am-630 and capsazepine

The endocannabinoid system modulates a wide variety of pain conditions. Systemically administered AM404, an endocannabinoid reuptake inhibitor, exerts antinociceptive effects via activation of the endocannabinoid system. However, the mechanism and site of AM404 action are not fully understood. Here, we explored the effect of AM404 on neuropathic pain at the site of the spinal cord.. Male Sprague-Dawley rats were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effects of intrathecal administration of AM404 on mechanical and cold hyperalgesia were examined using the electronic von Frey test and cold plate test, respectively. Motor coordination was assessed using the rotarod test. To understand the mechanisms underlying the action of AM404, we tested the effects of pretreatment with the cannabinoid type 1 (CB. AM404 attenuated mechanical and cold hyperalgesia with minimal effects on motor coordination. AM251 significantly inhibited the antihyperalgesic action of AM404, whereas capsazepine showed a potentiating effect.. These results indicate that AM404 exerts antihyperalgesic effects primarily via CB

Topics: Animals; Arachidonic Acids; Capsaicin; Constriction; Disease Models, Animal; Endocannabinoids; Hyperalgesia; Indoles; Male; Neuralgia; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rotarod Performance Test; Spinal Cord; TRPV Cation Channels

The role of cannabinoids in thrombosis remains controversial. In view of the primary importance of tissue factor (TF) in blood coagulation and its involvement in the pathology of several cardiovascular, inflammatory and neoplastic diseases, a regulation of this initial procoagulant signal seems to be of particular interest. Using human umbilical vein endothelial cells (HUVEC) the present study investigated the impact of the synthetic cannabinoid WIN 55,212-2 on interleukin (IL)-1β-induced TF expression and activity. WIN 55,212-2 caused a time- and concentration-dependent suppression of IL-1β-induced TF protein accompanied by decreases in TF mRNA and activity. Inhibition of TF protein expression by WIN 55,212-2 was mimicked by its cannabinoid receptor-inactive enantiomer WIN 55,212-3 but not by structurally unrelated phyto-, endo- and synthetic cannabinoids. In addition, the inhibitory effect of WIN 55,212-2 was not reversed by antagonists to cannabinoid receptors (CB1, CB2) or transient receptor potential vanilloid 1. Mechanistic approaches revealed WIN 55,212-2 to suppress IL-1β-induced TF expression via inhibition of ceramide formation and via decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases. Further inhibitor experiments demonstrated neutral sphingomyelinase (nSMase) to confer ceramide generation upon IL-1β treatment with the parallel IL-1β-mediated activation of MAPKs occurring via an nSMase-independent pathway. Finally, a receptor-independent inhibition of IL-1β-induced TF protein by WIN 55,212-2 was confirmed in human blood monocytes. Collectively, this data provide a hitherto unknown receptor-independent anticoagulatory action of the cannabinoid WIN 55,212-2.

Topics: Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Capsaicin; Cells, Cultured; Ceramides; Human Umbilical Vein Endothelial Cells; Humans; Indoles; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Monocytes; Morpholines; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Partial Thromboplastin Time; Phosphorylation; Piperidines; Pyrazoles; RNA, Messenger; Signal Transduction; Sphingomyelin Phosphodiesterase; Thromboplastin; TRPV Cation Channels

Endocannabinoids exhibit vasodilatory properties and reduce blood pressure in vivo. However, the influence and mechanism of action of the prominent endocannabinoid, anandamide (AEA), in pulmonary arteries are not known. The present study determined the vascular response to AEA in isolated rat pulmonary arteries. AEA relaxed rat pulmonary arteries that were pre-constricted with U-46619. This relaxation was reduced by the following conditions:removal of the endothelium; in KCl pre-constricted preparations; in the presence of the potassium channel (K(Ca)) blockers, tetraethylammonium and the combination of charybdotoxin and apamin, and the prostacyclin receptor antagonist, RO1138452. Inhibitors of cyclooxygenase (indomethacin), nitric oxide (NO) synthase (N(G)-nitro-l-arginine methyl ester) and fatty acid amide hydrolase (URB597) alone or in combination diminished AEA-induced relaxation in endothelium-intact vessels. The remaining experiments were performed in the presence of URB597 to eliminate the influence of AEA metabolites. Antagonists of the endothelial cannabinoid receptor (CB(x)), O-1918 and cannabidiol, attenuated the AEA-induced response. Antagonists of CB(1), CB(2) and TRPV1 receptors, AM251, AM630 and capsazepine, respectively, did not modify the AEA-induced response. A reference activator of CB(x) receptors, abnormal cannabidiol, mimicked the receptor-mediated AEA effects. The present study demonstrated that AEA relaxed rat pulmonary arteries in an endothelium-dependent fashion via the activation of the O-1918-sensitive CB(x) receptor and/or prostacyclin-like vasoactive products of AEA. One or both of these mechanisms may involve K(Ca) or the NO pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amidohydrolases; Animals; Anisoles; Apamin; Arachidonic Acids; Benzamides; Benzyl Compounds; Cannabinoid Receptor Antagonists; Capsaicin; Carbamates; Charybdotoxin; Cyclohexanes; Endocannabinoids; Endothelium, Vascular; Imidazoles; Indoles; Indomethacin; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Chloride; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Epoprostenol; Tetraethylammonium; TRPV Cation Channels; Vasodilation

Diabetes is often associated with painful neuropathy. The current treatments are symptomatic and ineffective. Cannabinoids have been proposed as promising drugs for chronic pain treatment and its antinociceptive effect has already been related in nerve injury models of neuropathic pain, but little has been investigated in painful diabetic neuropathy models. Thus, the current study aims to investigate the potential antinociceptive effect of drugs that alter endocannabinoid system when injected subcutaneously into the dorsal surface of the ipsilateral hind paw in chemical hyperalgesia induced by formalin in both normoglycemic (Ngl) and streptozotocin-diabetic (Dbt) rats. Diabetic rats exhibited exaggerated flinching behaviors during first and second phases of the formalin test, indicating the presence of hyperalgesia. AM404, an anandamide (AEA) re-uptake inhibitor, AEA (an agonist of CB1/CB2 receptors) or ACEA (a selective CB1 receptor agonist) induced antinociception in both phases of formalin test in Ngl and Dbt rats. In both groups, the antinociceptive effect of ACEA was prevented by AM251, a CB1 inverse agonist while the antinociceptive effect of AEA was prevented by AM251 or AM630, a CB2 receptor antagonist. In Ngl rats, the antinociceptive effect of AM404 was prevented by AM251 or capsazepine only during first phase of the formalin test while in Dbt rats, this effect was blocked by pretreatment with AM251 (both phases) or AM630 (second phase). Taken together, these results demonstrated broad-spectrum antinociceptive properties of cannabinoids in a model of painful diabetic neuropathy. Peripheral activation of both cannabinoid receptors seems to mediate the antinociceptive effect of exogenous or endogenous anandamide.

Topics: Analgesics; Animals; Arachidonic Acids; Behavior, Animal; Capsaicin; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Endocannabinoids; Formaldehyde; Hyperalgesia; Indoles; Male; Pain Measurement; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid

This study investigates the actions of N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (anandamide) on blood flow of the rat knee joint. Topical bolus administration of anandamide (10-1000 nmol) onto the exposed knee joint capsules produced dose-dependent increases in the knee joint blood flow. Various antagonists were tested on the vasodilator response to 100 nmol anandamide. Capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, given at 10 and 100 nmol, suppressed the response by a maximum of 71%. A cannabinoid CB(1) receptor antagonist AM281 (10 nmol) and a CB(2) receptor antagonist AM630 (10 nmol) shortened its duration from 15 min to 5 min. O-1918 (1 nmol), an antagonist of the putative endothelial anandamide/abnormal-cannabidiol receptor, on its own or combined with capsazepine and the two cannabinoid receptor antagonists produced 38% and 24% inhibition on the peak vasodilator response to anandamide, respectively. URB597 (1 nmol), an inhibitor of fatty acid amide hydrolase (FAAH) suppressed the response by 40%, and an anandamide transporter inhibitor [N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] (AM404; 1 nmol) or a cyclo-oxygenase (COX) inhibitor flurbiprofen (20 nmol) abolished the response. These findings suggest the vasodilator action of anandamide in the rat knee joint involved hydrolysis of the compound by FAAH, production of COX-derived eicosanoid(s), activation of TRPV1 receptors, and a small component involved activation of endothelial anandamide/abnormal-cannabidiol receptors; a minor delayed dilator response was mediated by activation of conventional cannabinoid receptors.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Blood Vessels; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Flurbiprofen; Hindlimb; Indoles; Joints; Morpholines; Polyunsaturated Alkamides; Pyrazoles; Rats; Regional Blood Flow; TRPV Cation Channels; Vasodilation

Cannabinoids, such as anandamide, are involved in pain transmission. We evaluated the effects of AM404 (N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), an anandamide reuptake inhibitor, monitoring the expression of c-fos, a marker of activated neurons and the pain-related behaviours using formalin test. The study was carried out in an experimental model of inflammatory pain made by a single injection of formalin in rat hind paws. Formalin test showed that the antinociceptive effect of AM404 was evident in phase I. We found that Fos-positive neurons in dorsal superficial and deep laminae of the lumbar spinal cord increased in formalin-injected animals and that AM404 significantly reduced Fos induction. Co-administration of cannabinoid CB(1) receptor antagonist (AM251), cannabinoid CB(2) receptor antagonist (AM630) and transient receptor potential vanilloid type 1 (TRPV-1) antagonist (capsazepine), attenuate the inhibitory effect of AM404 and this effect was higher using cannabinoid CB(2) and vanilloid TRPV-1 receptor antagonists. These results suggest that AM404 could be a useful drug to reduce inflammatory pain in our experimental model and that cannabinoid CB(2) receptor and vanilloid TRPV-1 receptor, and to a lesser extent, the cannabinoid CB(1) receptor are involved.

Topics: Animals; Arachidonic Acids; Capsaicin; Endocannabinoids; Immunohistochemistry; Indoles; Inflammation; Male; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord; TRPV Cation Channels

Calcitonin gene-related peptide (CGRP) is a sensory neurotransmitter in the rat mesenteric arterial bed. Certain cannabinoids can inhibit, via CB(1) receptors, vasorelaxant responses to electrical field stimulation (EFS) of sensory nerves in the rat mesentery, but the mechanism of the inhibitory effect of the cannabinoid delta 9-tetrahydrocannabinol (THC) is unclear. This study assessed directly the effect of THC on EFS-induced release of CGRP from sensory nerves in the rat mesenteric bed and investigated the possible involvement of cannabinoid receptors and transient receptor potential (TRP) ion channels.. Rat mesenteric beds were perfused with physiological salt solution. Sensory nerves were stimulated electrically and perfusate levels of CGRP measured by immunoassay. The effects of THC on EFS-induced CGRP release and vasorelaxant responses to sensory nerve stimulation were investigated in the absence and presence of cannabinoid antagonists and TRP channel blockers.. EFS evoked a release of CGRP and vasodilatation of the mesenteric beds. THC inhibited the electrically-evoked release of CGRP and sensory neurogenic vasorelaxation. The effect of THC was unaffected by the CB1 antagonist AM251, the CB2 antagonist AM630 or the TRPV1 receptor antagonist capsazepine, but was blocked by the TRP channel blocker ruthenium red.. THC inhibits the EFS-induced release of CGRP (and subsequent vasorelaxation), from capsaicin-sensitive sensory nerves in the rat perfused mesentery. The effect of THC was not mediated by CB1, CB2 or TRPV1 receptors, but was sensitive to ruthenium red, suggesting a possible involvement of TRP ion channels.

Topics: Animals; Calcitonin Gene-Related Peptide; Capsaicin; Dose-Response Relationship, Drug; Dronabinol; Electric Stimulation; Indoles; Male; Mesenteric Arteries; Methoxamine; Neurons, Afferent; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Ruthenium Red; Time Factors; Vasodilation

Hyperdynamic circulation and mesenteric hyperaemia are found in cirrhosis. To delineate the role of endocannabinoids in these changes, we examined the cardiovascular effects of anandamide, AM251 (CB(1) antagonist), AM630 (CB(2) antagonist) and capsazepine (VR1 antagonist), in a rat model of cirrhosis.. Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, diameters of mesenteric arteriole and venule (intravital microscopy), arterial pressure, cardiac output, systemic vascular resistance and superior mesenteric artery (SMA) flow were measured after anandamide, AM251 (with or without anandamide), AM630 and capsazepine administration. CB(1), CB(2) and VR1 receptor expression in SMA was assessed by western blot and RT-PCR.. Anandamide increased mesenteric vessel diameter and flow, and cardiac output in cirrhotic rats, but did not affect controls. Anandamide induced a triphasic arterial pressure response in controls, but this pattern differed markedly in cirrhotic rats. Pre-administration of AM251 blocked the effects of anandamide. AM251 (without anandamide) increased arterial pressure and systemic vascular resistance, constricted mesenteric arterioles, decreased SMA flow and changed cardiac output in a time-dependent fashion in cirrhotic rats. Capsazepine decreased cardiac output and mesenteric arteriolar diameter and flow, and increased systemic vascular resistance in cirrhotic rats, but lacked effect in controls. Expression of CB(1) and VR1 receptor proteins were increased in cirrhotic rats. AM630 did not affect any cardiovascular parameter in either group.. These data suggest that endocannabinoids contribute to hyperdynamic circulation and mesenteric hyperaemia in cirrhosis, via CB(1)- and VR1-mediated mechanisms.

Topics: Animals; Arachidonic Acids; Bile Ducts; Blood Flow Velocity; Blood Pressure; Blotting, Western; Capsaicin; Cardiac Output; Disease Models, Animal; Endocannabinoids; Hyperemia; Indoles; Liver Circulation; Liver Cirrhosis, Biliary; Male; Mesenteric Artery, Superior; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Splanchnic Circulation; Time Factors; TRPV Cation Channels; Vascular Resistance; Vasodilation; Vasodilator Agents

Cannabinoids like anandamide are involved in pain transmission. In this study we evaluated the effects of administrating N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), an inhibitor of anandamide reuptake and monitoring the expression of c-fos, a marker of activated neurons in an experimental model of neuropathic pain (sciatic nerve tying). Fos expression was monitored 14 days after tying of sciatic nerve and 2 h after non-noxious stimulation. We showed that non-noxious stimulation increased Fos-positivity in the dorsal superficial laminae of the lumbar spinal cord of tied animals but not in the control animals. AM404 significantly reduced Fos induction in tied animals. Co-administration of cannabinoid CB1 receptor, cannabinoid CB2 receptor and transient receptor potential vanilloid type 1 (TRPV-1) antagonists reduced the effect of AM404 and this reduction was higher using cannabinoid CB1 receptor antagonist. These results suggest that AM404 could be a useful drug to reduce neuropathic pain and that cannabinoid CB1 receptor, cannabinoid CB2 receptor and vanilloid TRPV-1 receptor are involved.

Topics: Animals; Arachidonic Acids; Capsaicin; Constriction; Dose-Response Relationship, Drug; Endocannabinoids; Immunohistochemistry; Indoles; Male; Physical Stimulation; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Spinal Cord

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.

Topics: Amides; Animals; Arachidonic Acids; Capsaicin; Coronary Vessels; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Ethanolamines; Gap Junctions; Glycyrrhetinic Acid; In Vitro Techniques; Indoles; Indomethacin; Male; Palmitic Acids; Peptides; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Serotonin; Tetraethylammonium; Vasodilation