Presence Of Anandamide Research Articles

Anandamide modulation of monocyte-derived Langerhans cells: implications for immune homeostasis and skin inflammation.

The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation. Human monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures. Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype. Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

The relaxation effect of endocannabinoid anandamide on isolated rat bladder and vas deferens tissues and possible mechanisms.

The endocannabinoid system plays important roles in various systems, including the genitourinary system; however, its mechanism of action is not fully understood. This study aimed to investigate the direct relaxant effects of anandamide and its possible mechanisms in isolated rat bladder and vas deferens tissues. Twenty-one adult male Wistar albino rats were used. Bladder and vas deferens (prostatic and epididymal portions) tissues were mounted in 10 mL of organ baths. Relaxation responses to anandamide were recorded at 3 and 10 μM concentrations. After the rest period, the procedures were repeated in the presence of cannabinoid (CB) and vanilloid receptor antagonists, various potassium channel blockers, cyclo-oxygenase, and nitric oxide synthase inhibitors. In different tissues to investigate the Ca2+-channel antagonistic effect of anandamide, concentration-response curves to CaCl2 were obtained in the absence and presence of anandamide. Anandamide caused a significant relaxation response in the bladder and epididymal vas deferens tissues, but not in the prostatic portion. The effect of anandamide was antagonized in the presence of the CB1 antagonist AM251 or the non-selective potassium channel blocker tetraethylammonium in bladder tissue. In the epididymal vas deferens, anandamide significantly inhibited the calcium contraction responses, especially at high concentrations. The CB2 antagonist AM630 reversed this inhibition. The results show that anandamide has a direct relaxant effect on the isolated rat bladder and epididymal vas deferens. Anandamide triggers different mechanisms in different types of tissues, and further studies are needed to elucidate the mechanism of action of anandamide.

Anti-inflammatory dopamine- and serotonin-based endocannabinoid epoxides reciprocally regulate cannabinoid receptors and the TRPV1 channel

The endocannabinoid system is a promising target to mitigate pain as the endocannabinoids are endogenous ligands of the pain-mediating receptors—cannabinoid receptors 1 and 2 (CB1 and CB2) and TRPV1. Herein, we report on a class of lipids formed by the epoxidation of N-arachidonoyl-dopamine (NADA) and N-arachidonoyl-serotonin (NA5HT) by epoxygenases. EpoNADA and epoNA5HT are dual-functional rheostat modulators of the endocannabinoid-TRPV1 axis. EpoNADA and epoNA5HT are stronger modulators of TRPV1 than either NADA or NA5HT, and epoNA5HT displays a significantly stronger inhibition on TRPV1-mediated responses in primary afferent neurons. Moreover, epoNA5HT is a full CB1 agonist. These epoxides reduce the pro-inflammatory biomarkers IL-6, IL-1β, TNF-α and nitrous oxide and raise anti-inflammatory IL-10 cytokine in activated microglial cells. The epoxides are spontaneously generated by activated microglia cells and their formation is potentiated in the presence of anandamide. Detailed kinetics and molecular dynamics simulation studies provide evidence for this potentiation using the epoxygenase human CYP2J2. Taken together, inflammation leads to an increase in the metabolism of NADA, NA5HT and other eCBs by epoxygenases to form the corresponding epoxides. The epoxide metabolites are bioactive lipids that are potent, multi-faceted molecules, capable of influencing the activity of CB1, CB2 and TRPV1 receptors.

Endocannabinoid Receptors Activity Modifies Insulin Secretion in Response to Glucose Exposition

Background: Recent studies suggest the participation of the endocannabinoid system in the development of type 2 diabetes mellitus, involved with the regulation of physiological and metabolic processes. The endocannabinoid system is expressed in pancreatic islets and presents a key role in gene expression and regulation of pancreatic hormones secretion. In type 2 diabetes patients and in obesity the blood levels of endocannabinoids are increased, however, little is known about the mechanism of actions in islets and its relation with glucose in media. Methods: Isolated pancreatics islets of mouse were incubated for 24 hours at 5.5mM and 15mM glucose concentration, in the presence of anandamide or THC as cannabinoid receptors agonists, or with antagonist AM251. Glucose-stimulated insulin secretion and intracellular cAMP were analyzed by ELISA and cytosolic calcium changes were measured by spectrofluorometry with FURA-2. Results: Activation of endocannabioid receptors significantly increases cAMP levels at low glucose concentration, meanwhile no further changes were observed at high-glucose concentration, correlated with a similar effect in insulin secretion. Shor-time exposure to the antagonist decreases insulin secretion under both glucose conditions. Anandamide does not induce changes in intracellular calcium but THC decreases it at high glucose concentration. Antagonist AM251 enhances calcium fluctuations, suggestion an indirect modulation of insulin secretion. Conclusions: Endocannabinoid receptors modulation regulates intracellular mechanisms involved in insulin secretion, according to glucose exposure, in isolated pancreatic mouse islets. Disclosure A. Vilches-Flores: None. G.M. Garcia-Luna: None.

CB1 cannabinoid receptor expression is regulated by glucose and feeding in rat pancreatic islets

Endocannabinoid system is involved in food intake and energy balance. Beside the hypothalamus, pancreatic islet also expresses CB1 cannabinoid receptor, however little is known about its physiological role and regulation. Since gene expression of many specific proteins of the islet depends on the concentration of glucose, we studied CB1 receptor expression in response to fasting and feeding. Whole pancreas or islets were isolated from food-deprived adult Wistar rats, with or without a previous 1.5 g/kg glucose oral-intake. CB1, insulin and glucagon expressions were analyzed by confocal immunofluorescence and PCR. In vitro, rat islets were cultured at different glucose concentrations, in the presence of anandamide, or with Rimonabant analog BAR-1. CB1, insulin, glucagon, glucokinase, and PDX-1 expression were determined by real-time RT-PCR, and insulin secretion and islet content by ELISA. CB1 expression in pancreatic islets is upregulated during food restriction, and decreases in response to glucose intake or feeding. In cultured islets, 16 mmol/l glucose, BAR-1, and anandamide at low glucose reduced CB1 mRNA. Insulin, glucagon, glucokinase and PDX-1 expression increased in islets treated with anandamide at low glucose, while BAR-1 modified PDX-1 and glucagon mRNA at high glucose. Basal insulin secretion and insulin content in islets increased with anandamide, but not the glucose-stimulated response. Our results suggest that the endocannabinoid system has an important role in gene expression on islets and its close relationship with glucose response.

Acid-sensitive TASK-like K+ conductances contribute to resting membrane potential and to orexin-induced membrane depolarization in rat thalamic paraventricular nucleus neurons

Orexin (hypocretin) peptides are known to depolarize rat thalamic paraventricular nucleus (PVT) neurons by suppression of one or more undefined potassium conductances. Here, we investigated a contribution of TWIK-related acid-sensitive K+ (TASK) channels to the resting membrane potential and orexin-induced depolarization of PVT neurons, using patch clamp recording techniques in brain slice preparations. Upon exposure to an acidic (pH 6.3) extracellular solution, PVT neurons displayed membrane depolarization. Under voltage-clamp and in the presence of tetrodotoxin (TTX, 0.5 μM), low pH solutions induced an inward shift in baseline membrane current, accompanied by a net decrease in membrane conductance, reversing close to the potassium equilibrium potential. By contrast, exposure to alkaline (pH 8.3) solutions resulted in membrane hyperpolarization, induced an outward shift in baseline membrane current and an increase in net conductance that reversed close to the potassium equilibrium potential. A local anesthetic bupivacaine (20–40 μM) and the endocannabinoid anandamide (5–10 μM) mimicked the effects of the acidic solution. Exposure to the volatile anesthetic isoflurane (0.2–0.5 mM) induced changes in resting membrane potential, baseline current and membrane conductance similar to those caused by the alkaline solution. Although responsiveness to orexins was preserved under each of the above conditions, the amplitude of the orexin B (0.5 μM)-induced inward current was depressed in the acidic solution and in the presence of anandamide, remained largely unchanged in the alkaline solution, and was enhanced by isoflurane when compared with that in normal artificial cerebrospinal solution. We conclude that pH-sensitive potassium channels, TASK-1 and TASK-3 channels, contribute substantially to the resting membrane conductance(s) and excitability in PVT neurons. The observations that orexin-induced currents were affected by putative TASK-specific drugs in a manner predictable from their effects on TASK channels also suggest that the orexin-induced excitation in PVT neurons is mediated by closure of TASK channels.

The endocannabinoids anandamide and 2-arachidonoylglycerol inhibit cholinergic contractility in the human colon

The effects of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) were determined on cholinergic contractility in strips of human colonic longitudinal muscle and circular muscle in vitro, in the presence of nitric oxide synthase blockade with N-nitro-l-arginine (10(-4) M). Anandamide and 2-AG inhibited longitudinal muscle and circular muscle contractile responses to acetylcholine (10(-9)-10(-4) M) in a concentration-dependent manner. This was unaltered following pretreatment with the cannabinoid CB(1) receptor-selective antagonist AM251 (10(-7) M), however in isolation AM251 elicited a significant rightward shift in the potency of acetylcholine-evoked contraction in both longitudinal muscle and circular muscle preparations. Pretreatment with an inhibitor of anandamide catabolism, arachidonoyl trifluoromethyl ketone (10(-5) M), alone caused a significant decrease in the potency of acetylcholine-evoked contraction in both longitudinal and circular muscle, but had no significant additional effect on the anandamide-induced (10(-5) M) suppression of contraction. Pretreatment with the cannabinoid CB(2) receptor inverse agonist JTE 907 (10(-6) M) neither influenced the potency of acetylcholine-evoked contraction alone nor prevented the potency shift in acetylcholine-evoked contraction in the presence of anandamide (10(-5) M). The findings of the present study indicate that the endocannabinoids anandamide and 2-arachidonoylglycerol suppress colonic cholinergic contractility via a non conventional cannabinoid or non-cannabinoid receptor-mediated pathway. Cholinergic contraction may be tonically modulated by endocannabinoids and/or products of arachidonate metabolism unrelated to endocannabinoid production. The extent of anandamide metabolism is not sufficient to influence the functional effects of its exogenous administration in human colonic tissue in vitro.

Inhibition of Neutrophil Activation by Lafutidine, an H2-receptor Antagonist, Through Enhancement of Sensory Neuron Activation Contributes to the Reduction of Stress-Induced Gastric Mucosal Injury in Rats

Sensory neuron activation reduces water-immersion restraint stress (WIR)-induced gastric mucosal injury by inhibiting neutrophil activation through increase in endothelial production of prostacyclin. This study was designed to examine whether lafutidine, which is an H(2)-receptor antagonist and activates sensory neurons, inhibits neutrophil activation, thereby reducing WIR-induced gastric mucosal injury. Lafutidine enhanced WIR-induced increases in gastric tissue levels of calcitonin gene-related peptide (CGRP) and 6-keto-PGF(1alpha), a stable metabolite of prostacyclin, whereas famotidine, another H(2)-receptor antagonist, did not. Such lafutidine-induced increases in gastric tissue levels of 6-keto-PGF(1alpha) were reversed by pretreatment with capsazepine, an inhibitor of sensory neuron activation, CGRP(8-37), a CGRP antagonist, and indomethacin. Lafutidine inhibited acid-induced exacerbation of gastric mucosal injury in animals subjected to WIR by inhibiting neutrophil activation, whereas famotidine did not. Lafutidine synergistically increased CGRP release from isolated rat dorsal root ganglion neurons in the presence of anandamide, but famotidine did not. These observations suggest that lafutidine might reduce WIR-induced gastric mucosal injury not only by inhibiting acid secretion but also by inhibiting neutrophil activation through enhancement of sensory neuron activation.

Estrogen and isoflavone attenuate stress-induced gastric mucosal injury by inhibiting decreases in gastric tissue levels of CGRP in ovariectomized rats

We have previously reported that CGRP plays a critical role in the reduction of stress-induced gastric mucosal injury by increasing gastric prostacyclin (PGI(2)) levels in rats. Estrogen has been shown to increase the production of CGRP in sensory neurons. Isoflavone has estrogen-like effects and is referred to as a phytoestrogen. Thus, we hypothesized that estrogen and isoflavone might inhibit ovariectomy (OVX)-induced decreases in gastric tissue levels of CGRP, thereby attenuating gastric mucosal injury. We examined these possibilities in the present study. The administration of estradiol and isoflavone for 4 wk completely reversed OVX-induced decreases in CGRP mRNA levels of dorsal root ganglion neurons (DRGs) in rats. OVX-induced decreases in gastric tissue levels of CGRP and 6-keto-PGF(1alpha), a stable metabolite of PGI(2), in rats were reversed by estradiol and isoflavone. Water-immersion restraint stress (WIR)-induced increases in gastric tissue levels of CGRP and 6-keto-PGF(1alpha) were inhibited in ovariectomized rats. This inhibition was completely reversed by estradiol and was partially, but significantly, reversed by isoflavone. WIR-induced gastric mucosal injury was exacerbated by OVX, which was reversed by estradiol and isofolavone. In vitro experiments using DRGs isolated from rats demonstrated that neither estradiol nor isoflavone enhanced CGRP release from DRGs, but the former enhanced it in the presence of anandamide, an endogenous agonist for vanilloid receptor-1. These observations suggest that estrogen and isoflavone might inhibit OVX-induced decreases in CGRP levels in DRGs by promoting transcription, thereby contributing to the attenuation of stress-induced gastric mucosal injury in OVX rats.

Effect of anandamide on nonadrenergic noncholinergic-mediated relaxation of rat corpus cavernosum

The purpose of this study was to investigate the effect of the endogenous cannabinoid anandamide on the nonadrenergic noncholinergic (NANC) relaxant responses to electrical field stimulation in isolated rat corpus cavernosum. The corporal strips were mounted under tension in a standard oxygenated organ bath with guanethidine sulfate (5 μM) and atropine (1 μM) (to produce adrenergic and cholinergic blockade). The strips were precontracted with phenylephrine hydrochloride (7.5 μM) and electrical field stimulation was applied at different frequencies to obtain NANC-mediated relaxation. The expression of CB 1, CB 2 and vanilloid receptor proteins within the rat corpus cavernosum was evaluated using western blot analysis. The results showed that the relaxant responses to electrical stimulation were significantly enhanced in the presence of anandamide at 1 and 3 μM. The potentiating effect of anandamide (1 μM) on relaxation responses was significantly attenuated by either the selective cannabinoid CB 1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide (AM251; 1 μM) or the vanilloid receptor antagonist capsazepine (3 μM), but not by the selective cannabinoid CB 2 receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl) ethyl]-1 H-indol-3-yl (4-methoxyphenyl)methanone (AM630; 1 μM). Neither of these antagonists had influence on relaxation responses. Indomethacin (20 μM) had no effect on NANC-mediated relaxation in the presence or absence of anandamide (1 μM). Preincubation with N w-Nitro- l-Arginine Methyl Ester ( l-NAME; 1 μM) significantly inhibited the relaxation responses in the presence or absence of 1 μM anandamide. Although at 30 nM, l-NAME did not cause a significant inhibition of relaxant responses individually, it significantly inhibited the potentiating effect of anandamide (1 μM) on relaxation responses. Anandamide (1 μM) had no influence on concentration-dependent relaxant responses to sodium nitroprusside (10 nM–1 mM), a nitric oxide (NO) donor. The western blotting of corporal tissues demonstrated the existence of both vanilloid and CB 1 receptors in corporal strips. In conclusion, our results showed that anandamide has a potentiating effect on NANC-mediated relaxation of rat corpus cavernosum through both CB 1 and vanilloid receptors and the NO-mediated component of the NANC relaxant responses to electrical stimulation is involved in this enhancement.

Antithrombin reduces ischemia/reperfusion-induced liver injury in mice by enhancing the activation of sensory neurons through protein kinase A activation

We recently demonstrated that antithrombin (AT) reduces ischemia/reperfusion (I/R)-induced liver injury in rats by increasing hepatic tissue levels of calcitonin gene-related peptide (CGRP), a neuropeptide released from the sensory nerve endings. In the present study, we examined the effect of AT on I/R-induced liver injury in wildtype mice (CGRP+/+) and congenital α CGRP-deficient mice (CGRP-/-). We further investigated whether AT affects CGRP release from dorsal root ganglion neurons (DRG) isolated from CGRP+/+. Based on results obtained in the present study, we attempted to determine whether the anti-inflammatory activity of AT in vivo is dependent mainly on sensory neuron activation. AT enhanced I/R-induced increases in hepatic tissue levels of CGRP and 6-keto-PGF1α a stable metabolite of PGI2, in CGRP+/+, while it did not enhance these increases in CGRP-/-. AT inhibited reperfusion-induced increases in serum alanine aminotransferase levels by increasing hepatic tissue blood flow and by attenuating increases in hepatic levels of tumor necrosis factor and myeloperoxidase in CGRP+/+, while it showed neither of these therapeutic effects in CGRP-/-. AT increased CGRP release from cultured DRGs only in the presence of anandamide, and the AT-induced increase in CGRP release was not observed in the presence of KT5720, an inhibitor of protein kinase A (PKA). AT markedly increased intracellular levels of cAMP in the presence of anandamide. In conclusion, these results strongly suggest that AT might reduce I/R-induced liver injury by enhancing activation of the sensory neurons through activation of PKA in sensory neurons.

Antithrombin reduces reperfusion-induced liver injury in mice by enhancing sensory neuron activation

We recently demonstrated that antithrombin (AT) reduces ischemia/reperfusion (I/R)-induced liver injury in rats by increasing hepatic tissue levels of calcitonin gene-related peptide (CGRP), a neuropeptide released from the sensory nerve endings. In the present study, we examined the effect of AT on I/R-induced liver injury in wild type mice (CGRP+/+) and congenitally alphaCGRP-deficient mice (CGRP-/-). We also investigated any effects of AT on CGRP release from dorsal root ganglion neurons (DRG) isolated from CGRP+/+. Based on results obtained in the present study, we attempted to determine if the anti-inflammatory activity of AT in vivo is dependent mainly on sensory neuron activation. AT enhanced ischemia/reperfusion-induced increases in hepatic tissue levels of CGRP and 6-keto-PGF(1alpha), a stable metabolite of PGI2, in CGRP+/+, but it did not enhance these increases in CGRP-/-. AT inhibited reperfusion-induced increases in serum alanine aminotransferase levels by increasing hepatic tissue blood flow and by attenuating increases in hepatic levels of tumor necrosis factor and myeloperoxidase in CGRP+/+, although it showed neither of these therapeutic effects in CGRP-/-. AT increased CGRP release from cultured DRGs only in the presence of anandamide, and AT-induced increase in CGRP release was not observed in the presence KT5720, an inhibitor of protein kinase A (PKA). AT markedly increased intracellular levels of cAMP in the presence of anandamide. These results strongly suggest that AT might reduce I/R-induced liver injury by enhancing activation of the sensory neurons through activation of PKA in sensory neurons.

Antithrombin reduces endotoxin-induced hypotension by enhancing pulmonary sensory neuron activation in rats

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

Misidentification of prostamides as prostaglandins

Prostaglandins and endogenous cannabinoid metabolites share the same lipid backbone with differing polar head groups at exactly the position through which a large molecule is attached to provide antigenicity and thus raise antisera. Hence, we hypothesized that antisera raised against prostaglandins linked to a large molecule such as BSA at the carboxyl functional group would also recognize endogenous cannabinoid metabolites and lead to highly misleading interpretations of data. We found major cross-reactivity of commercial antisera raised to prostaglandins with endocannabinoid metabolites. Furthermore, in a well-characterized cell line (WISH) or primary amnion tissue explants, endocannabinoid treatment led to increased production of endocannabinoid metabolites as opposed to primary prostaglandins. This was apparent only after separation of products by thin-layer chromatography, because they measured as prostaglandins by radioimmunoassay. These findings have major implications for our interpretation of data in situations in which these prostaglandin-like molecules are formed, and they stress the need for chromatographic or spectrometric confirmation of prostaglandin production detected by antibody-based methods.

Effects of anandamide on the binding and signaling properties of M 1 muscarinic acetylcholine receptors

This study investigated the effects of the endocannabinoid, anandamide, on M 1 muscarinic acetylcholine receptors (mAChRs) expressed in Chinese hamster ovary (CHO) cells. In the presence of anandamide, [ 3H] N-methylscopolamine ([ 3H]NMS) inhibition binding was characterized by Hill coefficients greater than 1 while saturation binding isotherms were characterized by a reduction in radioligand B max. Anandamide did not affect the potency of classic agonists, antagonists or allosteric modulators to inhibit [ 3H]NMS binding, indicating that the site of anandamide action did not involve receptor regions recognized by these compounds. Although the mode of binding of anandamide was reversible, the order of ligand addition was important; the inhibitory effect was greatest when anandamide was equilibrated with the receptor prior to radioligand addition, and weakest in the converse situation. Interestingly, the inhibitory potency of anandamide was reduced on pre-equilibration with non-transfected CHO cell membranes, prior to addition of M 1 mAChR-transfected membranes. In phosphoinositide (PI) hydrolysis assays, anandamide significantly reduced the maximal response to acetylcholine, but at higher concentrations than those needed to fully inhibit radioligand binding. Studies utilizing a range of agonists with varying intrinsic activities showed that the inhibitory effects of anandamide on agonist function were most pronounced with the lowest efficacy agonists. These findings suggest that the mechanism of action of anandamide at the M 1 mAChR involves perturbation of the receptor via the membrane in a manner that is sensitive to the conformation of the receptor (occupied versus vacant).

5-HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK-1-like K+ current in the adult turtle

The modulatory effects of serotonin mediated by 5-HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current-clamp mode, activation of 5-HT1A receptors by 8-OH-DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5-HT1A receptor antagonist WAY-100635, 8-OH-DPAT had no effect. In voltage-clamp mode, with 1 mm CsCl in the bathing medium, 8-OH-DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK-1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8-OH-DPAT had no effect. Our results show that activation of 5-HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK-1 potassium channel leading to depolarization and increased input resistance.

5-HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK-1-like K+ current in the adult turtle.

The modulatory effects of serotonin mediated by 5-HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current-clamp mode, activation of 5-HT1A receptors by 8-OH-DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5-HT1A receptor antagonist WAY-100635, 8-OH-DPAT had no effect. In voltage-clamp mode, with 1 mM CsCl in the bathing medium, 8-OH-DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK-1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8-OH-DPAT had no effect. Our results show that activation of 5-HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK-1 potassium channel leading to depolarization and increased input resistance.

Characterization of a novel endocannabinoid, virodhamine, with antagonist activity at the CB1 receptor.

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor. Virodhamine is arachidonic acid and ethanolamine joined by an ester linkage. Concentrations of virodhamine measured by liquid chromatography atmospheric pressure chemical ionization-tandem mass spectrometry in rat brain and human hippocampus were similar to anandamide. In peripheral tissues that express the CB2 cannabinoid receptor, virodhamine concentrations were 2- to 9-fold higher than anandamide. In contrast to previously described endocannabinoids, virodhamine was a partial agonist with in vivo antagonist activity at the CB1 receptor. However, at the CB2 receptor, virodhamine acted as a full agonist. Transport of [(14)C]anandamide by RBL-2H3 cells was inhibited by virodhamine. Virodhamine produced hypothermia in the mouse and acted as an antagonist in the presence of anandamide both in vivo and in vitro. As a potential endogenous antagonist at the CB1 receptor, virodhamine adds a new form of regulation to the endocannabinoid system.

Cardiovascular pharmacology of anandamide

The fatty acid amide anandamide produces hypotension and a decrease in systemic vascular resistance in vivo. A drop in blood pressure is also seen with synthetic cannabinoid (CB) receptor agonists. The hypotensive responses to anandamide and synthetic cannabinoids are absent in CB1 receptor gene knockout mice. In isolated arteries and perfused vascular beds, anandamide induces vasodilator responses, which cannot be mimicked by synthetic cannabinoids. Instead, vanilloid receptors on perivascular sensory nerves play a key role in these effects of anandamide. Activation of vanilloid receptors by anandamide triggers the release of sensory neuropeptides such as the vasodilator calcitonin gene-related peptide (CGRP). Anandamide is detected in blood and in many cells of the cardiovascular system, and macrophage-derived anandamide may be involved in several hypotensive clinical conditions. Interestingly, cannabinoid and vanilloid receptors display an overlap in ligand recognition properties, and the frequently used CB1 receptor antagonist SR141716A also inhibits vanilloid receptor-mediated responses. The presence of anandamide in endothelial cells, neurones and activated macrophages (monocytes), and its ability to activate CB and vanilloid receptors make this lipid a potential bioregulator in the cardiovascular system.