PURPOSE:Human bronchial smooth muscle cells (BSMCs) contribute to airway obstruction and hyperresponsiveness in patients with bronchial asthma. BSMCs also generate cytokines and matricellular proteins in response to extracellular acidification through the ovarian cancer G protein-coupled receptor 1 (OGR1). Cobalt (Co) and nickel (Ni) are occupational agents, which cause occupational asthma. We examined the effects of Co and Ni on interleukin-6 (IL-6) secretion by human BSMCs because these metals may act as ligands of OGR1.METHODS:Human BSMCs were incubated in Dulbecco's Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM) for 16 hours and stimulated for the indicated time by exchanging the medium with 0.1% BSA-DMEM containing any of the metals or pH-adjusted 0.1% BSA-DMEM. IL-6 mRNA expression was quantified via reverse transcription polymerase chain reaction (RT-PCR) using the real-time TaqMan technology. IL-6 was measured using an enzyme-linked immunosorbent assay. Dexamethasone (DEX) was added 30 minutes before each stimulation. To knock down the expression of OGR1 in BSMCs, small interfering RNA (siRNA) targeting OGR1 (OGR1-siRNA) was transfected to the cells and non-targeting siRNA (NT-siRNA) was used as a control.RESULTS:Co and Ni both significantly increased IL-6 secretion in human BSMCs at 300 μM. This significant increase in IL-6 mRNA expression was observed 5 hours after stimulation. BSMCs transfected with OGR1-siRNA produced less IL-6 than BSMCs transfected with NT-siRNA in response to either Co or Ni stimulation. DEX inhibited Co- and Ni-stimulated IL-6 secretion by human BSMCs as well as pH 6.3-stimulated IL-6 secretion in a dose-dependent manner. DEX did not decrease phosphorylation of ERK1/2, p38 MAP kinase, and NF-κB p65 induced by either Co or Ni stimulation.CONCLUSION:Co and Ni induce secretion of IL-6 in human BSMCs through activation of OGR1. Co- and Ni-stimulated IL-6 secretion is inhibited by DEX.

Prevalence of atopic dermatitis (AD), a chronic, pruritic, and relapsing inflammatory skin disorder, is growing. Because available therapeutics is limited, immune regulators from natural resources could be helpful for treating AD symptoms. The root of Salvia miltiorrhiza Bunge (Lamiaceae) has been studied for the treatment of inflammatory diseases, including dermatologic disorders in Korea. This study examined the effect of salvianolic acid A on AD-like symptoms. Sensitization on the dorsal skin and repeated application on the ears with 2,4-dinitrochlorobenzene (DNCB) were performed in BALB/c mice to induce AD-like skin lesions. After induction of atopic dermatitis, salvianolic acid A (5 and 10 mg/kg) or dexamethasone (10 mg/kg) were administrated via intraperitoneal injection for 3 weeks. Salvianolic acid A suppressed DNCB-induced AD-like symptoms like ear skin hypertrophy and decreased mast cell infiltration into skin lesions. Salvianolic acid A not only reduced DNCB-induced increase of serum IgE but also lowered levels of the Th2 cytokines (IL-4 and IL-13), Th1 cytokine (interferon-γ), and Th17 cytokine (IL-17A). Furthermore, salvianolic acid A blocked DNCB-induced lymph node enlargement. In summary, these results suggest that salvianolic acid A might have a therapeutic potential for the treatment of AD.

Extracellular acidification in the brain has been observed in ischemia; however, the physiological and pathophysiological implications of the pH reduction remain largely unknown. Here, we analyzed the roles of proton-sensing G protein-coupled receptors, including T-cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) in a mouse ischemia reperfusion model. Cerebral infarction and dysfunctional behavior with transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion were exacerbated by the deficiency of TDAG8, whereas no significant effect was observed with the deficiency of OGR1 or GPR4. We confirmed that the pH of the predicted infarction region was 6.5. TDAG8 mRNA was observed in Iba1-positive microglia in the mouse brain. The tMCAO increased the mRNA expression of tumor necrosis factor-α in the ipsilateral cerebral hemisphere and evoked morphological changes in microglia in an evolving cerebral injury. These tMCAO-induced actions were significantly enhanced by the TDAG8 deficiency. Administration of minocycline, which is known to inhibit microglial activation, improved the cerebral infarction and dysfunctional behavior induced by tMCAO in the TDAG8-deficient mouse. Thus, acidic pH/TDAG8 protects against cerebral infarction caused by tMCAO, at least due to the mechanism involving the inhibition of microglial functions.

Ogerin is a positive allosteric modulator of human and mouse ovarian cancer G protein-coupled receptors (OGR1s). In the present study, we found that ogerin differentially enhances the activation of OGR1 in various animal species. Amino acid residues of OGR1 that are associated with ogerin are conserved among the species. This suggests that other amino acid residues may be involved in the action of ogerin. Chimeric receptors between human and zebrafish OGR1s showed that the amino acid residues that determine the species specificity of ogerin-induced enhancement reside in the transmembrane and/or intracellular regions of OGR1. This result highlights the importance of first verifying the effectiveness of ogerin to the OGR1 of the species of interest at the cellular level prior to analyzing the physiological and pathophysiological roles of OGR1 in the species.

Ovarian cancer G protein-coupled receptor 1 (OGR1), also known as GPR68, is a proton-sensing G protein-coupled receptor (GPCR) coupling to Gq/11/phospholipase C/Ca2+ signaling pathways. The specific histidine residues at the extracellular surface of OGR1 are suggested to be involved in the proton sensing. Later, some metal ions, including nickel ion (Ni2+), are also indicated to be OGR1 ligands. OGR1 polymorphic variants have recently been found in three families with amelogenesis imperfecta, which suggested that OGR1 is required for the process of dental enamel formation. One of these families possesses a missense mutation from leucine to proline at 74 (L74P) of OGR1. In the present study, we characterized HEK293 cells with L74P OGR1 (L74P-OGR1) and hemagglutinin (HA)-tag, as compared with cells with wild-type OGR1 (WT-OGR1) and HA-tag. We found that either acidic pH or NiCl2 induced intracellular Ca2+ mobilization and morphological change in WT-OGR1-transfected cells; however, the extracellular stimulus-induced actions were severely damaged in L74P-OGR1-transfected cells. We further confirmed that either WT-OGR1 or L74P-OGR1 is localized mainly in the surface of the cells, but only WT-OGR1 is internalized in response to acidification or NiCl2. Thus, the L74P-OGR1 protein may be distributed in the plasma membranes but severely damaged in the receptor functions. We speculate that L74P in the second transmembrane domain in OGR1 may result in conformational changes in the receptor, thereby disturbing the sensing extracellular signals, i.e., protons or metal ions, and/or transducing them to the intracellular signaling machinery through G proteins.

Hormone-secreting pituitary adenomas show unregulated hormonal hypersecretion and cause hyperpituitarism. However, the mechanism of the unregulated hormone production and secretion has not yet been fully elucidated. Solid tumors show reduced extracellular pH, partly due to lactate secretion from anaerobic glycolysis. It is known that extracellular acidification affects hormone secretion. However, whether and how the extracellular acidification influences the unregulated hormone production and secretion remain unknown. In the present study, we found that GPR4, a proton-sensing G protein-coupled receptor, was highly expressed in MtT/S cells, a growth hormone-producing and prolactin-producing pituitary tumor cell line. When we reduced the extracellular pH, growth hormone and prolactin mRNA expressions increased in the cells. Both increased expressions were partially suppressed by a GPR4 antagonist. We also found that extracellular acidification enhanced growth hormone-releasing factor-induced growth hormone secretion from MtT/S cells. These results suggest that GPR4 may play a role in hypersecretion of the hormone from hormone-producing pituitary tumors. A GPR4 antagonist will be a useful tool for preventing the hypersecretion.

Mammalian T cell death-associated gene 8 (TDAG8)s are activated by extracellular protons. In the present study, we examined whether the TDAG8 homologs of other species are activated by protons as they are in mammals. We found that Xenopus TDAG8 also stimulated cAMP response element (CRE)-driven promoter activities reflecting the activation of G(s)/cAMP signaling pathways when they are stimulated by protons. On the other hand, the activities of chicken and zebrafish TDAG8s are hardly affected by protons. Results using chimeric receptors of human and zebrafish TDAG8s indicate that the specificity of the proton-induced activation lies in the extracellular region. These results suggest that protons are not an evolutionarily conserved agonist of TDAG8.

Human airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They also generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs.

Introduction: The pH of the bronchoalveolar space has been shown to fall in airway inflammatory disease. T cell death associated gene 8 (TDAG8) is one of the proton sensing G protein coupled receptors. The role of TDAG8 has not been elucidated sufficiently in the allergic airway inflammation. Aim: We explored the role of TDAG8 in the allergic airway inflammation with an ovalbumin (OVA)-induced asthma model and with the human bronchial epithelial cell line (NCl-H292). Methods: TDAG8-deficient (TDAG8-KO) and wild-type (WT) mice were assigned to the following two groups: control group, intraperitoneal sensitization and airway challenge with PBS, and OVA group, intraperitoneal sensitization and airway challenge with OVA. The number of bronchoalveolar lavage (BAL) fluid cells and the mRNA expression in lungs were measured. Histological analysis of lung sections was also performed. We examined the phorbol 12-myristate 13-acetate (PMA)-induced expression of the MUC5AC mRNA and the effect of the knock-down of TDAG8 on the expression in the NCl-H292 in acidic conditions. Result: The expression levels of Tdag8 and Muc5ac in lungs and eosinophil accumulation in BAL fluids were all increased in the OVA group. The OVA-induced eosinophil accumulation and the expression levels of Muc5ac in lungs were lower in TDAG8-KO mice than those in WT mice. The expression of TDAG8 and MUC5AC in the NCl-H292 were enhanced by the treatment with PMA. The PMA-induced MUC5AC expression was enhanced by acidification of medium pH and the acidic pH effect was suppressed by the knock down of TDAG8. Conclusion: Our results suggest that TDAG8 regulates the expression of MUC5AC in acidic conditions.

Extracellular acidification regulates endocrine cell functions. Ovarian cancer G protein-coupled receptor 1 (OGR1), also known as GPR68, is a proton-sensing G protein-coupled receptor and is activated by extracellular acidification, resulting in the activation of multiple intracellular signaling pathways. In the present study, we found that OGR1 was expressed in some gonadotropic cells in rat anterior pituitary and in LβΤ2 cells, which are used as a model of gonadotropic cells. When we reduced extracellular pH, a transient intracellular Ca2+ increase was detected in LβT2 cells. The Ca2+ increase was inhibited by a Gq/11 inhibitor and Cu2+, which is known as an OGR1 antagonist. We also found that extracellular acidification enhanced GnRH-induced Gaussia luciferase secretion from LβT2 cells. These results suggest that OGR1 may play a role in the regulation of gonadotropic cell function such as its hormone secretion.

Human, mouse, and zebrafish ovarian cancer G protein—coupled receptors (OGR1s) are activated by both metals and extracellular protons. In the present study, we examined whether pig, rat, chicken, and Xenopus OGR1 homologs could sense and be activated by protons and metals. We found that all homologs stimulated serum response element (SRE)-driven promoter activities when they are stimulated by protons. On the other hand, metals differentially activated the homologs. The results using chimeric receptors of human and zebrafish OGR1s indicate that the specificity of the metal-induced activation lies in the extracellular region. These results suggest that protons are an evolutionally conserved agonist of OGR1. However, the types of metals that activated the receptor differed among the homologs.

Myocardial infarction (MI) is an ischaemic heart condition caused by the occlusion of coronary arteries. Following MI, lactic acid from anaerobic glycolysis increases and infiltrating immune cells produce severe inflammation, which leads to acidosis in the ischaemic heart. However, the physiological implication of this pH reduction remains largely unknown. T-cell death-associated gene 8 (TDAG8) is a proton-sensing G protein-coupled receptor found on cardiac macrophages that recognise increases in extracellular protons. We demonstrated that TDAG8 negatively regulates the transcription of the chemokine Ccl20. The infarcted hearts of TDAG8 KO mice showed an increase in CCL20 expression and the number of infiltrating IL-17A-producing γδT cells that express CCR6, a receptor for CCL20. Accordingly, excessive IL-17A production, which is linked to the functional deterioration after MI, was observed in MI-operated TDAG8 KO mice. The survival rate and cardiac function significantly decreased in TDAG8 KO mice compared with those in wild-type mice after MI. Thus, our results suggest that TDAG8 is a key regulator of MI and a potential therapeutic target.

Objective Obesity is associated with an increased risk of diabetes mellitus, hypertension, and renal dysfunction. Angiotensin 1–7 and alamandine are heptameric renin angiotensin system peptide hormones. Further, alamandine levels increase with renal dysfunction. In the cardiovascular system, angiotensin 1–7 and alamandine produce similar improvements and counterbalance angiotensin II in regulating vascular function. We aimed to determine whether the effect of alamandine on leptin expression and secretion in adipocytes was similar to that of angiotensin 1–7. Approach and results We studied isolated peri-renal visceral adipose tissue and peri-renal isolated visceral adipocytes from male Wistar rats. Angiotensin II from 0.01 to 10nM had no effect on leptin expression. Angiotensin 1–7 (1 nM) increased leptin secretion and expression, whereas alamandine (1 nM) decreased leptin secretion and expression in adipose tissue and isolated adipocytes and reduced blood leptin levels in vivo. These effects were mediated by Gq, c-Src, p38 mitogen-activated protein, and IκB activation. Additionally, alamandine induced nitric oxide expression via inducible nitric oxidase synthase and plasminogen activator inhibitor 1 expression in adipose tissue and isolated adipocytes. Conclusions Angiotensin 1–7 and alamandine produced opposing effects on leptin expression and secretion in adipose tissue. This result suggests that the action of Mas (angiotensin 1–7 receptor) and Mas-related G-protein coupled receptor D in adipocytes exhibited opposing actions similar to angiotensin II type 1 and type 2 receptors.

本総説では私の40年間におよぶ研究をまとめた. 私は1973年に北海道大学薬学部の大学院生として研究活動を開始し, 2年間の米国でのポスドクを経て, 1981年から1986年まで助手 (助教) として研究を行った. その間の最も重要な発見はアデニル酸シクラーゼ系に連関した受容体系と同様にCa2+ 動員性受容体系にもGTP結合タンパク質が介在しているというものである. その後, 群馬大学に赴任し, 助教授 (准教授), 教授として細胞外シグナルリガンドとしてのATP, スフィンゴシン1-リン酸 (S1P), そして, プロトンの研究を行った. 受容体リガンドとしてのプロトンは必ずしも最初の発見とはいえないが, ATPやS1Pが細胞外シグナル分子であるということは当時としては新しい発想であった. これらの研究の中で, S1Pが高密度リポタンパク質 (HDL) 中に濃縮して, HDLの抗動脈硬化性の様々な作用を仲介しているという発見は国際的にも広く認められている. 一方, プロトン研究は比較的新しく多くの課題が残されたままである.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.

The original version of this Article contained an error in the spelling of the author Nobuhiko Ohno, which was incorrectly given as Noubuhiko Ohno. This has now been corrected in both the PDF and HTML versions of the Article.

Introduction: The pH of the interstitium and the bronchoalveolar space has been shown to fall in airway inflammatory disease, such as bronchial asthma. Proton-sensing T-cell death associated gene 8 (TDAG8) is mainly expressed in hematopoietic cells. We have previously reported that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production in the lipopolysaccharide-induced lung injury model. In the allergic airway inflammation, such as bronchial asthma, the role of TDAG8 has not been investigated in detail. Aims: We explored the role of TDAG8 in the allergic airway inflammation with an ovalbumin (OVA)-induced asthma model. Methods: To determine the expression profile of proton-sensing GPCRs, we measured the mRNA expression in lung tissues. TDAG8-deficient (TDAG8-KO) and wild-type (WT) mice were assigned to the following two groups: OVA group, intraperitoneal sensitization and airway challenge with OVA, and control group, those with PBS. Two days after the final challenge, we measured the number of bronchoalveolar lavage (BAL) fluid cells and mRNA expression in lungs. Histological analysis of lung sections was also performed. Results: TDAG8 was expressed in lung tissues. The mRNA expression of TDAG8 and MUC5AC in lungs and eosinophil accumulation in BAL fluids were all increased in OVA group. The OVA-induced eosinophil accumulation and MUC5AC expression in lungs were lower in TDAG8-KO mice than those in WT mice. Conclusions: Our results suggest that absence of TDAG8 protects against OVA-induced allergic airway inflammation and TDAG8 plays an important role in the pathogenesis of asthma.