sr-144528 and anandamide

Recent advances have dramatically increased our understanding of cannabinoid pharmacology: the psychoactive constituents of Cannabis sativa have been isolated, synthetic cannabinoids described and an endocannabinoid system identified, together with its component receptors, ligands and their biochemistry. Strong laboratory evidence now underwrites anecdotal claims of cannabinoid analgesia in inflammatory and neuropathic pain. Sites of analgesic action have been identified in brain, spinal cord and the periphery, with the latter two presenting attractive targets for divorcing the analgesic and psychotrophic effects of cannabinoids. Clinical trials are now required, but are hindered by a paucity of cannabinoids of suitable bioavailability and therapeutic ratio.

Topics: Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzoxazines; Brain; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Cell Membrane; Clinical Trials as Topic; Disease Models, Animal; Drug Design; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Injections, Spinal; Molecular Structure; Morpholines; Naphthalenes; Pain; Palmitates; Palmitic Acids; Piperidines; Plant Extracts; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Spinal Cord

Bladder afferents play a crucial role in urine storage and voiding, and conscious sensations from the bladder. Endocannabinoids, anandamide (AEA) and 2-arachidonolylglycerol (2-AG), are endogenous ligands of G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2) found in the CNS and peripheral organs. They also have off-target effects on some ligand- and voltage-gated channels. The aim of this study is to determine the role of AEA and 2-AG in regulation of mechanosensitivity of probable nociceptive neurons innervating the bladder - capsaicin-sensitive mucosal afferents. The activity of these afferents was determined by ex vivo single unit extracellular recordings in the guinea pig bladder. A stable analogue of anandamide, methanandamide (mAEA) evoked initial excitatory response of mucosal afferents followed by potentiation of their responses to mechanical stimulation. In the presence of TRPV1 antagonist (AMG9810), mAEA's effect on mechanosensitivity switched from excitatory to inhibitory. The inhibitory effect of mAEA is due to activation of both CB1 and CB2 cannabinoid receptors since it was abolished by combined application of selective CB1 (NESS0327) and CB2 (SR144528) antagonists. 2-AG application evoked a brief excitation of mucosal afferents, without potentiation of their mechanosensitivity, followed by the inhibition of their responses to mechanical stimulation. CB2 receptor antagonist, SR144528 abolished the inhibitory effect of 2-AG. Our data indicated that anandamide and 2-AG have opposite effects on mechanosensitivity of mucosal capsaicin-sensitive afferents in the guinea pig bladder; mAEA potentiated while 2-AG inhibited responses of mucosal afferents to mechanical stimulation. These findings are important for understanding of the role of endocannabinoids in regulating bladder sensation and function.

Topics: Animals; Capsaicin; Endocannabinoids; Guinea Pigs; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Urinary Bladder

Cannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. We therefore investigated the effect of cannabinoids on retinal capillaries and pericytes.. The effects of cannabinoids on capillary diameters were determined using an ex vivo whole-mount rat retinal model. Western blotting, quantitative PCR, and immunohistochemistry were performed to explore the underlying mechanism.. Endogenous cannabinoid 2-arachidonoylglycerol and anandamide and exogenous cannabinoid (R-(+)-WIN55212-2) dilated the noradrenaline-precontracted capillaries in a concentration-dependent manner (1 µM to 0.1 mM). The extent of vasorelaxation was positively correlated with changes in pericyte width. The effects of R-(+)-WIN55212-2 on vasorelaxation and pericyte width were inhibited by a cannabinoid receptor type-1 (CB1) antagonist, AM251 or rimonabant (SR141716A), the nitric oxide synthase inhibitor l-NAME, and the guanylate cyclase inhibitor ODQ. They were also abolished by the removal of the endothelium, but not by the cannabinoid receptor-2 antagonist SR144528, the endothelial cannabinoid receptor antagonist O-1918, or the cyclooxygenase inhibitor indomethacin.. The exogenous cannabinoid R-(+)-WIN55212-2 promotes the vasorelaxation of pericyte-containing rat retinal capillaries. This effect of R-(+)-WIN55212-2 is dependent on CB1 and the nitric oxide-cyclic guanosine monophosphate pathway, and requires an intact endothelium.

Topics: Animals; Arachidonic Acids; Blotting, Western; Camphanes; Cannabinoid Receptor Antagonists; Cannabinoids; Capillaries; Endocannabinoids; Guanylate Cyclase; Immunohistochemistry; Male; Nitric Oxide Synthase; Pericytes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Retina; Rimonabant

Anandamide, an endocannabinoid, prostaglandins derived from cyclooxygenase-2 and nitric oxide synthesized by nitric oxide synthase (NOS), are relevant mediators of embryo implantation. We adopted a pharmacological approach to investigate if anandamide modulated NOS activity in the receptive rat uterus and if prostaglandins mediated this effect. As we were interested in studying the changes that occur at the maternal side of the fetal-maternal interface, we worked with uteri obtained from pseudopregnant rats. Females were sacrificed on day 5 of pseudopregnancy, the day in which implantation would occur, and the uterus was obtained. Anandamide (2 ng/kg, i.p.) inhibited NOS activity (P<0.001) and increased the levels of prostaglandin E(2) (P<0.001) and prostaglandin F(2α) (P<0.01). These effects were mediated via cannabinoid receptor type 2, as the pre-treatment with SR144528 (10 mg/kg, i.p.), a selective cannabinoid receptor type 2 antagonist, completely reverted anandamide effect on NOS activity and prostaglandin levels. The pre-treatment with a non-selective cyclooxygenase inhibitor (indomethacin 2.5mg/kg, i.p.) or with selective cyclooxygenase-2 inhibitors (meloxicam 4 mg/kg, celecoxib 3mg/kg, i.p.) reverted anandamide inhibition on NOS, suggesting that prostaglandins are derived from cyclooxygenase-2 mediated anandamide effect. Thus, anandamide levels seemed to modulate NOS activity, fundamental for implantation, via cannabinoid receptor type 2 receptors, in the receptive uterus. This modulation depends on the production of cyclooxygenase-2 derivatives. These data establish cannabinoid receptors and cyclooxygenase enzymes as an interesting target for the treatment of implantation deficiencies.

Topics: Animals; Arachidonic Acids; Camphanes; Cannabinoid Receptor Modulators; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprost; Dinoprostone; Endocannabinoids; Female; Nitric Oxide Synthase; Polyunsaturated Alkamides; Pseudopregnancy; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Uterus

Hypothermia occurs in the most severe cases of systemic inflammation, but the mechanisms involved are poorly understood. This study evaluated whether the hypothermic response to bacterial lipopolysaccharide (LPS) is modulated by the endocannabinoid anandamide(AEA) and its receptors: cannabinoid-1 (CB1), cannabinoid-2 (CB2) and transient receptor potential vanilloid-1 (TRPV1). In rats exposed to an ambient temperature of 22◦C, a moderate dose of LPS (25 - 100 μg kg−1 I.V.) induced a fall in body temperature with a nadir at ∼100 minpostinjection. This response was not affected by desensitization of intra-abdominal TRPV1 receptors with resiniferatoxin (20 μg kg - 1 I.P.), by systemic TRPV1 antagonism with capsazepine(40mg kg−1 I.P.), or by systemic CB2 receptor antagonism with SR144528 (1.4 mg kg−1 I.P.).However, CB1 receptor antagonism by rimonabant (4.6mg kg−1 I.P.) or SLV319 (15mg kg−1 I.P.)blocked LPS hypothermia. The effect of rimonabant was further studied. Rimonabant blocked LPS hypothermia when administered I.C.V. at a dose (4.6 μg) that was too low to produce systemic effects. The blockade of LPS hypothermia by I.C.V. rimonabant was associated with suppression of the circulating level of tumour necrosis factor-α. In contrast to rimonabant,the I.C.V. administration of AEA (50 μg) enhanced LPS hypothermia. Importantly, I.C.V. AEAdid not evoke hypothermia in rats not treated with LPS, thus indicating that AEA modulates LPS-activated pathways in the brain rather than thermo effector pathways. In conclusion, the present study reveals a novel, critical role of brain CB1 receptors in LPS hypothermia. Brain CB1 receptors may constitute a new therapeutic target in systemic inflammation and sepsis.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Body Temperature Regulation; Brain; Camphanes; Capsaicin; Disease Models, Animal; Diterpenes; Endocannabinoids; Female; Hypothermia; Injections, Intraperitoneal; Injections, Intravenous; Injections, Intraventricular; Lipopolysaccharides; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Long-Evans; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Signal Transduction; Sulfonamides; Time Factors; TRPV Cation Channels

Comparison of human, rat and mouse cannabinoid CB(2) receptor primary sequences has shown significant divergence at the mRNA and protein sequence level, raising the possibility of species specific pharmacological properties. Additionally, given the importance of the dog as a non-rodent species for predicting human safety during the drug development process, we cloned the dog CB(2) receptor gene and characterized its in-vitro pharmacological properties in a recombinant expression system. A 1.1 kb dog peripheral cannabinoid receptor (dCB(2)) fragment encoding a 360 amino acid protein was cloned from dog spleen cDNA. Analysis of the cloned dCB(2) polypeptide sequence revealed that it shares between 76 and 82% homology with rat, mouse, human and predicted chimpanzee cannabinoid CB(2) receptors. The dog CB(2) receptor expressed in CHO cells displayed similar binding affinities for various synthetic and endogenous cannabinoids as compared to those measured for the human and rat cannabinoid CB(2) receptors. However, these ligands exhibited altered functional potencies and efficacies for the dog cannabinoid CB(2) receptor, which was also found to be negatively coupled to adenylate cyclase activity. These complex pharmacological differences observed across species for the cannabinoid CB(2) receptor suggest that caution should be exerted when analyzing the outcome of animal efficacy and safety studies, notably those involving cannabinoid CB(2) receptor targeting molecules tested in the dog.

Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Base Sequence; Benzoxazines; Binding, Competitive; Camphanes; CHO Cells; Cloning, Molecular; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Dogs; Dronabinol; Endocannabinoids; Humans; Mice; Molecular Sequence Data; Morpholines; Naphthalenes; Pan troglodytes; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Sequence Alignment

Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB(1)R and CB(2)R, making AEA a prominent member of the "endocannabinoid" family. Also immune cells express CB(1) and CB(2) receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells.. In this investigation we used flow cytometry and ELISA tests, in order to show that AEA suppresses proliferation and release of cytokines like IL-2, TNF-alpha and INF-gamma from activated human peripheral T-lymphocytes. However, AEA did not exert any cytotoxic effect on T-cells. The immunosuppression induced by AEA was mainly dependent on CB(2)R, since it could be mimicked by the CB(2)R selective agonist JWH-015, and could be blocked by the specific CB(2)R antagonist SR144528. Instead the selective CB(1)R agonist ACEA, or the selective CB(1)R antagonist SR141716, were ineffective. Furthermore, we demonstrated an unprecedented immunosuppressive effect of AEA on IL-17 production, a typical cytokine that is released from the unique CD4+ T-cell subset T-helper 17.. Overall, our study investigates for the first time the effects of the endocannabinoid AEA on primary human T-lymphocytes, demonstrating that it is a powerful modulator of immune cell functions. In particular, not only we clarify that CB(2)R mediates the immunosuppressive activity of AEA, but we are the first to describe such an immunosuppressive effect on the newly identified Th-17 cells. These findings might be of crucial importance for the rational design of new endocannabinoid-based immunotherapeutic approaches.

Topics: Arachidonic Acids; Base Sequence; Camphanes; Cell Proliferation; Cytokines; DNA Primers; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Microscopy, Confocal; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; T-Lymphocytes

Anandamide binds to cannabinoid receptors and plays several central and peripheral functions. The aim of this work was to study the possible role for this endocannabinoid in controlling sperm-oviduct interaction in mammals. We observed that bull sperm and bovine oviductal epithelial cells express cannabinoid receptors, CB1 and CB2, and fatty acid amide hydrolase, the enzyme that controls intracellular anandamide levels. A quantitative assay to determine whether anandamide was involved in bovine sperm-oviduct interaction was developed. R(+)-methanandamide, a non-hydrolysable anandamide analog, inhibited sperm binding to and induced sperm release from oviductal epithelia. Selective CB1 antagonists (SR141716A or AM251) completely blocked R(+)-methanandamide effects. However, SR144528, a selective CB2 antagonist, did not exert any effect, indicating that only CB1 was involved in R(+)-methanandamide effect. This effect was not caused by inhibition of the sperm progressive motility or by induction of the acrosome reaction. Overall, our findings indicate for the first time that the endocannabinoid system is present in bovine sperm and oviductal epithelium and that anandamide modulates the sperm-oviduct interaction, by inhibition of sperm binding and induction of sperm release from oviductal epithelial cells, probably by activating CB1 receptors.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Blotting, Western; Camphanes; Carbamates; Cattle; Endocannabinoids; Epithelium; Fallopian Tubes; Female; Immunohistochemistry; Male; Microscopy, Fluorescence; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Sperm Motility; Sperm-Ovum Interactions; Spermatozoa

To study the effects of N-arachidonylethanolamide (anandamide [AEA]) on aqueous humor outflow and to investigate the existence and activity of fatty acid amide hydrolase (FAAH), an AEA metabolic enzyme in trabecular meshwork (TM) tissue.. The effects of AEA on aqueous humor outflow were measured using a porcine anterior segment-perfused organ culture model. Western blot analysis was used to study the expression of FAAH, and a thin-layer chromatography-based approach was used to measure the enzymatic activity of FAAH in TM tissue.. Administration of AEA caused a transient enhancement of aqueous humor outflow facility. In the presence of 100 nM URB597, an FAAH inhibitor, the effect of 10 nM AEA on outflow facility was prolonged by at least 4 hours. The AEA-induced enhancement of outflow facility was blocked by SR141716A, a CB1 antagonist, and was partially blocked by SR144528, a CB2 antagonist. In Western blot studies, positive signals were detected on TM tissues with an anti-FAAH antibody. In the enzyme activity studies, the enzymatic activity of AEA hydrolysis was detected in TM tissues, and this activity was reduced with the addition of 100 nM URB597.. Results from this study demonstrate that the administration of AEA increases aqueous humor outflow facility and that this effect of AEA involves CB1 and CB2 cannabinoid receptors. In addition, this study reveals the existence and the activity of FAAH, an AEA-metabolizing enzyme, in the TM tissues.

Topics: Amidohydrolases; Animals; Anterior Eye Segment; Aqueous Humor; Arachidonic Acids; Benzamides; Blotting, Western; Camphanes; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Carbamates; Chromatography, Thin Layer; Endocannabinoids; Organ Culture Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Swine; Trabecular Meshwork

2-arachidonoylglycerol (2-AG) is an endogenous ligand for the cannabinoid receptors with a variety of potent biological activities. In this study, we first examined the effects of potassium-induced depolarization on the level of 2-AG in rat brain synaptosomes. We found that a significant amount of 2-AG was generated in the synaptosomes following depolarization. Notably, depolarization did not affect the levels of other molecular species of monoacylglycerols. Furthermore, the level of anandamide was very low and did not change markedly following depolarization. It thus appeared that the depolarization-induced accelerated generation is a unique feature of 2-AG. We obtained evidence that phospholipase C is involved in the generation of 2-AG in depolarized synaptosomes: U73122, a phospholipase C inhibitor, markedly reduced the depolarization-induced generation of 2-AG, and the level of diacylglycerol was rapidly elevated following depolarization. A significant amount of 2-AG was released from synaptosomes upon depolarization. Interestingly, treatment of the synaptosomes with SR141716A, a CB1 receptor antagonist, augmented the release of glutamate from depolarized synaptosomes. These results strongly suggest that the endogenous ligand for the cannabinoid receptors, i.e. 2-AG, generated through increased phospholipid metabolism upon depolarization, plays an important role in attenuating glutamate release from the synaptic terminals by acting on the CB1 receptor.

Topics: Animals; Arachidonic Acids; Brain; Calcimycin; Calcium Channel Blockers; Camphanes; Diglycerides; Endocannabinoids; Estrenes; Fatty Acids; Glycerides; Male; Membrane Potentials; Neuromuscular Depolarizing Agents; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Cannabinoid; Rimonabant; Synaptosomes

We have previously shown that the endocannabinoid anandamide and its metabolically stable analog (R)-methanandamide produce vasorelaxation in rabbit aortic ring preparations in an endothelium-dependent manner that could not be mimicked by other CB(1) cannabinoid receptor agonists (Am J Physiol 282: H2046-H2054, 2002). Here, we show that (R)-methanandamide and abnormal cannabidiol stimulated nitric oxide (NO) production in rabbit aortic endothelial cells (RAEC) in a dose-dependent manner but that other CB(1) and CB(2) receptor agonists, such as cis-3R-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4R-3(3-hydroxypropyl)-1R-cyclohexanol (CP55940) and (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55212-2), failed to do so. CB(1) antagonists rimonabant [also known as SR141716; N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and 6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanophenyl]methanone (LY320135) and CB(2) antagonist N-[(1S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) failed to block (R)-methanandamide-mediated NO production in RAEC. However, anandamide receptor antagonist (-)-4-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol (O-1918) blocked (R)-methanandamide-mediated NO production in RAEC. Reverse transcriptase-polymerase chain reaction and Western blot analyses failed to detect the CB(1) receptor in RAEC, making this a good model to study non-CB(1) responses to anandamide. (R)-Methanandamide produced endothelial nitric-oxide synthase (eNOS) phosphorylation via the activation of phosphoinositide 3-kinase-Akt signaling. Inhibition of G(i) signaling with pertussis toxin, or phosphatidylinositol 3-kinase activity with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), resulted in a decrease in (R)-methanandamide-induced Akt phosphorylation and NO production. Results from this study suggest that in RAEC, (R)-methanandamide acts on a novel non-CB(1) and non-CB(2) anandamide receptor and signals through G(i) and phosphatidylinositol 3-kinase, leading to Akt activation, eNOS phosphorylation, and NO production.

Topics: Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Camphanes; Cannabinoid Receptor Modulators; Cells, Cultured; Chromones; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Endothelial Cells; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go; Morpholines; Naphthalenes; Nitric Oxide; Pertussis Toxin; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Pyrazoles; Rabbits; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Resorcinols; Rimonabant; Signal Transduction

Allergic contact dermatitis affects about 5% of men and 11% of women in industrialized countries and is one of the leading causes for occupational diseases. In an animal model for cutaneous contact hypersensitivity, we show that mice lacking both known cannabinoid receptors display exacerbated allergic inflammation. In contrast, fatty acid amide hydrolase-deficient mice, which have increased levels of the endocannabinoid anandamide, displayed reduced allergic responses in the skin. Cannabinoid receptor antagonists exacerbated allergic inflammation, whereas receptor agonists attenuated inflammation. These results demonstrate a protective role of the endocannabinoid system in contact allergy in the skin and suggest a target for therapeutic intervention.

Topics: Animals; Arachidonic Acids; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Chemokines; Dermatitis, Allergic Contact; Dinitrofluorobenzene; Disease Models, Animal; Down-Regulation; Dronabinol; Endocannabinoids; Female; Glycerides; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Skin; Up-Regulation

This study shows that two cannabinoids, Delta(9)-tetrahydrocannabinol (THC) and anandamide, induce dose-related immunosuppression in both the primary and secondary in vitro plaque-forming cell assays of antibody formation. The immunosuppression induced by both compounds could be blocked by SR144528, an antagonist specific for the CB(2) receptor, but not by SR141716, a CB(1) antagonist. These studies are novel in that they show that both anandamide and THC are active in the nanomolar to picomolar (for anandamide) range in these assays of immune function, and that both mediate their effects directly on cells of the immune system through the CB(2) receptor.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Camphanes; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Endocannabinoids; Female; Immune System; In Vitro Techniques; Lymphocytes; Mice; Mice, Inbred C3H; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB2; Rosette Formation

Noradrenaline and ATP are sympathetic co-transmitters. In the rat perfused mesenteric bed cannabinoids have been shown to modify the overall response to sympathetic nerve stimulation. This study has assessed whether cannabinoid receptor activation modulates differentially the noradrenergic and purinergic components of sympathetic vasoconstriction.. Rat mesenteric beds were perfused with physiological salt solution and the effects of cannabinoids on responses to nerve stimulation, or exogenous noradrenaline or alpha,beta-methylene ATP (alpha,beta-meATP; P2X receptor agonist) were determined after raising tone with U46619. The effects of cannabinoids on the noradrenaline and ATP components of sympathetic neurotransmission were assessed using the alpha 1-adrenoceptor antagonist, prazosin, or after P2X receptor desensitization with alpha,beta-meATP.. Anandamide, WIN 55,212-2 and CP55,940 attenuated sympathetic neurogenic vasoconstrictor responses. The inhibitory actions of anandamide and WIN 55,212-2 were blocked by LY320135, a CB1 receptor antagonist, but not by SR144528, a CB2 receptor antagonist. The inhibitory actions of CP55,940 were unaffected by LY320135 and SR144528. WIN 55,212-3, the inactive S(-) enantiomer of WIN 55,212-2, had no effect on sympathetic neurogenic responses. None of the cannabinoids affected contractile responses to exogenous noradrenaline or alpha,beta-meATP. Anandamide and WIN 55,212-2 inhibited both the noradrenaline and ATP components of the sympathetic neurogenic contractile responses, with effects on the ATP component being most marked.. These results indicate that prejunctional CB1-like receptors mediate the sympathoinhibitory action of anandamide and WIN 55,212-2, but not CP55,940, in the rat mesenteric bed. Cannabinoids inhibit both the noradrenergic and purinergic components of sympathetic neurotransmission.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Adrenergic Fibers; Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Camphanes; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; In Vitro Techniques; Male; Mesenteric Arteries; Morpholines; Muscle Tonus; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Perfusion; Polyunsaturated Alkamides; Prazosin; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Purinergic P2

The effect of the endogenous cannabinoid anandamide on K(+) currents activated by the ATP-sensitive potassium (K(ATP)) channel opener cromakalim was investigated in follicle-enclosed Xenopus oocytes using the two-electrode voltage-clamp technique. Anandamide (1-90 microM) reversibly inhibited cromakalim-induced K(+) currents, with an IC(50) value of 8.1 +/- 2 microM. Inhibition was noncompetitive and independent of membrane potential. Coapplication of anandamide with the cannabinoid type 1 (CB(1)) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) (1 microM), the CB(2) receptor antagonist N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), or pertussis toxin (5 microg/ml) did not alter the inhibitory effect of anandamide, suggesting that known cannabinoid receptors are not involved in anandamide inhibition of K(+) currents. Similarly, neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor indomethacin (5 microM) affected anandamide inhibition of K(+) currents, suggesting that the effects of anandamide are not mediated by its metabolic products. In radioligand binding studies, anandamide inhibited the specific binding of the K(ATP) ligand [(3)H]glibenclamide in the oocyte microsomal fractions, with an IC(50) value of 6.3 +/- 0.4 microM. Gonadotropin-induced oocyte maturation and the cromakalim-acceleration of progesterone-induced oocyte maturation were significantly inhibited in the presence of 10 microM anandamide. Collectively, these results indicate that cromakalim-activated K(+) currents in follicular cells of Xenopus oocytes are modulated by anandamide via a cannabinoid receptor-independent mechanism and that the inhibition of these channels by anandamide alters the responsiveness of oocytes to gonadotropin and progesterone.

Topics: Animals; Arachidonic Acids; Calcium; Camphanes; Cromakalim; Egtazic Acid; Endocannabinoids; Female; Glyburide; KATP Channels; Oocytes; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Pyrazoles; Rimonabant; Xenopus laevis

Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta.. Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide.. Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide.. Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.

Topics: Animals; Aorta, Abdominal; Apamin; Arachidonic Acids; Benzamides; Calcitonin Gene-Related Peptide; Camphanes; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Charybdotoxin; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Indomethacin; Isoindoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Sulfonamides; Vasodilation

Male Wistar rats were trained to discriminate (-)-nicotine (0.4 mg/kg) from saline under a two-lever, fixed-ratio 10 schedule of water reinforcement. During test sessions the following drugs were coadministered with saline (substitution studies) or nicotine (0.025-0.4 mg/kg; combination studies): the alpha4beta2 nicotinic acetylcholine receptor subtype antagonist dihydro-beta-erythroidine (DHbetaE), the non-selective nicotinic acetylcholine receptor subtype antagonist mecamylamine, the alpha7 nicotinic acetylcholine receptor subtype antagonist methyllycaconitine (MLA), the alpha4beta2 nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA), the cannabinoid CB1 receptor antagonist/partial agonist rimonabant, the cannabinoid CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methybenzyl)pyrazole-3-carboxamide (SR 144528), the cannabinoid CB1/2 receptor agonists (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)-methanone mesylate (WIN 55,212-2), the endogenous cannabinoid agonist and non-competitive alpha7 nicotinic acetylcholine receptor subtype antagonist anandamide, the anandamide uptake and fatty acid amide hydrolase inhibitor N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM-404), the fatty acid amide hydrolase inhibitor cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB 597), AM-404+anandamide or URB 597+anandamide. 5-IA (0.01 mg/kg) fully substituted for nicotine, while other drugs were inactive. In combination studies, DHbetaE and mecamylamine dose-dependently attenuated the discriminative stimulus effects of nicotine and the full substitution of 5-IA, while MLA, rimonabant, SR 144528, CP 55,940, WIN 55,212-2, and URB 597 did not alter the nicotine cue. Pretreatment with AM-404+anandamide or URB 597+anandamide weakly enhanced nicotine-lever responding. Our pharmacological analyses demonstrates that the expression of nicotine discrimination is under the control of nicotinic acetylcholine receptor subtypes composed of alpha4beta2 (but not of alpha7) subunits. Furthermore, we excluded the involvement of either cannabinoid CB1 and CB2 receptors or increases in the endocannabinoid tone in the nicotine discrimination.

Topics: Aconitine; Animals; Arachidonic Acids; Azetidines; Benzamides; Benzoxazines; Camphanes; Cannabinoid Receptor Antagonists; Carbamates; Cyclohexanols; Dihydro-beta-Erythroidine; Discrimination Learning; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Male; Mecamylamine; Morpholines; Naphthalenes; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Polyunsaturated Alkamides; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Nicotinic

Although neurogenic inflammation via the activation of C fibers in the airway must have an important role in the pathogenesis of asthma, their regulatory mechanism remains uncertain.. The pharmacological profiles of endogenous cannabinoid receptor agonists on the activation of C fibers in airway tissues were investigated and the mechanisms how cannabinoids regulate airway inflammatory reactions were clarified.. The effects of endogenous cannabinoid receptor agonists on electrical field stimulation-induced bronchial smooth muscle contraction, capsaicin-induced bronchoconstriction and capsaicin-induced substance P release in guinea pig airway tissues were investigated. The influences of cannabinoid receptor antagonists and K+ channel blockers to the effects of cannabinoid receptor agonists on these respiratory reactions were examined.. Both endogenous cannabinoid receptor agonists, anandamide and palmitoylethanolamide, inhibited electrical field stimulation-induced guinea pig bronchial smooth muscle contraction, but not neurokinin A-induced contraction. A cannabinoid CB2 antagonist, SR 144528, reduced the inhibitory effect of endogenous agonists, but not a cannabinoid CB1 antagonist, SR 141716A. Inhibitory effects of agonists were also reduced by the pretreatment of large conductance Ca2+ -activated K+ channel (maxi-K+ channel) blockers, iberiotoxin and charybdotoxin, but not by other K+ channel blockers, dendrotoxin or glibenclamide. Anandamide and palmitoylethanolamide blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea pig airway tissues. Additionally, intravenous injection of palmitoylethanolamide dose-dependently inhibited capsaicin-induced guinea pig bronchoconstriction, but not neurokinin A-induced reaction. However, anandamide did not reduce capsaicin-induced guinea pig bronchoconstriction.. These findings suggest that endogenous cannabinoid receptor agonists inhibit the activation of C fibers via cannabinoid CB2 receptors and maxi-K+ channels in guinea pig airways.

Topics: Amides; Animals; Arachidonic Acids; Bronchi; Bronchoconstriction; Calcium Channel Blockers; Camphanes; Cannabinoid Receptor Agonists; Capsaicin; Electric Stimulation; Endocannabinoids; Ethanolamines; Guinea Pigs; Male; Muscle Contraction; Muscle, Smooth; Nerve Fibers, Unmyelinated; Neurogenic Inflammation; Organ Culture Techniques; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Substance P

The aim of this study was to investigate the expression of cannabinoid receptors in human uterine smooth muscle during pregnancy and to evaluate the effects of endogenous and exogenous cannabinoids on myometrial contractility in vitro.. Human myometrial biopsy specimens were obtained at elective cesarean delivery and snap frozen or mounted for isometric recording under physiologic conditions. Cumulative doses of the endogenous cannabinoid anandamide or the exogenous cannabinoid Delta(9) (indicates a double bond between carbons 9 and 10) tetrahydrocannabinol were added in the range 1 nmol/L to 100 micromol/L. Selectivity of the cannabinoid receptor agonists was investigated with specific antagonists for the CB(1) and the CB(2) receptors. Reverse transcription-polymerase chain reaction with primers for the CB(1) and CB(2) receptors was performed on messenger RNA that was isolated from human pregnant myometrium.. Both anandamide and Delta(9)-tetrahydrocannabinol exerted a direct relaxant effect on human pregnant myometrium in vitro, which was of equal potency for both compounds. This relaxant effect was antagonized by the specific CB(1) receptor antagonist, SR 141716, but not by the specific CB(2) receptor antagonist, SR 144528 (n=6 specimens, P<.01). Both the CB(1) and CB(2) receptors are expressed in human myometrium.. Both endogenous and exogenous cannabinoids exert a potent and direct relaxant effect on human pregnant myometrium, which is mediated through the CB(1) receptor. This highlights a possible role for endogenous cannabinoids during human parturition and pregnancy. These results also support the view that the use of exogenous cannabinoids during pregnancy is not linked independently with preterm labor.

Topics: Adult; Arachidonic Acids; Camphanes; Dronabinol; Endocannabinoids; Female; Humans; In Vitro Techniques; Myometrium; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Uterine Contraction; Uterus

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Previously, we provided evidence that 2-arachidonoylglycerol, but not anandamide (N-arachidonoylethanolamine), is the true natural ligand for the cannabinoid receptors. In the present study, we examined in detail the effects of 2-arachidonoylglycerol on the production of chemokines in human promyelocytic leukemia HL-60 cells. We found that 2-arachidonoylglycerol induced a marked acceleration in the production of interleukin 8. The effect of 2-arachidonoylglycerol was blocked by treatment of the cells with SR144528, a cannabinoid CB2 receptor antagonist, indicating that the effect of 2-arachidonoylglycerol is mediated through the CB2 receptor. Augmented production of interleukin 8 was also observed with CP55940, a synthetic cannabinoid, and an ether-linked analog of 2-arachidonoylglycerol. On the other hand, neither anandamide nor the free arachidonic acid induced the enhanced production of interleukin 8. A similar effect of 2-arachidonoylglycerol was observed in the case of the production of macrophage-chemotactic protein-1. The accelerated production of interleukin 8 by 2-arachidonoylglycerol was observed not only in undifferentiated HL-60 cells, but also in HL-60 cells differentiated into macrophage-like cells. Noticeably, 2-arachidonoylglycerol and lipopolysaccharide acted synergistically to induce the dramatically augmented production of interleukin 8. These results strongly suggest that the CB2 receptor and its physiological ligand, i.e., 2-arachidonoylglycerol, play important regulatory roles such as stimulation of the production of chemokines in inflammatory cells and immune-competent cells. Detailed studies on the cannabinoid receptor system are thus essential to gain a better understanding of the precise regulatory mechanisms of inflammatory reactions and immune responses.

Topics: Arachidonic Acid; Arachidonic Acids; Blotting, Northern; Calcitriol; Camphanes; Cell Differentiation; Chemokine CCL2; Chemokines; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Gene Expression; Glycerides; HL-60 Cells; Humans; Interleukin-8; Lipopolysaccharides; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB2; RNA, Messenger; Time Factors

2-arachidonoylglycerol (2-AG) is an endogenous cannabinoid receptor ligand. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, abundantly expressed in the brain, and the CB2 receptor, expressed in various lymphoid tissues such as the spleen. The CB1 receptor has been assumed to play an important role in the regulation of synaptic transmission, whereas the physiological roles of the CB2 receptor remain obscure. In this study, we examined whether the CB2 receptor is present in human eosinophils and found that the CB2 receptor is expressed in human peripheral blood eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB2 receptor. We then examined the effect of 2-AG on the motility of eosinophils. We found that 2-AG induces the migration of human eosinophilic leukemia EoL-1 cells. The migration evoked by 2-AG was abolished in the presence of SR144528, a CB2 receptor antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB2 receptor and Gi/o are involved in the 2-AG-induced migration. The migration of EoL-1 cells induced by 2-AG was suggested to be a result of chemotaxis. In contrast to 2-AG, neither anandamide nor free arachidonic acid elicited the migration. Finally, we examined the effect of 2-AG on human peripheral blood eosinophils and neutrophils and found that 2-AG induces migration of eosinophils but not neutrophils. These results suggest that the CB2 receptor and its endogenous ligand 2-AG may be closely involved in allergic inflammation accompanied by the infiltration of eosinophils.

Topics: Arachidonic Acid; Arachidonic Acids; Camphanes; Cell Line, Tumor; Chemotaxis, Leukocyte; Endocannabinoids; Eosinophils; Glycerides; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Hypereosinophilic Syndrome; Hypersensitivity; Neutrophils; Pertussis Toxin; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB2; RNA, Messenger

Endocannabinoids are novel lipid mediators with hypotensive and cardiodepressor activity. Here, we examined the possible role of the endocannabinergic system in cardiovascular regulation in hypertension.. In spontaneously hypertensive rats (SHR), cannabinoid-1 receptor (CB1) antagonists increase blood pressure and left ventricular contractile performance. Conversely, preventing the degradation of the endocannabinoid anandamide by an inhibitor of fatty acid amidohydrolase reduces blood pressure, cardiac contractility, and vascular resistance to levels in normotensive rats, and these effects are prevented by CB1 antagonists. Similar changes are observed in 2 additional models of hypertension, whereas in normotensive control rats, the same parameters remain unaffected by any of these treatments. CB1 agonists lower blood pressure much more in SHR than in normotensive Wistar-Kyoto rats, and the expression of CB1 is increased in heart and aortic endothelium of SHR compared with Wistar-Kyoto rats.. We conclude that endocannabinoids tonically suppress cardiac contractility in hypertension and that enhancing the CB1-mediated cardiodepressor and vasodilator effects of endogenous anandamide by blocking its hydrolysis can normalize blood pressure. Targeting the endocannabinoid system offers novel therapeutic strategies in the treatment of hypertension.

Topics: Amidohydrolases; Angiotensin II; Animals; Arachidonic Acids; Benzamides; Benzyl Compounds; Blood Pressure; Camphanes; Carbamates; Dronabinol; Endocannabinoids; Endothelium, Vascular; Hypertension; Male; Models, Cardiovascular; Myocardial Contraction; Myocardium; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Inbred Dahl; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Up-Regulation; Vascular Resistance; Vasodilation; Ventricular Function, Left

The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

Topics: Arachidonic Acids; Blood-Brain Barrier; Brain; Camphanes; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Capsaicin; Cell Adhesion Molecules; Cells, Cultured; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Glycerides; Humans; Ion Channels; Microcirculation; Microfilament Proteins; Phosphoproteins; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; TRPV Cation Channels

Anandamide (AEA) is an endogenous cannabinoid ligand acting predominantly on the cannabinoid 1 (CB(1)) receptor, but it is also an agonist on the capsaicin VR(1)/TRPV(1) receptor. In the present study we examined the effects of AEA and the naturally occurring cannabinoid 2 (CB(2)) receptor agonist palmitylethanolamide (PEA) on basal and resiniferatoxin (RTX)-induced release of calcitonin gene-related peptide (CGRP) and somatostatin in vivo. Since these sensory neuropeptides play important role in the development of neuropathic hyperalgesia, the effect of AEA and PEA was also examined on mechanonociceptive threshold changes after partial ligation of the sciatic nerve. Neither AEA nor PEA affected basal plasma peptide concentrations, but both of them inhibited RTX-induced release. The inhibitory effect of AEA was prevented by the CB(1) receptor antagonist SR141716A. AEA abolished and PEA significantly decreased neuropathic mechanical hyperalgesia 7 days after unilateral sciatic nerve ligation, which was antagonized by SR141716A and the CB(2) receptor antagonist SR144528, respectively. Both SR141716A and SR144528 increased hyperalgesia, indicating that endogenous cannabinoids acting on CB(1) and peripheral CB(2)-like receptors play substantial role in neuropathic conditions to diminish hyperalgesia. AEA and PEA exert inhibitory effect on mechanonociceptive hyperalgesia and sensory neuropeptide release in vivo suggesting their potential therapeutical use to treat chronic neuropathic pain.

Topics: Amides; Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Camphanes; Cannabinoids; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Ethanolamines; Hyperalgesia; Injections, Intravenous; Male; Neuropeptides; Neurotoxins; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Sciatic Nerve; Sciatic Neuropathy; Somatostatin

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that 14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R. However, 56 nM anandamide is capable of inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that anandamide may play an essential role at the outset of implantation.

Topics: Animals; Arachidonic Acids; Blastocyst; Camphanes; Dose-Response Relationship, Drug; Embryo Implantation; Embryonic and Fetal Development; Endocannabinoids; Epithelial Cells; Female; Mice; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Uterus

Cannabinoids have been shown to increase the release of arachadonic acid, whereas nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to decrease the analgesic effects of cannabinoids. We evaluated the antinociceptive effects of chronic administration of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), anandamide (an endogenous cannabinoid), arachadonic acid, ethanolamine, and methanandamide on several NSAIDs via p.o. and/or i.p. routes of administration using the mouse p-phenylquinone (PPQ) test, a test for visceral nociception. Our studies with a cannabinoid receptor (CB1) antagonist [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A)], a CB2 antagonist [N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528)], and an another CB2 agonist [1,1-dimethylbutyl-1-deoxy-Delta(9)-THC (JWH-133)] were performed to better characterize PPQ interactions with cannabinoid receptors. The acute affects of Delta(9)-THC were blocked by SR141716A (i.p.) and partially blocked by SR144528 (i.p.). When NSAIDs (p.o.) were administered, the ED(50) values were as follows: 23 mg/kg aspirin, 3 mg/kg indomethacin, 5 mg/kg celecoxib, 3 mg/kg ketorolac, 57 mg/kg acetaminophen (32.3-99.8), and 0.8 mg/kg diclofenac (0.1-4.9). In animals given chronic Delta(9)-THC, only diclofenac and acetaminophen were active. Conversely, chronic methanandamide (i.p.) did not alter the antinociceptive effects of the NSAIDs. Neither the CB1 or CB2 antagonist blocked the effects of the NSAIDs. The effects of chronic arachadonic acid, ethanolamine, and anandamide could not be evaluated. In summary, our data indicate that chronic Delta(9)-THC alters the cyclooxygenase system. Alternatively, the data suggest that this alteration is not due to chronic endogenous cannabinoid release. Based upon these data, we hypothesize that human subjects who are chronic users of Delta(9)-THC may not respond to analgesic treatment with the above NSAIDs.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Arachidonic Acids; Aspirin; Camphanes; Diclofenac; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Ethanolamines; Indomethacin; Kinetics; Male; Mice; Mice, Inbred ICR; Pain; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug

1. Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea-pigs, and its effect on VR1 receptor function in isolated primary guinea-pig nodose ganglia neurons. 2. Anandamide (0.3-3 mg.ml(-1)), when given by aerosol, induced cough in conscious guinea-pigs in a concentration dependent manner. When guinea-pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide-induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide-induced cough. These results indicate that anandamide-induced cough is mediated through the activation of VR1 receptors. 3. Anandamide (10-100 micro M) increased intracellular Ca(2+) concentration estimated by Fluo-4 fluorescence change in isolated guinea-pig nodose ganglia cells. The anandamide-induced Ca(2+) response was inhibited by two different VR1 antagonists: capsazepine (1 micro M) and iodo-resiniferatoxin (I-RTX, 0.1 micro M), indicating that anandamide-induced Ca(2+) response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 micro M) and CB2 (SR 144528, 0.1 micro M) receptor antagonists had no effect on Ca(2+) response to anandamide. 4. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea-pig nodose ganglia cells and induces cough through activation of VR1 receptors.

Topics: Aerosols; Animals; Arachidonic Acids; Calcium; Camphanes; Capsaicin; Cells, Cultured; Consciousness; Cough; Dronabinol; Endocannabinoids; Excitatory Amino Acid Antagonists; Guinea Pigs; Male; Nodose Ganglion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The purpose of this study was to investigate the role of the central cannabinoid receptor (CB(1)) in mediating the actions of the endogenous cannabinoid agonist anandamide and the synthetic cannabinoid CP-55940. Activation of primary mouse astrocyte cultures by exposure to bacterial lipopolysaccharide (LPS) caused a marked (approximately tenfold) increase in nitric oxide (NO) release. Coincubation with the cannabinoid agonists anandamide or CP-55940 markedly inhibited release of NO (-12% to -55%). This effect was abolished by SR-141716A (1 microM), a CB1 receptor antagonist. SR-141716A alone also significantly increased NO release in response to LPS, suggesting that endogenous cannabinoids modify inflammatory responses. In contrast, coincubation with the CB2 receptor antagonist SR-144528 (1 microM) abolished the inhibitory effects of the endogenous cannabinoid anandamide on LPS-induced NO release, although this may reflect nonspecific effects of this ligand or cannabinoid actions through atypical receptors of anandamide. We also showed that endogenous or synthetic cannabinoids inhibit LPS-induced inducible NO synthase expression (mRNA and protein) in astrocyte cultures. These results indicate that CB1 receptors may promote antiinflammatory responses in astrocytes.

Topics: Analgesics; Animals; Arachidonic Acids; Astrocytes; Calcium Channel Blockers; Camphanes; Cannabinoids; Cells, Cultured; Cyclohexanols; Dronabinol; Endocannabinoids; Gene Expression; Lipopolysaccharides; Mice; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Cannabinoids have previously been shown to possess analgesic properties in a model of visceral hyperalgesia in which the neurotrophin, nerve growth factor (NGF), plays a pivotal role. The purpose of this study was to investigate the antihyperalgesic effects of two cannabinoids in NGF-evoked visceral hyperalgesia in order to test the hypothesis that endocannabinoids may modulate the NGF-driven elements of inflammatory hyperalgesia. Intra-vesical installation of NGF replicates many features of visceral hyperalgesia, including a bladder hyper-reflexia and increased expression of the immediate early gene c fos in the spinal cord. We investigated the action of anandamide and palmitoylethanolamide (PEA) on these parameters. Both anandamide (at a dose of 25 mg/kg) and PEA (at a dose of 2.5 mg/kg) attenuated the bladder hyper-reflexia induced by intra-vesical NGF. The use of cannabinoid CB1 receptor (SR141617A) and CB2 receptor (SR144528) antagonists suggested that the effect of anandamide was mediated by both CB1 and CB2 cannabinoid receptors whilst the action of PEA was via CB2 (or CB2-like) receptors only. Furthermore, anandamide (25 mg/kg) and PEA (2.5 mg/kg) reduced intra-vesical NGF-evoked spinal cord Fos expression at the appropriate level (L6) by 35 and 43%, respectively. However, neither CB1 nor CB2 receptor antagonists altered the action of anandamide. PEA-induced reduction in Fos expression was abrogated by SR144528. These data add to the growing evidence of a therapeutic potential for cannabinoids, and support the hypothesis that the endogenous cannabinoid system modulates the NGF-mediated components of inflammatory processes.

Topics: Amides; Animals; Arachidonic Acids; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Ethanolamines; Female; Hyperalgesia; Nerve Growth Factor; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Reflex, Abnormal; Rimonabant; Spinal Cord; Urinary Bladder; Visceral Afferents

The endocannabinoid 2-arachidonoylglycerol (2-Delta(4)Ach-Gro) activates human platelets in platelet-rich plasma at physiological concentrations. The activation was inhibited by selective antagonists of CB(1) and CB(2) cannabinoid receptors, but not by acetylsalicylic acid. Human platelets can metabolize 2-Delta(4)Ach-Gro by internalization through a high affinity transporter (K(m) = 300 +/- 30 nM, V(max) = 10 +/- 1 pmol.min(-1).mg protein(-1)), followed by hydrolysis by a fatty acid amide hydrolase (K(m) = 8 +/- 1 microM, V(max) = 400 +/- 50 pmol.min(-1).mg protein(-1)). The anandamide transport inhibitor AM404, and anandamide itself, were ineffective on 2-Delta(4)Ach-Gro uptake by platelets, whereas anandamide competitively inhibited 2-Delta(4)Ach-Gro hydrolysis (inhibition constant = 10 +/- 1 microM). Platelet activation by 2-Delta(4)Ach-Gro was paralleled by an increase of intracellular calcium and inositol-1,4,5-trisphosphate, and by a decrease of cyclic AMP. Moreover, treatment of preloaded platelet-rich plasma with 2-Delta(4)Ach-Gro induced an approximately threefold increase in [(3)H]2-Delta(4)Ach-Gro release, according to a CB receptor-dependent mechanism. On the other hand, ADP and collagen counteracted the activation of platelets by 2-Delta(4)Ach-Gro, whereas 5-hydroxytryptamine (serotonin) enhanced and extended its effects. Remarkably, ADP and collagen also reduced [(3)H]2-Delta(4)Ach-Gro release from 2-Delta(4)Ach-Gro-activated platelets, whereas 5-hydroxytryptamine further increased it. These findings suggest a so far unnoticed interplay between the peripheral endocannabinoid system and physiological platelet agonists.

Topics: Adenosine Diphosphate; Amidohydrolases; Arachidonic Acids; Aspirin; Biological Transport; Blood Platelets; Calcium Channel Blockers; Camphanes; Cannabinoid Receptor Modulators; Collagen; Cyclic AMP; Endocannabinoids; Glycerides; Humans; Hydrolysis; Inositol 1,4,5-Trisphosphate; Kinetics; Piperidines; Platelet Activation; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Serotonin; Time Factors

The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB2 receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide x HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB1 receptor antagonist SR141716A, not the cannabinoid CB2 receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.

Topics: Amides; Analgesics; Analysis of Variance; Animals; Arachidonic Acids; Calcium Channel Blockers; Camphanes; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Ethanolamines; Formaldehyde; Male; Mice; Pain; Palmitic Acids; Polyunsaturated Alkamides; Pyrazoles

Intravenous injection of 10 mg/kg anandamide reduces the incidence and duration of epinephrine-induced arrhythmias in rats. SR141716A and SR144528, antagonists of cannabinoid receptor I and II did not abolish the antiarrhythmic effect of anandamide. These data suggest that the antiarrhythmic effect of anandamide is nonspecific or mediated via unknown cannabinoid receptors, but not associated with activation of cannabinoid receptors I and II.

Topics: Animals; Arachidonic Acids; Arrhythmias, Cardiac; Camphanes; Electrocardiography; Endocannabinoids; Epinephrine; Heart; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.

Topics: Amides; Amidohydrolases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Arachidonic Acids; Blotting, Western; Breast Neoplasms; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Cell Division; Colforsin; COS Cells; Cyclic AMP; Dose-Response Relationship, Drug; Endocannabinoids; Ethanolamines; Glycerides; Humans; Hydrolysis; Inhibitory Concentration 50; Palmitic Acids; Polyunsaturated Alkamides; Protein Binding; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured

1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

Topics: Acetylcholine; Animals; Arachidonic Acids; Camphanes; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Guinea Pigs; Ileum; In Vitro Techniques; Male; Methacholine Chloride; Muscle Contraction; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

Topics: Acetylcholine; Animals; Arachidonic Acids; Benzofurans; Calcitonin Gene-Related Peptide; Calcium; Calcium Channel Blockers; Camphanes; Cannabinoids; Capsaicin; Cell Line; Dose-Response Relationship, Drug; Endocannabinoids; Humans; In Vitro Techniques; Mesenteric Arteries; Neurons, Afferent; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Ruthenium; Synaptic Transmission; Vasodilation; Vasodilator Agents

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

Topics: Amides; Arachidonic Acids; Calcium Signaling; Camphanes; Cannabinoids; Cyclohexanols; Cyclooxygenase Inhibitors; Drug Interactions; Endocannabinoids; Ethanolamines; Glycerides; HL-60 Cells; Humans; Ligands; Lipoxygenase Inhibitors; Molecular Mimicry; Palmitic Acids; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Structure-Activity Relationship; Virulence Factors, Bordetella

The role of cannabinoid CB(1) receptors in sympathetic neurotransmission was characterised in nerve-mediated responses of isolated right atria, vasa deferentia and small mesenteric resistance arteries using the cannabinoid CB(1) receptor agonists Delta(9)-tetrahydrocannabinol, CP 55,940 and anandamide and the cannabinoid CB(1)-selective antagonist SR 141716A. In the mouse vas deferens, the twitch response was completely inhibited by each of the putative cannabinoid receptor agonists with pIC(50) values of CP 55,940, 9.2+/-0.1; Delta(9)-tetrahydrocannabinol, 8.4+/-0.1; anandamide, 7.1+/-0.1. SR 141716A 10-100 nM was a competitive antagonist of all three agonists with a pK(B) value of 8.4-8.6, consistent with an interaction at the cannabinoid CB(1) receptor. In the rat vas deferens CP 55,940 (0.01-10 microM) inhibited the contractions to a significant extent (88.5+/-0.5% at 10 microM; pIC(50) of 7.1+/-0.1) while Delta(9)-tetrahydrocannabinol and anandamide (both up to 10 microM) were inactive. CP 55,940 exhibited low potency in rat compared with mouse vas deferens and the rat concentration-response curve was not competitively antagonised by SR 141716A (100 nM) or SR 144528 (10 nM-10 microM), suggesting an interaction at a receptor(s) distinct from cannabinoid CB(1) or CB(2). Sympathetic nerve-induced tachycardia in rat and mouse atria, and rat mesenteric artery smooth muscle contractile responses to perivascular nerve stimulation, were not inhibited by Delta(9)-tetrahydrocannabinol, CP 55,940 or anandamide up to 1 microM. These data indicate that cannabinoid CB(1) receptor activation inhibits sympathetic neurotransmission only in the mouse vas deferens and thus point to species and regional differences in cannabinoid CB(1) receptor involvement in pre-synaptic inhibition of sympathetic neurotransmission and CP 55,940 may have inhibitory actions in rat vas deferens unrelated to cannabinoid receptor activity.

Topics: Animals; Arachidonic Acids; Camphanes; Cannabinoids; Cyclohexanols; Dronabinol; Endocannabinoids; Heart Atria; In Vitro Techniques; Male; Mesenteric Artery, Inferior; Mice; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Sympathetic Nervous System; Synaptic Transmission; Vas Deferens; Vascular Resistance

Anandamide (ANA) is an endogenous ligand for the cannabinoid receptors Cb1 and Cb2 that is able to synergistically stimulate the proliferation of hematopoietic growth factor-dependent blood cells in serum-free culture. To elucidate the mechanisms by which ANA enhances the proliferative responses of hematopoietic cells, we investigated the ANA-mediated effects on proliferation, cell cycling, apoptosis and intracellular signaling of erythropoietin-stimulated 32D/EPO cells.. 32D/EPO cells were cultured serum free to determine the effects of EPO and anandamide on these cells. Proliferation was analyzed by tritiated thymidine incorporation. Apoptosis as well as cell cycle analysis was carried out by flow cytometry. MAPKinase activation was determined by Western blotting, using phospho-specific MAPK antibodies.. Simultaneous addition of erythropoietin (EPO) and ANA enhanced DNA synthesis and increased 32D/EPO cell numbers in serum-free culture. Interestingly, ANA did not alter the G1/S transition but it accelerated each of the successive cell cycle phases of EPO-stimulated 32D/EPO cells. Percentages of apoptotic 32D/EPO cells were equally low in cultures supplemented with EPO alone or a combination of EPO and ANA. Both cultures showed enhanced activation of two mitogen-activated protein kinases, namely, extracellular factor responsive kinases 1 and 2 (ERK1/2), as well as the MAPK-target gene protein c-Fos. This fully correlated with the synergistic stimulation of proliferation of 32D/EPO cells by EPO and ANA. ANA had no effect on EPO-induced STAT-5 activation of 32D/EPO cells. Experiments with the Cb2 receptor-specific antagonist SR144528 demonstrated that the synergistic stimulation of proliferation by ANA was partially Cb2 receptor-mediated.. These data suggest that the positive effects of ANA on the erythropoietin-induced proliferation of 32D/EPO cells are mediated by receptor-dependent as well as receptor-independent mechanisms, both of which involve activation of the mitogen-activated protein kinases, ERK1/2.

Topics: Animals; Apoptosis; Arachidonic Acids; Camphanes; Cell Cycle; Cell Line; Culture Media, Serum-Free; DNA Replication; DNA-Binding Proteins; Drug Synergism; Endocannabinoids; Enzyme Activation; Erythropoietin; Genes, fos; Mice; Milk Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; RNA, Messenger; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Up-Regulation

The effect of anandamide, an endogenous ligand for central (CB1) and peripheral (CB2) cannabinoid receptors, was investigated on the growth of the murine IL-6-dependent lymphoid cell line B9 and the murine IL-3-dependent myeloblastic cell line FDC-P1. In conditions of low serum level, anandamide potentiated the growth of both cytokine-dependent cell lines. Comparison with other fatty acid cannabinoid ligands such as (R)-methanandamide, a ligand with improved selectivity for the CB1 receptor, or palmitylethanolamide, an endogenous ligand for the CB2 receptor, showed a very similar effect, suggesting that cell growth enhancement by anandamide or its analogs could be mediated through either receptor subtype. However, several lines of evidence indicated that this growth-promoting effect was cannabinoid receptor-independent. First, the potent synthetic cannabinoid agonist CP 55940, which displays high affinity for both receptors, was inactive in this model. Second, SR 141716A and SR 144528, which are potent and specific antagonists of CB1 and CB2 receptors respectively, were unable, alone or in combination, to block the anandamide-induced effect. Third, inactivation of both receptors by pretreatment of cells with pertussis toxin did not affect the potentiation of cell growth by anandamide. These data demonstrated that neither CB1 nor CB2 receptors were involved in the anandamide-induced effect. Moreover, using CB2-transfected Chinese hamster ovary cells, we demonstrated that after complete blockade of the receptors by the specific antagonist SR 144528, anandamide was still able to strongly stimulate a mitogen-activated protein (MAP) kinase activity, clearly indicating that the endogenous cannabinoid can transduce a mitogenic signal in the absence of available receptors. Finally, arachidonic acid, a structurally related compound and an important lipid messenger without known affinity for cannabinoid receptors, was shown to trigger MAP kinase activity and cell growth enhancement similar to those observed with anandamide. These findings provide clear evidence for a functional role of anandamide in activating a signal transduction pathway leading to cell activation and proliferation via a non-cannabinoid receptor-mediated process.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinases; Camphanes; Cannabinoids; Cell Division; CHO Cells; Cricetinae; Cyclohexanols; Endocannabinoids; Enzyme Activation; Gene Expression Regulation; Mice; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

Topics: Amides; Analgesics; Animals; Arachidonic Acids; Camphanes; Cannabinoids; Endocannabinoids; Ethanolamines; Mice; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant