capsazepine and anandamide

The antiallodynic effect of PhAR-DBH-Me was evaluated on two models of neuropathic pain, and the potential roles of CB1, CB2, and TRPV1 receptors as molecular targets of PhAR-DBH-Me were studied. Female Wistar rats were submitted to L5/L6 spinal nerve ligation (SNL) or repeated doses of cisplatin (0.1 mg/kg, i.p.) to induce experimental neuropathy. Then, tactile allodynia was determined, and animals were treated with logarithmic doses of PhAR-DBH-Me (3.2-100 mg/kg, i.p.). To evaluate the mechanism of action of PhAR-DBH-Me, in silico studies using crystallized structures of CB1, CB2, and TRPV1 receptors were performed. To corroborate the computational insights, animals were intraperitoneally administrated with antagonists for CB1 (AM-251, 3 mg/kg), CB2 (AM-630, 1 mg/kg), and TRPV1 receptors (capsazepine, 3 mg/kg), 15 min before to PhAR-DBH-Me (100 mg/kg) administration. Vagal stimulation evoked on striated muscle contraction in esophagus, was used to elicited pharmacological response of PhAR-DBH-ME on nervous tissue. Systemic administration of PhAR-DBH-Me reduced the SNL- and cisplatin-induced allodynia. Docking studies suggested that PhAR-DBH-Me acts as an agonist for CB1, CB2, and TRPV1 receptors, with similar affinity to the endogenous ligand anandamide. Moreover antiallodynic effect of PhAR-DBH-Me was partially prevented by administration of AM-251 and AM-630, and completely prevented by capsazepine. Finally, PhAR-DBH-Me decreased the vagally evoked electrical response in esophagus rat. Taken together, results indicate that PhAR-DBH-Me induces an antiallodynic effect through partial activation of CB1 and CB2 receptors, as well as desensitization of TRPV1 receptors. Data also shed light on the novel vanilloid nature of the synthetic compound PhAR-DBH-Me.

Topics: Animals; Antineoplastic Agents; Arachidonic Acids; Azabicyclo Compounds; Cannabinoid Receptor Antagonists; Capsaicin; Cisplatin; Endocannabinoids; Female; Hyperalgesia; Injections, Intraperitoneal; Ligation; Models, Animal; Neuralgia; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Spinal Nerves; TRPV Cation Channels; Vagus Nerve Stimulation

The expression level of TRPV1 is high in hippocampus which is a main epileptic area in the brain. In addition to the actions of capsaicin (CAP) and reactive oxygen species (ROS), the TRPV1 channel is activated in neurons by endogenous cannabinoid, anandamide (AEA). In the current study, we investigated the role of inhibitors of TRPV1 (capsazepine, CPZ), AEA transport (AM404), and FAAH (URB597) on the modulation of Ca

Topics: Amidohydrolases; Animals; Apoptosis; Arachidonic Acids; Calcium Signaling; Capsaicin; Cell Line, Tumor; Disease Models, Animal; Endocannabinoids; Hippocampus; Humans; Male; Oxidative Stress; Polyunsaturated Alkamides; Rats; Rats, Wistar; Seizures; TRPV Cation Channels

In this study, a taste bud tissue biosensor was prepared by a starch-sodium alginate cross-linking fixation method. Capsaicin was used as a TRPV1 noxious ion channel activator to investigate the antagonism kinetics of six different substances on capsaicin. The results showed that capsazepine, AMG517, loureirin B, and tetrahydropalmatine were all competitive allosteric regulatory ligands for capsaicin, while aconitine and anandamide were mixed allosteric regulatory ligand that combines non-competition and competition effect. Through analyzing the kinetic parameters of capsaicin and its competitive allosteric regulatory ligands, and comparing the structures between spicy substances and endocannabinoids, the importance of amide groups and similar groups in the allosteric regulation of cannabinoids (CB) receptors and analgesic mechanism was elucidated. This indicates that vanilloid activators turn on the TRPV1 ion channel to transmit only pain and other nociceptive signals, while capsaicin and its competitive ligands are capable of activating intracellular G protein/PI3K/PIP2 signaling pathways by binding to endogenous cannabinoid receptors, and then increase intracellular PIP2 levels (the increasing PIP2 can competitively replace capsaicin and other vanilloid activators), thereby closing the TRPV1 channel and exerting the analgesic effect. The elucidation of this mechanism of pain and analgesia will lay the theoretical foundation and new ideas for investigating nociceptive signal and screening potential analgesic drugs.

Topics: Alginates; Allosteric Regulation; Analgesics; Animals; Arachidonic Acids; Biosensing Techniques; Body Temperature; Capsaicin; Cross-Linking Reagents; Electrochemical Techniques; Endocannabinoids; Fever; Pain; Polyunsaturated Alkamides; Rats, Sprague-Dawley; Starch; Taste Buds; TRPV Cation Channels

In view of the limited information available on functional significance of TRPV1 in regulating sperm functions, present study was undertaken on bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls and were used for molecular and functional characterisation of TRPV1. Immunoblotting using TRPV1 specific antibody revealed the presence of a single band of 104 kDa corresponding to TRPV1 in Hariana bull spermatozoa. Indirect immuno fluorescence revealed positive immune-reactivity to TRPV1 at acrosomal, pre-acrosomal, post acrosomal and flagellar regions of spermatozoa. Based on the results of pilot study dose-response analysis, doses of anandamide (AEA; 0.3 μM) and capsazepine (Cp; 10 μM) were selected for further studies. Three groups of semen samples (control 100 μL diluted semen having 1 × 10

Topics: Acrosome Reaction; Animals; Arachidonic Acids; Calcium Channel Blockers; Capsaicin; Cattle; Endocannabinoids; Male; Polyunsaturated Alkamides; Sperm Capacitation; Sperm Motility; Spermatozoa; TRPV Cation Channels

In the dura mater encephali a significant population of trigeminal afferents coexpress the nociceptive ion channel transient receptor potential vanilloid type 1 (TRPV1) receptor and calcitonin gene-related peptide (CGRP). Release of CGRP serves the central transmission of sensory information, initiates local tissue reactions and may also sensitize the nociceptive pathway. To reveal the possible activation of meningeal TRPV1 receptors by endogenously synthetized agonists, the effects of arachidonylethanolamide (anandamide) and N-arachidonoyl-dopamine (NADA) were studied on dural vascular reactions and meningeal CGRP release.. Changes in meningeal blood flow were measured with laser Doppler flowmetry in a rat open cranial window preparation following local dural applications of anandamide and NADA. The release of CGRP evoked by endovanilloids was measured with ELISA in an in vitro dura mater preparation.. Topical application of NADA induced a significant dose-dependent increase in meningeal blood flow that was markedly inhibited by pretreatments with the TRPV1 antagonist capsazepine, the CGRP antagonist CGRP8-37, or by prior systemic capsaicin desensitization. Administration of anandamide resulted in minor increases in meningeal blood flow that was turned into vasoconstriction at the higher concentration. In the in vitro dura mater preparation NADA evoked a significant increase in CGRP release. Cannabinoid CB1 receptors of CGRP releasing nerve fibers seem to counteract the TRPV1 agonistic effect of anandamide in a dose-dependent fashion, a result which is confirmed by the facilitating effect of CB1 receptor inhibition on CGRP release and its reversing effect on the blood flow.. The present findings demonstrate that endovanilloids are potential activators of meningeal TRPV1 receptors and, consequently the trigeminovascular nocisensor complex that may play a significant role in the pathophysiology of headaches. The results also suggest that prejunctional CB1 receptors may modulate meningeal vascular responses.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cannabinoid Receptor Agonists; Capsaicin; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Dura Mater; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Laser-Doppler Flowmetry; Nociceptors; Peptide Fragments; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Calcitonin Gene-Related Peptide; Regional Blood Flow; Trigeminal Nerve; TRPV Cation Channels

Topics: Animals; Anti-Dyskinesia Agents; Arachidonic Acids; Brain; Cannabidiol; Capsaicin; Cyclooxygenase 2; Dyskinesia, Drug-Induced; Endocannabinoids; Extracellular Signal-Regulated MAP Kinases; Histones; Levodopa; Male; Mice, Inbred C57BL; NF-kappa B; Oxidopamine; Parkinsonian Disorders; Polyunsaturated Alkamides; PPAR gamma; Receptor, Cannabinoid, CB1; TRPV Cation Channels; Tyrosine 3-Monooxygenase

The transient receptor potential vanilloid type 1 (TRPV1) is expressed in the cardiovascular system, and increased TRPV1 expression has been associated with cardiac hypertrophy. Nevertheless, the role of TRPV1 in the pathogenesis of cardiac hypertrophy and the underlying molecular mechanisms remain unclear.. In cultured cardiomyocytes, activation of TRPV1 increased cell size and elevated expression of atrial natriuretic peptide mRNA and intracellular calcium level, which was reversed by TRPV1 antagonist capsazepine. Increased expression of phosphorylated calmodulin-dependent protein kinase IIδ and mitogen-activated protein kinases were found in TRPV1 agonist capsaicin-treated cardiomyocytes. Selective inhibitor of calmodulin-dependent protein kinase IIδ decreased phosphorylation of extracellular signal-regulated kinases and p38. Capsaicin induced an increase in expression of ornithine decarboxylase protein, which is the key enzyme in polyamine biosynthesis in cardiomyocytes. Nevertheless, there was no obvious change of ornithine decarboxylase expression in TRPV1 knockdown cells after capsaicin treatment, and specific inhibitors of calmodulin-dependent protein kinase IIδ or p38 downregulated the capsaicin-induced expression of ornithine decarboxylase. Capsazepine alleviated the increase in cross-sectional area of cardiomyocytes and the ratio of heart weight to body weight and improved cardiac function, including left ventricular internal end-diastolic and -systolic dimensions and ejection fraction and fractional shortening percentages, in mice treated with transverse aorta constriction. Capsazepine also reduced expression of ornithine decarboxylase and cardiac polyamine levels. Transverse aorta constriction induced increases in phosphorylated calmodulin-dependent protein kinase IIδ and extracellular signal-regulated kinases, and p38 and Serca2a were attenuated by capsazepine treatment.. This study revealed that the mitogen-activated protein kinase signaling pathway and intracellular polyamines are essential for TRPV1 activation-induced cardiac hypertrophy.

Topics: Analysis of Variance; Animals; Aorta, Thoracic; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Capsaicin; Cardiomegaly; Cardiotonic Agents; Cells, Cultured; Down-Regulation; Endocannabinoids; Enzyme Inhibitors; Ligation; Male; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Kinases; Myocytes, Cardiac; Polyamines; Polyunsaturated Alkamides; Rats, Wistar; RNA, Small Interfering; Signal Transduction; TRPV Cation Channels

The endocannabinoid and endovanniloid anandamide (AEA) exerts biphasic effects when injected into the dorsolateral periaqueductal grey (dlPAG) in rats submitted to threatening situations. Whereas lower doses of AEA induce anxiolytic-like effects by activating cannabinoid CB1 receptors, no effects are observed with higher doses, possibly due to the simultaneous activation of transient receptor potential vanilloid type 1 (TRPV1) receptors. This activation would facilitate glutamatergic neurotransmission.. Considering that the blockade of TRPV1 or NMDA receptors in the dlPAG induces anxiolytic-like effects, we tested the hypothesis that facilitation of glutamate transmission through TRPV1 is responsible for the lack of anxiolytic-like effect observed with high AEA doses.. Male Wistar rats with a unilateral cannula aimed at the dlPAG received injections of an ineffective dose of AP7 (an NMDA antagonist, 1 nmol) or capsazepine (CPZ, a TRPV1 antagonist, 10 nmol), followed by a high dose of AEA (50 and 200 pmol) and were exposed to the elevated plus maze (EPM) or the Vogel conflict test (VCT).. AP7, CPZ, or AEA did not induce any significant effects when administered alone. However, AP7 or CPZ prior to AEA significantly increased the percentage of entries and time spent in the open arms of EPM and the number of punished licks in the VCT suggesting an anxiolytic-like effect.. These results suggest that the lack of anxiolytic-like effect of higher AEA doses is due to facilitation of glutamate release in the dlPAG, probably via activation of TRPV1 receptors in this structure.

Topics: 2-Amino-5-phosphonovalerate; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Capsaicin; Dose-Response Relationship, Drug; Endocannabinoids; Male; Maze Learning; Periaqueductal Gray; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; 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

Odontoblasts are involved in the transduction of stimuli applied to exposed dentin. Although expression of thermo/mechano/osmo-sensitive transient receptor potential (TRP) channels has been demonstrated, the properties of TRP vanilloid 1 (TRPV1)-mediated signaling remain to be clarified. We investigated physiological and pharmacological properties of TRPV1 and its functional coupling with cannabinoid (CB) receptors and Na(+)-Ca(2+) exchangers (NCXs) in odontoblasts. Anandamide (AEA), capsaicin (CAP), resiniferatoxin (RF) or low-pH evoked Ca(2+) influx. This influx was inhibited by capsazepine (CPZ). Delay in time-to-activation of TRPV1 channels was observed between application of AEA or CAP and increase in [Ca(2+)](i). In the absence of extracellular Ca(2+), however, an immediate increase in [Ca(2+)](i) was observed on administration of extracellular Ca(2+), followed by activation of TRPV1 channels. Intracellular application of CAP elicited inward current via opening of TRPV1 channels faster than extracellular application. With extracellular RF application, no time delay was observed in either increase in [Ca(2+)](i) or inward current, indicating that agonist binding sites are located on both extra- and intracellular domains. KB-R7943, an NCX inhibitor, yielded an increase in the decay time constant during TRPV1-mediated Ca(2+) entry. Increase in [Ca(2+)](i) by CB receptor agonist, 2-arachidonylglycerol, was inhibited by CB1 receptor antagonist or CPZ, as well as by adenylyl cyclase inhibitor. These results showed that TRPV1-mediated Ca(2+) entry functionally couples with CB1 receptor activation via cAMP signaling. Increased [Ca(2+)](i) by TRPV1 activation was extruded by NCXs. Taken together, this suggests that cAMP-mediated CB1-TRPV1 crosstalk and TRPV1-NCX coupling play an important role in driving cellular functions following transduction of external stimuli to odontoblasts.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Signaling; Cannabinoid Receptor Antagonists; Capsaicin; Cyclic AMP; Diterpenes; Endocannabinoids; Glycerides; Hydrogen-Ion Concentration; Odontoblasts; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Sodium-Calcium Exchanger; Thiourea; TRPV Cation Channels

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

Hypothermia occurs in the most severe cases of systemic inflammation, but the mechanisms involved are poorly understood. This study evaluated whether the hypothermic response to bacterial lipopolysaccharide (LPS) is modulated by the endocannabinoid anandamide(AEA) and its receptors: cannabinoid-1 (CB1), cannabinoid-2 (CB2) and transient receptor potential vanilloid-1 (TRPV1). In rats exposed to an ambient temperature of 22◦C, a moderate dose of LPS (25 - 100 μg kg−1 I.V.) induced a fall in body temperature with a nadir at ∼100 minpostinjection. This response was not affected by desensitization of intra-abdominal TRPV1 receptors with resiniferatoxin (20 μg kg - 1 I.P.), by systemic TRPV1 antagonism with capsazepine(40mg kg−1 I.P.), or by systemic CB2 receptor antagonism with SR144528 (1.4 mg kg−1 I.P.).However, CB1 receptor antagonism by rimonabant (4.6mg kg−1 I.P.) or SLV319 (15mg kg−1 I.P.)blocked LPS hypothermia. The effect of rimonabant was further studied. Rimonabant blocked LPS hypothermia when administered I.C.V. at a dose (4.6 μg) that was too low to produce systemic effects. The blockade of LPS hypothermia by I.C.V. rimonabant was associated with suppression of the circulating level of tumour necrosis factor-α. In contrast to rimonabant,the I.C.V. administration of AEA (50 μg) enhanced LPS hypothermia. Importantly, I.C.V. AEAdid not evoke hypothermia in rats not treated with LPS, thus indicating that AEA modulates LPS-activated pathways in the brain rather than thermo effector pathways. In conclusion, the present study reveals a novel, critical role of brain CB1 receptors in LPS hypothermia. Brain CB1 receptors may constitute a new therapeutic target in systemic inflammation and sepsis.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Body Temperature Regulation; Brain; Camphanes; Capsaicin; Disease Models, Animal; Diterpenes; Endocannabinoids; Female; Hypothermia; Injections, Intraperitoneal; Injections, Intravenous; Injections, Intraventricular; Lipopolysaccharides; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Long-Evans; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Signal Transduction; Sulfonamides; Time Factors; TRPV Cation Channels

The contributions of brain cannabinoid (CB) receptors, typically CB1 (CB type 1) receptors, to the behavioral effects of nicotine (NC) have been reported to involve brain transient receptor potential vanilloid 1 (TRPV1) receptors, and the activation of candidate endogenous TRPV1 ligands is expected to be therapeutically effective. In the present study, the effects of TRPV1 ligands with or without affinity for CB1 receptors were examined on NC-induced depression-like behavioral alterations in a mouse model in order to elucidate the "antidepressant-like" contributions of TRPV1 receptors against the NC-induced "depression" observed in various types of tobacco abuse.. Repeated subcutaneous NC treatments (NC group: 0.3 mg/kg, 4 days), like repeated immobilization stress (IM) (IM group: 10 min, 4 days), caused depression-like behavioral alterations in both the forced swimming (reduced swimming behaviors) and the tail suspension (increased immobility times) tests, at the 2 h time point after the last treatment. In both NC and IM groups, the TRPV1 agonists capsaicin (CP) and olvanil (OL) administered intraperitoneally provided significant antidepressant-like attenuation against these behavioral alterations, whereas the TRPV1 antagonist capsazepine (CZ) did not attenuate any depression-like behaviors. Furthermore, the endogenous TRPV1-agonistic CB1 agonists anandamide (AEA) and N-arachidonyldopamine (NADA) did not have any antidepressant-like effects. Nevertheless, a synthetic "hybrid" agonist of CB1 and TRPV1 receptors, arvanil (AR), caused significant antidepressant-like effects. The antidepressant-like effects of CP and OL were antagonized by the TRPV1 antagonist CZ. However, the antidepressant-like effects of AR were not antagonized by either CZ or the CB1 antagonist AM 251 (AM).. The antidepressant-like effects of TRPV1 agonists shown in the present study suggest a characteristic involvement of TRPV1 receptors in NC-induced depression-like behaviors, similar to those caused by IM. The strong antidepressant-like effects of the potent TRPV1 plus CB1 agonist AR, which has been reported to cause part of its TRPV1-mimetic and cannabimimetic effects presumably via non-TRPV1 or non-CB1 mechanisms support a contribution from other sites of action which may play a therapeutically important role in the treatment of NC abuse.

Topics: Animals; Antidepressive Agents; Arachidonic Acids; Capsaicin; Depression; Dopamine; Endocannabinoids; Hindlimb Suspension; Ligands; Male; Mice; Mice, Inbred ICR; Nicotine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Stress, Psychological; Swimming; TRPV Cation Channels

Transient receptor potential vanilloid 1 (TRPV1) channels participate in the modulation of synaptic transmission in the periphery and in central structures. Here, we investigated the role of TRPV1 channels in the control of both excitatory and inhibitory transmission in the striatum. Pharmacological stimulation of TRPV1 channels with capsaicin (10 nM) selectively enhanced the frequency of glutamate-mediated spontaneous (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) recorded from putative striatal medium spiny neurons. Capsaicin-mediated response underwent a rapid rundown, and was no longer detected in the majority of the neurons when the concentration of the drug was in the micromolar range, possibly due to receptor desensitization. Consistently, the totality of striatal neurons responded to capsaicin (10 nM or 10 microM) after prevention of desensitization of TRPV1 channels with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). PMA was able per se to increase sEPSC frequency. The effects of capsaicin and of PMA were absent after pharmacological or genetic inactivation of TRPV1 channels. Finally, we provided evidence for anandamide as an endovanilloid substance in the striatum, since genetic inhibition of anandamide degradation resulted in a tonic activation of TRPV1 channels modulating glutamate but not GABA release. TRPV1-mediated regulation of excitatory transmission in the striatum might be important for the final output to other basal ganglia structures, and might play a role in several physiological and pathological processes.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Capsaicin; Corpus Striatum; Endocannabinoids; gamma-Aminobutyric Acid; Glutamic Acid; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Patch-Clamp Techniques; Polyunsaturated Alkamides; Protein Kinase C; Sensory System Agents; Synaptic Transmission; Tetradecanoylphorbol Acetate; TRPV Cation Channels

Increasing evidence suggests that chronic stress plays an important role in the pathophysiology of several functional gastrointestinal disorders. We investigated whether cannabinoid receptor 1 (CB1) and vanilloid receptor 1 (TRPV1; transient receptor potential vanilloid 1) are involved in stress-induced visceral hyperalgesia.. Male rats were exposed to 1 h water avoidance (WA) stress daily for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Immunofluorescence and western blot analysis were used to assess the expression of CB1 and TRPV1 receptors in dorsal root ganglion (DRG) neurons.. WA stressed rats demonstrated a significant increase in the serum corticosterone levels and faecal pellet output compared to controls supporting stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. The VMR increased significantly at pressures of 40 and 60 mm Hg in WA stress rats compared with controls, respectively, and was associated with hyperalgesia. The endogenous CB1 agonist anandamide was increased significantly in DRGs from stressed rats. Immunofluorescence and western blot analysis showed a significant decrease in CB1 and a reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide in vitro. In contrast, treatment of control DRGs in vitro with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats in situ with WIN 55,212-2 or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the upregulation of TRPV1.. These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Capsaicin; Corticosterone; Defecation; Electromyography; Endocannabinoids; Ganglia, Spinal; Hyperalgesia; Immunohistochemistry; Intestinal Mucosa; Male; Microscopy, Fluorescence; Models, Animal; Morpholines; Naphthalenes; Pain Threshold; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Spectrum Analysis; Stress, Psychological; TRPV Cation Channels

Endogenous analogues of capsaicin, N-acyldopamines, were previously identified from striatal extracts, but the putative presynaptic role of their receptor, the TRPV(1)R (formerly: vanilloid or capsaicin receptor) in the caudate-putamen is unclear. We found that the endogenous TRPV(1)R agonists, N-arachidonoyldopamine (NADA) and oleoyldopamine (OLDA) with EC(50) values in the nanomolar range, as well as the synthetic TRPV(1)R activator 2-aminoethoxydiphenylborane (2APB), and palmytoyldopamine (PALDA, another endogenous N-acyldopamine inactive at the TRPV(1)R), but not capsaicin or other endogenous and synthetic cannabinoids, triggered a rapid Ca(2+) entry with the concomitant stimulation of glutamate and dopamine release. These effects persisted in the TRPV(1)R null-mutant mice, and were insensitive to antagonists of common ionotropic receptors, to several TRPV(1)R antagonists and to the absence of K(+). Furthermore, these N-acyldopamine receptors in glutamatergic and dopaminergic terminals are different based on their different sensitivity to anandamide, capsazepine and Gd(3+) at nanomolar concentrations. Altogether, novel ion channels instead of the TRPV(1)R mediate the presynaptic action of N-acyldopamines in the striatum of adult rodents.

Topics: Animals; Arachidonic Acids; Boron Compounds; Capsaicin; Cations; Corpus Striatum; Dopamine; Endocannabinoids; Glutamic Acid; Ion Channels; Ligands; Male; Mice; Mice, Mutant Strains; Polyunsaturated Alkamides; Presynaptic Terminals; Rats; Rats, Wistar; Synaptosomes; 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

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 ( approximately 56 kDa), TRPV1 ( approximately 95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) ( approximately 46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), approximately 66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.

Topics: Acrosome Reaction; Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Capsaicin; Cricetinae; Endocannabinoids; Humans; Infertility, Male; Male; Phospholipase D; Polyunsaturated Alkamides; Progesterone; Receptor, Cannabinoid, CB1; Spermatozoa; TRPV Cation Channels

Several studies have shown anxiolytic effects of cannabinoids after systemic or central injections. The periaqueductal gray matter is a midbrain structure involved in the control of anxiety states. Intra-cerebral administration of cannabidiol, a phytocannabinoid, or anandamide, an endocannabinoid, into the dorsolateral portion of periaqueductal gray (dlPAG) promotes anxiolytic-like effects in several animal models of anxiety with bell-shaped dose-response curves. The reasons for these curves are still unclear, but since these drugs can also activate TRPV1 receptors and increase glutamate release, we hypothesized that, at high doses, cannabidiol and WIN 55,212-2, a CB1 receptor agonist, could activate TRPV1 receptors, facilitating glutamate neurotransmission and anxiety responses. To test this hypothesis male Wistar rats with cannulae aimed toward the dlPAG were submitted to the following intra-dlPAG treatments: Experiment 1. Vehicle (0.2 microL) or WIN 55,212-2 (3-30 pmol); Experiment 2. Capsazepine (CPZ, 10 nmol, a TRPV1 receptor antagonist) or vehicle followed, 5 min later, by vehicle or WIN 55, 212-2 (10 or 30 pmol); Experiment 3. CPZ (10 nmol) or vehicle followed, 5 min later, by cannabidiol (30 or 60 nmol). Ten minutes after the last injection the animals were tested in the elevated plus maze (EPM). WIN 55,212-2 and cannabidiol induced anxiolytic effects at lower doses that disappeared at the higher dose. Although CPZ+WIN 10 or CPZ+WIN 30 pmol groups were not different from control (CPZ+V), capsazepine prevented the decrease in open arm exploration caused by the higher of dose of WIN 55,212-2 (30 nmol) relative to the lower dose of WIN 55,212-2 (10 nmol) and, in the case of cannabidiol (60 nmol), increased open arm exploration (V+CBD 60 group versus CPZ+CBD 60 group). These results suggest that TRPV1 receptors in the dlPAG modulate anxiety and that activation of these receptors by high doses of cannabinoids could be involved in the bell-shaped dose-response curves observed with these compounds.

Topics: Analgesics; Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Capsaicin; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Male; Microinjections; Morpholines; Naphthalenes; Periaqueductal Gray; Polyunsaturated Alkamides; Rats; Rats, Wistar; Time Factors; TRPV Cation Channels

While it is well known that the endogenous cannabinoid receptor ligand anandamide also activates the transient receptor potential vanilloid1 (TRPV1) receptors, there has been no in vivo study indicating the role of the TRPV1 receptors in the antinociceptive effect of anandamide at spinal level. The goal of this study was to determine the effect of inhibition of TRPV1 receptors by capsazepine on the antinociceptive potency of anandamide after intrathecal administration. Anandamide alone (1, 30 or 100 microg) dose-dependently decreased carrageenan-induced thermal hyperalgesia, however, the highest dose caused temporary excitation and vocalization, suggesting the pain-inducing potential of anandamide. Capsazepine (10 or 20 microg) by itself did not change the pain sensitivity markedly, but the lower dose increased it, and the higher dose decreased the antinociceptive effect of 30 microg anandamide. Furthermore, both doses of capsazepine decreased the efficacy of the largest dose of anandamide. These results show that TRPV1 receptor activation plays a substantial role in the antinociceptive effects of anandamide at spinal level. The effect of the inhibition on TRPV1 receptors depended on the dose applied. We presume that coactivation of the cannabinoid and TRPV1 receptors by anandamide provides elevated antinociception through the release of antinociceptive endogenous ligands at spinal level.

Topics: Analgesics; Animals; Arachidonic Acids; Capsaicin; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Hyperalgesia; Injections, Spinal; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Spinal Cord; TRPV Cation Channels

Migraine is a common and disabling neurological disorder that involves activation or the perception of activation of the trigeminovascular system. Cannabinoid (CB) receptors are present in brain and have been suggested to be antinociceptive. Here we determined the effect of cannabinoid receptor activation on neurons with trigeminovascular nociceptive input in the rat. Neurons in the trigeminocervical complex (TCC) were studied using extracellular electrophysiological techniques. Responses to both dural electrical stimulation and cutaneous facial receptive field activation of the ophthalmic division of the trigeminal nerve and the effect of cannabinoid agonists and antagonists were studied. Nonselective CB receptor activation with R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) (WIN55,212; 1 mg kg(-1)) inhibited neuronal responses to A-(by 52%) and C-fiber (by 44%) afferents, an effect blocked by the CB(1) receptor antagonist SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 3 mg kg(-1)] but not the CB2 receptor antagonist AM630 (6-iodopravadoline; 3 mg kg(-1)). Anandamide (10 mg kg(-1)) was able to inhibit both A- and C-fiber-elicited TCC firing, only after transient receptor potential vanilloid 1 receptor inhibition. Activation of cannabinoid receptors had no effect on cutaneous receptive fields when recorded from TCC neurons. The data show that manipulation of CB1 receptors can affect the responses of trigeminal neurons with A- and C-fiber inputs from the dura mater. This may be a direct effect on neurons in the TCC itself or an effect in discrete areas of the brain that innervate these neurons. The data suggest that CB receptors may have therapeutic potential in migraine, cluster headache, or other primary headaches, although the potential hazards of psychoactive side effects that accompany cannabinoid treatments may be complex to overcome.

Topics: Animals; Arachidonic Acids; Benzoxazines; Blood Pressure; Capsaicin; Endocannabinoids; Male; Migraine Disorders; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Trigeminal Nerve; TRPV Cation Channels

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

This study was designed to examine the role of the endocannabinoids in blood pressure regulation during high sodium (HS) intake. HS (4% Na+ by weight) intake for 3 weeks increased baseline mean arterial pressure (MAP, mm Hg) compared with normal sodium (NS, 0.4% Na+ by weight)-treated male Wistar rats. Capsazepine (3 mg/kg), a selective transient receptor potential vanilloid type 1 (TRPV1) antagonist, caused a greater increase in MAP (mm Hg) in HS-treated compared with NS-treated rats (13+/-3 versus 4+/-2, p<0.05), whereas calcitonin gene-related peptide (CGRP) dose-dependently decreased MAP in both HS- and NS-treated rats with a more profound effect in the former. HS increased plasma anandamide levels analyzed by liquid chromatography/electrospray tandem mass spectrometry (NS, 2.40+/-0.31 versus HS, 4.05+/-0.47 pmol/ml, p<0.05) and plasma CGRP levels determined by radioimmunoassay (NS, 36.6+/-3.8 versus HS, 55.7+/-6.4 pg/ml, p<0.05). Methanandamide, a metabolically stable analog of anandamide, caused a greater CGRP release in mesenteric arteries isolated from HS-treated compared with NS-treated rats. Western blot showed that expression of receptor activity-modifying protein 1, a subunit of the CGRP receptor, in mesenteric arteries was greater in HS-treated compared with NS-treated rats. These results show that HS intake increases production of anandamide, which may serve as an endovanilloid to activate TRPV1, leading to release of CGRP to blunt salt-induced increases in blood pressure. These data support the notion that TRPV1 may act as a molecular target for salt-induced elevation of endovanilloid compounds to regulate blood pressure.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Cannabinoid Receptor Modulators; Capsaicin; Endocannabinoids; Hypertension; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Sodium Chloride, Dietary; TRPV Cation Channels

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.

Topics: Arachidonic Acids; Calcium; Calcium Signaling; Capsaicin; Cell Culture Techniques; Cell Death; Cell Line, Tumor; Cell Proliferation; Diterpenes; Dopamine; Endocannabinoids; Humans; Models, Biological; Neuroblastoma; Neurons; Norepinephrine; Polyunsaturated Alkamides; Recombinant Proteins; Synaptic Transmission; Transfection; TRPV Cation Channels; Up-Regulation

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

As an endogenous agonist at the cannabinoid receptor CB1 and the capsaicin-receptor TRPV1, anandamide may exert both anti- and pronociceptive actions. Therefore we studied the effects of anandamide and other activators of both receptors on changes in free cytosolic calcium ([Ca(2+)](i)) in acutely dissociated small dorsal root ganglion neurons (diameter: < or =30 microm). Anandamide (10 microM) increased [Ca(2+)](i) in 76% of the neurons. The EC(50) was 7.41 microM, the Hill slope was 2.15 +/- 0.43 (mean +/- SE). This increase was blocked by the competitive TRPV1-antagonist capsazepine (10 microM) and in Ca(2+)-free extracellular solution. Neither exclusion of voltage-gated sodium channels nor additional blockade of voltage-gated calcium channels of the L-, N-, and/or T-type, significantly reduced the anandamide-induced [Ca(2+)](i) increase or capsaicin-induced [Ca(2+)](i) transients (0.2 microM). The CB1-agonist HU210 (10 microM) inhibited the anandamide-induced rise in [Ca(2+)](i). Conversely, the CB1-antagonist AM251 (3 microM) induced a leftward shift of the concentration-response relationship by approximately 4 microM (P < 0.001; Hill slope, 2.17 +/- 0.75). Intracellular calcium transients in response to noxious heat (47 degrees C for 10 s) were highly correlated with the anandamide-induced [Ca(2+)](i) increases (r = 0.84, P < 0.001). Heat-induced [Ca(2+)](i) transients were facilitated by preincubation with subthreshold concentrations of anandamide (3 microM), an effect that was further enhanced by 3 microM AM251. Although anandamide acts on both TRPV1 and CB1 receptors in the same nociceptive DRG neurons, its pronociceptive effects dominate. Anandamide triggers an influx of calcium through TRPV1 but no intracellular store depletion. It facilitates the heat responsiveness of TRPV1 in a calcium-independent manner. These effects of anandamide differ from those of the classical exogenous TRPV1-agonist capsaicin and suggest a primarily modulatory mode of action of anandamide.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Calcium; Calcium Channel Blockers; Capsaicin; Chelating Agents; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Egtazic Acid; Endocannabinoids; Extracellular Fluid; Ganglia, Spinal; Hot Temperature; Neurons; Physical Stimulation; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; TRPV Cation Channels

To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult.. Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used.. In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively).. The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Cell Count; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Hydrolysis; Intraocular Pressure; Male; Ocular Hypertension; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Thy-1 Antigens; TRPV Cation Channels

The endocannabinoid system mediates in the pharmacological actions of ethanol and genetic studies link endocannabinoid signaling to alcoholism. Drugs activating cannabinoid CB1 receptors have been found to promote alcohol consumption but their effects on self-administration of alcohol are less clear because of the interference with motor performance. To avoid this problem, a novel pharmacological approach to the study of the contribution of the cannabinoid system in alcoholism may be to use drugs that locally amplify the effects of alcohol on endogenous cannabinoids. In the present study we addressed this model by studying the effects of the anandamide transport inhibitor N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404) on both ethanol self-administration and reinstatement of alcohol-seeking behavior in rats. The results show that AM404 significantly reduced ethanol self-administration in a dose-dependent manner but failed to modify reinstatement for lever pressing induced by the stimulus associated with alcohol. This effect was not due to a motor depressant effect and was not related to a decrease in general motivational state, as it was not effective in other reward paradigms such as lever pressing for a saccharin solution. The mechanism of action of AM404 does not involve cannabinoid CB1 receptors as the CB1-selective antagonist SR141716A did not block the reduction of ethanol self-administration induced by the anandamide uptake blocker. Moreover, 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol (HU-210), a classical cannabinoid receptor agonist, did not affect ethanol self-administration. The effects of AM404 are not mediated by either vanilloid VR1 receptors or cannabinoid CB2 receptors because it is not antagonized by either the VR1 receptor antagonist capsazepine or the CB2 antagonist AM630. These results indicate that AM404 may be considered as an innovative approach to reduce alcohol consumption.

Topics: Alcohol Drinking; Animals; Arachidonic Acids; Capsaicin; Central Nervous System Depressants; Conditioning, Operant; Depression, Chemical; Endocannabinoids; Ethanol; Extinction, Psychological; Hypothermia; Male; Motor Activity; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reinforcement, Psychology; Saccharin; Self Administration; TRPV Cation Channels

Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta.. Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide.. Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide.. Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.

Topics: Animals; Aorta, Abdominal; Apamin; Arachidonic Acids; Benzamides; Calcitonin Gene-Related Peptide; Camphanes; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Charybdotoxin; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Indomethacin; Isoindoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Sulfonamides; Vasodilation

Levodopa is the most commonly prescribed drug for Parkinson's disease (PD). Although levodopa improves PD symptoms in the initial stages of the disease, its long-term use is limited by the development of side effects, including abnormal involuntary movements (dyskinesias) and psychiatric complications. The endocannabinoid system is emerging as an important modulator of basal ganglia functions and its pharmacologic manipulation represents a promising therapy to alleviate levodopa-induced dyskinesias. Rats with 6-OHDA lesions that are chronically treated with levodopa develop increasingly severe axial, limb, locomotor and oro-facial abnormal involuntary movements (AIMs). Administration of the cannabinoid agonist WIN 55,212-2 attenuated levodopa-induced axial, limb and oral AIMs dose-dependently via a CB(1)-mediated mechanism, whereas it had no effect on locomotive AIMs. By contrast, systemic administration of URB597, a potent FAAH inhibitor, did not affect AIMs scoring despite its ability to increase anandamide concentration throughout the basal ganglia. Unlike WIN, anandamide can also bind and activate transient receptor potential vanilloid type-1 (TRPV1) receptors, which have been implicated in the modulation of dopamine transmission in the basal ganglia. Interestingly, URB597 significantly decreased all AIMs subtypes only if co-administered with the TRPV1 antagonist capsazepine. Our data indicate that pharmacological blockade of TRPV1 receptors unmasks the anti-dyskinetic effects of FAAH inhibitors and that CB(1) and TRPV1 receptors play opposite roles in levodopa-induced dyskinesias.

Topics: Amidohydrolases; Animals; Antiparkinson Agents; Arachidonic Acids; Basal Ganglia; Benzamides; Benzoxazines; Cannabinoids; Capsaicin; Carbamates; Drug Therapy, Combination; Dyskinesia, Drug-Induced; Endocannabinoids; Levodopa; Male; Morpholines; Naphthalenes; Oxidopamine; Parkinson Disease, Secondary; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

Knockout (KO) mice invalidated for the dopamine transporter (DAT) constitute a powerful animal model of neurobiological alterations associated with hyperdopaminergia relevant to schizophrenia and attention-deficit/hyperactivity disorder (ADHD).. Because of continuously increasing evidence for a neuromodulatory role of endocannabinoids in dopamine-related pathophysiological responses, we assessed endocannabinoid signaling in DAT KO mice and evaluated the ability of endocannabinoid ligands to normalize behavioral deficits, namely spontaneous hyperlocomotion in these mice.. In DAT KO mice, we found markedly reduced anandamide levels, specifically in striatum, the dopamine nerve terminal region. Furthermore, three distinct indirect endocannabinoid agonists, the selective anandamide reuptake inhibitors AM404 and VDM11 and the fatty acid amidohydrolase inhibitor AA5HT, attenuated spontaneous hyperlocomotion in DAT KO mice. The hypolocomotor effects of AM404, VDM11, and AA5HT were significantly attenuated by co-administration of the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine but not the selective cannabinoid type 1 (CB1)receptor antagonist AM251. Interestingly, TRPV1 binding was increased in the striatum of DAT KO mice, while CB1 receptor binding was unaffected.. These data indicate a dysregulated striatal endocannabinoid neurotransmission associated with hyperdopaminergic state. Restoring endocannabinoid homeostasis in active synapses might constitute an alternative therapeutic strategy for disorders associated with hyperdopaminergia. In this process, TRPV1 receptors seem to play a key role and represent a novel promising pharmacological target.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Attention Deficit Disorder with Hyperactivity; Cannabinoid Receptor Modulators; Capsaicin; Corpus Striatum; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Endocannabinoids; Mice; Mice, Knockout; Motor Activity; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Serotonin; Signal Transduction; TRPV Cation Channels

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

We recently demonstrated that activation of the pulmonary sensory neurons plays a critical role in prevention of endotoxin-induced shock by releasing calcitonin gene-related peptide (CGRP) in rats. CGRP increased the endothelial production of prostacyclin (PGI(2)) in the lungs, thereby preventing endotoxin-induced shock response by inhibiting tumor necrosis factor-alpha (TNF-alpha) production. Since antithrombin (AT) enhances sensory neuron activation, we hypothesized that AT might reduce endotoxin-induced hypotension by enhancing the activation of pulmonary sensory neurons in rats. We examined this possibility using a rat model of endotoxin shock. AT-induced effects including reduction of hypotension (n = 5) and inhibition of induction of iNOS (n = 4 or 5) and TNF- alpha (n = 5) in the lungs of endotoxin-treated animals were completely reversed by pretreatment with capsazepine (CPZ) (n = 4 or 5), a vanilloid receptor antagonist, or CGRP(8-37), a CGRP receptor antagonist (n = 4 or 5). AT enhanced endotoxin-induced increases in lung tissue levels of CGRP (n = 4), but this effect of AT was not seen in animals pretreated with CPZ (n = 4). CGRP produced therapeutic effects (n = 5) similar to those induced by AT, and such therapeutic effects were completely abrogated by pretreatment with indomethacin (n = 4). AT increased CGRP release from cultured dorsal root ganglion neurons only in the presence of anandamide (n = 5), and AT-induced increase in CGRP release was not observed in the presence KT5720, an inhibitor of protein kinase A (n = 5). AT markedly increased intracellular levels of cAMP in the presence of anandamide (n = 5). These results strongly suggested that AT might reduce endotoxin-induced hypotension in rats by enhancing activation of sensory neurons via activation of protein kinase A.

Topics: Animals; Antithrombins; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Capsaicin; Carbazoles; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase Inhibitors; Disease Models, Animal; Endocannabinoids; Endotoxins; Ganglia, Spinal; Gene Expression Regulation; Hypotension; Indoles; Indomethacin; Lung; Male; Neurons, Afferent; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Peptide Fragments; Polyunsaturated Alkamides; Protein Kinase Inhibitors; Pyrroles; Rats; Rats, Wistar; Receptors, Calcitonin Gene-Related Peptide; RNA, Messenger; TRPV Cation Channels; Tumor Necrosis Factor-alpha

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit fatty-acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of anandamide, an endocannabinoid. It has been suggested that the mechanisms of action of NSAIDs could be due to inhibition of cyclooxygenase (COX) and also to an increase in endocannabinoid concentrations. In a previous study we have demonstrated that the local analgesic interaction between anandamide and ibuprofen (a non-specific COX inhibitor) was synergistic for the acute and inflammatory phases of the formalin test. To test this hypothesis further, we repeated similar experiments with rofecoxib (a selective COX-2 inhibitor) and also measured the local concentrations of anandamide, and of two fatty-acid amides, oleoylethanolamide and palmitoylethanolamide. We established the ED(50) for anandamide (34.52 pmol+/-17.26) and rofecoxib (381.72 pmol+/-190.86) and showed that the analgesic effect of the combination was synergistic. We also found that paw tissue levels of anandamide, oleoylethanolamide and palmitoylethanolamide were significantly higher when anandamide was combined with NSAIDs and that this effect was greater with rofecoxib. In conclusion, local injection of anandamide or rofecoxib was antinociceptive in a test of acute and inflammatory pain and the combination of anandamide with rofecoxib was synergistic. Finally, locally injected anandamide with either NSAID (ibuprofen or rofecoxib) generates higher amount of fatty-acid ethanolamides. The exact comprehension of the mechanisms involved needs further investigation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Capsaicin; Chromatography, High Pressure Liquid; Cyclooxygenase 2 Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Edema; Endocannabinoids; Formaldehyde; Ibuprofen; Lactones; Male; Mass Spectrometry; Nitrobenzenes; Pain Measurement; Peripheral Nervous System; Polyunsaturated Alkamides; Rats; Rats, Wistar; Sulfonamides; Sulfones; TRPV Cation Channels

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

Anandamide (AEA), one of the endocannabinoid compounds, has an important regulatory function by serving as an autocrine/paracrine or endocrine factor throughout the body via activation of the cannabinoid receptor 1 (CB1) and/or the transient receptor potential vanilloid type 1 (TRPV1) channels. However, the role of AEA in the regulation of renal excretory function is largely unknown. The present study was designed to test the hypothesis that intrarenal administration of AEA enhances renal excretory function leading to a decrease in blood pressure.. A metabolically stable analog of AEA, methanandamide (MethA, 300 nmol/kg per min), was infused into the left renal medulla of anesthetized Wistar rats with or without a selective TRPV1 antagonist, capsazepine (Capz, 150 nmol/kg per min) or a selective cannabinoid receptor 1 (CB1) antagonist, AM251 (Am, 150 nmol/kg per min). Ureters were cannulated for collection of urine. A laser-Doppler flowmeter was used to determine the changes of blood flow in the cortex and medulla of the infused kidney.. In the absence of the changes in cortical and medullary blood flow, unilateral intramedullary infusion of MethA significantly increased urine flow rate by 64% ipsilaterally and 62% contralaterally without changing sodium excretion when compared to vehicle controls (P < 0.01). Neither Capz nor Am blocked the MethA-induced increases in urine flow rate bilaterally. Intramedullary infusion of MethA significantly decreased mean arterial pressure (MAP) (P < 0.01), which was blocked by Am but not Capz. Denervation of the infused kidney blocked the MethA-induced increases in urine flow rate bilaterally without altering MethA-induced decreases in MAP.. Therefore, our data show that intramedullary infusion of AEA increases urine volume excretion and decreases blood pressure via distinct operational mechanisms. While activation of the CB1 receptor may underlie AEA-induced depressor effects, the diuretic effect of AEA appears to be mediated by neuronal reflex of the kidney, which is not sensitive to blockade of the TRPV1 or CB1 receptor.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcium Channel Blockers; Capsaicin; Diuresis; Dose-Response Relationship, Drug; Endocannabinoids; Kidney; Kidney Medulla; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

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

TRPV1 is a ligand-gated ion channel whose activation by capsaicin increases intracellular Ca(2+) ([Ca(2+)](i)). TRPV1 and cannabinoid CB(1) receptor activation are capable of eliciting analgesia. In this study, using recombinant human (h) and rat (r) TRPV1 receptors expressed in HEK293 cells, we have performed a comparison of both TRPV1 species at 22 and 37 degrees C and compared endo- and exocannabinoid activity at both receptors.. [Ca(2+)](i) was measured in Fura-2-loaded HEK293(hTRPV1) and HEK293(rTRPV1) cells. To assess native CB(1) receptor activity, [(35)S]GTPgammaS binding to membranes prepared from rat cerebellum was measured.. Both capsaicin (pEC(50) rat approximately 6.9 and pEC(50) human approximately 6.8 at 37 degrees C) and anandamide (pEC(50) rat approximately 5.3 and pEC(50) human approximately 5.8 at 37 degrees C) produced a concentration-dependent increase in [Ca(2+)](i) in rat and human systems and at 22 and 37 degrees C. In HEK293(rTRPV1) cells, anandamide appeared to be a partial agonist. Capsazepine demonstrated competitive antagonism at both human and rat TRPV1 receptors and at both temperatures studied. Capsazepine effects were not temperature dependent: pK(B) at rTRPV1 was 5.98 at 22 degrees C and 6.02 at 37 degrees C, and pK(B) at hTRPV1 was 6.76 at 22 degrees C and 6.75 at 37 degrees C. However, there was a consistent 6-fold increase in capsazepine potency for hTRPV1 relative to rTRPV1. The exocannabinoid Delta(9)-tetrahydrocannabinol failed to increase [Ca(2+)](i), although its solvent ethanol was an effective TRPV1 activator. In the [(35)S]GTPgammaS binding assay using rat cerebellar membranes, anandamide (pEC(50) approximately 5.8) and Delta(9)-tetrahydrocannabinol (pEC(50) approximately 7.1), but not capsaicin, stimulated binding. Delta(9)-tetrahydrocannabinol was a partial agonist. pEC(50) values for anandamide at rTRPV1 and rCB(1) were similar.. There were small differences in the pharmacology of rat and human TRPV1 receptors. Whilst capsaicin activated TRPV1 and Delta(9)-tetrahydrocannabinol activated CB(1), anandamide is an endogenous agonist for both receptor systems.

Topics: Animals; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Capsaicin; Cells, Cultured; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ion Channels; Polyunsaturated Alkamides; Rats; Recombinant Proteins; Species Specificity; Temperature; TRPV Cation Channels

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Topics: Animals; Apamin; Arachidonic Acids; Azepines; Barium Compounds; Biological Factors; Calcium; Cannabinoid Receptor Modulators; Capsaicin; Charybdotoxin; Chlorides; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Female; In Vitro Techniques; Indoles; Male; Mesenteric Arteries; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Vasodilation; Verapamil

The endocannabinoid anandamide is an emerging potential signalling molecule in the cardiovascular system. Anandamide causes vasodilatation, bradycardia and hypotension in animals and has been implicated in the pathophysiology of endotoxic, haemorrhagic and cardiogenic shock, but its vascular effects have not been studied in man. Human forearm blood flow and skin microcirculatory flow were recorded using venous occlusion plethysmography and laser-Doppler perfusion imaging (LDPI), respectively. Each test drug was infused into the brachial artery or applied topically on the skin followed by a standardized pin-prick to disrupt the epidermal barrier. Anandamide failed to affect forearm blood flow when administered intra-arterially at infusion rates of 0.3-300 nmol min(-1). The highest infusion rate led to an anandamide concentration of approximately 1 microM in venous blood as measured by mass spectrometry. Dermal application of anandamide significantly increased skin microcirculatory flow and coapplication of the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine inhibited this effect. The TRPV1 agonists capsaicin, olvanil and arvanil all induced concentration-dependent increases in skin blood flow and burning pain when administered dermally. Coapplication of capsazepine inhibited blood flow and pain responses to all three TRPV1 agonists. This study shows that locally applied anandamide is a vasodilator in the human skin microcirculation. The results are consistent with this lipid being an activator of TRPV1 on primary sensory nerves, but do not support a role for anandamide as a circulating vasoactive hormone in the human forearm vascular bed.

Topics: Adult; Arachidonic Acids; Benzylamines; Capsaicin; Endocannabinoids; Female; Forearm; Humans; Laser-Doppler Flowmetry; Male; Microcirculation; Middle Aged; Muscle, Skeletal; Plethysmography; Polyunsaturated Alkamides; Regional Blood Flow; Skin; TRPV Cation Channels

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

Recent research in our laboratory has demonstrated that stress activates an endogenous cannabinoid mechanism that suppresses sensitivity to pain [Nature 435 (2005) 1108]. In this work, CB(1) antagonists administered systemically blocked stress-induced analgesia induced by brief, continuous foot-shock. The present studies were conducted to examine the role of cannabinoid CB(1) receptors in the brainstem rostral ventromedial medulla (RVM) and midbrain dorsolateral periaqueductal gray (PAG) in cannabinoid stress-induced analgesia (SIA). Pharmacological blockade of vanilloid TRPV1 receptors with capsazepine, administered systemically, did not alter cannabinoid SIA, suggesting that cannabinoid SIA was not dependent upon TRPV1. Microinjection of the competitive CB(1) antagonist rimonabant (SR141716A) into either the RVM or dorsolateral PAG suppressed stress antinociception in this model. Rimonabant was maximally effective following microinjection into the dorsolateral PAG. The fatty-acid amide hydrolase (FAAH) inhibitor arachidonoyl serotonin (AA-5-HT) was subsequently used to block hydrolysis of endocannabinoids and enhance SIA. Systemic and site-specific injections of AA-5-HT into either the dorsolateral PAG or RVM induced CB(1)-mediated enhancements of SIA. Palmitoyltrifluoromethylketone, a potent inhibitor of FAAH and phospholipase A2 activity, administered systemically, exerted similar effects. In all conditions, the antinociceptive effects of each FAAH inhibitor were completely blocked by coadministration of the CB(1) antagonist rimonabant. The present results provide evidence that a descending cannabinergic neural system is activated by environmental stressors to modulate pain sensitivity in a CB(1)-dependent manner.

Topics: Amidohydrolases; Analgesia; Animals; Arachidonic Acids; Cannabinoids; Capsaicin; Carrier Proteins; Cytosol; Endocannabinoids; Male; Medulla Oblongata; Mice; Microinjections; Pain Measurement; Periaqueductal Gray; Phospholipases A; Phospholipases A2; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Stress, Psychological; TRPV Cation Channels

This study was designed to test the hypothesis that increased sensitivity of blood pressure to anandamide (AEA), an endocannabinoid compound, occurs during high-salt intake, which can be blocked by a selective vanilloid receptor 1(VR1) antagonist, capsazepine (CAPZ). Intravenous administration of a metabolically stable analog, methanandamide (MethA), dose-dependently decreased mean arterial pressure (MAP) in conscious rats fed a high-sodium diet (HS) for 3 weeks but it had a minimal effect in normal sodium (NS)-treated rats. The MethA-induced decrease in MAP was significantly attenuated but not abolished by CAPZ, or a selective cannabinoid receptor 1 (CB1) antagonist, SR141716A, administered separately in HS-treated rats. The MethA-induced depressor effect was prevented by the combined administration of CAPZ and SR141716A in HS-treated rats. Likewise, administration of capsaicin, a selective VR1 receptor agonist, dose-dependently decreased MAP in both HS- and NS-treated rats. The depressor effect of capsaicin was more profound in HS-treated rats, which was prevented by CAPZ. Western blot showed that expression of VR1 but not CB1 in mesenteric arteries was increased in HS-treated compared with NS-treated rats. Therefore, these data show that: (1) HS upregulates mesenteric VR1 expression; (2) HS increases sensitivity of blood pressure to AEA; and (3) HS-induced enhancement of the depressor effect of AEA can be prevented only when both VR1 and CB1 receptors are blocked. These results indicate that AEA contributes to the prevention of salt induced increases in blood pressure via, at least in part, activating the VR1 receptor.

Topics: Animals; Arachidonic Acids; Blood Pressure; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Dose-Response Relationship, Drug; Drug Combinations; Endocannabinoids; Male; Mesenteric Arteries; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Sodium, Dietary; Time Factors; TRPV Cation Channels

The endogenous ligand, anandamide activates at least two receptors on nociceptors; the excitatory vanilloid type 1 transient receptor potential receptor, the activity of which is indispensable for the development and maintenance of inflammatory heat hyperalgesia, and the inhibitory cannabinoid 1 receptor, the activity of which reduces that pathological pain sensation. Recent data are equivocal on whether increasing anandamide levels at the peripheral terminals of nociceptors in pathological conditions increases or decreases inflammatory heat hyperalgesia. Here, by using the cobalt-uptake technique we examined whether vanilloid type 1 transient receptor potential receptor activity evoked by 10 nM-100 microM anandamide is increased or decreased in inflammatory conditions. An inflammatory milieu for cultured rat primary sensory neurons was established by incubating the cells in the presence of the inflammatory mediators, bradykinin and prostaglandin E2. Anandamide, similarly to the archetypical vanilloid type 1 transient receptor potential receptor agonist, capsaicin induced concentration-dependent cobalt-uptake in a proportion of neurons. However, the potency of anandamide was significantly lower than that of capsaicin. While pre-incubation of cultures with bradykinin and prostaglandin E2 alone did not evoke cobalt-entry, the inflammatory mediators potentiated the effect of both capsaicin and anandamide. Application of the competitive vanilloid type 1 transient receptor potential receptor antagonist, capsazepine, or inhibitors of protein kinase A, protein kinase C or phospholipase C inhibited the anandamide-evoked cobalt-uptake both in the presence and absence of bradykinin and prostaglandin E2. These findings show that inflammatory mediators significantly increase the excitatory potency and efficacy of anandamide on vanilloid type 1 transient receptor potential receptor, thus, increasing the anandamide concentration in, or around the peripheral terminals of nociceptors might rather evoke than decrease inflammatory heat hyperalgesia.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Bradykinin; Capsaicin; Cells, Cultured; Cobalt; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Endocannabinoids; Enzyme Inhibitors; Inflammation Mediators; Neurons, Afferent; Nociceptors; Polyunsaturated Alkamides; Protein Kinase C; Rats; Rats, Sprague-Dawley; TRPV Cation Channels; Type C Phospholipases

1. Peripheral cannabinoids have been shown to suppress nociceptive neurotransmission in a number of behavioral and neurophysiological studies. It is not known, however, whether cannabinoids exert this action through direct interactions with nociceptors in the periphery and/or if other processes are involved. To gain a better understanding of the direct actions of cannabinoid-vanilloid agonists on sensory neurons, we examined the effects of these compounds on trigeminal ganglion (TG) neurons in vitro. 2. AEA (EC(50)=11.0 microM), NADA (EC(50)=857 nM) and arachidonyl-2-chloroethylamide ACEA (EC(50)=14.0 microM) each evoked calcitonin gene-related peptide (CGRP) release from TG neurons. The TRPV1 antagonists iodo-resiniferatoxin (I-RTX) and capsazepine (CPZ) each obtunded AEA-, NADA-, ACEA- and capsaicin (CAP)-evoked CGRP release with individually equivalent IC(50)'s for each of the compounds (I-RTX IC(50) range=2.6-4.0 nM; CPZ IC(50) range=523-1140 microM). 3. The pro-inflammatory mediator prostaglandin E(2) significantly increased the maximal effect of AEA-evoked CGRP release without altering the EC(50). AEA, ACEA and CAP stimulated cAMP accumulation in TG neurons in a calcium- and TRPV1-dependent fashion. Moreover, the protein kinase inhibitor staurosporine significantly inhibited AEA- and CAP-evoked CGRP release. 4. The pungency of AEA, NADA, ACEA and CAP in the rat eye-wipe assay was also assessed. Interestingly, when applied intraocularly, NADA or CAP each produced nocifensive responses, while AEA or ACEA did not. 5. Finally, the potential inhibitory effects of these cannabinoids on TG nociceptors were evaluated. Neither AEA nor ACEA decreased CAP-evoked CGRP release. Furthermore, neither of the cannabinoid receptor type 1 antagonists SR141716A nor AM251 had any impact on either basal or CAP-evoked CGRP release. AEA also did not inhibit 50 mM K(+)-evoked CGRP release and did not influence bradykinin-stimulated inositol phosphate accumulation. 6. We conclude that the major action of AEA, NADA and ACEA on TG neurons is excitatory, while, of these, only NADA is pungent. These findings are discussed in relation to our current understanding of interactions between the cannabinoid and vanilloid systems and nociceptive processing in the periphery.

Topics: Aminobutyrates; Animals; Arachidonic Acid; Arachidonic Acids; Calcitonin Gene-Related Peptide; Calcium Channels; Capsaicin; Dinoprostone; Diterpenes; Dopamine; Endocannabinoids; Ganglia, Spinal; Male; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Drug; Staurosporine; Trigeminal Ganglion; TRPC Cation Channels; TRPV Cation Channels

1. We have investigated the effects of the endocannabinoid anandamide (AEA) on neuronal excitability and vanilloid TRPV1 receptors in neonatal rat cultured dorsal root ganglion neurones. 2. Using whole-cell patch-clamp electrophysiology, we found that AEA inhibits high-voltage-activated Ca(2+) currents by 33+/-9% (five out of eight neurones) in the absence of the CB(1) receptor antagonist SR141716A (100 nM) and by 32+/-6% (seven out of 10 neurones) in the presence of SR141716A. 3. Fura-2 fluorescence Ca(2+) imaging revealed that AEA produced distinct effects on Ca(2+) transients produced by depolarisation evoked by 30 mM KCl. In a population of neurones of larger somal area (372+/-20 microM(2)), it significantly enhanced Ca(2+) transients (80.26+/-13.12% at 1 microM), an effect that persists after pertussis toxin pretreatment. In a population of neurones of smaller somal area (279+/-18 microM(2)), AEA significantly inhibits Ca(2+) transients (30.75+/-3.54% at 1 microM), an effect that is abolished by PTX pretreatment. 4. Extracellular application of 100 nM AEA failed to evoke TRPV1 receptor inward currents in seven out of eight neurones that responded to capsaicin (1 microM), with a mean inward current of -0.94+/-0.21 nA. In contrast, intracellular application of 100 nM AEA elicited robust inward currents in approximately 62% of neurones, the mean population response was -0.85+/-0.21 nA. When AEA was applied to the intracellular environment with capsazepine (1 microM), the mean population inward current was -0.01+/-0.01 nA. Under control conditions, mean population current fluctuations of -0.09+/-0.05 nA were observed.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Calcium Channels; Calcium Signaling; Capsaicin; Cells, Cultured; Drug Synergism; Endocannabinoids; Fura-2; Ganglia, Spinal; Neurons, Afferent; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Chloride; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, Drug; Rimonabant; TRPV Cation Channels

The administration of the endocannabinoid anandamide to rats produces hypokinesia in parallel to a decrease in the activity of nigrostriatal dopaminergic neurons. It was earlier hypothesized that this effect was mediated through the activation of CB(1) receptors, although these receptors have not been found in dopaminergic neurons, but in striatal projection neurons connected with them. However, two recent discoveries: (i) that anandamide is also able to activate vanilloid VR(1) receptors, and (ii) that VR(1) receptors are located on nigrostriatal dopaminergic neurons, allow to re-evaluate this hypothesis and suggest that the activation of vanilloid-like receptors rather than CB(1) receptors might be responsible of anandamide-induced hypokinesia and decreased nigrostriatal dopaminergic activity. To validate this new hypothesis, we carried out two different experiments. First, we explored whether the inhibitory effects of anandamide on motor activity and dopaminergic transmission were reversed by capsazepine, an antagonist for vanilloid-like receptors. Our data demonstrated that anandamide reduced ambulation, stereotypies and exploration, measured in the open-field test, whereas it increased the time spent in inactivity. All these effects were completely reversed by capsazepine, which had no effect by itself. Anandamide also caused a significant decrease in nigrostriatal dopaminergic activity, reflected by a reduction in DOPAC contents in the caudate-putamen, which was also reversed by capsazepine. As a second objective, we explored whether anandamide is able to directly influence nigrostriatal dopaminergic function by examining its effects on in vitro dopamine (DA) release using perifused striatal fragments. Our data confirmed that anandamide significantly decreased K(+)-stimulated dopamine release from nigrostriatal terminals and that this effect was vanilloid-like receptor-mediated since it was prevented by capsazepine. This in vitro inhibitory effect was not seen with a classic cannabinoid agonist that does not bind vanilloid-like receptors. In summary, anandamide behaves as a hypokinetic substance, thus producing motor depression in the open-field test, presumably related to a decrease in nigrostriatal dopaminergic activity. These effects were completely reversed by the vanilloid-like receptor antagonist capsazepine, thus indicating a role of these receptors, which are located on dopaminergic neurons, in mediating hypokinetic effects of anandamide. In

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Arachidonic Acids; Behavior, Animal; Cannabinoid Receptor Modulators; Capsaicin; Chromatography, High Pressure Liquid; Corpus Striatum; Dopamine; Drug Interactions; Electrochemistry; Endocannabinoids; In Vitro Techniques; Ion Channels; Male; Motor Activity; Neurons; Polyunsaturated Alkamides; Potassium; Rats; Rats, Wistar; Receptors, Drug; Stereotyped Behavior; TRPV Cation Channels; Tyrosine 3-Monooxygenase

Anandamide (0.01 to 10 microM) caused greater concentration-dependent reductions of the contractile-induced responses to noradrenaline in female than in male mesenteric vascular beds isolated from adult Sprague-Dawley rats. Greater relaxant responses in females were also induced by the vanilloid TRPV1 receptor agonist capsaicin (0.01 to 10 microM), whereas no sex differences were observed for the relaxations caused by either acetylcholine or sodium nitroprusside. The effect of anandamide in either sex was reduced by the vanilloid TRPV1 receptor antagonist capsazepine but not by the cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A). In males, the anandamide-induced relaxations were potentiated by in vitro exposure during 5 min to 0.5 microM 17beta-oestradiol and unmodified by the protein synthesis inhibitor cycloheximide. The vasorelaxant effects of anandamide in female rats were decreased by ovariectomy. This decrease was prevented by in vivo treatment with 17beta-oestradiol-3-benzoate (450 microg/kg i.m., once a week during 3 weeks) and counteracted by in vitro exposure to oestrogen. In vivo treatment with 17beta-oestradiol also potentiated anandamide-induced responses in males. In conclusion, this study shows an oestrogen-dependent sensitivity to the vanilloid TRPV1 receptor-mediated vasorelaxant effects of anandamide in the mesenteric vasculature of Sprague-Dawley rats, that could be mediated by both genomic and non-genomic mechanisms.

Topics: Acetylcholine; Animals; Arachidonic Acids; Argentina; Capsaicin; Chile; Cycloheximide; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Estradiol; Estrogens; Female; Male; Mesentery; Muscle, Smooth, Vascular; Nitroprusside; Norepinephrine; Ovariectomy; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Sex Characteristics; Time Factors; Vasodilation

In 84% of substantia gelatinosa (SG) neurones examined in adult rat spinal cord slices, an anandamide transport inhibitor, AM404, increased the frequency of spontaneous excitatory postsynaptic currents in a manner similar to that of capsaicin. AM404 was without actions in the presence of a vanilloid TRPV1 receptor antagonist, capsazepine. We conclude that AM404 enhances the spontaneous release of L-glutamate by activating TRPV1 receptors in the SG.

Topics: Anesthetics, Local; Animals; Arachidonic Acids; Biological Transport; Capsaicin; Drug Interactions; Endocannabinoids; Glutamic Acid; Male; Neurons; Polyunsaturated Alkamides; Rats; Receptors, Drug; Substantia Gelatinosa; Tetrodotoxin

The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

Topics: Arachidonic Acids; Blood-Brain Barrier; Brain; Camphanes; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Capsaicin; Cell Adhesion Molecules; Cells, Cultured; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Glycerides; Humans; Ion Channels; Microcirculation; Microfilament Proteins; Phosphoproteins; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; TRPV Cation Channels

The role of anandamide in the development of inflammatory hyperalgesia and visceral hyperreflexia was studied in the rat urinary bladder. Animals were given intraperitoneal cyclophosphamide injection, which evokes painful hemorrhagic cystitis accompanied by increased bladder reflex activity. The vanilloid receptor 1 [transient receptor potential vanilloid 1 (TRPV1)] antagonist capsazepine, applied onto the serosal surface of bladders, significantly reduced the hyperreflexia. Mass spectrometric analysis revealed that cyclophosphamide injection significantly and persistently increased the anandamide content of bladder tissues. The increase in the anandamide content paralleled the development of reflex hyperactivity. Anandamide (1-100 microm), applied onto the serosal surface of naive bladders, increased the reflex activity in a concentration-dependent manner. Repeated anandamide applications did not produce desensitization of the response. The anandamide-evoked effect was blocked by capsazepine or by instillation of resiniferatoxin, the ultrapotent TRPV1 agonist, into the bladders 24 hr before the anandamide challenge. The cannabinoid 1 receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] significantly increased the potency of anandamide in enhancing bladder reflex activity in naive but not in cyclophosphamide-injected animals. Application of the fatty acid amide hydrolyze inhibitor palmitoylisopropylamine onto the serosal surface of bladders also increased the reflex activity both in naive and cyclophosphamide-injected rats. This latter effect in naive animals was blocked by capsazepine and by resiniferatoxin pretreatment. Finally, intravesical instillation of anandamide (50 microm) increased c-fos expression in the spinal cord, which was reduced by capsazepine or by resiniferatoxin pretreatment. These results suggest that anandamide, through activating TRPV1, contributes to the development of hyperreflexia and hyperalgesia during cystitis.

Topics: Acrolein; Animals; Arachidonic Acids; Capsaicin; Cyclophosphamide; Cystitis; Endocannabinoids; Female; Hydrolysis; Ion Channels; Pain; Polyunsaturated Alkamides; Posterior Horn Cells; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Reflex, Abnormal; TRPV Cation Channels; Urinary Bladder

Most actions of anandamide (AEA) are mediated by the cannabinoid 1 (CB(1)) receptor activation, but on sensory neurones it is also an agonist on the vanilloid subtype 1 receptor (VR(1)). The aim of the present study was to analyse the effect of AEA (10(-6)-10(-4) M) on inhibitory CB(1) and excitatory VR(1) receptors by measuring sensory neuropeptide release such as somatostatin, substance P and calcitonin gene-related peptide, from isolated rat tracheae. AEA (10(-6) M) vas without significant effect, 10(-5) M inhibited neuropeptide release, which was abolished by the G protein-coupled receptor blocker pertussis toxin (100 ng/ml) and the CB(1) receptor antagonist SR141716A (5x10(-7) M). High concentrations of AEA (5x10(-5) M, 10(-4) M) increased the release of the peptides and this inhibition was prevented by the competitive VR(1) antagonist capsazepine (10(-5) M). These results indicate a dual, concentration-dependent action of AEA on CB(1) receptors and VR(1) on peripheral sensory nerve terminals.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Capsaicin; Dose-Response Relationship, Drug; Endocannabinoids; In Vitro Techniques; Male; Neuropeptides; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Reference Values; Reproducibility of Results; Rimonabant; Sensitivity and Specificity; Somatostatin; Substance P; Trachea

Whole-cell patch-clamp recordings were made from neurons in the trigeminal nucleus caudalis and trigeminal ganglion, in vitro, to investigate the cellular actions of the endogenous cannabinoid, anandamide. Anandamide has been shown to act through both the cannabinoid receptor 1 (CB1) and the vanilloid receptor 1 (VR1). Anandamide (30 microM) caused a 54 % increase in the rate of miniature excitatory post-synaptic currents (mEPSCs), without affecting their amplitude. The effect of anandamide was blocked by the VR1 antagonist capsazepine (20 microM), but not by the CB1-specific antagonist AM251 (3 microM). Application of the VR1 receptor agonist capsaicin (300 nM) caused a 4200 % increase in the mEPSC rate. In dissociated trigeminal ganglion neurons, both anandamide and capsaicin caused an outward current in neurons that were voltage clamped at +40 mV. The maximal outward current produced by anandamide (EC50, 10 microM) was 45 % of that produced by capsaicin (10 microM). Co-application of the VR1 antagonist capsazepine (30 microM) completely reversed the effects of both capsaicin and anandamide. The anandamide transport inhibitor, AM404 (30 microM) caused a 40 % increase in mEPSC rate in the slice preparation and an outward current in dissociated neurons. The latter current was reversed by the VR1 antagonist iodoresiniferatoxin (1 microM). The fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (20 microM) and OL53 (1 microM) did not enhance the effect of anandamide in either the slice or dissociated neuron preparations. These results suggest that within the superficial medullary dorsal horn, anandamide (30 microM) acts presynaptically to enhance the release of glutamate via activation of the VR1 receptor.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Capsaicin; Electric Stimulation; Electrophysiology; Endocannabinoids; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; In Vitro Techniques; Medulla Oblongata; Membrane Potentials; Nerve Endings; Patch-Clamp Techniques; Polyunsaturated Alkamides; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Trigeminal Ganglion

The effects of arvanil (N-arachidonoyl-vanillyl-amine), a structural hybrid between capsaicin and anandamide, on ion currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. Arvanil (0.2-50 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Arvanil produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of arvanil-induced inhibition of I(Ca,L) was 2 microM. Arvanil (5 microM) could shift the steady-state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. No effect of arvanil (20 microM) on delayed rectifier K(+) current (I(K(DR))) was observed; however, capsaicin (20 microM), glyceryl nonivamide (20 microM) and capsinolol (20 microM) suppressed it significantly. Arvanil (20 microM) caused a slight reduction in the amplitude of erg (ether-à-go-go-related)-mediated K(+) current (I(K(erg))) without modifying the activation curve of this current, while capsaicin and glyceryl nonivamide were more effective in suppressing I(K(erg)). Under current-clamp configuration, arvanil decreased the firing frequency of action potentials. Arvanil-mediated inhibition of I(Ca,L) appeared to be independent of its binding to either vanilloid or cannabinoid receptors. The channel-blocking properties of arvanil may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.

Topics: Action Potentials; Animals; Arachidonic Acids; Calcium Channels, L-Type; Capsaicin; Cell Line; Delayed Rectifier Potassium Channels; Drug Interactions; Endocannabinoids; Glycerol; Hybrid Cells; Mice; Neurons; omega-Conotoxin GVIA; Pertussis Toxin; Polyunsaturated Alkamides; Potassium Channels; Potassium Channels, Voltage-Gated; Rats

To test the hypothesis that activation of the vanilloid receptor (VR1) contributes to the anandamide-induced depressor effect in spontaneously hypertensive rats (SHR), we used a selective VR1 antagonist capsazepine (CAPZ) and a selective cannabinoid type 1 receptor antagonist SR141716A in conjunction with a VR1 agonist capsaicin in both SHR and Wistar-Kyoto rats (WKY). Mean arterial pressure was increased in SHR compared with WKY (P<0.05). Intravenous administration of capsaicin caused a greater depressor response in SHR compared with WKY (P<0.05), which was blocked by approximately 60% by CAPZ (P<0.05) in SHR only. Methanandamide caused a similar greater depressor response (P<0.05), which was blocked by approximately 50% and 60% by CAPZ and SR141716A, respectively, in SHR (P<0.05) but not in WKY. Radioimmunoassay showed that methanandamide increased plasma calcitonin gene-related peptide (CGRP) levels from baseline in both SHR and WKY (P<0.05), with no difference between 2 strains. Western blot showed that protein expression for the calcitonin receptor-like receptor-but not receptor activity modifying protein 1, VR1, and cannabinoid type 1 receptors-was increased in mesenteric resistance arteries in SHR compared with WKY (P<0.05). These data indicate that in addition to activation of cannabinoid type 1, anandamide may serve as an endogenous compound to stimulate VR1, leading to a decrease in blood pressure via CGRP release from sensory nerve terminals. Increased mesenteric CGRP receptor expression in SHR may account for increased sensitivity of blood pressure to anandamide and may serve as a compensatory response to buffer the increase in blood pressure in SHR.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Capsaicin; Endocannabinoids; Hypertension; Male; Mesenteric Arteries; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Growing evidence regarding the function of vanilloid receptor-1 (VR1) in the brain suggests potential central roles of this receptor, previously described to occur primarily in peripheral sensory neurons. In the present study, we used electrophysiological and biochemical techniques to investigate the function and the endogenous stimulation of VR1 in dopaminergic neurons of the substantia nigra pars compacta (SNc). The VR1 agonist capsaicin increased the frequency of both TTX-sensitive and -insensitive spontaneous EPSCs (sEPSCs) without affecting their amplitude, suggesting a presynaptic site of action. In contrast, no effect was detected with regard to GABAergic transmission. No increase in sEPSC frequency was observed in the presence of cadmium chloride, while the voltage-dependent calcium channel antagonist omega-conotoxin MVIIC did not prevent capsaicin action. The VR1 antagonists capsazepine and iodoresiniferatoxin (IRTX) blocked the effects of capsaicin. Importantly, IRTX per se reduced sEPSC frequency, suggesting a tonic activity of VR1. The endogenous VR1 agonist anandamide (AEA) produced an IRTX-sensitive increase in the frequency of sEPSCs on dopaminergic neurons that was more pronounced when protein kinase A had been activated. Furthermore, mass spectrometric analyses and binding experiments revealed high levels of endogenous AEA and specific binding of AEA to VR1 receptors in the SNc. These data suggest a tonic facilitation of glutamate release exerted by VR1 in the SNc through a stimulation of VR1 by endovanilloids, including anandamide. The increase in sEPSC frequency by VR1 onto midbrain dopaminergic neurons suggests the involvement of these receptors in motor and cognitive functions involving the dopaminergic system.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Blockers; Capsaicin; Diterpenes; Dopamine; Dose-Response Relationship, Drug; Endocannabinoids; Excitatory Postsynaptic Potentials; GABA Antagonists; Glutamic Acid; In Vitro Techniques; Mesencephalon; Neurons; Patch-Clamp Techniques; Polyunsaturated Alkamides; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, Drug; RNA, Messenger; Substantia Nigra; Synapses; Synaptic Transmission; Tetrodotoxin

This study examined the effect of intrathecal (i.t.) injection of the endocannabinoid anandamide in urethane-anesthetized rats. The tip of the i.t. cannula was positioned at the T(12)-L(1) level of the spinal cord. Either anandamide or its metabolically stable analogue methanandamide (25 to 100 nmol) produced dose-dependent decreases in the blood pressure that persisted at least for up to 30 min. The hypotensive responses to 100 nmol anandamide and to 100 nmol methanandamide were -17.7+/-1.6 mmHg ( n=5) and -17.9+/-2.0 mmHg ( n=4), respectively. Hypotensive effects were also obtained with the CB(1) cannabinoid receptor agonist WIN 55212-2 (20 nmol; i.t.) as well as with the vanilloid VR(1) receptor agonist capsaicin (3 nmol; i.t.). Nicotinic ganglionic blockade with hexamethonium bromide [10 mg/kg; intravenous(i.v.)] abolished the responses to both anandamide and capsaicin. The i.t. administration of the CB(1) receptor antagonist, 20 nmol SR 141716A, as well as the VR(1) receptor antagonist, 20 nmol capsazepine, prevented almost completely the hypotensive responses to both anandamide and methanandamide. SR 141716A prevented the hypotension caused by WIN 55212-2 but did not modify the response to the vanilloid receptor agonist capsaicin. On the contrary, capsazepine antagonized the hypotension caused by capsaicin but failed to affect the decrease in blood pressure caused by the CB1 cannabinoid receptor agonist WIN 55212-2. These results suggest that anandamide could modulate the blood pressure through the activation of cannabinoid CB(1) receptors and vanilloid VR(1) receptors localized at the spinal cord.

Topics: Anesthesia, Intravenous; Anesthetics, Intravenous; Animals; Antihypertensive Agents; Arachidonic Acids; Benzoxazines; Blood Pressure; Calcium Channel Blockers; Capsaicin; Dose-Response Relationship, Drug; Endocannabinoids; Ganglionic Blockers; Heart Rate; Hemodynamics; Hexamethonium; Injections, Spinal; Male; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Drug; Rimonabant; Spinal Cord; Urethane

Nerve growth factor (NGF) is central to processes involved in an inflammatory hyperalgesia. Administration of exogenous NGF induces a hyperalgesia that is dependent on local neutrophil influx. The effects of administration of the cannabinoid anandamide and the cannabimimetic palmitoylethanolamide on an NGF-induced hyperalgesia and neutrophil accumulation were examined in this study.. Baseline hind limb withdrawal latencies to a noxious heat stimulus were recorded before intraplantar administration of NGF (1 microg in 0.05 ml) to the hind paw of 75 male Wistar rats. Anandamide or palmitoylethanolamide (a substance that has cannabinoid-like actions but little affinity for cannabinoid receptors) at doses of 10 and 25 mg/kg were given (intraperitoneally) immediately after NGF. CB1 (SR141716A) and CB2 (SR144528) receptor antagonists were coadministered with the higher dose of cannabinoids. Withdrawal latencies were expressed as difference from baseline. Seventy rats received intraplantar NGF and intraperitoneal treatments. Neutrophil accumulation in the injected paw was assessed using a myeloperoxidase assay.. Administration of NGF reduced latencies consistent with hyperalgesia. Anandamide and palmitoylethanolamide significantly reduced this hyperalgesia. The action of anandamide was CB1 receptor-mediated. SR144528 abrogated the action of palmitoylethanolamide. NGF also provoked neutrophil accumulation in the injected paw, denoted by an increase in myeloperoxidase. Palmitoylethanolamide significantly reduced neutrophil accumulation by an SR144528-sensitive action, whereas anandamide was without effect.. NGF induced a thermal hyperalgesia that was attenuated by anandamide and palmitoylethanolamide. Only palmitoylethanolamide reduced neutrophil influx. Thus, cannabinoids show a neuronal CB1 receptor-mediated antihyperalgesic action and a separate inhibition of a proinflammatory neuroimmune process. Such a mechanism suggests a therapeutic site of analgesic action separable from central side effects.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoids; Capsaicin; Cell Movement; Endocannabinoids; Hyperalgesia; Male; Nerve Growth Factor; Neutrophils; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Rimonabant

Anandamide acts as a full vanilloid receptor agonist in many bioassay systems, but it is a weak activator of primary afferents in the airways. To address this discrepancy, we compared the effect of different vanilloid receptor agonists in isolated airways and mesenteric arteries of guinea pig using preparations containing different phenotypes of the capsaicin-sensitive sensory nerve. We found that anandamide is a powerful vasodilator of mesenteric arteries but a weak constrictor of main bronchi. These effects of anandamide are mediated by vanilloid receptors on primary afferents and do not involve cannabinoid receptors. Anandamide also contracts isolated lung strips, an effect caused by the hydrolysis of anandamide and subsequent formation of cyclooxygenase products. Although capsaicin is equally potent in bronchi and mesenteric arteries, anandamide, resiniferatoxin, and particularly olvanil are significantly less potent in bronchi. Competition experiments with the vanilloid receptor antagonist capsazepine did not provide evidence of vanilloid receptor heterogeneity. Arachidonoyl-5-methoxytryptamine (VDM13), an inhibitor of the anandamide membrane transporter, attenuates responses to olvanil and anandamide, but not capsaicin and resiniferatoxin, in mesenteric arteries. VDM13 did not affect responses to these agonists in bronchi, suggesting that the anandamide membrane transporter is absent in this phenotype of the sensory nerve. Computer simulations using an operational model of agonism were consistent, with differences in intrinsic efficacy and receptor content being responsible for the remaining differences in agonist potency between the tissues. This study describes differences between vanilloid receptor agonists regarding tissue selectivity and provides a conceptual framework for developing tissue-selective vanilloid receptor agonists devoid of bronchoconstrictor activity.

Topics: Animals; Arachidonic Acids; Bronchi; Bronchial Arteries; Calcium Channel Blockers; Capsaicin; Computer Simulation; Endocannabinoids; Guinea Pigs; Lung; Male; Mesenteric Arteries; Polyunsaturated Alkamides; Receptors, Drug

1. Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea-pigs, and its effect on VR1 receptor function in isolated primary guinea-pig nodose ganglia neurons. 2. Anandamide (0.3-3 mg.ml(-1)), when given by aerosol, induced cough in conscious guinea-pigs in a concentration dependent manner. When guinea-pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide-induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide-induced cough. These results indicate that anandamide-induced cough is mediated through the activation of VR1 receptors. 3. Anandamide (10-100 micro M) increased intracellular Ca(2+) concentration estimated by Fluo-4 fluorescence change in isolated guinea-pig nodose ganglia cells. The anandamide-induced Ca(2+) response was inhibited by two different VR1 antagonists: capsazepine (1 micro M) and iodo-resiniferatoxin (I-RTX, 0.1 micro M), indicating that anandamide-induced Ca(2+) response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 micro M) and CB2 (SR 144528, 0.1 micro M) receptor antagonists had no effect on Ca(2+) response to anandamide. 4. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea-pig nodose ganglia cells and induces cough through activation of VR1 receptors.

Topics: Aerosols; Animals; Arachidonic Acids; Calcium; Camphanes; Capsaicin; Cells, Cultured; Consciousness; Cough; Dronabinol; Endocannabinoids; Excitatory Amino Acid Antagonists; Guinea Pigs; Male; Nodose Ganglion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

This study was carried out to determine the effect of intravenous injection of anandamide on pulmonary C-fibre afferents and the cardiorespiratory reflexes. In anaesthetized, spontaneously breathing rats, intravenous bolus injection of anandamide near the right atrium immediately elicited the pulmonary chemoreflex responses, characterized by apnoea, bradycardia and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C-fibres, anandamide no longer evoked these reflex responses. In open-chest, and artificially ventilated rats, anandamide injection evoked an abrupt and intense discharge in vagal pulmonary C-fibres in a dose-dependent manner. After injection of the high dose, the fibre discharge generally started within 1 s, reached a peak in approximately 2 s, and returned to baseline within 7 s. The stimulation of C-fibres by anandamide was completely and reversibly blocked by pretreatment with capsazepine, a competitive antagonist of the vanilloid type 1 receptor. Anandamide (0.4 mg kg(-1)) stimulated approximately 93 % of pulmonary C-fibres that were activated by capsaicin at a much lower dose (0.6 microg kg(-1)); the response to anandamide showed similar intensity, but had slightly longer latency and duration than that to capsaicin. In conclusion, intravenous bolus injection of anandamide evokes a consistent and distinct stimulatory effect on pulmonary C-fibre terminals, and this effect appears to be mediated through an activation of the vanilloid type 1 receptor.

Topics: Afferent Pathways; Animals; Arachidonic Acids; Capsaicin; Cardiovascular System; Chemoreceptor Cells; Dose-Response Relationship, Drug; Electrophysiology; Endocannabinoids; Lung; Male; Morpholines; Nerve Fibers; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Drug; Reflex; TRPV Cation Channels; Vagus Nerve

The effects of the endogenous cannabinoid anandamide were studied on peripheral, polymodal nociceptors recorded from normal and chronically inflamed (Freund's adjuvant) knee joint afferents in rats anaesthetized with pentobarbitone. Anandamide (860 nmol) caused a rapid, short lasting excitation of a sub-population of capsaicin-sensitive nociceptive afferents in normal knee joints (7.2+/-2.3 impulses s(-1); n=15 units from five animals). In arthritic joints there were 9.7+/-3.0 impulses s(-1) (n=11 from six animals), which was not significantly different from normal joints. The excitation was dose dependent (8.6 - 2900 nmol) and mediated by activation of the vanilloid receptor (VR(1)) as it was abolished by the VR1 antagonist capsazepine (1 mg kg(-1)). Our results show that anandamide, at high doses, can activate nociceptive afferents innervating the rat knee joints, in contrast with its widely described analgesic actions.

Topics: Animals; Arachidonic Acids; Arthritis, Experimental; Calcium Channel Blockers; Capsaicin; Disease Models, Animal; Drug Interactions; Endocannabinoids; Knee Joint; Male; Nociceptors; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Drug; Treatment Outcome; TRPV Cation Channels

1. This study was directed at exploring the structure-activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB(1) and vanilloid receptors in the mouse vas deferens. 2. pK(i) values for displacement of [(3)H]-resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N-(4-hydroxyphenyl)-arachidonylamide (AM404; 6.18) and N-(3-methoxy-4-hydroxy)benzyl-arachidonylamide (arvanil; 6.77). 3. pEC(50) values for stimulating (45)Ca(2+) uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC(50)=7.37), a compound with the same substituted benzyl polar head group as arvanil. 4. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically-evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pK(B)=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 microM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pK(B)=6.02) and less susceptible to antagonism by SR141716A (pK(B)=8.66) than methanandamide (pK(B)=9.56). WIN55212 was antagonized by SR141716A (pK(B)=9.02) but not by capsazepine (10 microM). 5. In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB(1) receptors, both receptor types appear to contribute to anandamide-induced inhibition of evoked contractions.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Biological Transport; Calcium; Calcium Channel Blockers; Cannabinoids; Capsaicin; CHO Cells; Cricetinae; Drug Interactions; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Male; Mice; Morpholines; Muscle Contraction; Naphthalenes; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Structure-Activity Relationship; Transfection; TRPV Cation Channels; Vas Deferens

Recently, the cannabinoid (CB) receptor agonist anandamide (AEA) has been shown to excite perivascular terminals of primary sensory neurons via activation of the vanilloid receptor-1 (VR-1). To determine whether AEA stimulates central terminals of these neurons, via VR-1 activation, we studied the release of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities (LI) from slices of rat dorsal spinal cord. Mobilization of Ca(2+) in rat dorsal root ganglion (DRG) neurons in culture was also studied. AEA (0.1-10 micrometer) increased the outflow of CGRP-LI and SP-LI from slices of the rat dorsal spinal cord in a Ca(2+)-dependent manner and increased [Ca(2+)](i) in capsaicin-sensitive cultured DRG neurons. Both effects of AEA were abolished by capsaicin pretreatment and by the VR-1 antagonist capsazepine but not affected by the CB receptor antagonists AM281 or AM630. Both neuropeptide release and Ca(2+) mobilization induced by electrical field stimulation (EFS) were inhibited by a low concentration of AEA (10 nm). Inhibition by AEA of EFS-induced responses was reversed by AM281 and AM630, but was not affected by capsazepine. Results indicate that stimulation of VR-1 with high concentrations of AEA excites central terminals of capsaicin-sensitive DRG neurons, thus causing neuropeptide release in the dorsal spinal cord. This novel activity opposes the CB receptor-mediated inhibitory action of low concentrations AEA. However, only if large amounts of endogenous AEA could be produced at the level of the dorsal spinal cord, they may not inhibit, but rather activate, nociceptive sensory neurons.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Calcium; Capsaicin; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Ganglia, Spinal; In Vitro Techniques; Male; Neurons, Afferent; Polyunsaturated Alkamides; Presynaptic Terminals; Rats; Receptors, Cannabinoid; Receptors, Drug; Spinal Cord; Substance P; TRPV Cation Channels

The possibility that anandamide is an endothelium-derived hyperpolarizing factor was explored in the rat mesenteric vasculature by use of conventional microelectrode techniques. In the main mesenteric artery, anandamide and its more stable analog methanandamide hardly caused a measurable change in membrane potential of the smooth muscle cells, which promptly hyperpolarized to EDHF liberated by acetylcholine. Inhibition of endogenous anandamide breakdown by phenylmethylsulfonyl fluoride did not increase membrane responses to acetylcholine. The CB(1) receptor antagonist SR141716 did not significantly influence EDHF-mediated hyperpolarization except at extremely high concentrations. Smooth muscle cells of third to fourth order branches of the mesenteric artery, which have a more negative resting membrane potential and show smaller responses to acetylcholine, hyperpolarized by about 6 mV to both anandamide and methanandamide, whereas another CB(1) receptor agonist, WIN 55,212-2, had no effect. Mechanical endothelium removal or pre-exposure to SR141716A did not affect anandamide- and methanandamide-induced hyperpolarizations. However, in the presence of capsazepine, a selective vanilloid receptor antagonist, these membrane potential changes were reversed to a small depolarization, whereas EDHF-induced hyperpolarizations were not affected. Pretreating small vessels with capsaicin, causing desensitization of vanilloid receptors and/or depletion of sensory neurotransmitter, completely blocked methanandamide-induced hyperpolarizations. These findings show that anandamide cannot be EDHF. In smooth muscle cells of small arteries, anandamide-induced changes in membrane potential are mediated by vanilloid receptors on capsaicin-sensitive sensory nerves. The different membrane response to the cannabinoids between the main mesenteric artery and its daughter branches might be explained by the different density of perivascular innervation.

Topics: Animals; Arachidonic Acids; Benzoxazines; Biological Factors; Calcium Channel Blockers; Cannabinoids; Capsaicin; Electrophysiology; Endocannabinoids; Endothelium, Vascular; Membrane Potentials; Mesenteric Arteries; Morpholines; Muscle, Smooth, Vascular; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Drug; Rimonabant; Vasodilator Agents

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Topics: Alkaloids; Aniline Compounds; Arachidonic Acids; Benzophenanthridines; Calcium; Capsaicin; Cell Line; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Fluorescence; Fluorometry; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Phenanthridines; Polycyclic Sesquiterpenes; Polyunsaturated Alkamides; Protein Kinase C; Receptors, Drug; Ruthenium Red; Sesquiterpenes; Time Factors; Xanthenes

Capsaicin activates vanilloid (VR1) receptors found on sensory neurons. These ligand-gated ion channels are also sensitive to low pH, elevated temperature and the endocannabinoid, anandamide. In this study, we have measured capsaicin- and anandamide-induced elevations in intracellular calcium concentrations ([Ca(2+)](i)) in fura-2 loaded HEK293 cells stably expressing the rat VR1 receptor at 22, 37 and 50 degrees C. Both capsaicin and anandamide produced a concentration-dependent elevation in [Ca(2+)](i) at all temperatures. pEC(50) values were 7.74 and 5.69 at 22 degrees C and 6.90 and 5.15 at 37 degrees C for capsaicin and anandamide, respectively. At 50 degrees C, the pEC(50) value for capsaicin was 6.36 but the response to anandamide did not saturate. Responses to both agonists were sensitive to ruthenium red and capsazepine at all temperatures. This temperature-dependent reduction in potency may result from desensitization.

Topics: Animals; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Capsaicin; Cell Line; Dose-Response Relationship, Drug; Endocannabinoids; Gene Expression; Humans; Polyunsaturated Alkamides; Rats; Receptors, Drug; Recombinant Proteins; Ruthenium Red; Temperature

1. In the absence of indomethacin, anandamide did not contract the guinea-pig bronchus at concentrations up to 100 microM. In the presence of indomethacin (10 microM), anandamide induced concentration-related contractions with a pEC(50) value of 5.18+/-0.11. It was significantly less potent than capsaicin (pEC(50) 7.01+/-0.1). The anandamide uptake inhibitor AM404, produced only a 14.1+/-3.22% contraction at 100 microM. All experiments were conducted in the presence of PMSF (20 microM). 2. The vanilloid receptor antagonist, capsazepine (10 microM), significantly attenuated the contractile effect of anandamide, the response to 100 microM anandamide being 40.53+/-7.04% in the presence of vehicle and 1.57+/-8.93% in the presence of 10 microM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan. 3. The log concentration-response curve of anandamide was unaltered by the CB1 receptor antagonist, SR141716A. The pEC(50) values for anandamide were 4.88+/-0.08 and 5.17+/-0.19 in the presence of vehicle and SR141716A (1 microM) respectively. 4. The lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 microM anandamide from 34.7+/-1.9% (vehicle) to 7.7+/-5% (ETYA, 10 microM) and from 41.85+/-4.25% (n=6) (vehicle) to 10.31+/-3.54 (n=6) (ETI, 20 microM). Neither inhibitor significantly affected contraction of the tissue by substance P. 5. This study provides evidence that anandamide acts on vanilloid receptors in the guinea-pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acids; Bronchi; Calcium Channel Blockers; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Fatty Acids, Unsaturated; Guinea Pigs; Hydrazines; In Vitro Techniques; Indomethacin; Lipoxygenase; Muscle Contraction; Oxazepines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Drug; Rimonabant; Thiorphan

1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

Topics: Acetylcholine; Animals; Arachidonic Acids; Camphanes; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Guinea Pigs; Ileum; In Vitro Techniques; Male; Methacholine Chloride; Muscle Contraction; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

1. (-)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA). 2. CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5' pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca(2+) concentrations in cells over-expressing human VR1; (b) [(14)C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [(14)C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase. 3. Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC(50)=3.2 - 3.5 microM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 - 70% of the effect obtained with ionomycin (4 microM). CBD (10 microM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive. 4. (+)-5'-DMH-CBD and (+)-7-hydroxy-5'-DMH-CBD inhibited [(14)C]-AEA uptake (IC(50)=10.0 and 7.0 microM); the (-)-enantiomers were slightly less active (IC(50)=14.0 and 12.5 microM). 5. CBD and (+)-CBD were also active (IC(50)=22.0 and 17.0 microM). CBD (IC(50)=27.5 microM), (+)-CBD (IC(50)=63.5 microM) and (-)-7-hydroxy-CBD (IC(50)=34 microM), but not the other analogues (IC(50)>100 microM), weakly inhibited [(14)C]-AEA hydrolysis. 6. Only the (+)-isomers exhibited high affinity for CB(1) and/or CB(2) cannabinoid receptors. 7. These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB(1) and CB(2) receptors, (-)-5'-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.

Topics: Amidohydrolases; Arachidonic Acids; Binding, Competitive; Biological Transport; Calcium; Cannabidiol; Capsaicin; Cell Line; Cell Membrane; Cytosol; Dose-Response Relationship, Drug; Endocannabinoids; Gene Expression; Humans; Hydrolysis; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug

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

1. The effects of the endocannabinoid, anandamide, and its metabolically stable analogue, methanandamide, on induced tone were examined in sheep coronary artery rings in vitro. 2. In endothelium-intact rings precontracted to the thromboxane A(2) mimetic, U46619, anandamide (0.01 - 30 microM) induced slowly developing concentration-dependent relaxations (pEC(50) [negative log of EC(50)]=6.1+/-0.1; R(max) [maximum response]=81+/-4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration-relaxation curve without modifying R(max). Methanandamide was without effect on U46619-induced tone. 3. The anandamide-induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 microM), the vanilloid receptor antagonist, capsazepine (3 and 10 microM) or the nitric oxide synthase inhibitor, L-NAME (100 microM). 4. The cyclo-oxygenase inhibitor, indomethacin (3 and 10 microM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 microM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 microM), shifted the anandamide concentration-response curve to the right. 5. Precontraction of endothelium-intact rings with 25 mM KCl attenuated the anandamide-induced relaxations (R(max)=7+/-7%), as did K(+) channel blockade with tetraethylammonium (TEA; 3 microM) or iberiotoxin (100 nM). Blockade of small conductance, Ca(2+)-activated K(+) channels, delayed rectifier K(+) channels, K(ATP) channels or inward rectifier K(+) channels was without effect. 6. These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Animals; Apamin; Arachidonic Acid; Arachidonic Acids; Barium; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Coronary Vessels; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glyburide; In Vitro Techniques; Indomethacin; Miconazole; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peptides; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Potassium; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Receptors, Drug; Rimonabant; Sheep; Tetraethylammonium; Vasoconstrictor Agents; Vasodilation

Topics: Animals; Arachidonic Acids; Cannabinoids; Capsaicin; Endocannabinoids; Humans; Ligands; Polyunsaturated Alkamides; Receptors, Drug

The structural similarities between the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonylamide (AM404) and the synthetic vanilloid agonist olvanil [(N-vanillyl)-9-oleamide], prompted us to investigate the possibility that olvanil may interfere with anandamide transport. The intracellular accumulation of [3H]anandamide by human astrocytoma cells was prevented by olvanil with a Ki value of 14.1+/-7.1 microM. By contrast, capsaicin [(8-methyl-N-vanillyl)-6-noneamide], a plant-derived vanilloid agonist, and capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2 H-2-benzazepine-2-carbothioamide), a vanilloid antagonist, had no such effect (Ki > 100 microM). These results indicate that, although less potent than AM404 (Ki 2.1+/-0.2 microM), olvanil may reduce anandamide clearance at concentrations similar to those needed for vanilloid receptor activation.

Topics: Amidohydrolases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Astrocytoma; Biological Transport; Brain; Capsaicin; Depression, Chemical; Endocannabinoids; Humans; Polyunsaturated Alkamides; Rats; Receptors, Drug; Tumor Cells, Cultured

The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that 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, inhibited the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibited anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our 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.

Topics: Animals; Arachidonic Acids; Basilar Artery; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Calcium Channels; Capsaicin; Cell Line; Endocannabinoids; Female; Guinea Pigs; Hepatic Artery; In Vitro Techniques; Neurons, Afferent; Peptide Fragments; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Recombinant Proteins; Vasodilator Agents; Xenopus