piperidines and arachidonyl-dopamine

Endocannabinoids (eCBs) modulate synaptic transmission in the brain, but little is known of their regulatory role in nigral dopaminergic neurons, and whether transmission to these neurons is tonically inhibited by eCBs as seen in some other brain regions. Using whole-cell recording in midbrain slices, we observed potentiation of evoked IPSCs (eIPSCs) in these neurons after blocking CB1 receptors with rimonabant or LY-320,135, indicating the presence of an eCB tone reducing inhibitory synaptic transmission. Increased postsynaptic calcium buffering and block of mGluR1 or postsynaptic G-protein coupled receptors prevented this potentiation. Increasing spillover of endogenous glutamate by inhibiting uptake attenuated eIPSC amplitude, while enhancing the potentiation by rimonabant. Group I mGluR activation transiently inhibited eIPSCs, which could be prevented by GDP-β-S, increased calcium buffering or rimonabant. We explored the possibility that the dopamine-derived eCB N-arachidonoyl dopamine (NADA) is involved. The eCB tone was abolished by preventing dopamine synthesis, and enhanced by l-DOPA. It was not detected in adjacent non-dopaminergic neurons. Preventing 2-AG synthesis did not affect the tone, while inhibition of NADA production abolished it. Quantification of ventral midbrain NADA suggested a basal level that increased following prolonged depolarization or mGluR activation. Since block of the tone was not always accompanied by attenuation of depolarization-induced suppression of inhibition (DSI) and vice versa, our results indicate DSI and the eCB tone are mediated by distinct eCBs. This study provides evidence that dopamine modulates the activity of SNc neurons not only by conventional dopamine receptors, but also by CB1 receptors, potentially via NADA.

Topics: Animals; Arachidonic Acids; Benzofurans; Calcium; Cannabinoid Receptor Antagonists; Central Nervous System Agents; Dopamine; Dopamine Agents; Dopaminergic Neurons; Endocannabinoids; Glutamic Acid; Guanosine Diphosphate; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Levodopa; Neurons; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Metabotropic Glutamate; Rimonabant; Substantia Nigra; Synaptic Transmission; Thionucleotides

Action potentials trigger synaptic terminals to synchronously release vesicles, but some vesicles release spontaneously. G-protein-coupled receptors (GPCRs) can modulate both of these processes. At cranial primary afferent terminals, the GPCR cannabinoid 1 (CB1) is often coexpressed with transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel present on most afferents. Here we tested whether CB1 activation modulates synchronous, action potential-evoked (eEPSCs) and/or spontaneous (sEPSCs) EPSCs at solitary tract nucleus neurons. In rat horizontal brainstem slices, activation of solitary tract (ST) primary afferents generated ST-eEPSCs that were rapidly and reversibly inhibited from most afferents by activation of CB1 with arachidonyl-2'-chloroethylamide (ACEA) or WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate]. The CB1 antagonist/inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] blocked these responses. Despite profound depression of ST-eEPSCs during CB1 activation, sEPSCs in these same neurons were unaltered. Changes in temperature changed sEPSC frequency only from TRPV1(+) afferents (i.e., thermal sEPSC responses only occurred in TRPV1(+) afferents). CB1 activation failed to alter these thermal sEPSC responses. However, the endogenous arachidonate metabolite N-arachidonyldopamine (NADA) promiscuously activated both CB1 and TRPV1 receptors. NADA inhibited ST-eEPSCs while simultaneously increasing sEPSC frequency, and thermally triggered sEPSC increases in neurons with TRPV1(+) afferents. We found no evidence for CB1/TRPV1 interactions suggesting independent regulation of two separate vesicle pools. Together, these data demonstrate that action potential-evoked synchronous glutamate release is modulated separately from TRPV1-mediated glutamate release despite coexistence in the same central terminations. This two-pool arrangement allows independent and opposite modulation of glutamate release by single lipid metabolites.

Topics: Action Potentials; Analgesics; Animals; Arachidonic Acids; Benzoxazines; Brain Stem; Capsaicin; Dopamine; Excitatory Postsynaptic Potentials; Glutamic Acid; Hot Temperature; In Vitro Techniques; Male; Morpholines; Naphthalenes; Neurons; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission; TRPV Cation Channels

Endocannabinoids exert numerous effects in the CNS under physiological and pathological conditions. The aim of the present study was to examine whether the endocannabinoid N-arachidonoyldopamine (NADA) may protect neurons in excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). OHSC were excitotoxically lesioned by application of N-methyl-d-aspartate (NMDA, 50 μM) for 4 h and subsequently treated with different NADA concentrations (0.1 pM-50 μM) alone or in combination with cannabinoid receptor antagonists. NADA protected dentate gyrus granule cells and caused a slight reduction in the number of microglial cells. The number of degenerated neurons significantly decreased between 100 pM and 10 μM NADA (p < 0.05). To identify the responsive receptor type of NADA mediated neuroprotection, we applied the cannabinoid (CB) receptor 1 (CB(1)) inverse agonist/antagonist AM251, CB(2) inverse agonist/antagonist AM630, abnormal-cannabidiol (abn-CBD)-sensitive receptor antagonist O-1918, transient receptor potential channel V1 (TRPV1) antagonist 6-iodonordihydrocapsaicin and A1 (TRPA1) antagonist HC-030031. Neuroprotective properties of low (1 nM) but not high (10 μM) NADA concentrations were solely blocked by AM251 and were absent in CB(1)(-/-) mice. AM630, O-1918, 6-iodonordihydrocapsaicin and HC-030031 showed no effects at all NADA concentrations applied. Our findings demonstrate that NADA protects dentate gyrus granule cells by acting via CB(1). NADA reduced the number of microglial cells at distinct concentrations. TRPV1 and TRPA1 were not involved in NADA mediated neuroprotection. Thus, our data implicate that NADA mediated activation of neuronal CB(1) may serve as a novel pharmacological target to mitigate symptoms of neuronal damage.

Topics: Animals; Arachidonic Acids; Cells, Cultured; Dopamine; Excitatory Postsynaptic Potentials; Hippocampus; Mice; Nerve Degeneration; Neurons; Neuroprotective Agents; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

The endogenous N-arachidonoyl-dopamine (NADA) activates both transient receptor potential vanilloid1 (TRPV1) and cannabinoid-1 (CB(1)) receptors. The goal of this study was to characterize the antinociceptive potential of NADA on inflammatory thermal hyperalgesia in rats at spinal level, and to determine its interaction with endomorphin-1 (EM) at the spinal level. The effects of NADA and EM on thermal hyperalgesia were evaluated in rats with a unilateral hind paw carrageenan-induced inflammation. Intrathecal injection of either EM (0.03-10 μg) or NADA (1.5-50 μg) caused dose-dependent antihyperalgesia, but NADA was 5.4 times less potent than EM. The antihyperalgesia caused by 15 μg NADA was inhibited by the TRPV1 antagonist AMG9810, but not by CB(1) antagonist/inverse agonist AM 251, whereas the effect of 50 μg NADA was decreased by both drugs. Co-administration of EM with NADA in 1:15 and 1:50 ratios produced a short-lasting potentiation, but isobolographic analysis for the whole investigated period revealed additive interaction between the two endogenous ligands. The results show that both TRPV1 and CB(1) receptor activation play a substantial role in the antinociceptive effects of NADA at spinal level, while co-administration of NADA with EM did not show potentiation.

Topics: Acrylamides; Analgesics; Animals; Arachidonic Acids; Area Under Curve; Bridged Bicyclo Compounds, Heterocyclic; Dopamine; Dose-Response Relationship, Drug; Drug Synergism; Hot Temperature; Hyperalgesia; Injections, Spinal; Male; Oligopeptides; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Spinal Cord; 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

Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays.. MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated.. Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates.. 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Chromans; Cyclohexanols; Dopamine; Enzyme Assays; Glycerides; Humans; Monoacylglycerol Lipases; Nitrophenols; Piperidines; PPAR gamma; Rats; Rats, Sprague-Dawley; Rats, Wistar; Thiazolidinediones; Troglitazone

We investigated the effect of N-arachidonyl dopamine (NADA), an endogenous agonist of both transient receptor potential vanilloid 1 (TRPV1) and cannabinoid CB1 receptors, on the sensitivity of rat capsaicin-sensitive lung vagal afferent (CSLVA) fibers. In artificially ventilated rats, an intravenous infusion of NADA (400 microg/kg/ml, 0.5 ml/min for 2 min) mildly elevated the baseline CSLVA fiber activity, whereas it markedly potentiated CSLVA fiber responses to a right atrial injection of capsaicin or adenosine, and to lung inflation. The potentiating effect on CSLVA fiber sensitivity to an adenosine injection or lung inflation was blocked by capsazepine pretreatment (a TRPV1 receptor antagonist), but was unaffected by AM251 pretreatment (a CB1 receptor antagonist). In spontaneously breathing rats, a NADA infusion similarly potentiated the CSLVA fiber-mediated apneic response evoked by an adenosine injection, and this potentiating effect was also prevented by capsazepine pretreatment. We concluded that NADA at the dose tested non-specifically increases CSLVA fiber sensitivity to chemical and mechanical stimulation via activation of TRPV1 receptors.

Topics: Adenosine; Animals; Arachidonic Acids; Capsaicin; Dopamine; Hemodynamics; Male; Nerve Fibers; Neurons, Afferent; Physical Stimulation; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Respiration, Artificial; Respiratory Mechanics; TRPV Cation Channels; Vagus Nerve

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

This study was designed to test the hypothesis that the systemic administration of N-arachidonoyl-dopamine (NADA), an endovanilloid, causes a depressor effect via activation of transient receptor potential vanilloid type 1 (TRPV1) channels during high-salt intake.. Wistar rats were fed a normal (0.4%) or high (4%) sodium diet for 10 days, and arteries and veins were cannulated for measurement of mean arterial pressure (MAP) or injection of drugs and collection of plasma. Radioimmunoassay and western blot were used to determine the plasma calcitonin gene-related peptide (CGRP) level and TRPV1 protein content, respectively.. The NADA-induced dose-dependent decrease in MAP was greater in high-sodium than normal-treated rats, and was abolished by capsazepine, a selective TRPV1 antagonist, or CGRP8-37, a selective CGRP receptor antagonist, but not by SR141716A, a selective cannabinoid 1 receptor antagonist. Capsaicin, a selective TRPV1 receptor agonist, or CGRP dose-dependently decreased MAP in normal or high-sodium-treated rats, with a greater effect in the latter. Baseline and NADA-induced increases in plasma CGRP levels were higher in high-sodium than normal-treated rats. TRPV1 protein expression in mesenteric arteries was higher in high-sodium than normal-treated rats. In vitro, NADA caused a greater CGRP release from mesenteric arteries of high-sodium than normal-treated rats, which was blocked by capsazepine.. High sodium increases the sensitivity of blood pressure responses to NADA. The enhanced depressor effect induced by NADA during high-sodium intake is prevented by blockade of the TRPV1 or CGRP receptors, but not cannabinoid 1 receptor. High sodium upregulates mesenteric TRPV1 expression, and increases NADA-induced CGRP release in vitro and in vivo.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Capsaicin; Dopamine; Male; Peptide Fragments; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Calcitonin Gene-Related Peptide; Rimonabant; Sodium Chloride, Dietary; TRPV Cation Channels

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

N-arachidonoyl-dopamine (NADA) is an endogenous ligand at TRPV1 and CB(1) receptors, which are expressed on primary afferent nociceptors. The aim of this study was to determine contributions of proposed pronociceptive TRPV1 and antinociceptive CB(1) receptors to effects of peripheral NADA on primary afferent fibre function. Effects of NADA on primary afferent nociceptor function, determined by whole cell patch clamp and calcium imaging studies of adult dorsal root ganglion (DRG) neurons, were determined. Application of NADA (1 microm) to DRG neurons depolarized the resting membrane potential (Vm) from -58 +/- 1 to -44 +/- 3 mV (P < 0.00001) and evoked a significant increase (P < 0.0001) in intracellular calcium (74 +/- 11% of response to 60 mm KCl), compared to basal. The TRPV1 receptor antagonist capsazepine abolished NADA-evoked depolarization of Vm (P < 0.0001) and NADA-evoked calcium responses (P < 0.001), which were also blocked by the CB(1) receptor antagonist SR141716A (P < 0.001). Effects of NADA (1.5 microg and 5 microg/50 microL) on mechanically evoked responses of dorsal horn neurons in anaesthetized Sprague-Dawley rats were studied. Intraplantar injection of the higher dose of NADA (5 microg/50 microL) studied significantly inhibited innocuous (8, 10 g) mechanically evoked responses of dorsal horn neurons compared to vehicle, effects blocked by intraplantar injection of SR141716A. Higher weight (26-100 g) noxious-evoked responses of dorsal horn neurons were also significantly inhibited by NADA (5 microg/50 microL), effects blocked by intraplantar injection of the TRPV1 antagonist, iodo-resiniferatoxin. NADA has a complex pattern of effects on DRG neurons and primary afferent fibres, which is likely to reflect its dual site of action at TRPV1 and CB(1) receptors and the differential expression of these receptors by primary afferent fibres.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Calcium; Capsaicin; Cells, Cultured; Dopamine; Drug Interactions; Electrophysiology; Male; Membrane Potentials; Nerve Fibers; Pain Measurement; Physical Stimulation; Piperidines; Posterior Horn Cells; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Drug; Rimonabant; Sensory Thresholds; Spinal Cord