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.

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...