arachidonyl-dopamine and anandamide

Endovanilloids are defined as endogenous ligands and activators of transient receptor potential (TRP) vanilloid type 1 (TRPV1) channels. The first endovanilloid to be identified was anandamide (AEA), previously discovered as an endogenous agonist of cannabinoid receptors. In fact, there are several similarities, in terms of opposing actions on the same intracellular signals, role in the same pathological conditions, and shared ligands and tissue distribution, between TRPV1 and cannabinoid CB(1) receptors. After AEA and some of its congeners (the unsaturated long chain N-acylethanolamines), at least 2 other families of endogenous lipids have been suggested to act as endovanilloids: (i) unsaturated long chain N-acyldopamines and (ii) some lipoxygenase (LOX) metabolites of arachidonic acid (AA). Here we discuss the mechanisms for the regulation of the levels of the proposed endovanilloids, as well as their TRPV1-mediated pharmacological actions in vitro and in vivo. Furthermore, we outline the possible pathological conditions in which endovanilloids, acting at sometimes aberrantly expressed TRPV1 receptors, might play a role.

Topics: Animals; Arachidonic Acids; Cannabinoids; Dopamine; Endocannabinoids; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Polyunsaturated Alkamides; Receptors, Cannabinoid; TRPV Cation Channels

Topics: Amides; Amidohydrolases; Amines; Animals; Arachidonic Acids; Binding Sites; Cannabinoid Receptor Modulators; Drug Design; Endocannabinoids; Esters; Ethers; Glycerides; Humans; Ligands; Monoacylglycerol Lipases; Polyunsaturated Alkamides; Receptors, Cannabinoid

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

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics.

Topics: Aging; Amides; Amidohydrolases; Animals; Antineoplastic Agents; Apoptosis; Arachidonic Acids; Brain; Brain Neoplasms; Capsaicin; Cell Movement; Culture Media, Conditioned; Dopamine; Endocannabinoids; Ethanolamines; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Neoplasm Proteins; Neural Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; TRPV Cation Channels; Tumor Cells, Cultured

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 subtype 1 (TRPV1) receptor is a primary pain-sensing relay at peripheral sensory nerve endings and is also widespread in the brain, where it is implicated in neurodegeneration. Previous studies of TRPV1 neurotoxicity have utilized heterogeneous receptor populations, non-selective ligands, or non-neuronal cell types. Here, we explored the pharmacology of TRPV1-induced cytotoxicity in a homogeneous, neurone-like cellular environment.. Cell death was examined in a human neurone-like cell line, stably expressing recombinant human TRPV1. Cytotoxicity was quantified in terms of nuclear morphology and mitochondrial complex II activity. Immunocytochemical markers of apoptotic cell death were also examined.. The TRPV1-selective agonist capsaicin, and the endovanilloids anandamide and N-arachidonoyl-dopamine (NADA), induced TRPV1-dependent delayed cell death in a concentration- and time-dependent manner. Capsaicin exposure time was significantly correlated with potency (r(2)=0.91, P=0.01). Release of cytochrome c from mitochondria, activation of caspase-3, and condensed nuclear chromatin were evident 6 h after capsaicin exposure, but cytotoxicity was unaffected by a pan-caspase inhibitor (zVAD-fmk, 50 microM).. We conclude that capsaicin, anandamide, and NADA can initiate TRPV1-dependent delayed cell death in neurone-like cells. This is an apoptosis-like process, but independent of caspase activity.

Topics: Apoptosis; Arachidonic Acids; Capsaicin; Dopamine; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Neurons; Polyunsaturated Alkamides; TRPV Cation Channels; Tumor Cells, Cultured

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

It is widely accepted that neuroinflammation is a key player in various pathological events associated with brain injury. More specifically, glial activation and the subsequent release of pro-inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins play a role of paramount importance in cerebral damage. In this study, we examined the role of two endocannabinoids, anandamide (AEA) and N-arachidonoyldopamine (NADA) in the regulation of prostaglandin E(2) (PGE(2)) synthesis in primary glial cells. We show that NADA is a potent inhibitor of PGE(2) synthesis in lipopolysaccharide (LPS) stimulated cells, without modifying the expression or enzymatic activity of COX-2 and the production of prostaglandin D(2). We also show that NADA has the ability to prevent the free radical formation in primary microglial cells. The key findings of this investigation are our observation that AEA and NADA have opposite effects on glial cells and, most importantly, the first description of NADA as a potential antioxidative and anti-inflammatory agent acting through a mechanism that involves reduction in the synthesis of microsomal prostaglandin E synthase in LPS-activated microglia. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA in the CNS and its potential to design novel therapeutic strategies to manage neuroinflammatory diseases.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Arachidonic Acids; Cell Survival; Dinoprostone; Dopamine; Endocannabinoids; Isomerism; Isoprostanes; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuroglia; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley

The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Arachidonic Acids; Benzaldehydes; Cannabinoid Receptor Modulators; Cell Line; Cell Membrane; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Humans; Ion Channel Gating; Menthol; Polyunsaturated Alkamides; Pyrimidinones; Receptor, Cannabinoid, CB1; Signal Transduction; Transfection; TRPM Cation Channels

Inhibitory CB(1) cannabinoid receptors and excitatory TRPV(1) vanilloid receptors are abundant in the hippocampus. We tested if two known hybrid endocannabinoid/endovanilloid substances, N-arachidonoyl-dopamine (NADA) and anandamide (AEA), presynapticaly increased or decreased intracellular calcium level ([Ca(2+)](i)) and GABA and glutamate release in the hippocampus.. Resting and K(+)-evoked levels of [Ca(2+)](i) and the release of [(3)H]GABA and [(3)H]glutamate were measured in rat hippocampal nerve terminals.. NADA and AEA per se triggered a rise of [Ca(2+)](i) and the release of both transmitters in a concentration- and external Ca(2+)-dependent fashion, but independently of TRPV(1), CB(1), CB(2), or dopamine receptors, arachidonate-regulated Ca(2+)-currents, intracellular Ca(2+) stores, and fatty acid metabolism. AEA was recently reported to block TASK-3 potassium channels thereby depolarizing membranes. Common inhibitors of TASK-3, Zn(2+), Ruthenium Red, and low pH mimicked the excitatory effects of AEA and NADA, suggesting that their effects on [Ca(2+)](i) and transmitter levels may be attributable to membrane depolarization upon TASK-3 blockade. The K(+)-evoked Ca(2+) entry and Ca(2+)-dependent transmitter release were inhibited by nanomolar concentrations of the CB(1) receptor agonist WIN55212-2; this action was sensitive to the selective CB(1) receptor antagonist AM251. However, in the low micromolar range, WIN55212-2, NADA and AEA inhibited the K(+)-evoked Ca(2+) entry and transmitter release independently of CB(1) receptors, possibly through direct Ca(2+) channel blockade.. We report here for hybrid endocannabinoid/endovanilloid ligands novel dual functions which were qualitatively similar to activation of CB(1) or TRPV(1) receptors, but were mediated through interactions with different targets.

Topics: Animals; Arachidonic Acids; Calcium; Dopamine; Endocannabinoids; Fluorometry; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Dopamine; 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

Cannabinoid (CB) agonists suppress nausea and vomiting (emesis). Similarly, transient receptor potential vanilloid-1 (TRPV1) receptor agonists are anti-emetic. Arvanil, N-(3-methoxy-4-hydroxy-benzyl)-arachidonamide, is a synthetic 'hybrid' agonist of CB1 and TRPV1 receptors. Anandamide and N-arachidonoyl-dopamine (NADA) are endogenous agonists at both these receptors. We investigated if arvanil, NADA and anandamide were anti-emetic in the ferret and their mechanism of action. All compounds reduced the episodes of emesis in response to morphine 6 glucuronide. These effects were attenuated by AM251, a CB1 antagonist that was pro-emetic per se, and TRPV1 antagonists iodoresiniferatoxin and AMG 9810, which were without pro-emetic effects. Similar sensitivity to arvanil and NADA was found for prodromal signs of emesis. We analysed the distribution of TRPV1 receptors in the ferret brainstem and, for comparison, the co-localization of CB1 and TRPV1 receptors in the mouse brainstem. TRPV1 immunoreactivity was largely restricted to the nucleus of the solitary tract of the ferret, with faint labeling in the dorsal motor nucleus of the vagus and sparse distribution in the area postrema. A similar distribution of TRPV1, and its extensive co-localization with CB1, was observed in the mouse. Our findings suggest that CB1 and TRPV1 receptors in the brainstem play a major role in the control of emesis by agonists of these two receptors. While there appears to be an endogenous 'tone' of CB1 receptors inhibiting emesis, this does not seem to be the case for TRPV1 receptors, indicating that endogenously released endocannabinoids/endovanilloids inhibit emesis preferentially via CB1 receptors.

Topics: Acrylamides; Animals; Antiemetics; Arachidonic Acids; Area Postrema; Autonomic Pathways; Brain Stem; Bridged Bicyclo Compounds, Heterocyclic; Cannabinoids; Capsaicin; Dopamine; Emetics; Endocannabinoids; Ferrets; Male; Mice; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Solitary Nucleus; TRPV Cation Channels; Vagus Nerve; Vomiting

The aim of the present study was to investigate the relationship between TRPV1 stimulation and endocannabinoid-driven CB(1) receptor-mediated inhibition of activity in adult rat dorsal root ganglion (DRG) neurons, a model of primary afferent nociceptors. Calcium-imaging studies were performed to compare the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 microm) vs. the anandamide (AEA) uptake inhibitor UCM707 (1 microm) on capsaicin (100 nm) and N-arachidonoyl dopamine (NADA; 1 microm)-evoked changes in intracellular calcium [Ca(2+)](i) in DRG neurons. The ability of the CB(1) receptor antagonist AM251 (1 microm) to modulate the effects of URB597 and UCM707 was also determined. Suprafusion of NADA and capsaicin evoked robust increases in [Ca(2+)](i) in DRG neurons (89 +/- 4% and 132 +/- 6% of the depolarizing KCl response, respectively). Co-incubation with URB597 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (39 +/- 3% and 79 +/- 4% of KCl response, respectively). Similarly, co-incubation with UCM707 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (59 +/- 7% and 72 +/- 4% of KCl response, respectively). The CB(1) receptor antagonist AM251 significantly attenuated the effects of URB597 on NADA-evoked increases in [Ca(2+)](i) but not the effects of URB597 on capsaicin-evoked increases in [Ca(2+)](i). By contrast, AM251 significantly attenuated the inhibitory effects of UCM707 on both NADA and capsaicin-evoked increases in [Ca(2+)](i.) These data suggest that transport of both NADA and capsaicin into DRG neurons and the subsequent activation of TRPV1 is partly governed by FAAH-dependent mechanisms as well as via the putative AEA membrane transporter.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Calcium; Capsaicin; Carbamates; Cells, Cultured; Dopamine; Drug Interactions; Endocannabinoids; Fura-2; Furans; Ganglia, Spinal; Male; Membrane Potentials; Membrane Transport Proteins; Neurons; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; TRPV Cation Channels

The vascular effects of cannabinoids have been compared in the rat isolated aorta. Delta9-Tetrahydrocannabinol (THC), anandamide and N-arachidonoyl-dopamine (NADA) all caused vasorelaxation to similar degrees in pre-constricted aortae. Vasorelaxation to THC was inhibited by in vivo pre-treatment with pertussis toxin (10 microg/kg) or with the synthetic cannabinoid CP55,940 (((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), acutely or chronically), exposure to capsaicin in vitro (10 microM for 1 h), and de-endothelialisation. Vasorelaxation to anandamide was only inhibited by pertussis toxin and chronic CP55,940 pre-treatment (0.4 mg/kg for 11 days). Vasorelaxation to NADA was inhibited by pertussis toxin and chronic CP55,940 pre-treatment, and by de-endothelialisation. The vasorelaxant effects of the cannabinoids were not inhibited by cannabinoid CB1 receptor antagonism; however, vasorelaxation to both CP55,940 and THC was inhibited by cannabinoid CB2 receptor antagonism. Vasorelaxation to all cannabinoids was enhanced in the presence of indomethacin (10 microM). THC also caused vasoconstriction of the aorta while anandamide, NADA, CP55,940 and WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) did not. The vasoconstrictor effects of THC were inhibited by in vivo pre-treatment with pertussis toxin or CP55,940, acute exposure to CP55,940, cannabinoid CB1 receptor antagonism and cyclooxygenase inhibition. These results demonstrate the opposing vascular effects of cannabinoids in the rat aorta, and although vasorelaxation to each of the cannabinoids is of similar magnitude, it is mediated through different pathways. This gives further indication of the different vascular actions of cannabinoid compounds.

Topics: Animals; Aorta; Arachidonic Acids; Cannabinoids; Dopamine; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; In Vitro Techniques; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Vasomotor System

As persistent behavioural changes, such as increased anxiety-related behaviours, can be predicted based on the phenomenon of psychostimulant-induced neuronal plasticity, the time course (3-, 5- and 10-day time points) of the effects of both a single and repeated (daily for 7 days) i.p. administrations of cocaine (COC) and methamphetamine (MA) on anxiety-related behavioural symptoms in the elevated plus-maze test were examined in mice. Furthermore, based on the reported interactions between brain dopamine versus cannabinoid (CB) receptors and the contribution of CB receptors to the occurrence of persistent anxiety-related behavioural symptoms, the interactions of the agonist CP 55940 (CP) and the endogenous ligands anandamide (arachidonylethanolamide: AEA), 2-arachidonylglycerol (ARA), N-arachidonyldopamine (NADA), noladin ether (NL), and virodhamine (VA) with the COC- or MA-induced anxiety-related behaviours were also studied. In both an acute experiment using a single COC (30 mg/kg) or MA (4 mg/kg) dose and a chronic experiment using repeated COC (15 mg/kg) or MA (2 mg/kg) doses, anxiety-related behavioural symptoms were observed similarly at 3- and 5-day time points, but disappeared at the 10-day time point. Among the CB ligands, the agonists CP, AEA, ARA, NADA, and NL provided strong protective effects against each parameter at 3- and 5-day time points. Therefore, it was concluded that both COC and MA caused persistent anxiety-related behavioural symptoms following both a single and repeated treatments. Since these anxiogenic effects were attenuated by the endogenous CB agonists, the involvement of brain CB receptors was suspected.

Topics: Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Cannabinoids; Cocaine; Cyclohexanols; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Glycerides; Injections, Intraperitoneal; Male; Maze Learning; Methamphetamine; Mice; Mice, Inbred ICR; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Time Factors

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

Four long-chain, linear fatty acid dopamides (N-acyldopamines) have been identified in nervous bovine and rat tissues. Two unsaturated members of this family of lipids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine, were shown to potently activate the transient receptor potential channel type V1 (TRPV1), also known as the vanilloid receptor type 1 for capsaicin. However, the other two congeners, N-palmitoyl- and N-stearoyl-dopamine (PALDA and STEARDA), are inactive on TRPV1. We have investigated here the possibility that the two compounds act by enhancing the effect of NADA on TRPV1 ('entourage' effect). When pre-incubated for 5 min with cells, both compounds dose-dependently enhanced NADA's TRPV1-mediated effect on intracellular Ca(2+) in human embryonic kidney cells overexpressing the human TRPV1. In the presence of either PALDA or STEARDA (0.1-10 microm), the EC(50) of NADA was lowered from approximately 90 to approximately 30 nm. The effect on intracellular Ca(2+) by another endovanilloid, N-arachidonoyl-ethanolamine (anandamide, 50 nm), was also enhanced dose-dependently by both PALDA and STEARDA. PALDA and STEARDA also acted in synergy with low pH (6.0-6.7) to enhance intracellular Ca(2+) via TRPV1. When co-injected with NADA (0.5 micrograms) in rat hind paws, STEARDA (5 micrograms) potentiated NADA's TRPV1-mediated nociceptive effect by significantly shortening the withdrawal latencies from a radiant heat source. STEARDA (1 and 10 micrograms) also enhanced the nocifensive behavior induced by carrageenan in a typical test of inflammatory pain. These data indicate that, despite their inactivity per se on TRPV1, PALDA and STEARDA may play a role as 'entourage' compounds on chemicophysical agents that interact with these receptors, with possible implications in inflammatory and neuropathic pain.

Topics: Animals; Arachidonic Acids; Calcium; Carrageenan; Cell Line; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Synergism; Drug Therapy, Combination; Endocannabinoids; Hindlimb; Humans; Hyperalgesia; Inflammation; Italy; Kidney; Male; Pain Measurement; Palmitates; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Drug; Stearates; TRPV Cation Channels

The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Calcium Channels; Capsaicin; Cell Line; CHO Cells; Cricetinae; Diterpenes; Dopamine; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Kinetics; Ligands; Models, Chemical; Polyunsaturated Alkamides; Protein Binding; Rats; Receptors, Drug; Spinal Cord; Time Factors; Transfection