painless does not encode a Drosophila ortholog of TRPV1 (see Capsaicin receptors below) but instead represents a second member of the TRPN gene family whose founding member, no mechanoreceptor potential-C (NOMP-C), is essential for Drosophila mechanosensation (Tracey, 2003 and references therein). Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, Drosophila mechanoreceptive mutants were analyzed for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolishes mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs (Walker, 2000).
Although cyclic nucleotide-gated channels mediate sensory transduction in olfaction and vision, other forms of sensory transduction are independent of these channels. Caenorhabditis elegans cyclic nucleotide-gated channel mutants respond normally to some olfactory stimuli and to osmotic stimuli, suggesting that these chemosensory responses use an alternative sensory transduction pathway. One gene that may act in this pathway is osm-9, which is required for each of these responses as well as a mechanosensory response to nose touch. osm-9 encodes a protein with ankyrin repeats and multiple predicted transmembrane domains that has limited similarity to the Drosophila phototransduction channels transient receptor potential (TRP) and TRP-like (TRPL). The sequence of OSM-9 and other TRP-like genes reveals a previously unsuspected diversity of mammalian and invertebrate genes in this family. osm-9 is required for the activity of the predicted G-protein-coupled odorant receptor ODR-10, which acts in the AWA olfactory neurons; its similarity to other G-protein-regulated transduction channels suggests that OSM-9 is involved in AWA signaling. osm-9:: GFP fusion genes are expressed in a subset of chemosensory, mechanosensory, and osmosensory neurons. osm-9 also affects olfactory adaptation within neurons that require the cyclic nucleotide-gated channel for olfaction; in these neurons, the gene has a regulatory function and not a primary role in sensory transduction (Colbert, 1997).
C. elegans OSM-9 is a TRPV channel protein involved in sensory transduction and adaptation. Distinct sensory functions arise from different combinations of OSM-9 and related OCR TRPV proteins. Both OSM-9 and OCR-2 are essential for several forms of sensory transduction, including olfaction, osmosensation, mechanosensation, and chemosensation. In neurons that express both OSM-9 and OCR-2, tagged OCR-2 and OSM-9 proteins reside in sensory cilia and promote each other's localization to cilia. In neurons that express only OSM-9, tagged OSM-9 protein resides in the cell body and acts in sensory adaptation rather than sensory transduction. Thus, alternative combinations of TRPV proteins may direct different functions in distinct subcellular locations. Animals expressing the mammalian TRPV1 (VR1) channel in ASH nociceptor neurons avoid the TRPV1 ligand capsaicin, allowing selective, drug-inducible activation of a specific behavior (Tobin, 2002).
Natural Caenorhabditis elegans isolates exhibit either social or solitary feeding on bacteria. Social feeding is induced by nociceptive neurons that detect adverse or stressful conditions. Ablation of the nociceptive neurons ASH and ADL transforms social animals into solitary feeders. Social feeding is probably due to the sensation of noxious chemicals by ASH and ADL neurons; it requires the genes ocr-2 and osm-9, which encode TRP-related transduction channels, and odr-4 and odr-8, which are required to localize sensory chemoreceptors to cilia. Other sensory neurons may suppress social feeding, since social feeding in ocr-2 and odr-4 mutants is restored by mutations in osm-3, a gene required for the development of 26 ciliated sensory neurons. These data suggest a model for regulation of social feeding by opposing sensory inputs: aversive inputs to nociceptive neurons promote social feeding, whereas antagonistic inputs from neurons that express osm-3 inhibit aggregation (de Bono, 2002).
All animals detect osmotic and mechanical stimuli, but the molecular basis for these responses is incompletely understood. The vertebrate transient receptor potential channel vanilloid subfamily 4 (TRPV4) (VR-OAC) cation channel has been suggested to be an osmo/mechanosensory channel. To assess its function in vivo, TRPV4 was expressed in Caenorhabditis elegans sensory neurons and its ability to generate behavioral responses to sensory stimuli was examined. C. elegans ASH neurons function as polymodal sensory neurons that generate a characteristic escape behavior in response to mechanical, osmotic, or olfactory stimuli. These behaviors require the TRPV channel OSM-9 because osm-9 mutants do not avoid nose touch, high osmolarity, or noxious odors. Expression of mammalian TRPV4 in ASH neurons of osm-9 worms restores avoidance responses to hypertonicity and nose touch, but not the response to odorant repellents. Mutations known to reduce TRPV4 channel activity also reduces its ability to direct nematode avoidance behavior. TRPV4 function in ASH requires the endogenous C. elegans osmotic and nose touch avoidance genes ocr-2, odr-3, osm-10, and glr-1, indicating that TRPV4 is integrated into the normal ASH sensory apparatus. The osmotic and mechanical avoidance responses of TRPV4- expressing animals are different in their sensitivity and temperature dependence from the responses of wild-type animals, suggesting that the TRPV4 channel confers its characteristic properties on the transgenic animals' behavior. These results provide evidence that TRPV4 can function as a component of an osmotic/mechanical sensor in vivo (Liedtke, 2003).
Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. ANKTM1 is a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. ANKTM1 is the closest vertebrate homolog of painless. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, noxious cold-sensitive sensory neurons have been identified that also respond to capsaicin but not to menthol (Story, 2003).
Capsaicin, the main pungent ingredient in 'hot' chilli peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. An expression cloning strategy based on calcium influx was used to isolate a functional cDNA encoding a capsaicin receptor from sensory neurons. This receptor is a non-selective cation channel that is structurally related to members of the TRP family of ion channels. The cloned capsaicin receptor is also activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo (Caterina, 1997).
Capsaicin, the main pungent ingredient in 'hot' chili peppers, elicits buming pain by activating specific (vanilloid) receptors on sensory nerve endings. The cloned vanilloid receptor (VR1) is a cation channel that is also activated by noxious heat. Analysis of heat-evoked single channel currents in excised membrane patches suggest that heat gates VR1 directly. Protons decrease the temperature threshold for VR1 activation such that even moderately acidic conditions (pH < or = 5.9) activate VR1 at room temperature. VR1 can therefore be viewed as a molecular integrator of chemical and physical stimuli that elicit pain. Immunocytochemical analysis indicates that the receptor is located in a neurochemically heterogeneous population of small diameter primary afferent fibers. A role for VR1 in injury-induced hypersensitivity at the level of the sensory neuron is presented (Tominaga, 1998).
Pain-producing heat is detected by several classes of nociceptive sensory neuron that differ in their thermal response thresholds. The cloned capsaicin receptor, also known as the vanilloid receptor subtype 1 (VR1), is a heat-gated ion channel that has been proposed to mediate responses of small-diameter sensory neurons to moderate (43°C) thermal stimuli. VR1 is also activated by protons, indicating that it may participate in the detection of noxious thermal and chemical stimuli in vivo. A structurally related receptor, VRL-1, has been identified that does not respond to capsaicin, acid or moderate heat. Instead, VRL-1 is activated by high temperatures, with a threshold of approximately 52 degrees C. Within sensory ganglia, VRL-1 is most prominently expressed by a subset of medium- to large-diameter neurons, making it a candidate receptor for transducing high-threshold heat responses in this class of cells. VRL-1 transcripts are not restricted to the sensory nervous system, indicating that this channel may be activated by stimuli other than heat. It is proposed that responses to noxious heat involve these related, but distinct, ion-channel subtypes that together detect a range of stimulus intensities (Caterina, 1999).
The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the 'pain' pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43°C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Sensory neurons from mice lacking VR1 have been demonstrated to be severely deficient in their responses to each of these noxious stimuli. VR1-/- mice show normal responses to noxious mechanical stimuli but exhibit no vanilloid-evoked pain behavior. They are impaired in the detection of painful heat, and show little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia (Caterina, 2000).
The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chili peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviors (that is, responses to painful stimuli in animals) have led to VR1 being considered important for pain sensation. The mouse VR1 gene has been disrupted using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lack many of the capsaicin-, acid- and heat-gated responses that have been well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appear normal in a wide range of behavioral tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. It is concluded that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat (Davis, 2000).
A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Described here is the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. The identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection (Peier, 2002a).
Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. A novel thermosensitive TRP channel, TRPV3, has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons (Peier, 2002b).
The cellular and molecular mechanisms that enable sensing of cold are not well understood. Insights into this process have come from the use of pharmacological agents, such as menthol, that elicit a cooling sensation. A menthol receptor from trigeminal sensory neurons that is also activated by thermal stimuli in the cool to cold range, has been cloned and characterized. This cold- and menthol-sensitive receptor, CMR1, is a member of the TRP family of excitatory ion channels, and it is proposed that this receptor functions as a transducer of cold stimuli in the somatosensory system. These findings, together with the identification of the heat-sensitive channels VR1 and VRL-1, demonstrate that TRP channels detect temperatures over a wide range and are the principal sensors of thermal stimuli in the mammalian peripheral nervous system (McKemy, 2002).
Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments, such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons, suggest the existence of thermosensitive receptors distinct from VR1. A member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3) is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39°C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels (Smith, 2002).
Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin, noxious heat, and cooling (TRPM8) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22°C and 40°C. A member of the vanilloid channel family, human TRPV3 (hTRPV3), has been identified that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22°C to 40°C in mammalian cells transfected with hTRPV3 elevates intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current is steeply dependent on temperature, sensitized with repeated heating, and displays a marked hysteresis on heating and cooling. On the basis of these properties, it is proposed that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1 (Xu, 2002).
The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. Ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells respond to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiates the response of VR1 to capsaicin, protons and heat and lowers the threshold for heat activation of VR1 from approximately 42°C to approximately 34°C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds (Trevisani, 2002).
Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using VRL1 (its capsaicin-insensitive homolog), key amino acids that determine ligand binding were localized (Arg-114 and Glu-761, in the N- and C-cytosolic tails, respectively). Point mutations of the two key residues result in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [(3)H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids block capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket (Jung, 2002).
The capsaicin receptor transient receptor potential V1 (TRPV1; also known as vanilloid receptor 1) is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that extracellular ATP potentiates the TRPV1 currents evoked by capsaicin or protons and reduces the temperature threshold for its activation through metabotropic P2Y receptors in a PKC-dependent pathway, suggesting that TRPV1 activation could trigger the sensation of pain at normal body temperature in the presence of ATP. ATP-induced thermal hyperalgesia is abolished in mice lacking TRPV1, suggesting the functional interaction between ATP and TRPV1 at a behavioral level. However, thermal hyperalgesia is preserved in P2Y1 receptor-deficient mice. Patch-clamp analyses using mouse dorsal root ganglion neurons indicate the involvement of P2Y2 rather than P2Y1 receptors. Coexpression of TRPV1 mRNA with P2Y2 mRNA, but not P2Y1 mRNA, occurs in the rat lumbar DRG using in situ hybridization histochemistry. These data indicate the importance of metabotropic P2Y2 receptors in nociception through TRPV1 (Moriyama, 2003).
The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca2+-dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. The Ca2+-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain (Numazaki, 2003).
The capsaicin receptor, VR1 (also known as TRPV1), is a ligand-gated ion channel expressed on nociceptive sensory neurons; the receptor responds to noxious thermal and chemical stimuli. Capsaicin responses in sensory neurons exhibit robust potentiation by cAMP-dependent protein kinase (PKA). PKA reduces VR1 desensitization and directly phosphorylates VR1. In vitro phosphorylation, phosphopeptide mapping, and protein sequencing of VR1 cytoplasmic domains delineate several candidate PKA phosphorylation sites. Electrophysiological analysis of phosphorylation site mutants clearly pinpoints Ser116 as the residue responsible for PKA-dependent modulation of VR1. Given the significant roles of VR1 and PKA in inflammatory pain hypersensitivity, VR1 phosphorylation at Ser116 by PKA may represent an important molecular mechanism involved in the regulation of VR1 function after tissue injury (Bhave, 2002).
The responses of vanilloid receptor (VR) channels to changing membrane potential were studied in Xenopus oocytes and rat dorsal root ganglion (DRG) neurons. In oocytes, capsaicin-evoked VR currents increase instantaneously upon a step depolarization and thereafter rise biexponentially with time constants of approximately 20 and 1000 ms. Similarly, upon repolarization the current abruptly decreases, followed by a biexponential decay with time constants of approximately 4 and 200 ms. Qualitatively similar effects are observed in single channel recordings of native VR channels from DRG neurons and with endogenous VR activators, including heat (43 degrees C), H(+), anandamide and protein kinase C (PKC). The magnitude of the time-dependent current rise increases with membrane depolarization. This effect is accompanied by an increase in the relative proportion of the fast kinetic component, A(1). In contrast, the time constants of the activation and deactivation processes are not strongly voltage dependent. Increasing the agonist concentration both reduces the magnitude of the current rise and increases its overall rate, without significantly altering the deactivation rate. In contrast, PKC both speeds the current rise and slows its decay. These results suggest that voltage interacts with agonists in a synergistic manner to augment VR current and this mechanism will be enhanced under conditions of inflammation when VRs are likely to be phosphorylated (Ahern, 2002).
Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activates Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu is antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist. This indicates that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulates PKC(alpha) in either the dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlates with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (1) direct ligand binding (anandamide, vanilloids) and (2) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes show that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia (Olah, 2002).
Inflammatory mediators not only activate 'pain-sensing' neurons (the nociceptors), to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. At the cellular level heat-activated ionic currents are potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation is prevented in the presence of a selective PKA inhibitor, suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits translocated to the cell periphery when activated. The translocation and the current potentiation are abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation is greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia (Rathee, 2002).
Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, direct sequencing was used to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. The ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1 was tested. Mutation of S800 to alanine significantly reduces the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduces PMA enhancement of capsaicin-evoked currents, but has no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine has no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduces direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation (Bhave, 2003).
Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. The influence of PKA on Ca(2+)-dependent desensitization of capsaicin-activated currents was investigated. By using site-directed mutagenesis, point mutations at PKA consensus sites were created and wild-type (WT) and mutant channels transiently expressed in HEK293t cells were studied under whole-cell voltage clamp. Forskolin, a stimulator of adenylate cyclase, decreases desensitization of TRPV1. The selective PKA inhibitor H89 inhibits this effect. Mimicking phosphorylation at PKA consensus sites by replacing S6, S116, T144, T370, S502, S774, or S820 with aspartate (D) results in five mutations (S116D, T144D, T370D, S774D, S820D) that exhibit decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine (A) results in four mutations (S6A, T144A, T370A, S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca(2+) over Na(+) or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D pre-treatment of cells with forskolin do not reduce desensitization as compared to WT and other mutant channels. It is concluded that S116 and possibly T370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents (Mohapatra, 2003).
The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. The vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibits the vasodilator effect of anandamide. Other endogenous and synthetic CB1 and CB2 receptor agonists do not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibits anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induces a capsazepine-sensitive current in whole cells and isolated membrane patches. These results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems (Zygmunt, 1999).
Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, it has been shown that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possess the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids fail to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction (Hwang, 2000).
The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitate transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibit and enhance, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhances anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following: (1) anandamide activates VR1 receptors by acting at an intracellular site; (2) degradation by fatty acid amide hydrolase limits anandamide activity on VR1 and (3) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A also inhibits the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism (De Petrocellis, 2001a).
The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. The potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect is decreased five-fold in the presence of forskolin or the cAMP analog, 8-Br-cAMP. The effects of 8-Br-cAMP and FRSK are blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhances the sensory neuron- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect is abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induces release of substance P (an effect mediated by VR1 activation) is enhanced three-fold by FRSK. Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions (De Petrocellis, 2001b).
The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and capsaicin, the pungent ingredient of 'hot' chili peppers. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. The study examines the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous 'capsaicin-like' substance in mammalian nervous tissues. NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. Evidence was gained for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency [EC(50) approximately 50 nM] and efficacy similar to that of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia. These data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors (Huang, 2002).
N-Arachidonoyldopamine (NADA) is an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicates the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Mass spectrometric analysis of bovine striatal extracts reveals the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA fail to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there is no evidence of spontaneous pain behavior. By contrast, OLDA induces calcium influx, reduces the latency of paw withdrawal from a radiant heat source in a dose-dependent manner, and produces nocifensive behavior. These effects are blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin. These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA is only a weak ligand for rat CB1 receptors; but like NADA, it is recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamines indicates the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors (Chu, 2003).
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