Protons can modulate the activity of a number of receptors and ion channels expressed in nociceptors (1). Among such entities, they can directly activate vanilloid receptor subtype-1 (VR1) and acid-sensing ion channel ASICs (2, 3). VR1 is selectively expressed in polymodal nociceptors, which are responsive to noxiously thermal, mechanical, and chemical stimuli, and is broadly regarded as a major detector of multiple pain-producing stimuli. The contribution of VR1 to the pH sensitivity of nociceptors has been established in vitro by gene knockout experiments (4). However, the activation of the VR1 channel requires extremely severe acidification to pH less than 6.0 (4, 5), raising the possibility that another signal sensor that is more sensitive to protons than VR1 may be present in nociceptors, because, for example, skin nociceptors have activation thresholds as high as pH 6.9 (6). In muscle and cardiac ischemia, an extracellular pH drop from 7.4 to 7.0 is sufficient to induce persistent activation of a subset of nociceptors (7-9). Recent electrophysiological experiments have strongly suggested the involvement of ASICs (amiloride-blockable proton-gated channel subunits expressed in mammalian central and peripheral nervous systems) (10) in nociception linked to acidoses. Sensory neurons from mice lacking ASIC-3 (nomenclature as in ref. 3) are severely deficient in their responses to acidic stimuli in vitro (11). The heterologously expressed ASIC-2b/ASIC-3 channel generates a biphasic inward current that is similar to the native proton-activated current in dorsal root ganglion (DRG) neurons (12). The ASIC-3 channel is capable of reproducing the features of acid-evoked currents in cardiac nociceptors (13). Despite these observations, there is still controversy about the functional roles of ASICs in mammals, because proton detection through ASICs has not yet been demonstrated in vivo. In this report, we evaluated the efficacy of amiloride (an inhibitor of ASICs) and capsazepine (an inhibitor of VR1) on acid-evoked pain in humans using a psychophysical method. To confirm the specificities of both drugs, we investigated their effects on capsaicinevoked pain using a similar psychophysical approach. Our results indicate the involvement of ASICs and VR1 in proton-induced pain in humans and show their relative importance in the nociception. Methods Psychophysical experiments. The following experiments were approved by the Ethics Committee of the Nagoya City University Medical School and conducted in accordance with the Declaration of Helsinki. A total of 56 healthy men, 21-41 years of age, participated in the study. All subjects stated that they had not used drugs of any kind within one week preceding the experiments.

Bitter taste perception is a conserved chemical sense against the ingestion of poisonous substances in mammals. A multigene family of G-protein-coupled receptors, T2R (so-called TAS2R or TRB) receptors and a G-protein alpha subunit (Galpha), gustducin, are believed to be key molecules for its perception, but little is known about the molecular basis for its interaction. Here, we use a heterologous expression system to determine a specific domain of gustducin necessary for T2R coupling. Two chimeric Galpha16 proteins harboring 37 and 44 gustducin-specific sequences at their C termini (G16/gust37 and G16/gust44) responded to different T2R receptors with known ligands, but G16/gust 23, G16/gust11, and G16/gust5 did not. The former two chimeras contained a predicted beta6 sheet, an alpha5 helix, and an extreme C terminus of gustducin, and all the domains were indispensable to the expression of T2R activity. We also expressed G16 protein chimeras with the corresponding domain from other Galpha(i) proteins, cone-transducin (Galpha(t2)), Galpha(i2), and Galpha(z) (G16/t2, G16/i2, and G16/z). As a result, G16/t2 and G16/i2 produced specific responses of T2Rs, but G16/z did not. Because Galpha(t2) and Galpha(i2) are expressed in the taste receptor cells, these G-protein alpha(i) subunits may also be involved in bitter taste perception via T2R receptors. The present Galpha16-based chimeras could be useful tools to analyze the functions of many orphan G-protein-coupled taste receptors.

Aurones are plant flavonoids that provide yellow color to the flowers of some popular ornamental plants, such as snapdragon and cosmos. In this study, we have identified an enzyme responsible for the synthesis of aurone from chalcones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation and/or oxidative cyclization of the precursor chalcones, 2',4',6',4-tetrahydroxychalcone and 2',4',6',3,4-pentahydroxychalcone. The complementary DNA encoding aureusidin synthase is expressed in the petals of aurone-containing varieties. DNA sequence analysis revealed that aureusidin synthase belongs to the plant polyphenol oxidase family, providing an unequivocal example of the function of the polyphenol oxidase homolog in plants, i.e., flower coloration.

Acid-sensing ion channel-2a (ASIC2a) is an amiloride-blockable proton-gated cation channel, probably contributing to sour-taste detection in rat taste cells. To isolate another subtype of the sour-taste receptor, we screened a rat circumvallate papilla cDNA library and identified ASIC2b, an N-terminal splice variant of ASIC2a. Reverse transcription-PCR analyses confirmed the expression of ASIC2b transcripts in the circumvallate papilla and, moreover, demonstrated its expression in the foliate and fungiform papillae. Immunohistochemical analyses revealed that ASIC2b, as well as ASIC2a, was expressed in a subpopulation of taste cells in the circumvallate, foliate, and fungiform papillae, and some of the cells displayed both ASIC2a and ASIC2b immunoreactivities. Subsequent coimmunoprecipitation studies with circumvallate papillae extracts indicated that ASIC2b associated with ASIC2a to form assemblies and, together with our immunohistochemical findings, strongly suggested that both ASIC2 subunits formed heteromeric channels in taste cells in the circumvallate, foliate, and fungiform papillae. Oocyte electrophysiology demonstrated that the ASIC2a/ASIC2b channel generated maximal inward currents at a pH of < or =2.0, which is in agreement with the in vivo pH sensitivity of rat taste cells, and that the amiloride sensitivity of the heteromer decreased with decreasing pH and was almost completely abolished at a pH of 2.0. These findings provide persuasive explanations for the amiloride insensitivity of acid-induced responses of rat taste cells.

Relapse is the most serious limitation of effective medical treatment of opiate addiction. Opiate-related behaviors appear to be modulated by cannabinoid CB1 receptors (CB1) through poorly understood cross-talk mechanisms. Opiate and CB1 receptors are coexpressed in the nucleus accumbens (NAc) and dorsal striatum. These regions also have the highest density of adenosine A2a receptors (A2a) in the brain. We have been investigating the postsynaptic signaling mechanisms of -opiate receptors (MORs) and CB1 receptors in primary NAc͞striatal neurons. In this article, we present evidence that MOR and CB1 act synergistically on cAMP͞PKA signaling in NAc͞striatal neurons. In addition, we find that synergy requires adenosine and A2a. Importantly, an A2a antagonist administered either directly into the NAc or indirectly by i.p. injection eliminates heroin-induced reinstatement in rats trained to self-administer heroin, a model of human craving and relapse. These findings suggest that A2a antagonists might be effective therapeutic agents in the management of abstinent heroin addicts.PKA ͉ addiction ͉ nucleus accumbens ͉ gene activation O piate addiction is a world-wide public health problem with serious socioeconomic ramifications. A major limitation of effective medical treatment is the craving and relapse that develops during attempted abstinence. Opiates bind to three opioid receptors: the ␦-opioid receptor (DOR), -opioid receptor (MOR), and -opioid receptor (KOR) receptors. MOR and DOR are implicated in reward for heroin and morphine, whereas KOR is implicated in aversion (1). MOR antagonists reduce opiate selfadministration, and constitutive deletion of the MOR attenuates opiate-induced conditioned place preference (CPP) (2, 3). Moreover, selective MOR blockade is sufficient to induce conditioned aversion in morphine-dependent animals, presumably because of unopposed activation of KOR (1). The nucleus accumbens (NAc) mediates reward and reinforcement of addictive agents. Thus, inactivation of the NAc core inhibits heroin self-administration (4). We have been investigating postsynaptic signaling mechanisms activated by opiate receptors in NAc͞striatal neurons. We reported that brief exposure of primary NAc͞striatal neurons to MOR agonists for 10 min activates cAMP͞PKA signal transduction followed by stimulation of cAMP response element (CRE)-mediated gene expression hours later (5). We also found that paradoxical stimulation of cAMP͞PKA signaling by G i -coupled MOR depends on preferential binding of the MOR to G␣ i3 ␤␥. Thus, activation of the MOR appears to release ␤␥ subunits from G␣ i3 ; released unbound ␤␥ subunits stimulate adenylyl cyclase (AC) II and IV to increase cAMP production. In turn, this transient increase in cAMP activates PKA and CRE-dependent gene transcription (5).Signaling of G protein-coupled receptors can be modulated by G protein regulators. Recent evidence suggests that an activator of G protein signaling 3 (AGS3), regulates G␣ i3 -coupled receptor signaling by competing with ␤␥ subunits for bin...

The transient receptor potential melastatin subfamily (TRPM), which is a mammalian homologue of cell death-regulated genes in Caenorhabditis elegans and Drosophila, has potential roles in the process of the cell cycle and regulation of Ca(2+) signaling. Among this subfamily, TRPM8 (also known as Trp-p8) is a Ca(2+)-permeable channel that was originally identified as a prostate-specific gene upregulated in tumors. Here we showed that the TRPM8 channel was expressed in human melanoma G-361 cells, and activation of the channel produced sustainable Ca(2+) influx. The application of menthol, an agonist for TRPM8 channel, elevated cytosolic Ca(2+) concentration in a concentration-dependent manner with an EC(50) value of 286 microM in melanoma cells. Menthol-induced responses were significantly abolished by the removal of external Ca(2+). Moreover, inward currents at a holding potential of -60 mV in melanoma cells were markedly potentiated by the addition of 300 microM menthol. The most striking finding was that the viability of melanoma cells was dose-dependently depressed in the presence of menthol. These results reveal that a functional TRPM8 protein is expressed in human melanoma cells to involve the mechanism underlying tumor progression via the Ca(2+) handling pathway, providing us with a novel target of drug development for malignant melanoma.

S U M M A R Y We studied the localization and physiological functions of the transient receptor potential (TRP) channels TRPV1 (TRP vanilloid 1) and TRPV4 (TRP vanilloid 4) in the mouse bladder, because both channels are thought to be mechanosensors for bladder distention. RT-PCR specifically amplified TRPV4 transcripts from the urothelial cells, whereas TRPV1 transcripts were barely detectable. ISH experiments showed that TRPV4 transcripts were abundantly expressed in the urothelium, whereas TRPV1 transcripts were not detectable in the urothelial cells. Immunoblotting and IHC studies showed that TRPV4 proteins were mainly localized at the basal plasma membrane domains of the basal urothelial cells. In contrast, TRPV1-immunoreactivities were found not in the urothelial cells but in the nerve fibers that innervate the urinary bladder. In Ca 21-imaging experiments, 4a-phorbol 12,13-didecanoate, a TRPV4 agonist, and hypotonic stimuli induced significant increases in intracellular calcium ion concentration ([Ca 21 ] i ) in isolated urothelial cells, whereas capsaicin, a TRPV1 agonist, showed no marked effect on the cells. These findings raise the possibility that, in mouse urothelial cells, TRPV4 may contribute to the detection of increases in intravesical pressure related to the micturition reflex. (J Histochem Cytochem 57:277-287, 2009)

The amiloride-sensitive epithelial Na ؉ channel (ENaC) controls Na ؉ transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as ␣, ␤, ␥, and ␦. ENaC␦ can associate with ␤ and ␥ subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaC␦. However, the distribution pattern of ENaC␦ is not consistent with that of the ␤ and ␥ subunits. ENaC␦ is expressed mainly in the brain in contrast to ␤ and ␥ subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaC␦ and investigated the detailed tissue distribution in humans. When human ENaC␦ was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by ϳ10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na ؉ channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaC␦ mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaC␦ was expressed in the cerebellum and the hippocampus. In conclusion, human ENaC␦ activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain. Four homologous epithelial Naϩ channel (ENaC) 1 subunits (␣, ␤, ␥, and ␦), members of the degenerin/epithelial Na ϩ channel superfamily, have been cloned in mammals (1-5). There is an overall ϳ37% amino acid identity between the ␣, ␤, ␥, and ␦ subunits. The ␦ subunit of ENaC was originally described as mainly being expressed in the human brain (5). ENaC␦ can associate with ␤ and ␥ subunits to form a heteromeric channel because the coexpression of these three subunits increases the Na ϩ current (5). The tissue distribution pattern of ENaC␦, however, is quite different from that of ␤ and ␥ subunits. ENaC␦ is expressed mainly in the brain, pancreas, testis, and ovary (5), whereas ␤ and ␥ subunits are expressed mainly in the kidney, lung, and colon (3, 4). In addition, the expressed sequence tag data base shows that an ENaC␦ gene has been found in humans and chimpanzees (GenBank TM accession numbers U38254 and O46547, respectively), but for now, there is no evidence for the orthologues in rats and mice. This suggests that ENaC␦ associates with unknown subunits or that homomeric ENaC␦ has its own unknown physiological function in humans. Our goal was to identify the novel functional properties of human ENaC␦ (hENaC␦) using electrophysiological techniques and to investigate the more detailed tissue distribution of ENaC␦ in humans by Northern blot and RNA dot-blot analyses.Here we describe how ...