sb-366791 and anandamide

The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

Topics: Activating Transcription Factor 3; Amidohydrolases; Anilides; Animals; Arachidonic Acids; Benzamides; Capsaicin; Carbamates; Cinnamates; Disease Models, Animal; Endocannabinoids; Ganglia, Spinal; Gene Expression; Hyperalgesia; Inflammation; Interleukin-6; Lumbar Vertebrae; Male; Osteoarthritis; Pain; Pain Management; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1α, IL-1β, and PGE(2), which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain.

Topics: Aminoquinolines; Anilides; Animals; Arachidonic Acids; Blotting, Western; Calcium; Capsaicin; Cells, Cultured; Cinnamates; Cytokines; Dinoprostone; Dose-Response Relationship, Drug; Endocannabinoids; Ganglia, Spinal; Humans; Imidazoles; Mice; Microscopy, Confocal; Neurons; Pain; Poly I-C; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Toll-Like Receptor 3; Toll-Like Receptor 7; Toll-Like Receptor 9; Toll-Like Receptors; TRPV Cation Channels

Anandamide has been characterized as both an endocannabinoid and endovanilloid. Consistent with its actions as an endovanilloid, previous studies have demonstrated that anandamide can excite primary sensory neurons in vitro via transient receptor potential vanilloid type one (TRPV1) receptors. In the present study, we sought to determine if anandamide excited cutaneous C nociceptors in vivo and if this excitation correlated with nocifensive behaviors. Using teased-fiber electrophysiological methods in the rat, C nociceptors isolated from the tibial nerve with receptive fields (RFs) on the plantar surface of the hindpaw were studied. Injection of anandamide into the RF dose-dependently excited nociceptors at doses of 10 and 100 microg. The TRPV1 receptor antagonists, capsazepine or SB 366791, were applied to the RF to determine if excitation by anandamide was mediated through TRPV1 receptors. Intraplantar injection of either capsazepine (10 microg) or SB 366791 (3 microg) attenuated the excitation produced by 100 microg anandamide. We also determined whether excitation of C nociceptors by anandamide was associated with nocifensive behaviors. Intraplantar injection of 100 microg anandamide produced nocifensive behaviors that were attenuated by pre-treatment with either capsazepine or SB 366791. Furthermore, we determined if intraplantar injection of anandamide altered withdrawal responses to radiant heat. Neither intraplantar injection of anandamide nor vehicle produced antinociception or hyperalgesia to radiant heat. Our results indicate that anandamide excited cutaneous C nociceptors and produced nocifensive behaviors via activation of TRPV1 receptors.

Topics: Action Potentials; Anilides; Animals; Arachidonic Acids; Calcium Channel Blockers; Capsaicin; Cinnamates; Dose-Response Relationship, Drug; Endocannabinoids; Hindlimb; Hot Temperature; Male; Nociceptors; Pain; Pain Measurement; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Skin; Tibial Nerve; TRPV Cation Channels

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonists L-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in the TRPV1(-/-) mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

Topics: Amino Acids; Anilides; Animals; Arachidonic Acids; Capsaicin; Cinnamates; Endocannabinoids; Excitatory Amino Acid Antagonists; Glutamic Acid; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Polyunsaturated Alkamides; Propionates; Receptors, Metabotropic Glutamate; Retinal Bipolar Cells; Ruthenium Red; Synaptic Transmission; Transient Receptor Potential Channels; TRPM Cation Channels; TRPV Cation Channels; Xanthenes

AM 404 inhibits endocannabinoid uptake and enhances the cannabinoid CB(1)-mediated effects of endogenous cannabinoids. Accumulating evidence also suggests that AM 404 acts at sites other than the endocannabinoid system. One site is the transient receptor potential vanilloid 1 cation channel (TRPV1). A useful endpoint for discriminating between TRPV1- or CB(1)-mediated effects of AM 404 is hypothermia. This is because TRPV1 or CB(1) receptor activation produces a significant hypothermia in rats. The present study investigated the effects of AM 404 (1, 5, 10 and 20 mg/kg, i.p.) on body temperature in rats and the involvement of TRPV1 and CB(1) receptors in the effects of AM 404. Doses of 10 and 20 mg/kg of AM 404 produced significant hypothermia. Pre-treatment with capsazepine (30 mg/kg, i.p.) blocked the hypothermia caused by 10 and 20 mg/kg of AM 404. Pre-treatment with SB 366791 (2 mg/kg, i.p.), a new TRPV1 antagonist, also abolished the hypothermia evoked by AM 404 (20 mg/kg, i.p.). In contrast, pre-treatment with SR 141716A (Rimonabant), a CB(1) antagonist, or AA-5-HT, a fatty acid amide hydrolase (FAAH) blocker, did not affect AM 404-evoked hypothermia. The present data demonstrate that AM 404 evokes a significant hypothermia in rats that is dependent on TRPV1 receptor activation.

Topics: Amidohydrolases; Anilides; Animals; Arachidonic Acids; Body Temperature; Capsaicin; Cinnamates; Endocannabinoids; Hypothermia; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; TRPV Cation Channels

Transient receptor potential (TRP) receptors are, typically, calcium-permeant cation channels that transduce environmental stimuli. Both kidney epithelial and inner ear sensory cells express TRPV1, are mechanosensors and accumulate the aminoglycoside antibiotic gentamicin. Recently, we showed that Texas Red-conjugated gentamicin (GTTR) enters kidney cells via an endosome-independent pathway. Here, we used GTTR to investigate this non-endocytotic mechanism of gentamicin uptake. In serum-free buffers, GTTR penetrated MDCK cells within 30 s and uptake was modulated by extracellular, multivalent cations (Ca2+, La3+, Gd3+) or protons. We verified the La3+ modulation of GTTR uptake using immunocytochemical detection of unconjugated gentamicin. Membrane depolarization, induced by high extracellular K+ or valinomycin, also reduced GTTR uptake, suggesting electrophoretic permeation through ion channels. GTTR uptake was enhanced by the TRPV1 agonists, resiniferatoxin and anandamide, in Ca2+-free media. Competitive antagonists of the TRPV1 cation current, iodo-resiniferatoxin and SB366791, also enhanced GTTR uptake independently of Ca2+, reinforcing these antagonists' potential as latent agonists in specific situations. Ruthenium Red blocked GTTR uptake in the presence or absence of these TRPV1-agonists and antagonists. In addition, GTTR uptake was blocked by RTX in the presence of more physiological levels (2 mM) of Ca2+. Thus gentamicin enters cells via cation channels, and gentamicin uptake can be modulated by regulators of the TRPV1 channel.

Topics: Anilides; Animals; Anti-Bacterial Agents; Arachidonic Acids; Calcium; Calcium Channel Blockers; Cell Line; Cinnamates; Diterpenes; Dogs; Endocannabinoids; Fluorescent Dyes; Gadolinium; Gentamicins; Hydrogen-Ion Concentration; Indicators and Reagents; Ionophores; Kidney Tubules, Distal; Lanthanum; Membrane Potentials; Polyunsaturated Alkamides; Ruthenium Red; TRPV Cation Channels; Valinomycin; Xanthenes

In the isolated rat mesenteric bed, the 1 min perfusion with 100 nm anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 +/- 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5'-iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB(1) receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

Topics: Anilides; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Capsaicin; Cinnamates; Cyclic GMP; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Artery, Superior; Nitric Oxide; Nitroarginine; Perfusion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; RNA, Messenger; TRPV Cation Channels; Vasodilation

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by

Topics: Anilides; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cell Division; Cells, Cultured; Cinnamates; Endocannabinoids; Esters; Glioma; Glycerides; Humans; Ketones; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Solubility; Tumor Cells, Cultured