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. Here we identify a member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3), which 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 degrees 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.

The endogenous cannabinoid anandamide was identi®ed as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 mM) and capsaicin (1 mM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 mM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC 50 values of 5.94+0.06 (n=5) and 7.13+0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK B of 7.40+0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the ®rst time that anandamide acts as a full agonist at the human VR1.

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. ] i inhibits the channel with an IC 50 of 406 nM. Ruthenium Red at a concentration of 1 M completely blocks inward currents at ؊80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.

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. We report here that 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 responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees 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.

The pharmacology of various peptide and non-peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin-1 (OX 1 ) or orexin-2 (OX 2 ) receptors by measuring intracellular calcium ([Ca 2+ ] i ) using Fluo-3AM. Orexin-A and orexin-B increased [Ca 2+ ] i in CHO-OX 1 (pEC 50 =8.38+0.04 and 7.26+0.05 respectively, n=12) and CHO-OX 2 (pEC 50 =8.20+0.03 and 8.26+0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM ± 10 mM) displayed neither agonist nor antagonist properties in either cell-line. SB-334867-A (1-(2-Methyylbenzoxanzol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride) inhibited the orexin-A (10 nM) and orexin-B (100 nM)-induced calcium responses (pK B =7.27+0.04 and 7.23+0.03 respectively, n=8), but had no eect on the UTP (3 mM)-induced calcium response in CHO-OX 1 cells. SB-334867-A (10 mM) also inhibited OX 2 mediated calcium responses (32.7+1.9% versus orexin-A). SB-334867-A was devoid of agonist properties in either cell-line. In conclusion, SB-334867-A is a non-peptide OX 1 selective receptor antagonist.

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The cellular mechanisms underlying the physiological eects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca 2+ ] i ) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX 1 ) or orexin-2 (OX 2 ) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca 2+ ] i in a concentration dependent manner in CHO-OX 1 (pEC 50 =8.03+0.08 and 7.30+0.08 respectively, n=5) and CHO-OX 2 (pEC 50 =8.18+0.10 and 8.43+0.09 respectively, n=5) cells. This response was typi®ed as a rapid peak in [Ca 2+ ] i (maximal at 6 ± 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 mM) or U73122 (3 mM) abolished the response. In calcium-free conditions the peak response was unaected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium in¯ux.

Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH-133 (5 and 15 microg/50 microL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan-induced hindpaw inflammation, sham operated rats and spinal nerve-ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6-80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan-inflamed, sham-operated and SNL rats. Intraplantar injection of JWH-133 (15 microg), but not vehicle, significantly (P < 0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 microg/50 microL), attenuated the inhibitory effects of JWH-133 (15 microg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH-133 on these responses. Intraplantar preadministration of JWH-133 also inhibited (P < 0.05) carrageenan-induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous- and noxious-evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.