am-630 and anandamide

Cholestatic diseases are often accompanied by elevated plasma levels of endogenous opioid peptides, but it is still unclear whether central or peripheral mechanisms are involved in this process, and little is known about the change of pain threshold in these patients. The purpose of this study was to determine the preoperative pain threshold, postoperative morphine consumption, and central and peripheral β-endorphin levels in patients with obstructive jaundice. This study also tests the hypothesis that activation of the cannabinoid receptor-2 (CB2R) in skin keratinocytes by endocannabinoids is the mechanism underlying circulating β-endorphin elevation in patients with obstructive jaundice.. The electrical pain thresholds, 48-hour postoperative morphine consumption, concentrations of β-endorphin in plasma and cerebrospinal fluid, skin and liver β-endorphin expression, and plasma levels of endocannabinoids were measured in jaundiced (n = 32) and control (n = 32) patients. Male Sprague-Dawley rats and human keratinocytes (human immortalized keratinocyte cell line [HaCaT]) were used for the in vivo and in vitro experiments, respectively. Mechanical and thermal withdrawal latency, plasma level, and skin expression of β-endorphin were measured in CB2R-antagonist-treated and control bile duct-ligated (BDL) rats. In cultured keratinocytes, the effect of CB2R agonist AM1241-induced β-endorphin expression was observed and the phosphorylation of extracellular-regulated protein kinases 1/2, p38, and signal transducer and activator of transcription (STAT) pathways were investigated.. This study found (1) the plasma level of β-endorphin (mean ± standard error of the mean [SEM]) was 193.9 ± 9.6 pg/mL in control patients, while it was significantly increased in jaundiced patients (286.6 ± 14.5 pg/mL); (2) the electrical pain perception threshold and the electrical pain tolerance threshold were higher in patients with obstructive jaundice compared with controls, while the 48-hour postoperative morphine consumption was lower in the jaundiced patients; (3) there was no correlation between plasma β-endorphin levels, electrical pain thresholds, and 48-hour postoperative morphine consumption in patients with obstructive jaundice; (4) the plasma level of the endogenous cannabinoid anandamide was increased in the jaundiced patients; (5) CB2R antagonist treatment of the BDL rats reduced β-endorphin levels in plasma and skin keratinocytes, while it did not alter the nociceptive thresholds in BDL and control rats; (6) the endocannabinoid anandamide-induced β-endorphin synthesis and release via CB2R in cultured keratinocytes; and (7) phosphorylation of extracellular-regulated protein kinases 1/2 is involved in the CB2R-agonist-induced β-endorphin expression in keratinocytes.. CB2R activation in keratinocytes by the endocannabinoid anandamide may play an important role in the peripheral elevation of β-endorphin during obstructive jaundice.

Topics: Animals; Arachidonic Acids; beta-Endorphin; Cannabinoid Receptor Agonists; Cell Line, Transformed; Cells, Cultured; Endocannabinoids; Humans; Indoles; Jaundice, Obstructive; Keratinocytes; Male; Middle Aged; Morphine; Pain Measurement; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2

Oxidative stress is one of the main pathogenic factors of neurodegenerative diseases. As the ligand of cannabinoid type 1 (CB1) and 2 (CB2) receptors, anandamide (AEA) exerts benign antioxidant activities. However, the instability of AEA results in low levels in vivo, which limit its further application. Based on the structure of AEA, N‑linoleyltyrosine (NITyr) was synthesized in our laboratory and was hypothesized to possess a similar function to that of AEA. To the best of our knowledge, the present study demonstrates for the first time, the activities and mechanisms of NITyr. NITyr treatment attenuated hydrogen peroxide (H2O2)‑induced cytotoxicity, with the most promiment effect observed at 1 µmol/l. Treatment with NITyr also suppressed the H2O2‑induced elevation of reactive oxygen species (ROS) and enhanced the expression of the autophagy‑related proteins, LC3‑II, beclin‑1, ATG 5 and ATG13. The autophagic inhibitor, 3‑methyladenine, reversed the effects of NITyr on ROS levels and cellular viability. Furthermore, AM251, a CB1 receptor antagonist, but not AM630 (a CB2 receptor antagonist), diminished the effects of NITyr on cell viability, ROS generation and autophagy‑related protein expression. However, NITyr increased the protein expression of both the CB1 and CB2 receptors. Therefore, NITyr was concluded to protect PC12 cells against H2O2‑induced oxidative injury by inducing autophagy, a process which may involve the CB1 receptor.

Topics: Adenine; Animals; Arachidonic Acids; Autophagy; Cell Survival; Endocannabinoids; Hydrogen Peroxide; Indoles; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Reactive Oxygen Species; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Tyrosine

The endocannabinoid (eCB) system is a particularly important neuronal mechanism implicated in alcohol use disorders. Animal models are key to broadening our knowledge of the neurobiological mechanisms underlying alcohol dependence. This study has two main aims: i) to assess how eCB levels in different brain areas are modified by alcohol-induced conditioning place preference (CPP), and ii) to study how cannabinoid type 2 receptor (CB2R) is involved in alcohol-rewarding properties, using pharmacological manipulation in C57BL/6 mice. Our results suggest that the eCB system is dysregulated throughout the mesocorticolimbic system by repeated alcohol exposure during the CPP paradigm, and that levels of anandamide (AEA) and several other N-acylethanolamines are markedly decreased in the medial prefrontal cortex and ventral midbrain of alcohol-CPP mice. We also observed that the administering an antagonist/inverse agonist of the CB2R (AM630) during the acquisition phase of CPP reduced the rewarding effects of alcohol. However, activating CB2R signalling using the agonist JWH133 seems to reduce both alcohol- and food-rewarding behaviours. Therefore, our findings indicate that the rewarding effects of alcohol are related to its disruptive effect on AEA and other N-acylethanolamine signalling pathways. Thus, pharmacological manipulation of CB2R is an interesting candidate treatment for alcohol use disorders.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Central Nervous System Depressants; Conditioning, Classical; Dopaminergic Neurons; Endocannabinoids; Ethanol; Indoles; Locomotion; Male; Mesencephalon; Mice; Mice, Inbred C57BL; Polyunsaturated Alkamides; Prefrontal Cortex; Receptor, Cannabinoid, CB2; Reward

Chronic pain and hyperalgesia, as well as pain resulting from episodes of vaso-occlusion, are characteristic features of sickle cell disease (SCD) and are difficult to treat. Since there is growing evidence that increasing local levels of endocannabinoids can decrease hyperalgesia, we examined the effects of URB597, a fatty acid amide hydrolase (FAAH) inhibitor, which blocks the hydrolysis of the endogenous cannabinoid anandamide, on hyperalgesia and sensitization of cutaneous nociceptors in a humanized mouse model of SCD. Using homozygous HbSS-BERK sickle mice, we determined the effects of URB597 on mechanical hyperalgesia and on sensitization of C-fiber nociceptors in vivo. Intraplantar administration of URB597 (10 μg in 10 μL) decreased the frequency of withdrawal responses evoked by a von Frey monofilament (3.9 mN bending force) applied to the plantar hind paw. This was blocked by the CB1 receptor antagonist AM281 but not by the CB2 receptor antagonist AM630. Also, URB597 decreased hyperalgesia in HbSS-BERK/CB2R sickle mice, further confirming the role of CB1 receptors in the effects produced by URB597. Electrophysiological recordings were made from primary afferent fibers of the tibial nerve in anesthetized mice. The proportion of Aδ- and C-fiber nociceptors that exhibited spontaneous activity and responses of C-fibers to mechanical and thermal stimuli were greater in HbSS-BERK sickle mice as compared to control HbAA-BERK mice. Spontaneous activity and evoked responses of nociceptors were decreased by URB597 via CB1 receptors. It is suggested that enhanced endocannabinoid activity in the periphery may be beneficial in alleviating chronic pain associated with SCD.

Topics: Anemia, Sickle Cell; Animals; Arachidonic Acids; Benzamides; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Evoked Potentials; Hemoglobins; Humans; Hydrolysis; Hyperalgesia; Indoles; Male; Mice; Mice, Transgenic; Morpholines; Nerve Fibers, Unmyelinated; Nociceptors; Pain Threshold; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

While activation of cannabinoid CB1 receptor (CB1R) regulates a variety of retinal neuronal functions by modulating ion channels in these cells, effect of activated cannabinoid receptors on Ca(2+) channels in retinal Müller cells is still largely unknown. In the present work we show that three subunits of T-type Ca(2+) channels, CaV3.1, CaV3.2 and CaV3.3, as well as one subunit of L-type Ca(2+) channels, CaV1.2, were expressed in rat Müller cells by immunofluorescent staining. Consistently, nimodipine- and mibefradil-sensitive Na(+) currents through L- and T-type Ca(2+) channels could be recorded electrophysiologically. The cannabinoid receptor agonist WIN55212-2 significantly suppressed Ca(2+) channel currents, mainly the T-type one, in acutely isolated rat Müller cells in a dose-dependent manner, with an IC50 of 3.98μM. The WIN55212-2 effect was not blocked by AM251/SR141716, specific CB1R antagonists. Similar suppression of the currents was observed when anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), endogenous ligands of cannabinoid receptors, were applied. Moreover, even though CB2 receptors (CB2Rs) were expressed in rat Müller cells, the effects of WIN55212-2 and 2-AG on Ca(2+) channel currents were not blocked by AM630, a selective CB2R antagonist. However, the effect of AEA could be partially rescued by AM630. These results suggest that WIN55212-2 and 2-AG receptor-independently suppressed the Ca(2+) channel currents in Müller cells, while AEA suppressed the currents partially through CB2Rs. The existence of receptor-dependent and -independent mechanisms suggests that cannabinoids may modulate Müller cell functions through multiple pathways.

Topics: Animals; Arachidonic Acids; Benzoxazines; Calcium; Calcium Channels; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cells, Cultured; Dose-Response Relationship, Drug; Endocannabinoids; Ependymoglial Cells; Glycerides; Indoles; Male; Membrane Potentials; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

The aim of this study was to investigate the effects of an anandamide transporter inhibitor that can increase endogenous anandamide concentration on the micturition reflex in urethane-anesthetized rats.. Continuous cystometrograms were performed in female Sprague-Dawley rats under urethane anesthesia. After stable micturition cycles were established, VDM11 (1, 3 and 10 mg/kg), an anandamide membrane transporter inhibitor, was administered intravenously to evaluate changes in bladder activity. In experiments examining the effects of cannabinoid (CB) receptor antagonists, VDM11 (10 mg/kg) was injected intravenously when the first bladder contraction was observed after intravenous administration of AM251, a CB1 receptor antagonist (3 mg/kg), or AM630, a CB2 receptor antagonist (3 mg/kg).. Intravenous administration of VDM11 increased intercontraction intervals and threshold pressure at doses of 3 mg/kg or higher in dose-dependent fashion. When AM251 was administered one voiding cycle before VDM11 administration, the increases in intercontraction intervals and threshold pressure induced by VDM11 administration alone were not seen. In contrast, when AM630 was administered before VDM11 administration, increases in intercontraction intervals and threshold pressure were observed, as they were after VDM11 alone.. These results suggest that anandamide, an endogenous CB ligand, can modulate the micturition reflex and that anandamide transporters play an important role in this modulation. In urethane-anesthetized rats, inhibition of the uptake of anandamide can inhibit the micturition reflex and these inhibitory effects of VDM11 are at least in part mediated by the CB1 receptor.

Topics: Administration, Intravenous; Anesthetics, Intravenous; Animals; Arachidonic Acids; Endocannabinoids; Female; Indoles; Membrane Transport Modulators; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reflex; Urethane; Urination; Urodynamics

The pathophysiology associated with increased prevalence of depression in diabetics is not completely understood, although studies have pointed the endocannabinoid system as a possible target. Then, we aimed to investigate the role of this system in the pathophysiology of depression associated with diabetes. For this, diabetic (DBT) male Wistar rats were intraperitoneally treated with cannabinoid CB1 (AM251, 1mg/kg) or CB2 (AM630, 1mg/kg) receptor antagonists followed by anandamide (AEA, 0.005mg/kg) and then submitted to the forced swimming test (FST). Oxidative stress parameters, CB1 receptor expression and serotonin (5-HT) and noradrenaline levels in the hippocampus (HIP) and prefrontal cortex (PFC) were also performed. It was observed that DBT animals presented a more pronounced depressive-like behavior and increase of CB1 receptor expression in the HIP. AEA treatment induced a significant improvement in the depressive-like behavior, which was reversed by the CB1 antagonist AM251, without affecting the hyperglycemia or weight gain. AEA was also able to restore the elevated CB1 expression and also to elevate the reduced level of 5-HT in the HIP from DBT animals. In addition, AEA restored the elevated noradrenaline levels in the PFC and induced a neuroprotective effect by restoring the decreased reduced glutathione and increased lipid hydroperoxides levels along with the decreased superoxide dismutase activity observed in HIP or PFC. Together, our data suggest that in depression associated with diabetes, the endocannabinoid anandamide has a potential to induce neuroadaptative changes able to improve the depressive-like response by its action as a CB1 receptor agonist.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Brain Chemistry; Calcium Channel Blockers; Depression; Diabetes Mellitus, Experimental; Endocannabinoids; Indoles; Male; Norepinephrine; Oxidative Stress; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Serotonin; Swimming

The endocannabinoid system (ECS) is associated with an alteration of glucose homeostasis dependent on cannabinoid receptor-1 (CB1R) activation. However, very little information is available concerning the consequences of ECS activation on intestinal glucose absorption. Mice were injected intraperitoneally with anandamide, an endocannabinoid binding both CB1R and CB2R. We measured plasma glucose and xylose appearance after oral loading, gastrointestinal motility, and glucose transepithelial transport using the everted sac method. Anandamide improved hyperglycemia after oral glucose charge whereas glucose clearance and insulin sensitivity were impaired, pointing out some gastrointestinal events. Plasma xylose appearance was delayed in association with a strong decrease in gastrointestinal transit, while anandamide did not alter transporter-mediated glucose absorption. Interestingly, transit was nearly normalized by coinjection of SR141716 and AM630 (CB1R and CB2R antagonist, respectively), and AM630 also reduced the delay of plasma glucose appearance induced by anandamide. When gastric emptying was bypassed by direct glucose administration in the duodenum, anandamide still reduced plasma glucose appearance in wild-type but not in CB1R(-/-) mice. In conclusion, our findings demonstrated that acute activation of intestinal ECS reduced postprandial glycemia independently on intestinal glucose transport but rather inhibiting gastric emptying and small intestine motility and strongly suggest the involvement of both CB1R and CB2R.

Topics: Animals; Arachidonic Acids; Blood Glucose; Endocannabinoids; Gastrointestinal Motility; Gastrointestinal Transit; Hyperglycemia; Indoles; Male; Mice; Mice, Inbred C57BL; Piperidines; Polyunsaturated Alkamides; Postprandial Period; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant

Painful neuropathy frequently develops as a consequence of commonly used chemotherapy agents for cancer treatment and is often a dose-limiting side effect. Currently available analgesic treatments are often ineffective on pain induced by neurotoxicity. Although peripheral administration of cannabinoids, endocannabinoids, and inhibitors of endocannabinoid hydrolysis has been effective in reducing hyperalgesia in models of peripheral neuropathy, including chemotherapy-induced peripheral neuropathy (CIPN), few studies have examined cannabinoid effects on responses of nociceptors in vivo. In this study we determined whether inhibition of fatty acid amide hydrolase (FAAH), which slows the breakdown of the endocannabinoid anandamide (AEA), reduced sensitization of nociceptors produced by chemotherapy. Over the course of a week of daily treatments, mice treated with the platinum-based chemotherapy agent cisplatin developed robust mechanical allodynia that coincided with sensitization of cutaneous C-fiber nociceptors as indicated by the development of spontaneous activity and increased responses to mechanical stimulation. Administration of the FAAH inhibitor URB597 into the receptive field of sensitized C-fiber nociceptors decreased spontaneous activity, increased mechanical response thresholds, and decreased evoked responses to mechanical stimuli. Cotreatment with CB1 (AM281) or CB2 (AM630) receptor antagonists showed that the effect of URB597 was mediated primarily by CB1 receptors. These changes following URB597 were associated with an increase in the endocannabinoid anandamide in the skin. Our results suggest that enhanced signaling in the peripheral endocannabinoid system could be utilized to reduce nociceptor sensitization and pain associated with CIPN.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Cisplatin; Endocannabinoids; Hydrolysis; Indoles; Male; Mice; Morpholines; Nerve Fibers, Unmyelinated; Nociception; Nociceptors; Peripheral Nervous System Diseases; Polyunsaturated Alkamides; Pyrazoles; Skin

Enhancement of the endocannabinoid (EC) system may reduce anticipatory nausea (AN).. The experiments evaluated the potential of the dual fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase (MAGL) inhibitor, JZL195, on its own and combined with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) to reduce contextually elicited gaping, a measure of AN in rats.. Following four context lithium chloride (LiCl) pairings, rats were injected with vehicle (VEH) or JZL195 (10 mg kg(-1), intraperitoneally) 105 min before an injection of VEH, 2-AG (1.25 mg kg(-1)), or AEA (5.0 mg kg(-1)). Fifteen minutes later, all rats were placed in the LiCl-paired context for 5 min and in a different context for a 15-min locomotor test. Whole brains were extracted for EC analysis. The potential of the CB1 antagonist, SR141716, to reverse the suppression of AN by both JZL195 and AEA and of the CB2 antagonist, AM630, to reverse the suppression of AN by JZL195 was then evaluated.. JZL195 suppressed gaping and elevated AEA, palmitoylethanolamine, and oleoylethanolamide. As the suppression of gaping was reversed by SR141716, but not by AM630, the effect was CB1 mediated. The suppressive effect of JZL195 on gaping, as well as elevation of AEA and 2-AG, was amplified by pretreatment with either AEA or 2-AG. On its own, AEA, but not 2-AG, also suppressed gaping-an effect that was also prevented by CB1 antagonism.. JZL195 reduces AN primarily by acting as a FAAH inhibitor, but MAGL inhibition is also indicated.

Topics: Amidohydrolases; Animals; Anticipation, Psychological; Arachidonic Acids; Brain; Cannabinoid Receptor Antagonists; Carbamates; Endocannabinoids; Enzyme Inhibitors; Glycerides; Indoles; Lithium Chloride; Male; Monoacylglycerol Lipases; Motor Activity; Nausea; Oleic Acids; Piperazines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB1 , CB2 , TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non-selective), JWH-015 (CB2 ) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre-treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T9 -T12 ) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α-CGRP produced frequency-dependent or dose-dependent vasodepressor responses. The infusions of anandamide in a dose-dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH-015 or lysophosphatidylinositol), but not those by α-CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31-100 μg/kg NIDA41020 (CB1 ), (ii) unaffected by 180 μg/kg AM630 (CB2 ), 31 μg/kg cannabidiol (GPR55) or 31-100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre-junctional activation of CB1 receptors, with no pharmacological evidence for the role of CB2 , TRPV1 or GPR55 receptors.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cannabinoid Receptor Agonists; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Indoles; Lysophospholipids; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Vasodilation

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+ -uptake and Ca2+ -ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10 μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 μg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.

Topics: Action Potentials; Animals; Arachidonic Acids; Caffeine; Calcium; Calcium Signaling; Endocannabinoids; Fura-2; Heart Ventricles; In Vitro Techniques; Indoles; Male; Myocardial Contraction; Myocytes, Cardiac; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Sarcoplasmic Reticulum; Transport Vesicles

Cisplatin has been used effectively to treat a variety of cancers but its use is limited by the development of painful peripheral neuropathy. Because the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is anti-hyperalgesic in several preclinical models of chronic pain, the anti-hyperalgesic effect of JZL184, an inhibitor of 2-AG hydrolysis, was tested in a murine model of cisplatin-induced hyperalgesia. Systemic injection of cisplatin (1mg/kg) produced mechanical hyperalgesia when administered daily for 7 days. Daily peripheral administration of a low dose of JZL184 in conjunction with cisplatin blocked the expression of mechanical hyperalgesia. Acute injection of a cannabinoid (CB)-1 but not a CB2 receptor antagonist reversed the anti-hyperalgesic effect of JZL184 indicating that downstream activation of CB1 receptors suppressed the expression of mechanical hyperalgesia. Components of endocannabinoid signaling in plantar hind paw skin and lumbar dorsal root ganglia (DRGs) were altered by treatments with cisplatin and JZL184. Treatment with cisplatin alone reduced levels of 2-AG and AEA in skin and DRGs as well as CB2 receptor protein in skin. Combining treatment of JZL184 with cisplatin increased 2-AG in DRGs compared to cisplatin alone but had no effect on the amount of 2-AG in skin. Evidence that JZL184 decreased the uptake of [(3)H]AEA into primary cultures of DRGs at a concentration that also inhibited the enzyme fatty acid amide hydrolase, in conjunction with data that 2-AG mimicked the effect of JZL184 on [(3)H]AEA uptake support the conclusion that AEA most likely mediates the anti-hyperalgesic effect of JZL184 in this model.

Topics: Amides; Analgesics; Animals; Antineoplastic Agents; Arachidonic Acids; Benzodioxoles; Cells, Cultured; Cisplatin; Disease Models, Animal; Endocannabinoids; Ethanolamines; Ganglia, Spinal; Glycerides; Hyperalgesia; Indoles; Male; Mesencephalon; Mice; Mice, Inbred C3H; Monoacylglycerol Lipases; Morpholines; Neuralgia; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin; Spinal Cord

Endocannabinoid anandamide (AEA) has protective effect on the heart against ischemia/reperfusion injury and arrhythmia, but the electrophysiological mechanism is unclear yet. In this study, the sinoatrial node (SAN) samples from New Zealand rabbits were prepared, and intracellular recording technique was used to elucidate the effect of AEA on the action potential (AP) of SAN pacemaker cells of rabbits and the mechanism. Different concentrations of AEA (1, 10, 100, 200, 500 nmol/L) were applied cumulatively. For some SAN samples, cannabinoid type 1 (CB1) receptor antagonist AM251, cannabinoid type 2 (CB2) receptor antagonist AM630, potassium channel blocker tetraethylammonium (TEA) and nitric oxide (NO) synthase inhibitor L-nitro-arginine methylester (L-NAME) were used before AEA treatment, respectively. We found that: (1) AEA (100, 200 and 500 nmol/L) not only shortened AP duration (APD), but also decreased AP amplitude (APA) (P < 0.05). (2) AM251, but not AM630, abolished the effect of AEA on APD shortening. (3) TEA and L-NAME had no influence on the AEA effect. These findings suggest that anandamide can decrease APA and shorten APD in SAN pacemaker cells of rabbits, which may be mediated by activation of CB1 receptors, and is related to blockade of calcium channels but not potassium channels and NO.

Topics: Action Potentials; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Endocannabinoids; Indoles; Myocytes, Cardiac; NG-Nitroarginine Methyl Ester; Nitric Oxide; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Pyrazoles; Rabbits; Sinoatrial Node

Opioids do not effectively manage pain in many patients with advanced cancer. Because anandamide (AEA) activation of cannabinoid type-1 receptors (CB1R) on nociceptors reduces nociception, manipulation of AEA metabolism in the periphery may be an effective alternative or adjuvant therapy in the management of cancer pain. AEA is hydrolyzed by the intracellular enzyme fatty acid amide hydrolase (FAAH), and this enzyme activity contributes to uptake of AEA into neurons and to reduction of AEA available to activate CB1R. We used an in vitro preparation of adult murine dorsal root ganglion (DRG) neurons co-cultured with fibrosarcoma cells to investigate how tumors alter the uptake of AEA into neurons. Evidence that the uptake of [(3)H]AEA into dissociated DRG cells in the co-culture model mimicked the increase in uptake that occurred in DRG cells from tumor-bearing mice supported the utility of the in vitro model to study AEA uptake. Results with the fluorescent AEA analog CAY10455 confirmed that an increase in uptake in the co-culture model occurred in neurons. One factor that contributed to the increase in [(3)H]AEA uptake was an increase in total cellular cholesterol in the cancer condition. Treatment with the FAAH inhibitor URB597 reduced CAY10455 uptake in the co-culture model to the level observed in DRG neurons maintained in the control condition (i.e., in the absence of fibrosarcoma cells), and this effect was paralleled by OMDM-1, an inhibitor of AEA uptake, at a concentration that had no effect on FAAH activity. Maximally effective concentrations of the two drugs together produced a greater reduction than was observed with each drug alone. Treatment with BMS309403, which competes for AEA binding to fatty acid binding protein-5, mimicked the effect of OMDM-1 in vitro. Local injection of OMDM-1 reduced hyperalgesia in vivo in mice with unilateral tumors in and around the calcaneous bone. Intraplantar injection of OMDM-1 (5μg) into the tumor-bearing paw reduced mechanical hyperalgesia through a CB1R-dependent mechanism and also reduced a spontaneous nocifensive behavior. The same dose reduced withdrawal responses evoked by suprathreshold mechanical stimuli in naive mice. These data support the conclusion that OMDM-1 inhibits AEA uptake by a mechanism that is independent of inhibition of FAAH and provide a rationale for the development of peripherally restricted drugs that decrease AEA uptake for the management of cancer pain.

Topics: Animals; Arachidonic Acids; Benzamides; Brain Neoplasms; Cannabinoid Receptor Antagonists; Carbamates; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Fibrosarcoma; Fluorescent Dyes; Ganglia, Spinal; Hyperalgesia; Indoles; Lactones; Male; Mice; Mice, Inbred C3H; Pain; Pain Threshold; Polyunsaturated Alkamides; Sensory Receptor Cells; Statistics, Nonparametric; Tritium

Anandamide, one of the endocannabinoids, has been reported to exhibit cardioprotective properties, particularly in its ability to limit the damage produced by ischemia reperfusion injury. However, the mechanisms underlying the effect are not well known. This study is to investigate whether anandamide alter Na(+)/Ca(2+) exchanger and the intracellular free Ca(2+) concentration ([Ca(2+)]i).. Na(+)/Ca(2+) exchanger current (I(NCX)) was recorded and analysed by using whole-cell patch-clamp technique and [Ca(2+)]i was measured by loading myocytes with the fluorescent Ca(2+) indicator Fura-2/AM.. We found that I(NCX) was enhanced significantly after perfusion with simulated ischemic external solution; [Ca(2+)]i was also significantly increased by simulated ischemic solution. The reversal potential of I(NCX) was shifted to negative potentials in simulated ischemic external solution. Anandamide (1-100 nM) failed to affect I(NCX) and [Ca(2+)]i in normal solution. However, anandamide (1-100 nM) suppressed the increase in INCX in simulated ischemic external solution concentration-dependently and normalized INCX reversal potential. Furthermore, anandamide (100 nM) significantly attenuated the increase in [Ca(2+)]i in simulated ischemic solution. Blocking CB1 receptors with the specific antagonist AM251 (500 nM) failed to affect the effects of anandamide on I(NCX) and [Ca(2+)]i in simulated ischemic solution. CB2 receptor antagonist AM630 (100 nM) eliminated the effects of anandamide on I(NCX) and [Ca(2+)]i in simulated ischemic solution, and CB2 receptor agonist JWH133 (100 nM) simulated the effects of anandamide that suppressed the increase in I(NCX) and [Ca(2+)]i in simulated ischemic solution. In addition, pretreatment with the Gi/o-specific inhibitor pertussis toxin (PTX, 500 ng/ml) eliminated the effects of anandamide and JWH133 on I(NCX) in simulated ischemic solution.. Collectively, these findings suggest that anandamide suppresses calcium overload through inhibition of I(NCX) during perfusion with simulated ischemic solution; the effects may be mediated by CB2 receptor via PTX-sensitive Gi/o proteins. This mechanism is importantly involved in the anti-ischemia injury caused by endocannabinoids.

Topics: Aniline Compounds; Animals; Arachidonic Acids; Calcium; Cannabinoids; Cell Separation; Endocannabinoids; GTP-Binding Protein alpha Subunits, Gi-Go; Heart Ventricles; Indoles; Intracellular Space; Ion Channel Gating; Male; Myocardial Ischemia; Myocytes, Cardiac; Pertussis Toxin; Phenyl Ethers; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Sodium-Calcium Exchanger; Solutions

Recently, we demonstrated that peripheral antinociception induced by δ opioid receptor is dependent of Ca(2+)-activated Cl(-) channels (CaCCs). Because opioid and cannabinoid receptors share some common mechanisms of action, our objective was to identify a possible relationship between CaCCs and the endocannabinoid system.. To induce hyperalgesia, rat paws were treated with intraplantar prostaglandin E2 (PGE2, 2μg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3h following injection. Probabilities were calculated using ANOVA/Bonferroni's test, and values that were less than 5% were considered to be statistically significant.. Administration of the cannabinoid agonist CB1 anandamide (12.5, 25 and 50μg/paw) and the cannabinoid agonist CB2 PEA (5, 10 and 20μg/paw) decreased the PGE2-induced hyperalgesia in a dose-dependent manner. The possibility of the higher doses of anandamide (50μg) and PEA (20μg) having a central or systemic effect was excluded because the administration of the drug into the contralateral paw did not elicit antinociception in the right paw. As expected, the antinociceptive effects induced by anandamide and PEA were blocked by the CB1 and CB2 receptor antagonists AM251 and AM630, respectively. The peripheral antinociception was induced by anandamide but not PEA and was dose-dependently inhibited by the CaCC blocker niflumic acid (8, 16 and 32μg).. These results provide the first evidence for the involvement of CaCCs in the peripheral antinociception induced by activation of the CB1 cannabinoid receptor.

Topics: Amides; Analysis of Variance; Animals; Arachidonic Acids; Calcium Channel Blockers; Cannabinoid Receptor Agonists; Chloride Channels; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Ethanolamines; Hyperalgesia; Indoles; Male; Niflumic Acid; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

The energy required for active Na chloride reabsorption in the thick ascending limb (TAL) depends on oxygen consumption and oxidative phosphorylation (OXP). In other cells, Na transport is inhibited by the endogenous cannabinoid anandamide through the activation of the cannabinoid receptors (CB) type 1 and 2. However, it is unclear whether anandamide alters TAL transport and the mechanisms that could be involved. We hypothesized that anandamide inhibits TAL transport via activation of CB1 receptors and NO. For this, we measured oxygen consumption (Q(O(2))) in TAL suspensions to monitor the anandamide effects on transport and OXP. Anandamide reduced Q(O(2)) in a concentration-dependent manner. During Na-K-2Cl cotransport and Na/H exchange inhibition, anandamide did not inhibit TAL Q(O(2)). To test the role of the cannabinoid receptors, we used specific agonists and antagonists of CB1 and CB2 receptors. The CB1-selective agonist WIN55212-2 reduced Q(O(2)) in a concentration-dependent manner. Also, the CB1 receptor antagonist rimonabant blocked the effect of anandamide on Q(O(2)). In contrast, the CB2-selective agonist JHW-133 had no effect on Q(O(2)), while the CB2 receptor antagonist AM-630 failed to block the anandamide effects on Q(O(2)). To confirm these results, we measured CB1 and CB2 receptor expression and only CB1 expression was detected. Because CB1 receptors are strong nitric oxide synthase (NOS) stimulators and NO inhibits transport in TALs, we evaluated the role of NO. Anandamide stimulated NO production and the NOS inhibitor N(G)-nitro-L-arginine methyl ester blocked the anandamide effects on Q(O(2)). We conclude that anandamide inhibits TAL Na transport-related Q(O(2)) via activation of CB1 receptor and NOS.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cell Culture Techniques; Endocannabinoids; Indoles; Ion Transport; Loop of Henle; Male; Morpholines; Naphthalenes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxygen Consumption; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.

Topics: Animals; Anti-Arrhythmia Agents; Arachidonic Acids; Arrhythmias, Cardiac; Cardiac Electrophysiology; Dose-Response Relationship, Drug; Endocannabinoids; Heart Ventricles; Indoles; KATP Channels; Male; Myocytes, Cardiac; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

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

Periodontitis is an infectious disease leading to inflammation and destruction of tissue surrounding and supporting the tooth. The progress of the inflammatory response depends on the host's immune system and risk factors such as stress. The aim of the present study was to investigate the role of the endocannabinoid anandamide (AEA) in experimental periodontitis with restraint stress, since the endocannabinoid system is known to modulate the hypothalamo-pituitary-adrenal axis as well as immune functions and has been found in human gingival tissues.. Experimental periodontitis was induced by ligature around first inferior molars and immobilization stress for 2 h twice daily for 7 days in a rat model.. Corticosterone plasma levels, locomotor activity, adrenal gland weight and bone loss were increased in periodontitis and stress groups, and there was also less weight gain. The inflammatory parameters such as prostaglandin E(2) (radioimmunoassay), nitric oxide (radioconversion of (14)C-arginine), tumor necrosis factor (TNF)-α (ELISA) and interleukin (IL)-1β (Western blot) measured in the gingival tissue were significantly increased in the periodontitis groups compared to the control group. Local injection of AEA (10(-8)M, 30 µl) decreased corticosterone plasma levels and the content of the cytokines TNF-α and IL-1β in gingival tissue in periodontitis-stress groups. These AEA-induced inhibitions were mediated by CB(1) and CB(2) cannabinoid receptors since the injection of both antagonists together, AM251 (10(-6)M) and AM630 (10(-6)M) in 30 µl, prevented these effects.. The endocannabinoid AEA diminishes the inflammatory response in periodontitis even during a stressful situation.

Topics: Alveolar Bone Loss; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Body Weight; Cannabinoid Receptor Agonists; Corticosterone; Disease Models, Animal; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Exploratory Behavior; Indoles; Interleukin-1beta; Male; Nitric Oxide Synthase; Periodontitis; Piperidines; Polyunsaturated Alkamides; Prostaglandins E; Pyrazoles; Rats; Rats, Wistar; Statistics, Nonparametric; Stress, Psychological; Tumor Necrosis Factor-alpha

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

Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) G protein coupled receptors. Docking of agonists and antagonists to CB(1) and CB(2) cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch and its possible participation in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB(1) and CB(2) receptor models were constructed based on the adenosine A(2A) receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β(2)AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation.

Topics: Amino Acid Motifs; Arachidonic Acids; Binding Sites; Dronabinol; Endocannabinoids; Fenoterol; Humans; Hydrogen Bonding; Indoles; Ligands; Molecular Dynamics Simulation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Adrenergic, beta-2; Stereoisomerism; Thermodynamics

To study the influences of endocannabinoid-anandamide (AEA) on the proliferation and apoptosis of the colorectal cancer cell line (CaCo-2) and to elucidate the effects of CB1 and lipid rafts, and to further elucidate the molecular mechanism and the effect of AEA on the generation and development of colorectal cancer.. Human colorectal cancer cell line CaCo-2 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in 5% CO(2) atmosphere at 37°C. CaCo-2 cells were divided into different groups and treated with different concentrations of AEA, AEA + SR141716A, AEA + AM630 and AEA + methyl-β-cyclodextrin (MCD). MTT assay was used to determine the effects of AEA, its putative CB1, CB2 receptor antagonists (SR141716A and AM630) and MCD on the proliferation of CaCo-2 cells. Annexin V-PE/7AAD binding assay was used to detect apoptosis in the CaCo-2 cells. Western-blot was applied to check the expressions of CB1, CB2, p-AKT and caspase-3 proteins in different groups of CaCo-2 cells.. AEA inhibited the proliferation of CaCo-2 cells in a concentration-dependent manner and the effect could be antagonized by SR141716A and MCD. The inhibiting rates were (21.52 ± 0.45)%, (42.16 ± 0.21)%, (73.64 ± 0.73)% and (83.28 ± 0.71)%, respectively, at different concentrations of AEA (5, 10, 20 and 40 µmol/L). The three groups (20 µmol/L AEA, 20 µmol/L AEA + 10 µmol/L SR141716A and 20 µmol/L AEA + 1 mmol/L MCD) showed different inhibiting rates [(73.64 ± 0.73)%, (16.15 ± 0.75)% and (12.58 ± 0.63)%], respectively. Annexin V-PE/7AAD binding assay showed that AEA induced apoptosis in the CaCo-2 cells and MCD could antagonize this effect. The apoptosis rates of the three groups (control, 20 µmol/L AEA and 20 µmol/L AEA + 1 mmol/L MCD) were (2.95 ± 0.73)%, (39.61 ± 0.73)% and (14.10 ± 0.64)%, respectively. The expressions of CB1, CB2, p-AKT and Caspase-3 proteins were all observed in the CaCo-2 cells. AEA inhibited p-AKT protein expression and induced caspase-3 protein expression. The two actions were also antagonized by MCD.. AEA can strongly suppress the proliferation of colorectal cancer CaCo-2 cells via the CB1 receptor and membrane cholesterol-LRs and induce apoptosis via lipid rafts. Anandamide plays a very important role in the carcinogenesis and development of colorectal cancer. MCD is a critical member in this system.

Topics: Antineoplastic Agents; Apoptosis; Arachidonic Acids; beta-Cyclodextrins; Caco-2 Cells; Cannabinoid Receptor Modulators; Caspase 3; Cell Proliferation; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Indoles; Membrane Microdomains; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

This study investigated the participation of the hypothalamic endocannabinoid system in the response to lipopolysaccharide (LPS) challenge evaluating oxytocin (OXT) and tumor necrosis factor-alpha (TNF-alpha) plasma levels in vivo and their release from hypothalamic fragments in vitro. LPS increased OXT and TNF-alpha release through anandamide-activation of hypothalamic cannabinoid receptor CB(1,) since the antagonist AM251 blocked this effect. Anandamide, through its receptors, also increased hypothalamic nitric oxide (NO) which inhibited OXT release, ending the stimulatory effect of the endocannabinoid. Our findings reveal a hypothalamic interaction between oxytocin, endocannabinoid and NO-ergic systems providing a regulation of the hypothalamic-neurohypophyseal axis under basal and stress conditions.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Hypothalamus; Indoles; Lipopolysaccharides; Male; Nitric Oxide; Oxytocin; Polyunsaturated Alkamides; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Tumor Necrosis Factor-alpha

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Proliferation; Endocannabinoids; Fibroblasts; Gingival Crevicular Fluid; Glycerides; Humans; Indoles; p38 Mitogen-Activated Protein Kinases; Periodontium; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Wound Healing

The aim of the present paper was to assess whether lipopolysaccharide (LPS)-induced inhibition of salivary secretion involves the activation of the endocannabinoid system and the participation of tumor necrosis factor (TNF)alpha in the submandibular gland.. Pharmacological approaches were performed by using CB1 and/or CB2 cannabinoid receptor antagonists, AM251 and AM630, respectively, injected into the submandibular gland, to study the participation of the endocannabinoid system in LPS inhibitory effects on metacholine-induced salivary secretion. To assess the participation of TNFalpha on LPS inhibitory effects, salivary secretion was studied in LPS treated rats after the intraglandular injection of etanercept, a soluble form of TNF receptor which blocks TNFalpha action. Finally, to evaluate the possible interplay between endocannabinoids and TNFalpha on the submandibular gland function reduced during LPS challenge, the salivary secretion was studied after the intraglandular injection of this cytokine alone or concomitantly with AM251 and AM630.. AM251 and AM630, injected separately or concomitantly, partially prevented LPS-induced inhibition of salivation. Also, anandamide synthase activity was increased in submandibular glands extracted from rats 3h after LPS injection, suggesting that the endocannabinoid system was activated in response to this challenge. On the other hand, etanercept, prevented the inhibitory effect of LPS on salivary secretion and moreover, TNFalpha injected intraglandularly inhibited salivary secretion, being this effect prevented by AM251 and AM630 injected concomitantly.. The present results demonstrate the participation of the endocannabinoid system and TNFalpha on salivary responses during systemic inflammation induced by LPS.

Topics: Amidohydrolases; Animals; Arachidonic Acid; Arachidonic Acids; Cannabinoid Receptor Modulators; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Endocannabinoids; Etanercept; Immunoglobulin G; Immunologic Factors; In Vitro Techniques; Indoles; Lipopolysaccharides; Male; Methacholine Chloride; Muscarinic Agonists; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; Saliva; Salivation; Secretory Rate; Submandibular Gland; Tumor Necrosis Factor-alpha

Anandamide and sphingosine-1-phosphate (S1P) both regulate vascular tone in a variety of vessels. This study aimed to examine the mechanisms involved in the regulation of coronary vascular tone by anandamide and S1P, and to determine whether any functional interaction occurs between these receptor systems.. Mechanisms used by anandamide and S1P to regulate rat coronary artery (CA) reactivity were investigated using wire myography. Interactions between S1P and the cannabinoid (CB)(2) receptor were determined using human embryonic kidney 293 (HEK293) cells that stably over-express recombinant CB(2) receptor.. Anandamide and S1P induced relaxation of the rat CA. CB(2) receptor antagonists attenuated anandamide-induced relaxation, while S1P-mediated relaxation was dependent on the vascular endothelium and S1P(3). Anandamide treatment resulted in an increase in the phosphorylation of sphingosine kinase-1 within the CA. Conversely, anandamide-mediated relaxation was attenuated by inhibition of sphingosine kinase. Moreover, S1P(3), specifically within the vascular endothelium, was required for anandamide-mediated vasorelaxation. In addition to this, S1P-mediated relaxation was also reduced by CB(2) receptor antagonists and sphingosine kinase inhibition. Further evidence that S1P functionally interacts with the CB(2) receptor was also observed in HEK293 cells over-expressing the CB(2) receptor.. In the vascular endothelium of rat CA, anandamide induces relaxation via a mechanism requiring sphingosine kinase-1 and S1P/S1P(3). In addition, we report that S1P may exert some of its effects via a CB(2) receptor- and sphingosine kinase-dependent mechanism, where subsequently formed S1P may have privileged access to S1P(3) to induce vascular relaxation.

Topics: Animals; Arachidonic Acids; Calcium Channel Blockers; Cardiovascular Agents; Cell Line; Coronary Vessels; Dronabinol; Endocannabinoids; Humans; Indoles; Indomethacin; Lysophospholipids; Male; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Sphingosine; Vasodilation

Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB₁ cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB₁ receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Carrageenan; Chromatography, Liquid; Disease Models, Animal; Drug Administration Routes; Drug Administration Schedule; Endocannabinoids; Enzyme Inhibitors; Escape Reaction; Ethylene Glycols; Feeding Behavior; Formaldehyde; Gene Expression Regulation; Hyperalgesia; Indoles; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Knockout; Monoacylglycerol Lipases; Motor Activity; Oncogene Proteins v-fos; Pain; Pain Measurement; Pain Threshold; Peripheral Nervous System Diseases; Piperidines; Polyunsaturated Alkamides; PPAR alpha; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Sciatica; Spinal Cord; Statistics, Nonparametric; Time Factors; Tissue Distribution; Tritium

Endogenous endocannabinoids bind to cannabinoid receptors; namely CB1, CB2, TRPV1 and GPR55, to activate intracellular pathways that control many cellular functions. Elevated levels of endocannabinoids have been identified in diseases such as obesity and diabetes, with the onset of diabetic nephropathy associated with proximal tubule hypertrophy. Recent research has identified a role for CB1 in apoptosis in human proximal tubular (HK2) cells, however the role of the other receptors has not been investigated. We investigated if the cannabinoid receptors played a role in hypertrophy in HK2 cells. Characterisation of HK2 cells demonstrated that mRNA and protein for CB1, CB2, TRPV1 and GPR55 occurs in these cells. Importantly, activation of the cannabinoid receptors with anandamide significantly increases hypertrophy in HK2 cells. In general, treatment with CB1 antagonist AM-251, reduces hypertrophy while treatment with CB2 (AM-630) and TRPV1 (SB-366791) antagonists increases hypertrophy. Targeting a cannabinoid receptor sensitive to O-1918 in HK2 cells did not alter proximal tubule cell hypertrophy. Therefore it is likely that in human proximal tubule, these receptors regulate cellular function by activating different cell signalling pathways. Nonetheless, we have identified a role for cannabinoid receptors in proximal tubule cells which may provide novel therapeutic targets for the treatment of diabetes and obesity.

Topics: Animals; Arachidonic Acids; Cell Enlargement; Cell Line; Endocannabinoids; Humans; Indoles; Kidney Tubules, Proximal; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; TRPV Cation Channels

Folic acid (FA) is an essential micronutrient that is particularly important during pregnancy for normal placental and fetal development and growth. The placenta and the fetus become frequently exposed to drugs of abuse such as cannabinoids because of maternal use of these substances. The aim of this study was to investigate the influence of cannabinoids on the uptake of FA by BeWo cells. Acute treatment with anandamide (1-10 micromol/l) caused a 15% decrease in (3)H-FA uptake at pH 7.5. Moreover, tetrahydrocannabinol (THC) (1-10 micromol/l) caused a 30% increase, and AM630 (1 mumol/l) a 15% decrease in this parameter at pH 6.5. Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed in the presence of cannabi- noid receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronic treatment (48 h) with THC (100 nmol/l) and AM251 (100 nmol/l) decreased the uptake of (3)H-FA by 20% at pH 7.5, and anandamide (1 micromol/l) and AM630 (10-500 nmol/l) increased it by 30%. Moreover, CP55,940 (10 nmol/l) increased the uptake of (3)H-FA by 30% at pH 6.5. RT-PCR analysis showed that the mRNA levels of the reduced folate transporter 1 increased by 9% after chronic treatment with AM630 (500 nmol/l). The mRNA levels of the proton-coupled folate transporter decreased by 17% and increased by 18% after chronic treatment with THC (0.1 mumol/l) and AM251 (100 nmol/l), respectively. In conclusion, (3)H-FA uptake by BeWo cells is significantly, although not very markedly, changed by several distinct CB receptor agonists and antagonists, both after acute and chronic exposure of the cells. The acute effects of cannabinoid receptor agonists do not seem to be mediated by the cannabinoid receptor, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters.

Topics: Arachidonic Acids; Carrier Proteins; Cell Culture Techniques; Cell Line, Tumor; Cyclohexanols; Dronabinol; Drug Administration Schedule; Endocannabinoids; Female; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Hydrogen-Ion Concentration; Indoles; Membrane Transport Proteins; Piperidines; Placenta; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cell Surface

Acupuncture is widely used clinically to treat acute and chronic pain conditions, but the mechanisms underlying its effect are not fully understood. Although endocannabinoids are involved in modulation of nociception in animal models and in humans, their role in acupuncture analgesia has not been assessed. In this report, we determined the effect of electroacupuncture (EA) on the level of anandamide in the skin tissue and the role of cannabinoid CB1 and CB2 receptors in the analgesic effect of EA in an animal model of inflammatory pain. Inflammatory pain was induced by local injection of complete Freund's adjuvant (CFA) into the hind paw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified with von Frey filaments. The anandamide concentration in the skin tissue was measured by using high-performance liquid chromatography. EA, applied to GB30 and GB34, at 2 and 100Hz significantly reduced thermal hyperalgesia and mechanical allodynia induced by CFA injection. Compared with the sham group, EA significantly increased the anandamide level in the inflamed skin tissue. Local pretreatment with a specific CB2 receptor antagonist, AM630, significantly attenuated the antinociceptive effect of EA. However, the effect of EA was not significantly altered by AM251, a selective CB1 receptor antagonist. These findings suggest that EA potentiates the local release of endogenous anandamide from inflamed tissues. Activation of peripheral CB2 receptors contributes to the analgesic effect of EA on inflammatory pain.. This study shows that electroacupuncture increases the anandamide level in inflammatory skin tissues, and CB2 receptors contribute to the analgesic effect of electroacupuncture in a rat model of inflammatory pain. This information improves our understanding of the mechanisms involved in the analgesic effect of acupuncture.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Electroacupuncture; Endocannabinoids; Freund's Adjuvant; Hindlimb; Hot Temperature; Hyperalgesia; Indoles; Male; Pain; Pain Management; Physical Stimulation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin

Cannabinoids have long been known to be potent inhibitors of intestinal and colonic propulsion. This effect has generally been attributed to their ability to prejunctionally inhibit release of acetylcholine from excitatory motor neurons that mediate, in part, the ascending contraction phase of the peristaltic reflex. In the present study we examined the effect of cannabinoids on the other transmitters known to participate in the peristaltic reflex using a three-compartment preparation of rat colon that allows separation of ascending contraction, descending relaxation, and the sensory components of the reflex. On addition to the orad motor compartment, anandamide decreased and AM-251, a CB-1 antagonist, increased ascending contraction and the concomitant substance P (SP) release. Similarly, on addition to the caudad motor compartment, anandamide decreased and AM-251 increased descending relaxation and the concomitant vasoactive intestinal peptide (VIP) release. On addition to the central sensory compartment, anandamide decreased and AM-251 increased both ascending contraction and SP release orad, and descending relaxation and VIP release caudad. This suggested a role for CB-1 receptors in modulation of sensory transmission that was confirmed by the demonstration that central addition of anandamide decreased and AM-251 increased release of the sensory transmitter, calcitonin gene-related peptide (CGRP). We conclude that the potent antipropulsive effect of cannabinoids is the result of inhibition of both excitatory cholinergic/tachykininergic and inhibitory VIPergic motor neurons that mediate ascending contraction and descending relaxation, respectively, as well as inhibition of the intrinsic sensory CGRP-containing neurons that initiate the peristaltic reflex underlying propulsive motility.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cannabinoid Receptor Modulators; Colon; Dose-Response Relationship, Drug; Endocannabinoids; Guinea Pigs; In Vitro Techniques; Indoles; Motor Neurons; Neural Inhibition; Peristalsis; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reflex; Sensory Receptor Cells; Substance P; Vasoactive Intestinal Peptide

To study the effects of endogenous cannabinoid anandamide (AEA) and its putative endocannabinoid receptors (CBR) on the activation and proliferation of hepatic stellate cells (HSC) and to study the role played by AEA during liver fibrosis.. By using immunofluorescence and cell culture, the expression of CBR 1 and 2 in the PDGF-stimulated HSCs was investigated. By using PCR and Western-blot, the effects of 10, 20mumol/L AEA and CBR2 antagonist AM630 on the cultured and activated HSC were observed. Methyl thiazolyl tetrazolium and flow cytometry were used to investigate whether AEA induces growth inhibition or apoptosis in the activated HSCs.. Both CBR1 and CBR2 receptors were detectable in cultured HSCs with a higher level of CBR2 than CBR1 (F = 116.797, P less than 0.01). When HSCs were stimulated by PDGF, the expression of CBR2 receptors was significantly enhanced (F = 7.878, P less than 0.05). HSC proliferation was dose-dependently inhibited by 10, 20, and 50micromol/L AEA, with the rates of 7.12%+/-0.34%, 12.52%+/-0.78%, 80.13%+/-1.57% respectively (F = 533.41, P less than 0.01). However, it did not induce apoptosis, but necrosis. The expressions of alpha-SMA, TGFb1, a1(I), a1(III) and TIMP-1 were significantly suppressed by 20micromol/L AEA, but CBR2 antagonist AM630 reversed this suppressor action of AEA.. AEA may inhibit activation and proliferation of HSCs; CBR2 receptors mediate AEA-induced inhibitory action on the activation of HSCs. This CBR2 receptor-mediated action and AEA on HSCs could be used as a therapeutic target against liver fibrosis.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Proliferation; Cells, Cultured; Endocannabinoids; Hepatic Stellate Cells; Indoles; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2

The purpose of the present study was to examine whether cannabinoid receptor agonists influence spontaneous contractile activity of longitudinal muscle in mouse ileum in vitro. Isolated segments of mouse ileum displayed spontaneous contractions with an amplitude and frequency of about 300 mg and 30 cpm, respectively. The endocannabinoid anandamide (1-100 microM), the selective cannabinoid CB(1) receptor agonist, ACEA (0.1 microM-10 microM), but not the selective cannabinoid CB(2) receptor agonist, JWH 133 (0.1 microM-10 microM), reduced in a concentration-dependent manner the spontaneous mechanical activity. The inhibitory effect consisted in a decrease of the mean amplitude of longitudinal spontaneous contractions, without changes in the resting tone. The inhibitory effect induced by cannabinoids was significantly antagonized by the selective cannabinoid CB(1) receptor antagonist, SR141716A (0.1 microM), but not by the selective cannabinoid CB(2) receptor antagonist, AM630 (0.1 microM). None of the cannabinoid antagonists, at the concentration used, did affect the spontaneous mechanical activity. The ACEA-induced reduction of spontaneous contractions was almost abolished by tetrodotoxin, atropine or apamin and it was unaffected by hexamethonium or N(omega)-nitro-l-arginine methyl ester (l-NAME), inhibitor of nitric oxide synthase. The myogenic contractions evoked by carbachol were not affected by ACEA. In conclusion, the present results suggest that activation of neural cannabinoid CB(1) receptors may play a role in the control of spontaneous mechanical activity through inhibition of acetylcholine release from cholinergic nerve. Activation of small conductance Ca(2+)-dependent K(+) channels is involved in this action.

Topics: Animals; Apamin; Arachidonic Acids; Atropine; Cannabinoid Receptor Modulators; Cannabinoids; Dose-Response Relationship, Drug; Endocannabinoids; Hexamethonium; Ileum; In Vitro Techniques; Indoles; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Tetrodotoxin

The endogenous cannabinoid system plays a role in the regulation of energy homeostasis acting through central pathways, and its dysregulation may be implicated in the pathogenesis of obesity. Recent evidence is accumulating showing that the endogenous cannabinoid system is also present in peripheral tissues. The aim of this work was to investigate the effect of cannabinoids upon the intestinal absorption of glucose. For this, we investigated the effect of some cannabinoid receptor agonists and antagonists upon the apical uptake of 3H-2-deoxy-D-glucose by the human intestinal epithelial Caco-2 cells. Uptake of a low concentration of 3H-2-deoxy-D-glucose (1 micromol/l) was both cytochalasin B- and phloridzin-sensitive. The maximal inhibition obtained with each of these inhibitors was 50%, and their effect was not cumulative. On the other hand, uptake of a high concentration of 3H-2-deoxy-D-glucose (20 mmol/l) was partially inhibited by cytochalasin B (+/-20%) and phloridzin had no effect. We verified that neither the cannabinoid receptor agonists [tetrahydrocannabinol (1-10 micromol/l), anandamide (0.1-10 micromol/l) and CP 55,940 (5 nmol/l to 1 micromol/l)], nor the specific CB1 and CB2 antagonists [AM251 (10-500 nmol/l) and AM630 (50 nmol/l to 1 micromol/l), respectively] had a significant effect upon 3H-2-deoxy-D-glucose uptake by Caco-2 cells. This was true for both the uptake of a low (1 micromol/l) and of a high (20 mmol/l) concentration of 3H-2-deoxy-D-glucose. From these results, we may hypothesize that cannabinoids do not interfere with the intestinal GLUT2-mediated apical uptake of glucose.

Topics: Arachidonic Acids; Caco-2 Cells; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cyclohexanols; Deoxyglucose; Dronabinol; Endocannabinoids; Humans; Indoles; Intestinal Absorption; Piperidines; Polyunsaturated Alkamides; Pyrazoles

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

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

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

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

It is well accepted that endogenous cannabinoids and CB1 receptors are involved in the regulation of smooth muscle contractility and intestinal motility, through a mechanism mainly related to reduction of acetylcholine release from cholinergic nerve endings. Because, few data exist on a possible modulatory action of the cannabinoid agents on the non-adrenergic non-cholinergic (NANC) excitatory and inhibitory neurotransmission, the aim of the present study was to investigate the effects of cannabinoid drugs on the NANC responses elicited by electrical field stimulation (EFS) in the circular muscle of mouse proximal colon. Colonic contractions were monitored as changes in endoluminal pressure. In NANC conditions, EFS evoked TTX-sensitive responses, characterized by a relaxation, nitrergic in origin, followed by a contraction. The EFS-evoked contraction was significantly reduced by SR48968, NK2 receptor antagonist, and abolished by co-administration of SR48968 and SR140333, NK1 receptor antagonist, suggesting that it was due to release of tachykinins. The cannabinoid receptor synthetic agonist, WIN55,212-2, the putative endogenous ligand, anandamide, the selective CB1 receptor agonist ACEA, but not the selective CB2 receptor agonist JWH-015, produced a concentration-dependent reduction of the NANC contractile responses, without affecting the NANC relaxation. ACEA or anandamide did not modify the contractions induced by exogenous [beta-Ala(8)]-NKA(4-10), agonist of NK2 receptors. The selective antagonist of CB1 receptors, SR141716A, per se failed to affect the EFS-evoked responses, but antagonized the inhibitory effects of WIN55,212-2, anandamide and ACEA on NANC contractile responses. AM630, CB2 receptor antagonist, did not modify the inhibitory effects of WIN55,212-2 or anandamide. URB597, inhibitor of the fatty acid amide hydrolase, enzyme which catalyze the hydrolysis of anandamide, was without any effect on the NANC evoked responses. We conclude that the activation of prejunctional CB1 receptors produces inhibition of NANC contractile responses in mouse colonic preparations. However, endogenous ligands do not seem to modulate tonically the NANC transmission in mouse colon.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Cannabinoids; Colon; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Enteric Nervous System; Excitatory Postsynaptic Potentials; Gastrointestinal Motility; In Vitro Techniques; Indoles; Inhibitory Postsynaptic Potentials; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Neuromuscular Junction; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Tachykinin; Rimonabant; Synaptic Transmission; Tachykinins

While most of the studies concerning the role of cannabinoids on gastric motility have focused the attention on the gastric emptying in in vivo animal models, there is little information about the cannabinoid peripheral influence in the stomach. In addition, the functional features of CB2 receptors in the gastrointestinal tract have been poorly characterized. The purpose of the present study was to investigate the effects of cannabinoid drugs on the excitatory cholinergic and inhibitory non-adrenergic non-cholinergic (NANC) neurotransmission in mouse isolated gastric preparations. Intraluminal pressure from isolated whole stomach was recorded and mechanical responses induced by electrical field stimulation (EFS) were analyzed in different experimental conditions. EFS (0.5ms duration, supramaximal voltage, in trains of 5s, 2-16Hz) caused a cholinergic contraction, which was abolished by atropine or tetrodotoxin (TTX). The cannabinoid receptor agonist, WIN 55,212-2, the endogenous ligand, anandamide, the selective CB1 receptor agonist ACEA, and the selective CB2 receptor agonists, JWH015 and JWH133, produced a concentration-dependent reduction of the EFS-evoked cholinergic contractions. SR141716A, CB1 receptor antagonist, significantly attenuated the inhibitory effects induced by WIN 55,212-2, anandamide or ACEA, without affecting those caused by JWH133. AM630, CB2 receptor antagonist, reduced the inhibitory effects induced by WIN 55,212-2, anandamide, JWH015 or JWH133, without affecting those caused by ACEA. The joint application of SR141716A and AM630 was able of fully preventing the WIN 55,212-2 and anandamide actions. The cannabinoid antagonists failed per se to affect the neurally evoked responses. Cannabinoids did not modify the contractions produced by exogenous carbachol. In the presence of atropine and guanethidine (NANC conditions) EFS-induced TTX-sensitive relaxation consisting in an early and rapid component followed by a second slow phase, which were unaffected by cannabinoid drugs. In conclusion, the present results suggest that cannabinoids play a prejunctional modulatory role on the cholinergic excitatory transmission without affecting the NANC inhibitory transmission. In addition, this study provides experimental evidence that also the activation of CB2 receptors is able to reduce cholinergic neurotransmission in the mouse stomach.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Cannabinoids; Cholinergic Fibers; Electric Stimulation; Endocannabinoids; Excitatory Postsynaptic Potentials; Gastrointestinal Motility; In Vitro Techniques; Indoles; Inhibitory Postsynaptic Potentials; Male; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Neuromuscular Junction; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Presynaptic; Rimonabant; Stomach; Synaptic Transmission

Anandamide, an endocannabinoid, is degraded by the enzyme fatty acid amide hydrolase which can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). The present work was designed to study the peripheral interactions between anandamide and ibuprofen (a non-specific cyclooxygenase inhibitor) in the rat formalin test. We first determined the ED50 for anandamide (0.018 microg +/- 0.009), ibuprofen (0.18 microg +/- 0.09), and their combination (0.006 microg +/- 0.002). Drugs were given 15 min before a 2.5% formalin injection into the dorsal surface of the right hind paw. Results were analyzed using isobolographic analysis. The antinociceptive interaction between anandamide and ibuprofen was synergistic. To further investigate the mechanisms by which the combination of anandamide with ibuprofen produced their antinociceptive effects, we used specific antagonists for the cannabinoid CB1 (AM251; 80 microg) and CB2 (AM630; 25 microg) receptors. We demonstrated that the antinociceptive effects of ibuprofen were not antagonized by either AM251 or AM630 and that those of anandamide were antagonized by AM251 but not by AM630. The synergistic antinociceptive effects of the combination of anandamide with ibuprofen were completely antagonized by AM251 but only partially inhibited by AM630. In conclusion, locally (hind paw) injected anandamide, ibuprofen or combination thereof decreased pain behavior in the formalin test. The combination of anandamide with ibuprofen produced synergistic antinociceptive effects involving both cannabinoid CB1 and CB2 receptors. Comprehension of the mechanisms involved needs further investigation.

Topics: Acute Disease; Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Area Under Curve; Cannabinoid Receptor Modulators; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Endocannabinoids; Ibuprofen; Indoles; Inflammation; Male; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Random Allocation; Rats

Endocannabinoids have been implicated in protective effects in the heart and brain, but the mechanism of possible infarct-size-reducing effects remains controversial. Using a model of delayed preconditioning (PC), rats received the nitric oxide (NO) donor nitroglycerin (0.15 mg/h/kg) for 24 hours via transdermal application. Two days later, rat isolated perfused hearts were subjected to global, no-flow ischemia (20 min), and reperfusion (120 min). Cannabinoid receptor antagonists were given before no-flow throughout the protocol. Endocannabinoids were detected by liquid chromatography and mass spectrometry. NO-induced PC reduced the left ventricular infarct size from 40.9 +/- 3.9% to 27.5 +/- 3.8% (P < 0.05). Treatment with the specific CB1 cannabinoid receptor antagonist AM-251 (0.3 microM) prevented the protective effect of PC on infarct size (40.2 +/- 4.7%, P > 0.05 vs. controls). On the contrary, the specific CB2 receptor antagonist AM-630 (0.3 microM) did not alter infarct size (31.6 +/- 6.3%, P > 0.05 vs. PC alone). Recovery of left ventricular developed pressure and coronary flow was incomplete in control and NO-pretreated hearts and not consistently altered by cannabinoid receptor antagonists. PC increased the heart tissue content of the endocannabinoid 2-arachidonylglycerol (2-AG) from 4.6 +/- 1.0 nmol/g in controls to 12.0 +/- 2.1 nmol/g (P < 0.05). Tissue levels of the endocannabinoid arachidonylethanolamide (anandamide) remained unchanged (19.8 +/- 3.9 pmol/g vs. 19.5 +/- 4.8 pmol/g). 2-AG (1 microM) or its metabolically stable derivative noladinether (0.1 microM), given 30 minutes before ischemia/reperfusion in unpreconditioned hearts, mimicked the cardioprotective effects of PC and reduced infarct size. We conclude that delayed PC through transdermal nitroglycerin application increases the production of the endocannabinoid 2-AG which elicits protective effects against myocardial infarction via CB1 cannabinoid receptors which represents one new mechanism of NO-mediated PC.

Topics: Animals; Arachidonic Acids; Blood Pressure; Cannabinoid Receptor Modulators; Coronary Vessels; Endocannabinoids; Glycerides; Heart; Heart Rate; Indoles; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Regional Blood Flow

Cannabinoids are known to possess both anti-inflammatory and neuroprotective effects. In the present study, we have investigated the ability of cannabinoids to inhibit the transmigration of neutrophils in response to chemotaxic stimuli. The cannabinoid receptor agonist WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate) significantly decreased the number of migrating neutrophils across a monolayer of tumour necrosis factor alpha (TNF-alpha) activated ECV304 cells at concentrations >or=1 microM. In contrast, the agonists HU210 and CP 55,940 (0.01-1 microM) and the endocannabinoid anandamide (0.1-10 microM) were without significant effect on the response to TNF-alpha. The ability of WIN 55,212-2 to reduce the neutrophil transmigration was still seen in the presence of the cannabinoid CB(1) receptor antagonist/inverse agonist AM251 (0.1-1 microM) and the cannabinoid CB(2) receptor antagonist/inverse agonist AM630 (0.1-1 microM). TNF-alpha treatment of ECV304 cells caused release of interleukin-8 (IL-8), but WIN 55,212-2 did not affect either the ability of neutrophils to migrate across chemotaxis plates in response to an IL-8 stimulus, or to change the percentage of CXC 1 and CXC 2 receptors expressed by the neutrophils. WIN 55,212-2 at a concentration of 1 microM, but not at lower concentrations, produced a significant inhibition of IL-8 release from ECV304 cells in response to TNF-alpha-stimulation. Thus WIN 55,212-2 reduces the transmigration of neutrophils across a monolayer of TNF-alpha-activated ECV304 cells by an indirect action upon the release of IL-8 and/or other chemokine release from the ECV304 cells, and that this effect is brought about mainly by a cannabinoid CB receptor-independent mechanism.

Topics: Analysis of Variance; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Line; Cell Movement; Chemotaxis; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Endothelial Cells; Flow Cytometry; Humans; Indoles; Interleukin-8; L-Lactate Dehydrogenase; Morpholines; Naphthalenes; Neutrophils; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Thapsigargin; Tumor Necrosis Factor-alpha

It is known that marijuana use decreases saliva secretion. Therefore, we hypothesized that cannabinoid receptors (CBs) are located in salivary glands to mediate that effect. In these experiments, we used the submandibular gland (SMG) of male rats, which is one of the major salivary glands. Mammalian tissues contain at least two types of CBs, CB1 and CB2, mainly located in the nervous system and peripheral tissues, respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase. We demonstrated that both CB1 and CB2 are present in the SMG, each showing specific localizations. The best-known endocannabinoid is anandamide (AEA), which binds with high affinity to CB1 and CB2. We showed that AEA markedly reduced forskolin-induced increase of cAMP content in vitro. This effect was blocked by AM251 and AM630 (CB1 and CB2 antagonists, respectively), indicating that both receptors are implicated in SMG physiology. In addition, we showed that AEA injected intraglandularly to anesthetized rats inhibited norepinephrine (NE)- and methacholine (MC)-stimulated saliva secretion in vivo and that both AM251 or AM630 prevented the inhibitory action of AEA. Also, the intraglandular injection of AM251 increased saliva secretion induced by lower doses of NE or MC. This increase was synergized after coinjection with AM630. Therefore, we concluded that AEA decreases saliva secretion in the SMG acting through CB1 and CB2 receptors.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Colforsin; Cyclic AMP; Endocannabinoids; Immunohistochemistry; Indoles; Male; Methacholine Chloride; Norepinephrine; Parasympathomimetics; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Saliva; Submandibular Gland; Sympathomimetics

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

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

Cannabinoids have been reported to have anti-inflammatory effects and reduce joint damage in animal models of arthritis. This suggests a potential therapeutic role in arthritis of this group of compounds. Cannabinoids were studied to determine whether they have direct effects on chondrocyte metabolism resulting in cartilage protection. Synthetic cannabinoids, R-(+)-Win-55,212 (Win-2) and S-(-)-Win-55,212 (Win-3) and the endocannabinoid, anandamide, were investigated on unstimulated or IL-1-stimulated nitric oxide (NO) production in bovine articular chondrocytes as well as on cartilage proteoglycan breakdown in bovine nasal cartilage explants. Win-2 significantly inhibited (P < 0.05) NO production in chondrocytes at 1-10 microM concentrations. The combined CB(1) and CB(2) cannabinoid receptor antagonists, AM281 and AM630, respectively, at 100 microM did not block this effect, but instead they potentiated it. Anandamide and Win-2 (5-50 microM) also inhibited the release of sulphated glycosaminoglycans in bovine cartilage explants. The results suggest that some cannabinoids may prevent cartilage resorption, in part, by inhibiting cytokine-induced NO production by chondrocytes and also by inhibiting proteoglycan degradation.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cartilage; Cattle; Chondrocytes; Endocannabinoids; Indoles; Interleukin-1; Morpholines; Naphthalenes; Nitric Oxide; Polyunsaturated Alkamides; Proteoglycans; Pyrazoles

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

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

1. In the present study, the effects of anandamide and WIN 55,212-2, cannabinoid receptor agonists, were investigated on electrical field stimulation (EFS)-induced biphasic twitch responses obtained from the epididymal and prostatic portions of rabbit vas deferens strips. 2. Anandamide and WIN 55,212-2 dose-dependently inhibited both the first and second phases of the EFS-induced twitch responses recorded from epididymal and prostatic portions of the vas deferens over the concentration range 10(-9) to 3 x 10(-6) mol/L. 3. The cannabinoid CB1 receptor antagonist AM 251 (10(-6) mol/L) and the cannabinoid CB2 receptor antagonist AM 630 (10(-6) mol/L) had no effect on the inhibitory action of anandamide on the biphasic twitch responses in the prostatic and epididymal portions of the rabbit vas deferens. 4. In both the prostatic and epididymal portions of the rabbit vas deferens, AM 251 significantly, but not completely, reversed the inhibitory effect of WIN 55,212-2 on the first phase of the twitch response. In contrast, AM 630 did not have any effect on the inhibitory action of WIN 55,212-2 in the rabbit vas deferens strips. 5. The inhibitory effects of anandamide or WIN 55,212-2 on EFS-induced twitch responses of both the prostatic and epididymal portions of the rabbit vas deferens were not altered in the presence of 10(-5) mol/L naloxone. 6. These results suggest that cannabinoid receptors may have a modulatory role in the regulation of sympathetic transmission in the rabbit vas deferens. However, further investigation is required to characterize the receptors involved.

Topics: Animals; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cannabinoid Receptor Agonists; Dose-Response Relationship, Drug; Endocannabinoids; In Vitro Techniques; Indoles; Male; Morpholines; Muscle Contraction; Muscle, Smooth; Naloxone; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rabbits; Receptors, Cannabinoid; Sympathetic Nervous System; Vas Deferens

Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Calcium; Cell Line; Chromones; Colforsin; Cyclic AMP; Cyclohexanols; DNA, Complementary; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Humans; Indoles; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Radioligand Assay; Rats; Receptor, Cannabinoid, CB2; Species Specificity; Transfection; Tritium

In rat isolated mesenteric beds, anandamide induced a concentration-dependent reduction (0.01-50 microM) of the contractile responses elicited by bolus administration of noradrenaline. The anandamide-induced reductions of noradrenaline responses were unmodified by the in vitro exposure to the nitric oxide synthase (NOS) inhibitor, 100 microM L-N(G)-nitro-L-arginine methyl ester (L-NAME), whereas they were significantly potentiated after the long-term in vivo administration of L-NAME (70 mg/kg/day during 4 weeks). Responses to anandamide were not potentiated and even reduced in mesenteric beds from rats made hypertensive by aortic coarctation. In mesenteric beds isolated from either untreated or in vivo L-NAME treated rats, concentration-response curves to anandamide were significantly attenuated by the non-selective K+ channel blocker tetraethylammonium (TEA) but were not modified by either endothelium removal, or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) or the cannabinoid receptor antagonists 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl] (4-methoxyphenyl) methanone (AM630) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281). On the other hand, the vanilloid receptor agonist (E)-N-[4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide (capsaicin) induced a concentration-dependent inhibition of noradrenaline-induced vasoconstriction, and the vanilloid receptor antagonist N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine) caused a significant reduction of anandamide-induced responses in mesenteric beds isolated from both control and chronic L-NAME treated rats. The non-metabolizable analogue of anandamide, methanandamide, produced higher reductions of noradrenaline responses than anandamide in mesenteric beds isolated from controls but not from the L-NAME treated rats. Moreover, in mesenteric beds from untreated but not from L-NAME treated rats, the effects of anandamide were significantly potentiated by the inhibitor of endocannabinoid degradation, 200 microM phenylmethylsulphonyl fluoride (PMSF), and by the inhibitor of anandamide uptake, 5 microM (all Z)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404). It is concluded that long-term inhibition of NOS potentiates anandamide-induced relaxations probably through changes in either endocannabinoid metabolism or uptake. A possibl

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Capsaicin; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Indoles; Male; Mesenteric Arteries; Morpholines; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Norepinephrine; Oxadiazoles; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Pyrazoles; Quinoxalines; Rats; Rats, Wistar; Time Factors; Vasoconstriction; Vasoconstrictor Agents

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

AM630 (iodopravadoline), a novel aminoalkylindole, has been found to attenuate the ability of a number of cannabinoids to inhibit electrically-evoked twitches of the mouse isolated vas deferens. It did not block the inhibitory effects of morphine or clonidine on the twitch response. AM630 behaved as a competitive antagonist of CP 55,940, WIN 55,212-2, anandamide and (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide (AM356), producing rightward shifts in the log concentration response curves of these cannabinoid receptor agonists that were concentration-dependent, essentially parallel and not accompanied by any decrease in the size of maximal response. AM630 also produced concentration-dependent, parallel rightward shifts in the log concentration-response curve of delta 9-THC. However, these shifts were accompanied by a decrease in the maximal response. AM630 was markedly more potent as an antagonist of delta 9-THC and CP 55,940 (Kd = 14.0 and 17.3 nM respectively) than as an antagonist of WIN 55,212-2, AM356 or anandamide (Kd = 36.5, 85.9 and 278.8 nM respectively). These differences in dissociation constant imply that the mouse vas deferens may contain more than one type of cannabinoid receptor. The data also indicate that the receptors for which AM630 has the highest affinity may not be CB1 cannabinoid receptors as the CB1 selective antagonist, SR141716A, is known to be equally potent in attenuating the inhibitory effects of CP 55,940 and anandamide on the twitch response of the mouse vas deferens.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Cannabinoids; Cyclohexanols; Endocannabinoids; In Vitro Techniques; Indoles; Male; Mice; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug