airway acidification has been shown to be attained by either the exogenous way , that is , the microaspiration of acid contents into the airway during gastroesophageal reflux and inhalation of low ph pollutant aerosol , or the endogenous way , that is , ischemia and inflammation of the airways in inflammatory diseases , such as asthma , cystic fibrosis , and chronic obstructive pulmonary disease ( copd ) [ 13 ] . in ischemic and inflammatory situations , , it has been reported that airway ph reaches 5.2 to 7.1 , depending on the severity : ph is normalized with corticosteroid therapy . although alteration of airway ph may serve an innate host defense capacity , that is , inhibiting the survival of bacteria in an acidic environment , it is also implicated in the pathophysiology of obstructive airway diseases . thus , exposure to acids evokes a cough , bronchoconstriction , airway hyperreactivity ( ahr ) , and microvascular leakage and stimulates mucus production . however , molecular mechanisms underlying the extracellular acidic ph - induced actions in the airways have not been fully understood . in the present review , we discuss the proton - sensing mechanisms , focusing on proton - sensing ionotropic receptors , such as transient receptor potential vanilloid-1 ( trpv1 ) and acid - sensing ion channels ( asics ) , and metabotropic ovarian cancer g protein - coupled receptor 1 ( ogr1)-family g protein - coupled receptors ( gpcrs ) , in the airway inflammation and ahr in asthma and respiratory diseases . the mammalian transient receptor potential ( trp ) superfamily of nonselective cation channels encompasses 28 isotypes and is divided into six subfamilies , that is , trpc , trpv , trpm , trpa , trpp , and trpml . these channels are expressed in neurons and a wide range of cell types in many biological systems [ 46 ] . trp channels have six putative transmembrane domains and a pore - forming loop between the fifth and sixth segments . they are thought to be composed of homo- or heterotetramers . among them , capsaicin - sensitive trpv1 is activated by a diverse range of chemical and noxious stimuli , including protons [ 57 ] . trpv1 senses relatively strong acidic ph of 4 to 5 through glutamic acid in the extracellular domain of the channel   ( figure 1 ) . trp channels other than trpv1 , including trpa1 , trpv4 , and trpm8 , are expressed in the respiratory system and involved in the regulation of airway functions [ 810 ] ; however , whether protons practically trigger their channel activation remains unknown . asics are proposed to assemble as tetramers with homomeric or heteromeric subunits ; each subunit consists of two transmembrane domains   ( figure 1 ) . six asic subunit proteins , encoded by four genes , have been identified : asic1a , asic1b , asic2a , asic2b , asic3 , and asic4 . recent studies have demonstrated that asics activated by acidic ph play an important role in a wide range of physiological and pathophysiological processes such as nociception , mechanosensation , synaptic plasticity , and acidosis - mediated neuronal injury . histidine , glutamic acid , and aspartic acid may determine a broad range of optical ph of 4 to 7 for activation , depending on the subtypes [ 4 , 13 , 14 ] . in addition to trpv1 and asics , there is increasing evidence that further acid - sensitive ion channels are involved in monitoring acidosis . these include trp family ion channels other than trpv1 , including trpv4 , trpc4 , trpc5 , and trpp2 ( pkd2l1 ) ; two - pore domain k ( k2p ) channels ; ionotropic purinoceptors ( p2x ) ; inward rectifier k channels ; voltage - activated k channels ; l - type ca channels ; hyperpolarization - activated cyclic nucleotide gated channels ; gap junction channels ; and cl channels . although most of these channels seem to be expressed and play important roles in the respiratory system , their extracellular proton - sensitivity has not , to our knowledge , been demonstrated for specific airway responses [ 10 , 1517 ] . recent studies suggest that ogr1-family gpcrs , including ogr1 ( gpr68 ) , gpr4 , and t cell - death associated gene 8 ( tdag8 or gpr65 ) , also sense extracellular protons and , thereby , stimulates a variety of cellular activities through several types of g proteins [ 1821 ] ( figure 1 ) . ogr1-family gpcrs were previously described as gpcrs for lysolipids , such as sphingosylphosphorylcholine [ 1921 ] . , however , first discovered that ogr1 and gpr4 sense extracellular protons and are coupled to gq and gs , leading to activation of the phospholipase c / ca signaling pathway and the adenylyl cyclase / camp signaling pathway , respectively . later it was found that tdag8 similarly senses extracellular protons , leading to the activation of the camp signaling pathway [ 22 , 23 ] . ogr1-family gpcrs sense weak acidic to weak alkaline ph of 6 to 8 through histidine residues [ 18 , 19 , 24 , 25 ] . g2a is also classified in this receptor family and is expressed by a broad range of immunoregulatory cell types , including macrophages , dendritic cells , neutrophils , mast cells , and t and b cells , and it is suggested to play an important role in innate and adaptive immunity . however , the proton sensitivity of the receptor is very small and its role as a proton sensor has been questioned . in the present review , therefore , we do not focus further on g2a . ogr1-family gpcrs are expressed in many cell types localized in the airways , and the cases in which their roles are demonstrated are summarized in figure 2 . the proton - sensing trpv1 channel and/or asics in sensory nerves have first been proposed to be involved in acidic ph - induced airway responses [ 2 , 28 , 29 ] . it has been established that trpv1 on capsaicin - sensitive primary sensory neurons plays an important role in nociception and transmission of pain as a sensor of noxious stimuli [ 2 , 4 , 30 ] . the activation of trpv1 on the sensory neurons by irritant compounds , including capsaicin and citric acid , generates reflex responses that , in turn , stimulate the release of tachykinins from the terminals of the sensory nerves , causing cough and bronchoconstriction [ 2 , 5 , 29 ] . the possibility that the afferent signals and neuropeptide release are coupled at the same nerve endings has also been proposed . the neurogenic role of trpv1 was based mainly upon experiments with selective agonists and antagonists . for example , capsazepine , a trpv1-selective antagonist , and sr48968 , a selective nk2 receptor antagonist , blocked citric acid inhalation - induced bronchoconstriction in guinea pig airways in vivo . the role of trpv1 in ahr has recently been demonstrated by the finding that oral administration of trpv1-specific antagonists significantly attenuated ahr in the asthma model of ova - sensitized guinea pigs . in addition to trpv1 , involvement of asics in acidic ph - induced ahr was suggested by using selective asic inhibitors in the guinea pig tracheal rings ex vivo . these results suggest that bronchoconstriction and ahr under acidic environments are indirectly mediated by sensory neurons through proton - sensing channels , such as trpv1 and asics [ 6 , 30 ] . in fact , no report has shown that trpv1 and asics are expressed and are directly functioning in asm cells , although vascular smooth muscle cells seem to express trpv1   and asics . the previous studies , however , did not rule out the direct actions of acidic ph on asm cells . .   showed that extracellular acidification stimulates mrna expression and protein production of il-6 , a proinflammatory cytokine , in association with the phosphorylation of extracellular signal - regulated kinase ( erk ) and p38mapk , in human asm cells . they also showed that extracellular acidification induced an increase in intracellular ca concentration ( [ ca]i ) , which was accompanied by asm cell contraction . acidification also induced expression of connective tissue growth factor ( ctgf ) , a critical factor involved in the formation of extracellular matrix proteins and , hence , airway remodeling . tgf--induced ctgf expression was also enhanced by acidic ph . in asm cells , ogr1 is expressed at by far the highest levels among proton - sensing gpcrs . the knockdown of ogr1 and gq with their specific small interfering rnas and an inhibition of gq with ym-254890 attenuated the acidification - induced actions [ 35 , 37 ] . these results suggest that extracellular acidification stimulates ca mobilization , inflammatory cytokine il-6 and ctgf production , and contraction through ogr1/gq in human asm cells ( figure 2 ) . the role of ogr1 in ahr is supported by recent findings with rgs2-   and rgs5-deficient mice . rgs2 and rgs5 are gtpase - activating proteins that inactivate gq stimulated by gq - coupled receptors , including ogr1 . .   showed that rgs2 deficiency caused spontaneous ahr in response to methacholine without any prior antigen sensitization / challenge . they also showed that the loss of rgs2 augmented ca mobilization and the contraction of asm cells , increased asm mass , and stimulated asm cell growth via erk and phosphatidylinositol 3-kinase ( pi3k ) pathways . these results are highly consistent with those of in vitro studies with human asm cells [ 35 , 36 ] . interestingly , asthma patients display lower expression of rgs2 in epithelial cells and asm cells in lung and circulating monocytes . rgs5-deficient mice also showed spontaneous ahr , even without antigen sensitization in association with enhanced ca mobilization , erk activation , and asm cell contraction . in both cases , however , appreciable infiltration of inflammatory cells in the lung and production of cytokines , such as il-4 and il-5 , was not observed [ 38 , 39 ] . these results suggest that t - cell activation is not always required for gpcr - mediated asthma pathophysiology . liu et al .   have recently shown that an acidic ph of 6.4 stimulates mucin5ac ( muc5ac ) secretion in the human bronchial epithelial cell line ( 16hbe ) through the ogr1/gq / phospholipase c pathway ( figure 2 ) . knockdown of ogr1 and gq expression with small interfering rnas inhibited acidification - induced increases in [ ca]i and mucin production . similarly , overexpression of rgs2 protein attenuated acidic ph - induced cellular responses , whereas knockdown of rgs2 slightly but significantly enhanced these responses . these results suggest that airway acidification induces mucin production through the ogr1 and gq , of which activation can be regulated by rgs2 . the same group has also shown that trpv1 is expressed and citric acid ( ph 5.0)-induced mucin secretion is mediated by ca influx via trpv1 in the same cells . these results suggest that epithelial cells utilize different proton - sensing machineries depending on the acidity of microenvironments ; ogr1-family gpcrs sense mild alkaline to acidic ph of 8 to 6 , whereas trpv1 senses more acidic ph of 6 to 4 , as described above . gpr4 has been shown to be expressed in endothelial cells and to be activated by extracellular acidification , leading to accumulation of camp , which may be relevant to normal blood vessel formation . chen et al . have recently shown that activation of gpr4 by acidosis increases endothelial cell adhesion of monocytes in association with the expression of adhesion molecules , such as vcam-1 and icam-1 , through the camp / epac pathway . since vcam-1 on the endothelium has been shown to mediate eosinophil adhesion and transmigration , this suggests that gpr4 is involved in the perivascular accumulation of eosinophils in the lung with inflammatory asthma ( figure 2 ) . however , it remains unknown whether gpr4 deficiency affects eosinophilia in the airways with asthma . have reported that acidic ph increased eosinophil viability due to an inhibition of apoptosis , which was associated with an increase in camp accumulation . the acidic ph - induced increase in cell viability was attenuated by the adenylyl cyclase inhibitor and , in contrast , the phosphodiesterase inhibitor , the cell - permeable camp analog , and forskolin mimicked the acidic ph effect . the acidification - induced increase in eosinophil viability and camp accumulation these results suggest that eosinophil viability is increased in acidic microenvironments through the tdag8/camp pathway ( figure 2 ) . however , the mechanism by which camp inhibits eosinophil apoptosis remains unknown . in macrophages , tdag8 and ogr1 are expressed . extracellular acidification inhibited lipopolysaccharide ( lps)-induced tnf- and il-6 protein and mrna production in mouse peritoneal macrophages . the inhibitory action on cytokine production by acidic ph was significantly attenuated in macrophages from tdag8-deficient mice but not in those from ogr1-deficient mice . further characterization revealed that acidic ph inhibited proinflammatory cytokine production through the tdag8/gs / camp / pka signaling pathway in mouse macrophages . the expression of tdag8 was increased by glucocorticoid in the macrophage , which was associated with the enhancement of acidification - induced inhibition of tnf- production . thus , tdag8 seems to be indirectly involved in glucocorticoid - induced anti - inflammatory actions . extracellular acidification has been shown to induce human neutrophil activation , inducing an increase in [ ca]i , a shape - changing response , upregulation of the expression of cd18 , an inhibition of apoptosis , and enhancement of agonist - induced h2o2 production . these acidic ph - induced responses are accompanied by the activation of akt and erk . on the other hand , although intracellular acidification was supposed to , in part , explain the acidic ph - induced actions [ 48 , 50 ] , the precise proton - sensing mechanisms remained largely unknown . murata et al .   have shown that acidic ph inhibited fmlp- and c5a - induced superoxide anion production . the acidic ph effect was mimicked by camp increasing agents and attenuated by a pka inhibitor . moreover , these data suggest that tdag8 may mediate extracellular acidification - induced inhibition of o2 production through camp . thus , tdag8 is coupled to anti - inflammatory camp signaling pathways in macrophages and neutrophils . consistently with an anti - inflammatory role , tdag8-deficient mice showed exacerbation of anti - type ii collagen antibody - induced arthritis , in which macrophages and neutrophils have been shown to play a critical role . however , it remains uncharacterized whether the anti - inflammatory actions of tdag8 in macrophages and neutrophils are involved in the pathophysiology of airway inflammation of asthma . geffner and his colleagues have shown that extracellular acidification ( ph 6.5 ) stimulates internalization of antigens ; upregulated the expression of cell surface proteins , including cd11c , mhc class ii , cd40 , and cd86 , involved in antigen presentation ; and promoted an efficient mhc class i - restricted presentation of antigen peptides in dendritic cells ( dcs ) of c57bl/6 mice . antigen - pulsed dcs under acidic ph showed an improved efficacy for inducing both specific cd8 cytotoxic t lymphocytes and specific antibody responses in vivo . they further characterized acidic ph effects using human dcs and found that the transient exposure of human dcs to ph of 6.5 markedly increases several costimulatory proteins and improves the t - cell priming ability of dcs , which was associated with a dramatic increase in p38mak - dependent il-12 production . dc maturation by acidic ph stimulated the production of ifn- , but not of il-4 , by antigen - specific cd4 t cells . these results suggest that extracellular acidification may contribute to the initiation of adaptive immune responses by dcs , favoring the development of the th1 phenotype in humans . basu and srivastava suggested that trpv1 is expressed in mouse dcs and involved in their maturation . trpv1 expression was not confirmed and either capsaicin or acidic ph failed to elicit a change in [ ca]i or the membrane current in mouse dcs . recent studies have shown that trpv1 is expressed in human dcs ; however , in this case , the channel seems to inactivate rather than activate their maturation . asic1 , asic2 , and asic3 are expressed in mouse dcs , and selective inhibitors for asics , such as amiloride and nonsteroidal anti - inflammatory drugs ( nsaids ) , inhibited acidic ph 6.5-induced expression of cell - surface molecules cd11c , mhc class ii , cd80 , and cd86 . mouse dcs express ogr1-family receptors , including ogr1 ; the functional expression of ogr1 was confirmed by the extracellular acidic ph- and ogr1-dependent increase in [ ca]i . ova - sensitized dcs from ogr1-deficient mice showed the reduction in the expression of ccr7 , a chemokine receptor for mature dcs , and the migration responses to ccl19 and ccl21 , ligands for ccr7 , as compared with those from wild - type mice . thus , ogr1 seems to be functioning in the migratory process of dcs to draining lymph nodes ( see the next section ) . although the contribution of trpv1 to acidic ph - induced cough and bronchoconstriction is well demonstrated as discussed , it remains to be proven whether trpv1-mediated neurogenic inflammation plays a central role in asthma and other respiratory diseases . the inarticulate conclusion is based on the lack of obvious effects by gene targeting experiments ; trpv1 deficiency did not attenuate or rather enhanced the airway inflammation and ahr as induced by lps , antigens [ 61 , 62 ] , and cigarette smoke   in trpv1 knockout mice . the anti - inflammatory role of trpv1 might be , in part , explained by the release of anti - inflammatory somatostatin from the sensory nerve terminals in response to trpv1 stimulation . regardless , the role of trpv1 in the airway inflammation and ahr remains to be established in mice . however , uncertain results with mice may be partly explained by the strain difference : the previous studies [ 6063 ] used c57bl/6 . have recently shown that trpv1 knockdown with sirna attenuates il-13- and antigen - induced asthmatic features including airway inflammation and ahr in balb / c mice . in addition to proton - sensing channels , proton - sensing gpcrs also play an important role in the pathophysiology of asthma . as described above , kottyan et al . have shown that tdag8 deficiency causes the stimulation of eosinophil apoptosis and , thereby , reduces airway eosinophilia in ova- and aspergillus fumigatus - sensitization models of mice in vivo . they have also shown increased expression of tdag8 in lungs from ova - sensitized mice and in nasal brushing samples from pediatric asthma patients . unfortunately , whether ahr and airway inflammation as cardinal features of asthma are modulated by tdag8 in vivo has not been examined ; however , it is noted that il-13 production , which plays a role in these processes , was not affected by tdag8 deficiency . the findings of the expression of proton - sensing ogr1 in dc , a critical cell for antigen recognition and its presentation to t cells , and the involvement of ogr1 in the dc migration process   suggest participation of ogr1 in the pathophysiology of allergic asthma . indeed , ogr1-deficient mice are resistant to the cardinal features of asthma , including airway eosinophilia , ahr , and goblet cell metaplasia , in association with a remarkable inhibition of the production of th2 cytokines , including il-4 , il-5 , and il-13 , and ova - specific ige in an ova - induced asthma model . intratracheal transfer to wild - type mice of ova - primed bone marrow - derived dcs from ogr1-deficient mice developed lower ahr and eosinophilia as compared with the transfer of those from wild - type mice , which was associated with lower migratory activity to the peribronchial lymph nodes in ogr1-deficient dcs than in wild - type dcs . these results suggest that stimulation of ogr1 on dcs is critical for the early processes , that is , migration to lymph nodes and initiation of th2 polarization , and , thereby , induces eosinophilia , airway inflammation , and ahr . since ogr1 is expressed in structural cells , including asm cells and epithelial cells , and functions in a variety of cell - specific responses , the reduction of ahr and goblet hyperplasia in ogr1-deficient mice may be partly attributed to the reduction of acidification - induced constriction of asm cells [ 35 , 36 ] and mucin production in epithelial cells . finally , it should be noted that acidic ph modulates dcs leading to th1 polarization in humans as described , which contrasts with the role of ogr1 in th2 polarization in mice . the reason for the difference in the fate of t cells , that is , th1 or th2 , is currently unknown . dcs express proton - sensing gpcrs other than ogr1 , and ogr1-family gpcrs are expressed in neutrophils and macrophages as well . therefore , differences in experimental conditions , for example , species , stimulants , antigens , and ph , may modify the state of dc activation and dc - t cell interaction , making nave t cells polarize to th1 , th2 , th17 , or other phenotypes . how change in the ph microenvironment modulates dc function and t cell polarization warrants further study . proton - sensing channels , such as trpv1 and asics , and ogr1-family gpcrs are expressed in structural cells , including asm cells and epithelial cells , and inflammatory and immune cells , including eosinophils and dcs , and play a variety of roles in airway responses , depending on the optimum ph of proton - sensing channels and gpcrs . cough and bronchoconstriction are activated by severe acidic ph of 4 to 5 and are mainly mediated by ph - sensing channels through sensory neurons , although ph - sensing ogr1 on asm cells may also be involved in the cell constriction . at mild alkaline or mild acidic ph of more than 6 , however , ogr1-family gpcrs may be the main receptors involved in the regulation of airway responses under pathophysiological situations , such as allergic asthma . thus , ionotropic and metabotropic proton - sensing receptors may be therapeutic targets for inflammatory and ischemic diseases , such as asthma , for which drugs that are more specific and have fewer side effects are still required .