Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

High-Concentration Piperine: Capsaicin-Sensitive and -Insensitive Effects on Isolated Organs.

Piperine (P), a sensory stimulant in black pepper, is an agonist on TRPV1 receptors. Earlier work has showed capsaicin-sensitive and -insensitive mechanisms of the contractile action of P on the intestine. The current isolated organ study in the guinea-pig ileum, urinary bladder and trachea (a) confirms the presence of such components of effect (ileum and bladder); (b) indicates TRPV1 involvement in the effect of 5 or 30 µmol/l of P on the basis of an inhibitory action of the antagonist BCTC (ileum); (c) indicates that HC 030031-sensitive TRPA1 receptors and nifedipine-sensitive Ca(2+) channels contribute to the capsaicin-resistant contraction to 30 µmol/l P (ileum) and (d) shows that the contractile effect of P up to 100 µmol/l (guinea-pig trachea) or 30 µmol/l (guinea-pig urinary bladder) is capsaicin-sensitive and mediated by TRPV1 receptors/channels.

Cannabinoid modulation of predator fear: involvement of the dorsolateral periaqueductal gray.

The present study investigated the effects of systemic or intra-dorsolateral periaqueductal gray (dlPAG) administration of CB1 agonists on behavioural changes induced in rats by predator (a live cat) exposure, a model of panic responses. Since nitric oxide (NO) and cannabinoid neurotransmission are proposed to interact in the dlPAG to modulate defensive responses, we also investigated if NO is involved in the biphasic effects of anandamide (AEA) injected into the dlPAG. The results showed that systemic administration of WIN55,212-2 or intra-dlPAG AEA attenuated the defensive behaviours caused by cat exposure. Both compounds produced biphasic curves. The cannabinoid receptor type 1 (CB1) antagonist AM251 prevented the panicolytic effect of AEA whereas a neuronal NOS inhibitor turned the ineffective high dose of AEA into an effective one. These results suggest that modulation of the cannabinoid system could be a target in the treatment of panic disorders. However, the biphasic effects of these compounds could limit their therapeutic potential.

CB1 and CB2 cannabinoid receptor agonists induce peripheral antinociception by activation of the endogenous noradrenergic system.

BACKGROUND: Cannabinoid agonists induce norepinephrine release in central, spinal, and peripheral sites. Previous studies suggest an interaction between the cannabinoid and adrenergic systems on antinociception. In this study, we sought to verify whether the CB1 and CB2 cannabinoid receptor agonists anandamide and N-palmitoyl-ethanolamine (PEA), respectively, are able to induce peripheral antinociception via an adrenergic mechanism. METHODS: All drugs were administered locally into the right hindpaw of male Wistar rats. The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2 (2 µg). RESULTS: Anandamide, 12.5 ng/paw, 25 ng/paw, and 50 ng/paw elicited a local peripheral antinociceptive effect that was antagonized by CB1 cannabinoid receptor antagonist AM251, 20 µg/paw, 40 µg/paw, and 80 µg/paw, but not by CB2 cannabinoid receptor antagonist AM630, 100 µg/paw. PEA, 5 µg/paw, 10 µg/paw, and 20 µg/paw, elicited a local peripheral antinociceptive effect that was antagonized by AM630, 25 µg/paw, 50 µg/paw, and 100 µg/paw, but not by AM251, 80 µg/paw. Antinociception induced by anandamide or PEA was antagonized by the nonselective α2 adrenoceptor antagonist yohimbine, 05 µg/paw, 10 µg/paw, and 20 µg/paw, and by the selective α2C adrenoceptor antagonist rauwolscine, 10 µg/paw, 15 µg/paw, and 20 µg/paw, but not by the selective antagonists for α2A, α2B, and α2D adrenoceptor subtypes, 20 µg/paw. The antinociceptive effect of the cannabinoids was also antagonized by the nonselective α1 adrenoceptor antagonist prazosin, 0.5 µg/paw, 1 µg/paw, and 2 µg/paw, and by the nonselective ß adrenoceptor antagonist propranolol, 150 ng/paw, 300 ng/paw, and 600 ng/paw. Guanethidine, which depletes peripheral sympathomimetic amines (30 mg/kg/animal, once a day for 3 days), restored approximately 70% the anandamide-induced and PEA-induced peripheral antinociception. Furthermore, acute injection of the norepinephrine reuptake inhibitor reboxetine, 30 µg/paw, intensified the antinociceptive effects of low-dose anandamide, 12.5 ng/paw, and PEA, 5 µg/paw. CONCLUSIONS: This study provides evidence that anandamide and PEA induce peripheral antinociception activating CB1 and CB2 cannabinoid receptors, respectively, stimulating an endogenous norepinephrine release that activates peripheral adrenoceptors inducing antinociception.

Endogenous cannabinoid signaling is essential for stress adaptation.

Secretion of glucocorticoid hormones during stress produces an array of physiological changes that are adaptive and beneficial in the short term. In the face of repeated stress exposure, however, habituation of the glucocorticoid response is essential as prolonged glucocorticoid secretion can produce deleterious effects on metabolic, immune, cardiovascular, and neurobiological function. Endocannabinoid signaling responds to and regulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis that governs the secretion of glucocorticoids; however, the role this system plays in adaptation of the neuroendocrine response to repeated stress is not well characterized. Herein, we demonstrate a divergent regulation of the two endocannabinoid ligands, N-arachidonylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), following repeated stress such that AEA content is persistently decreased throughout the corticolimbic stress circuit, whereas 2-AG is exclusively elevated within the amygdala in a stress-dependent manner. Pharmacological studies demonstrate that this divergent regulation of AEA and 2-AG contribute to distinct forms of HPA axis habituation. Inhibition of AEA hydrolysis prevented the development of basal hypersecretion of corticosterone following repeated stress. In contrast, systemic or intra-amygdalar administration of a CB(1) receptor antagonist before the final stress exposure prevented the repeated stress-induced decline in corticosterone responses. The present findings demonstrate an important role for endocannabinoid signaling in the process of stress HPA habituation, and suggest that AEA and 2-AG modulate different components of the adrenocortical response to repeated stressor exposure.

Effects of alterations in cannabinoid signaling, alone and in combination with morphine, on pain-elicited and pain-suppressed behavior in mice.

Inhibitors of fatty acid amide hydrolase (FAAH) and anandamide (AEA) uptake, which limit the degradation of endogenous cannabinoids, have received interest as potential therapeutics for pain. There is also evidence that endogenous cannabinoids mediate the antinociceptive effects of opioids. Assays of pain-elicited and pain-suppressed behavior have been used to differentiate the effects of drugs that specifically alter nociception from drugs that alter nociception caused by nonspecific effects such as catalepsy or a general suppression of activity. Using such procedures, this study examines the effects of the direct cannabinoid type 1 (CB1) agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940), the FAAH inhibitor cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), and the AEA uptake inhibitor N-(4-hydroxyphenyl) arachidonylamide (AM404). Additional experiments examined these compounds in combination with morphine. CP55940 produced antinociception in assays of pain-elicited, but not pain-suppressed, behavior and disrupted responding in an assay of schedule-controlled behavior. URB597 and AM404 produced antinociception in assays of pain-elicited and pain-suppressed behavior in which acetic acid was the noxious stimulus, but had no effect on the hotplate and schedule-controlled responding. CP55940 in combination with morphine resulted in effects greater than those of morphine alone in assays of pain-elicited and scheduled-controlled behavior but not pain-suppressed behavior. URB597 in combination with morphine resulted in enhanced morphine effects in assays of pain-elicited and pain-suppressed behavior in which diluted acetic acid was the noxious stimulus, but did not alter morphine's effects on the hotplate or schedule-controlled responding. These studies suggest that, compared with direct CB1 agonists, manipulations of endogenous cannabinoid signaling have enhanced clinical potential; however, their effects depend on the type of noxious stimulus.

[Anandamide inhibits the growth of colorectal cancer cells through CB1 and lipid rafts].

OBJECTIVE: 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. METHODS: 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-ß-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. RESULTS: 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. CONCLUSIONS: 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.

Endogenous opioid and cannabinoid systems modulate the muscle pain: A pharmacological study into the peripheral site.

The participation of the peripheral opioid and cannabinoid endogenous systems in modulating muscle pain and inflammation has not been fully explored. Thus, the aim of this study was to investigate the involvement of these endogenous systems during muscular-tissue hyperalgesia induced by inflammation. Hyperalgesia was induced by carrageenan injection into the tibialis anterior muscles of male Wistar rats. We padronized an available Randal-Sellito test adaptation to evaluate nociceptive behavior elicited by mechanical insult in muscles. Western blot analysis was performed to evaluate the expression levels of opioid and cannabinoid receptors in the dorsal root ganglia. The non-selective opioid peptide receptor antagonist (naloxone) and the selective mu opioid receptor MOP (clocinnamox) and kappa opioid receptor KOP (nor-binaltorphimine) antagonists were able to intensify carrageenan-induced muscular hyperalgesia. On the other hand, the selective delta opioid receptor (DOP) antagonist (naltrindole) did not present any effect on nociceptive behavior. Moreover, the selective inhibitor of aminopeptidases (Bestatin) provoked considerable dose-dependent analgesia when intramuscularly injected into the hyperalgesic muscle. The CB1 receptor antagonist (AM251), but not the CB2 receptor antagonist (AM630), intensified muscle hyperalgesia. All irreversible inhibitors of anandamide hydrolase (MAFP), the inhibitor for monoacylglycerol lipase (JZL184) and the anandamide reuptake inhibitor (VDM11) decreased carrageenan-induced hyperalgesia in muscular tissue. Lastly, MOP, KOP and CB1 expression levels in DRG were baseline even after muscular injection with carrageenan. The endogenous opioid and cannabinoid systems participate in peripheral muscle pain control through the activation of MOP, KOP and CB1 receptors.

Retrograde endocannabinoid signaling at striatal synapses requires a regulated postsynaptic release step.

Endocannabinoids (eCBs) mediate short- and long-term depression of synaptic strength by retrograde transsynaptic signaling. Previous studies have suggested that an eCB mobilization or release step in the postsynaptic neuron is involved in this retrograde signaling. However, it is not known whether this release process occurs automatically upon eCB synthesis or whether it is regulated by other synaptic factors. To address this issue, we loaded postsynaptic striatal medium spiny neurons (MSNs) with the eCBs anandamide (AEA) or 2-arachidonoylglycerol and determined the conditions necessary for presynaptic inhibition. We found that presynaptic depression of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) induced by postsynaptic eCB loading required a certain level of afferent activation that varied between the different synaptic types. Synaptic depression at excitatory synapses was temperature-dependent and blocked by the eCB membrane transport blockers, VDM11 and UCM707, but did not require activation of metabotropic glutamate receptors, l-calcium channels, nitric oxide, voltage-activated Na(+) channels, or intracellular calcium. Application of the CB(1)R antagonist, AM251, after depression was established, reversed the decrease in EPSC, but not in IPSC, amplitude. Direct activation of the CB(1) receptor by WIN 55,212-2 initiated synaptic depression that was independent of afferent stimulation. These findings indicate that retrograde eCB signaling requires a postsynaptic release step involving a transporter or carrier that is activated by afferent stimulation/synaptic activation.

Divergent effects of anandamide transporter inhibitors with different target selectivity on social play behavior in adolescent rats.

The endocannabinoid system plays an important role in the modulation of affect, motivation, and emotion. Social play behavior is a natural reinforcer in adolescent rats, and we have recently shown that interacting endocannabinoid, opioid, and dopamine systems modulate social play. In the present study, we tested the hypothesis that, in contrast to administration of exogenous cannabinoid agonists, increasing local endocannabinoid signaling through anandamide transporter inhibition enhances social play. To this aim, we tested the effects of two anandamide transporter inhibitors with different target selectivity on social play behavior in adolescent rats. Interestingly, we found that the prototypical anandamide transporter inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) reduced social play, whereas its more selective analog N-arachidonoyl-(2-methyl-4-hydroxyphenyl)amine (VDM11) enhanced it. The effects of AM404 were not mediated through its known pharmacological targets, since they were not blocked by the CB(1) cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), the CB(2) cannabinoid receptor antagonist N-(1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide (SR144528), or by the transient receptor potential vanilloid 1 receptor antagonist capsazepine. In contrast, the increase in social play induced by VDM11 was dependent on cannabinoid, opioid, and dopaminergic neurotransmission, since it was blocked by the CB(1) cannabinoid receptor antagonist SR141716A, the opioid receptor antagonist naloxone, and the dopamine receptor antagonist alpha-flupenthixol. These findings support the notion that anandamide plays an important role in the modulation of social interaction in adolescent rats, and they suggest that selective anandamide transporter inhibitors might be useful for the treatment of social dysfunctions. Furthermore, these results suggest that off-target effects may be responsible for some of the conflicting effects of anandamide transporter inhibitors on behavior.

The endocannabinoid system as a target for the treatment of cannabis dependence.

The endocannabinoid system modulates neurotransmission at inhibitory and excitatory synapses in brain regions relevant to the regulation of pain, emotion, motivation, and cognition. This signaling system is engaged by the active component of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC), which exerts its pharmacological effects by activation of G protein-coupled type-1 (CB1) and type-2 (CB2) cannabinoid receptors. During frequent cannabis use a series of poorly understood neuroplastic changes occur, which lead to the development of dependence. Abstinence in cannabinoid-dependent individuals elicits withdrawal symptoms that promote relapse into drug use, suggesting that pharmacological strategies aimed at alleviating cannabis withdrawal might prevent relapse and reduce dependence. Cannabinoid replacement therapy and CB1 receptor antagonism are two potential treatments for cannabis dependence that are currently under investigation. However, abuse liability and adverse side-effects may limit the scope of each of these approaches. A potential alternative stems from the recognition that (i) frequent cannabis use may cause an adaptive down-regulation of brain endocannabinoid signaling, and (ii) that genetic traits that favor hyperactivity of the endocannabinoid system in humans may decrease susceptibility to cannabis dependence. These findings suggest in turn that pharmacological agents that elevate brain levels of the endocannabinoid neurotransmitters, anandamide and 2-arachidonoylglycerol (2-AG), might alleviate cannabis withdrawal and dependence. One such agent, the fatty-acid amide hydrolase (FAAH) inhibitor URB597, selectively increases anandamide levels in the brain of rodents and primates. Preclinical studies show that URB597 produces analgesic, anxiolytic-like and antidepressant-like effects in rodents, which are not accompanied by overt signs of abuse liability. In this article, we review evidence suggesting that (i) cannabis influences brain endocannabinoid signaling and (ii) FAAH inhibitors such as URB597 might offer a possible therapeutic avenue for the treatment of cannabis withdrawal.

Role of the endocannabinoid and endovanilloid systems in an animal model of schizophrenia-related emotional processing/cognitive deficit.

Studies suggest that the endocannabinoid and endovanilloid systems are implicated in the pathophysiology of schizophrenia. The Spontaneously Hypertensive Rats (SHR) strain displays impaired contextual fear conditioning (CFC) attenuated by antipsychotic drugs and worsened by pro-psychotic manipulations. Therefore, SHR strain is used to study emotional processing/associative learning impairments associated with schizophrenia and effects of potential antipsychotic drugs. Here, we evaluated the expression of CB1 and TRPV1 receptors in some brain regions related to the pathophysiology of schizophrenia. We also assessed the effects of drugs that act on the endocannabinoid/endovanilloid systems on the CFC task in SHRs and control animals (Wistar rats - WRs). The following drugs were used: AM404 (anandamide uptake/metabolism inhibitor), WIN55-212,2 (non-selective CB1 agonist), capsaicin (TRPV1 agonist), and capsazepine (TRPV1 antagonist). SHRs displayed increased CB1 expression in prelimbic cortex and cingulate cortex area 1 and in CA3 region of the dorsal hippocampus. Conversely, SHRs exhibited decreases in TRPV1 expression in prelimbic and CA1 region of dorsal hippocampus and increases in the basolateral amygdala. AM404, WIN 55,212-2 and capsaicin attenuated SHRs CFC deficit, although WIN 55,212-2 worsened SHRs CFC deficit in higher doses. WRs and SHRs CFC were modulated by distinct doses, suggesting that these strains display different responsiveness to cannabinoid and vanilloid drugs. Treatment with capsazepine did not modify CFC in either strains. The effects of AM404 on SHRs CFC deficit was not blocked by pretreatment with rimonabant (CB1 antagonist) or capsazepine. These results reinforce the involvement of the endocannabinoid/endovanilloid systems in the SHRs CFC deficit and point to these systems as targets to treat the emotional processing/cognitive symptoms of schizophrenia.

RNA interference-mediated knockdown of dynamin 2 reduces endocannabinoid uptake into neuronal dCAD cells.

The precise mechanism by which the cellular uptake of the endocannabinoid anandamide (AEA) occurs has been the source of much debate. In the current study, we show that neuronal differentiated CAD (dCAD) cells accumulate anandamide by a process that is inhibited in a dose-dependent manner by N-(4-hydroxyphenyl)arachidonylamide (AM404). We also show that dCAD cells express functional fatty acid amide hydrolase, the enzyme primarily responsible for anandamide metabolism. Previous data from our laboratory indicated that anandamide uptake occurs by a caveolae-related endocytic mechanism in RBL-2H3 cells. In the current study, we show that anandamide uptake by dCAD cells may also occur by an endocytic process that is associated with detergent-resistant membrane microdomains or lipid rafts. Nystatin and progesterone pretreatment of dCAD cells significantly inhibited anandamide accumulation. Furthermore, RNA interference (RNAi)-mediated knockdown of dynamin 2, a protein involved in endocytosis, blocked the internalization of the fluorescently labeled anandamide analog SKM 4-45-1 ([3',6'-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen-5-yl]-2-[[1-oxo-5Z,8Z,11Z,14Z-eicosatetraenyl]amino]ethyl ester carbamic acid). RNAi-mediated knockdown of the beta2 subunit of the clathrin-associated activator protein 2 complex had no effect on SKM 4-45-1 internalization. We were surprised to find that dynamin 2 knockdown in dCAD cells did not affect [3H]AEA uptake. However, dynamin 2 knockdown caused a significant increase in the overall levels of intact [3H]AEA associated with the cells, suggesting that trafficking of [3H]AEA to FAAH had been disrupted. This finding may be the result of an accumulation of the anandamide carrier protein in detergent-resistant membranes after dynamin 2 knockdown. Our studies provide evidence that the cellular uptake of anandamide may occur by a dynamin 2-dependent, caveolae-related endocytic process in dCAD cells.

Endocannabinoid modulation of male rat sexual behavior.

RATIONALE: Synthetic and plant-derived cannabinoid CB(1) receptor agonists have consistently been shown to impair sexual behavior in male rodents; however, the role of the endocannabinoid system in regulating copulatory processes is largely unknown. The aim of this experiment was to determine the effect of pharmacological facilitation or antagonism of endocannabinoid signaling on male rat sexual behavior. MATERIALS AND METHODS: Male Long-Evans rats were administered a single injection of either the cannabinoid CB(1) receptor antagonist AM251 (1, 2, or 5 mg/kg), the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.1, 0.3, or 0.5 mg/kg), or the anandamide uptake inhibitor/FAAH inhibitor AM404 (1, 2, and 5 mg/kg), or their respective vehicles, and examined on parameters of appetitive and consummatory sexual behavior. RESULTS: Inhibition of anandamide metabolism through URB597 had no effect on any parameter of sexual behavior. However, the highest dose of AM404 increased the latency to engage in intromitting behavior, but had no other effect on sexual behavior, suggesting that this effect may be due to the sedative-suppressive effects of this drug. AM251 produced a dose-dependent facilitation of ejaculation, such that the number of intromissions required to achieve ejaculation and the ejaculation latency were reduced by AM251 administration. CONCLUSIONS: These data suggest that antagonism of the CB(1) receptor facilitates ejaculatory processes, an effect which may be due to interactions with neuropeptidergic systems in the hypothalamus, and further, suggest a novel target for pharmacological agents aimed at treating ejaculatory-based sexual dysfunction.

Activation of cannabinoid CB1 receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test.

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB(1) receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n=5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. In this box an electrical shock (0.5 mA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 microl) or AM251 (a cannabinoid CB(1) receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB(1) receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses.

New tetrazole-based selective anandamide uptake inhibitors.

A new series of 1,5- and 2,5-disubstituted tetrazoles have been synthesized and evaluated as inhibitors of anandamide cellular uptake. Some of them inhibit the uptake process with a relatively high potency (IC(50)=2.3-5.1microM) and selectively over other proteins involved in endocannabinoid action and metabolism.

Carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: a critical revisitation.

We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, 2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50=2.1-5.4nM) and selectively over all the other targets tested (IC50 >or= 10microM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.

AM404 decreases Fos-immunoreactivity in the spinal cord in a model of inflammatory pain.

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.

Anandamide: an update.

Endocannabinoid system has attracted the researchers to investigate into its ever fascinating roles governing many of the physiological functions in the human body. The prime endogenous cannabinoids are arachidonoylethanolamide also called anandamide and 2-arachidonoylglycerol. Recent pharmacological advances point out that this system of molecules are in initial stages and by updating our current knowledge, we could innovatively design molecules for suitable interventions that could be potentially beneficial to the mankind.

New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis.

We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB(1) and CB(2) receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca(2+)](i)); (c) [(14)C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse 'tetrad' tests (analgesia on a hot plate, immobility on a 'ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O-2093, either synthesized by us or commercially available, was inactive in the 'tetrad' up to a 20 mg kg(-1) dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB(1) (K(i) from 1.3 to >10 microM) and CB(2) binding assays (1.310 microM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC(50)>25 microM) and were inactive in the 'tetrad' up to a 30 mg kg(-1) dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [(14)C]AEA cellular uptake (IC(50)>40 microM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC(50)=1.4 microM) and is 12-fold more potent than O-2093. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake inhibitors as anti-spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.