR1 under conditions in which the proton-induced actions were largely inhibited (Fig. 5). Thus, the [Ca2⫹]i increase by D-SPC is unlikely mediated by OGR1 in AoSMCs and, rather, may be mediated by S1P receptors (37, 38). Supporting this, S1P strongly increased [Ca2⫹]i in a manner insusceptible to OGR1 siRNA. OGR1 and GPR4 have been shown to be coupled to the phospholipase C/Ca2⫹ and adenylyl cyclase/cAMP pathways, respectively (1). Although mRNA expression does not always reflect the protein expression, mRNA expression data suggested that GPR4 is expressed to a similar extent to OGR1 in AoSMCs (Fig. 2A). We, therefore, expected that cAMP accumulation in response to extracellular acidification might be mediated by GPR4 rather than OGR1. A small interfering RNA technique, however, revealed that OGR1 is a major receptor responsible for the acid-induced cAMP accumulation and the contribution of GPR4 is very small (Fig. 2E). Our results showed that cAMP was indirectly accumulated through COX/PGs (at least PGI2) production, and the resultant activation of adenylyl cyclase. At present, the marginal contribution of GPR4 in the cAMP accumulation in AoSMCs remains unknown. It should be noted, however, that GPR4 coupling to the adenylyl cyclase system has been demonstrated as yet only in a cell system overexpressed with GPR4. In the present study, we have not characterized in detail the mechanisms underlying OGR1 receptor-mediated PGI2 production. However, we speculate that cAMP is accumulated via the phospholipase C/ERK/ phospholipase A2/COX/PGI2 pathway in a similar way to that observed

measured as described in the legend to Fig. 1. Where indicated, 1 ␮M isoproterenol (ISP) was added to the incubation medium. A representative result is shown. The data are means ⫾ S.D. of three values. The other two independent experiments gave similar results.


Acid-induced PGI2 and cAMP Accumulation through OGR1 in CASMCs (27). In CASMCs, the S1P receptor activation of phospholipase C induced the activation of ERK and phospholipase A2 and, thereby, stimulated the subsequent production of arachidonic acid, which was metabolized to PGI2 by COX and PGI2 synthase. In fact, in AoSMCs as well, acid-induced cAMP accumulation was inhibited by the specific inhibitors for phospholipase C, ERK kinase, and COX. We also found that Ca2⫹ ionophore and PMA induced cAMP accumulation, supporting the role of phospholipase C; however, on the basis of the results of the PMA effect, any conclusion of the participation of protein kinase C should be made with caution. More experiments are needed to clarify the intracellular signaling pathways involved in the acid-induced PG synthesis in AoSMCs. It is well known that acidosis may cause vasodilation of systemic circulation, while pulmonary arteries are resistant to the vasodilator effect of extracellular acidosis (15). Both extracellular pH and intracellular pH seem to be responsible for acid-induced vasodilation of systemic circulation (15, 39). Recent studies revealed an important role of ATP-sensitive potassium channels in acid-induced vasodilation (39). Intracellular histidine residues have been suggested to play a role in the activation of the channel in response to intracellular acidosis (40). The potassium channel activation and the consequent hyperpolarization may cause the inhibition of voltage-dependent Ca2⫹ channels and reduction of [Ca2⫹]i. Thus, potassium channel inhibition is thought to be one of the mechanisms of the intracellular pH effect on vasodilation. On the other hand, the mechanism by which extracellular pH induces the relaxation of vascular SMC remains unclear. Proton-sensing GPCRs could be one of the missing players in this process. The acid-induced [Ca2⫹]i increase appears unfavorable for the relaxation of vascular SMC. However, careful consultation of the previous literature showed that extracellular acidification is sometimes associated with a transient vasoconstriction followed by a sustained vasorelaxation (41). Moreover, a transient [Ca2⫹]i increase has been reported in response to extracellular acidification (21). Such a transient [Ca2⫹]i increase might be responsible for opening the Ca2⫹-activated potassium channel and, thereby, might induce hyperpolarization (16). Furthermore, it is well known that PGI2 and its stable derivatives, such as beraprost, through cAMP accumulation, induce the relaxation of human uterine arteries (42). Thus, OGR1-mediated PGI2 production and the resultant cAMP accumulation might be involved in vasodilation. In addition, in vascular SMC, an increase in intracellular cAMP has been shown to be inhibitory for proliferation and migration (43). Moreover, PGI2 is a potent inhibitor for platelet aggregation (44). Taken together, these results suggest that the proton/OGR1-mediated stimulation of PGI2/cAMP system is a novel vasodilation and anti-atherogenic mechanism. In vascular walls under the states of vasoconstriction and inflammation-associated neointima formation, the extracellular or interstitial pH may be decreased because of the high production of lactic acid. Thus, OGR1 activation in response to acidic circumstances may be considered to counteract vasoconstriction and inflammation. Elucidating the role of proton-sensing GPCRs, especially OGR1, in vascular SMCs, will provide not only a novel insight into the regulatory mechanisms of vascular systems but also a novel potential target for vascular diseases, such as ischemic heart disease and atherosclerosis. Acknowledgment—We thank Chisuko Uchiyama for technical assistance.


REFERENCES 1. Ludwig, M. G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., and Seuwen, K. (2003) Nature 425, 93–98 2. Xu, Y., Zhu, K., Hong, G., Wu, W., Baudhuin, L. M., Xiao, Y., and Damron, D. S. (2000) Nat. Cell Biol. 2, 261–267 3. Zhu, K., Baudhuin, L. M., Hong, G., Williams, F. S., Cristina, K. L., Kabarowski, J. H., Witte, O. N., and Xu, Y. (2001) J. Biol. Chem. 276, 41325– 41335 4. Radu, C. G., Nijagal, A., McLaughlin, J., Wang, L., and Witte, O. N. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1632–1637 5. Murakami, N., Yokomizo, T., Okuno, T., and Shimizu, T. (2004) J. Biol. Chem. 279, 42484 – 42491 6. Wang, J. Q., Kon, J., Mogi, C., Tobo, M., Damirin, A., Sato, K., Komachi, M., Malchinkhuu, E., Murata, N., Kimura, T., Kuwabara, A., Wakamatsu, K., Koizumi, H., Uede, T., Tsujimoto, G., Kurose, H., Sato, T., Harada, A., Misawa, N., Tomura, H., and Okajima, F. (2004) J. Biol. Chem. 279, 45626 – 45633 7. Ishii, S., Kihara, Y., and Shimizu, T. (2005) J. Biol. Chem. 280, 9083–9087 8. Yang, L. V., Radu, C. G., Wang, L., Riedinger, M., and Witte, O. N. (2005) Blood 105, 1127–1134 9. Lin, P., and Ye, R. D. (2003) J. Biol. Chem. 278, 14379 –14386 10. Han, K. H., Hong, K. H., Ko, J., Rhee, K. S., Hong, M. K., Kim, J. J., Kim, Y. H., and Park, S. J. (2004) J. Leukoc. Biol. 76, 195–202 11. Yan, J. J., Jung, J. S., Lee, J. E., Lee, J., Huh, S. O., Kim, H. S., Jung, K. C., Cho, J. Y., Nam, J. S., Suh, H. W., Kim, Y. H., and Song, D. K. (2004) Nat. Med. 10, 161–167 12. Im, D. S., Heise, C. E., Nguyen, T., O’Dowd, B. F., and Lynch, K. R. (2001) J. Cell Biol. 153, 429 – 434 13. Bektas, M., Barak, L. S., Jolly, P. S., Liu, H., Lynch, K. R., Lacana, E., Suhr, K. B., Milstien, S., and Spiegel, S. (2003) Biochemistry 42, 12181–12191 14. Walley, K. R., Lewis, T. H., and Wood, L. D. (1990) Circ. Res. 67, 628 – 635 15. Hyvelin, J. M., O’Connor, C., and McLoughlin, P. (2004) Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L673– 684 16. Hayabuchi, Y., Nakaya, Y., Matsuoka, S., and Kuroda, Y. (1998) Pflugers. Arch. 436, 509 –514 17. Tian, R., Vogel, P., Lassen, N. A., Mulvany, M. J., Andreasen, F., and Aalkjaer, C. (1995) Circ. Res. 76, 269 –275 18. Ishizaka, H., Gudi, S. R., Frangos, J. A., and Kuo, L. (1999) Circulation 99, 558 –563 19. Wang, X., Wu, J., Li, L., Chen, F., Wang, R., and Jiang, C. (2003) Circ. Res. 92, 1225–1232 20. Leffler, C. W., Balabanova, L., and Williams, K. K. (1999) Am. J. Physiol. 277, H1878 –1883 21. Batlle, D. C., Peces, R., LaPointe, M. S., Ye, M., and Daugirdas, J. T. (1993) Am. J. Physiol. 264, C932–943 22. Iwasawa, K., Nakajima, T., Hazama, H., Goto, A., Shin, W. S., Toyo-oka, T., and Omata, M. (1997) J. Physiol. 503, 237–251 23. Weber, A. A., Zucker, T. P., Hasse, A., Bonisch, D., Wittpoth, M., and Schror, K. (1998) Basic Res. Cardiol. 93, 54 –57 24. Bunemann, M., Liliom, K., Brandts, B. K., Pott, L., Tseng, J. L., Desiderio, D. M., Sun, G., Miller, D., and Tigyi, G. (1996) EMBO J. 15, 5527–5534 25. Kon, J., Sato, K., Watanabe, T., Tomura, H., Kuwabara, A., Kimura, T., Tamama, K., Ishizuka, T., Murata, N., Kanda, T., Kobayashi, I., Ohta, H., Ui, M., and Okajima, F. (1999) J. Biol. Chem. 274, 23940 –23947 26. Sato, K., Tomura, H., Igarashi, Y., Ui, M., and Okajima, F. (1999) Mol. Pharmacol. 55, 126 –133 27. Damirin, A., Tomura, H., Komachi, M., Tobo, M., Sato, K., Mogi, C., Nochi, H., Tamoto, K., and Okajima, F. (2005) Mol. Pharmacol. 67, 1177–1185 28. Yamada, T., Sato, K., Komachi, M., Malchinkhuu, E., Tobo, M., Kimura, T., Kuwabara, A., Yanagita, Y., Ikeya, T., Tanahashi, Y., Ogawa, T., Ohwada, S., Morishita, Y., Ohta, H., Im, D. S., Tamoto, K., Tomura, H., and Okajima, F. (2004) J. Biol. Chem. 279, 6595– 6605 29. Xu, Y., and Casey, G. (1996) Genomics 35, 397– 402 30. Weng, Z., Fluckiger, A. C., Nisitani, S., Wahl, M. I., Le, L. Q., Hunter, C. A., Fernal, A. A., Le Beau, M. M., and Witte, O. N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12334 –12339 31. Kyaw, H., Zeng, Z., Su, K., Fan, P., Shell, B. K., Carter, K. C., and Li, Y. (1998) DNA Cell Biol. 17, 493–500 32. Ozkara, H. A. (2004) Brain Dev. 26, 497–505 33. Tokumura, A. (2004) J. Cell. Biochem. 92, 869 – 881 34. Okajima, F. (2002) Biochim. Biophys. Acta 1582, 132–137 35. Le, L. Q., Kabarowski, J. H., Weng, Z., Satterthwaite, A. B., Harvill, E. T., Jensen, E. R., Miller, J. F., and Witte, O. N. (2001) Immunity 14, 561–571 36. Brinkmann, V., Davis, M. D., Heise, C. E., Albert, R., Cottens, S., Hof, R., Bruns, C., Prieschl, E., Baumruker, T., Hiestand, P., Foster, C. A., Zollinger, M., and Lynch, K. R. (2002) J. Biol. Chem. 277, 21453–21457 37. Murata, N., Sato, K., Kon, J., Tomura, H., and Okajima, F. (2000) Anal. Biochem. 282, 115–120


34463

Acid-induced PGI2 and cAMP Accumulation through OGR1 38. Meyer zu Heringdorf, D., Himmel, H. M., and Jakobs, K. H. (2002) Biochim. Biophys. Acta 1582, 178 –189 39. Rosenblum, W. I. (2003) Stroke 34, 1547–1552 40. Xu, H., Cui, N., Yang, Z., Wu, J., Giwa, L. R., Abdulkadir, L., Sharma, P., and Jiang, C. (2001) J. Biol. Chem. 276, 12898 –12902 41. Krampetz, I. K., and Rhoades, R. A. (1991) Am. J. Physiol. 260, L516 –521


42. Kimura, T., Okamura, T., Yoshida, Y., and Toda, N. (1995) J. Cardiovasc. Pharmacol. 26, 333–338 43. Klemm, D. J., Watson, P. A., Frid, M. G., Dempsey, E. C., Schaack, J., Colton, L. A., Nesterova, A., Stenmark, K. R., and Reusch, J. E. (2001) J. Biol. Chem. 276, 46132– 46141 44. Fitzgerald, G. A., Catella, F., and Oates, J. A. (1987) Hum. Pathol. 18, 248 –252
