Animals ; Cannabinoid Receptor Antagonists ; therapeutic use ; Cannabinoids ; pharmacology ; Fibrosis ; metabolism ; Humans ; Liver Cirrhosis ; etiology ; metabolism ; therapy ; Piperidines ; therapeutic use ; Pyrazoles ; therapeutic use ; Receptor, Cannabinoid, CB1 ; metabolism ; Receptor, Cannabinoid, CB2 ; metabolism ; Receptors, Cannabinoid ; metabolism ; Scleroderma, Diffuse ; metabolism ; Signal Transduction ; drug effects ; Skin ; metabolism ; Smad Proteins ; metabolism ; Transforming Growth Factor beta1 ; metabolism

To observe a PPAR-alpha agonist effect of N-oleoylethanolamine (OEA) on CB2 (cannabinoid receptor 2), an anti-inflammatory receptor in vascular endothelial cell, healthy HUVECs and TNF-alpha induced HUVECs were used to establish a human vascular endothelial cell inflammatory model. Different doses of OEA (10, 50 and 100 micromol x L(-1)) had been given to HUVECs, cultured at 37 degrees C for 7 h and then collected the total protein and total mRNA. CB2 protein expression was detected by Western blotting and CB2 mRNA expression was assayed by real-time PCR. As the results shown, OEA (10 and 50 micromol x L(-1)) could induce the CB2 protein and mRNA expression, but not 100 micromol x L(-1). To detect if anti-inflammation effect of OEA is partly through CB2, CB2 inhibitor AM630 was used to inhibit HUVEC CB2 expression, then the VCAM-1 expression induced by TNF-alpha was detected, or THP-1 adhere to TNF-alpha induced HUVECs was examined. OEA (50 micromol x L(-1)) could inhibit TNF-alpha induced VCAM-1 expression and THP-1 adhere to HUVECs, these effects could be partly inhibited by a CB2 inhibitor AM630. The anti-inflammation effect of OEA is induced by PPAR-alpha and CB2, suggesting that CB2 signaling could be a target for anti-atherosclerosis, OEA have wide effect in anti-inflammation, it may have better therapeutic potential in anti-inflammation in HUVECs, thus achieving anti-atherosclerosis effect.
Anti-Inflammatory Agents ; pharmacology ; Atherosclerosis ; pathology ; Cell Adhesion ; drug effects ; Cells, Cultured ; Endocannabinoids ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Ethanolamines ; pharmacology ; Humans ; Indoles ; pharmacology ; Monocytes ; drug effects ; Oleic Acids ; pharmacology ; PPAR alpha ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Receptor, Cannabinoid, CB2 ; antagonists & inhibitors ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo investigate whether cannabinoid receptor 1 (CB1R) is involved in nerve growth in endometriosis-associated ectopic cyst.

METHODSThe effect of CB1R agonist and antagonist on the expression of pan-neuronal marker protein gene product (PGP) 9.5 in ectopic cyst was examined by immunofluorescence and Western blot in endometriosis model of 18 rats.

RESULTSImmunofluorescence revealed that PGP 9.5 was expressed in the nerve fibers and was mainly distributed in the cyst hilum. Western blot revealed that the protein density of either PGP 9.5 (2 week: 0.38 ± 0.05; 4 week: 0.63 ± 0.03; 8 week: 0.80 ± 0.07, P < 0.01) or CB1R (2 week: 0.48 ± 0.04; 4 week: 0.68 ± 0.01; 8 week: 0.80 ± 0.03, P < 0.01) in the ectopic cyst increased with cyst size. In addition, compared to control group (0.75 ± 0.01), PGP 9.5 expression in the ectopic cyst was promoted by CB1R agonist ACPA (0.81 ± 0.01, P < 0.05), and inhibited by CB1R antagonist AM251 (0.67 ± 0.03, P < 0.01).

CONCLUSIONSCB1R was involved in the nerve growth of ectopic cyst associated with endometriosis.

Animals ; Blotting, Western ; Cysts ; metabolism ; Disease Models, Animal ; Endometriosis ; metabolism ; Female ; Peripheral Nerves ; growth & development ; metabolism ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Rats ; Receptor, Cannabinoid, CB1 ; antagonists & inhibitors ; physiology ; Ubiquitin Thiolesterase ; metabolism

Endocannabinoid anandamide (AEA) has protective effect on the heart against ischemia/reperfusion injury and arrhythmia, but the electrophysiological mechanism is unclear yet. In this study, the sinoatrial node (SAN) samples from New Zealand rabbits were prepared, and intracellular recording technique was used to elucidate the effect of AEA on the action potential (AP) of SAN pacemaker cells of rabbits and the mechanism. Different concentrations of AEA (1, 10, 100, 200, 500 nmol/L) were applied cumulatively. For some SAN samples, cannabinoid type 1 (CB1) receptor antagonist AM251, cannabinoid type 2 (CB2) receptor antagonist AM630, potassium channel blocker tetraethylammonium (TEA) and nitric oxide (NO) synthase inhibitor L-nitro-arginine methylester (L-NAME) were used before AEA treatment, respectively. We found that: (1) AEA (100, 200 and 500 nmol/L) not only shortened AP duration (APD), but also decreased AP amplitude (APA) (P < 0.05). (2) AM251, but not AM630, abolished the effect of AEA on APD shortening. (3) TEA and L-NAME had no influence on the AEA effect. These findings suggest that anandamide can decrease APA and shorten APD in SAN pacemaker cells of rabbits, which may be mediated by activation of CB1 receptors, and is related to blockade of calcium channels but not potassium channels and NO.
Action Potentials ; Animals ; Arachidonic Acids ; pharmacology ; Cannabinoid Receptor Antagonists ; pharmacology ; Endocannabinoids ; pharmacology ; Indoles ; pharmacology ; Myocytes, Cardiac ; drug effects ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Piperidines ; pharmacology ; Polyunsaturated Alkamides ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Pyrazoles ; pharmacology ; Rabbits ; Sinoatrial Node ; cytology

BACKGROUND: WIN55212-2 is a synthetic cannabinoid agonist and selective to cannabinoid 1 (CB1) receptors, which are distributed mainly in the central nervous system. Opioid receptors and CB1 receptors have several similarities in terms of their intracellular signal transduction mechanisms, distributions, and pharmacological action. Several studies have therefore sought to describe the functional interactions between opioids and cannabinoids at the cellular and behavioral levels. The present study investigated agonist-stimulated [35S]GTPgammaS binding by WIN55212-2 in rat brain membranes and determined the antagonism by selective opioid antagonists at the level of receptor-ligand interaction and intracellular signal transduction. METHODS: Sprague-Dawley rats (male, n = 20) were euthanized for the preparation of brain membranes. In agonist-stimulated [35S]GTPgammaS binding by WIN55212-2, the values of EC50 and maximum stimulation (% over basal) were determined in the absence or presence of the micro, kappa and delta opioid receptor antagonists naloxone (20 nM), norbinaltorphimine (3 nM), and naltrindole (3 nM), respectively. Ke values for opioid antagonist inhibition in the absence or presence of each opioid receptor antagonist were calculated using the following equation: [nanomolar antagonist] / (dose ratio of EC50 - 1). RESULTS: In WIN55212-2-stimulated [35S]GTPgammaS binding in the rat brain membranes, the values of EC50 and maximum stimulation (% over basal) were 154 +/- 39.5 nM and 27.6 +/- 5.3% over basal, respectively. Addition of selective opioid antagonists did not produce a significant rightward shift in the WIN55212-2 concentration-response curve, and Ke values were not applicable. CONCLUSIONS: Our results suggest that the functional activity of WIN55212-2-stimulated [35S]GTPgammaS binding was not affected by opioid antagonists in the rat brain membranes. Although the exact mechanism remains unclear, our results may partially elucidate their actions.
Analgesics, Opioid ; Animals ; Benzoxazines ; Brain ; Cannabinoids ; Central Nervous System ; Membranes ; Morpholines ; Naloxone ; Naltrexone ; Naphthalenes ; Narcotic Antagonists ; Rats ; Rats, Sprague-Dawley ; Receptor, Cannabinoid, CB1 ; Receptors, Opioid ; Receptors, Opioid, delta ; Signal Transduction

BACKGROUNDThe cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.

METHODSRAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.

RESULTSAM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.

CONCLUSIONAM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Animals ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Indoles ; pharmacology ; Mice ; Osteoclasts ; cytology ; drug effects ; metabolism ; RANK Ligand ; pharmacology ; Receptor, Cannabinoid, CB2 ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

OBJECTIVETo study the influences of endocannabinoid-anandamide (AEA) on the proliferation and apoptosis of the colorectal cancer cell line (CaCo-2) and to elucidate the effects of CB1 and lipid rafts, and to further elucidate the molecular mechanism and the effect of AEA on the generation and development of colorectal cancer.

METHODSHuman colorectal cancer cell line CaCo-2 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in 5% CO(2) atmosphere at 37°C. CaCo-2 cells were divided into different groups and treated with different concentrations of AEA, AEA + SR141716A, AEA + AM630 and AEA + methyl-β-cyclodextrin (MCD). MTT assay was used to determine the effects of AEA, its putative CB1, CB2 receptor antagonists (SR141716A and AM630) and MCD on the proliferation of CaCo-2 cells. Annexin V-PE/7AAD binding assay was used to detect apoptosis in the CaCo-2 cells. Western-blot was applied to check the expressions of CB1, CB2, p-AKT and caspase-3 proteins in different groups of CaCo-2 cells.

RESULTSAEA inhibited the proliferation of CaCo-2 cells in a concentration-dependent manner and the effect could be antagonized by SR141716A and MCD. The inhibiting rates were (21.52 ± 0.45)%, (42.16 ± 0.21)%, (73.64 ± 0.73)% and (83.28 ± 0.71)%, respectively, at different concentrations of AEA (5, 10, 20 and 40 µmol/L). The three groups (20 µmol/L AEA, 20 µmol/L AEA + 10 µmol/L SR141716A and 20 µmol/L AEA + 1 mmol/L MCD) showed different inhibiting rates [(73.64 ± 0.73)%, (16.15 ± 0.75)% and (12.58 ± 0.63)%], respectively. Annexin V-PE/7AAD binding assay showed that AEA induced apoptosis in the CaCo-2 cells and MCD could antagonize this effect. The apoptosis rates of the three groups (control, 20 µmol/L AEA and 20 µmol/L AEA + 1 mmol/L MCD) were (2.95 ± 0.73)%, (39.61 ± 0.73)% and (14.10 ± 0.64)%, respectively. The expressions of CB1, CB2, p-AKT and Caspase-3 proteins were all observed in the CaCo-2 cells. AEA inhibited p-AKT protein expression and induced caspase-3 protein expression. The two actions were also antagonized by MCD.

CONCLUSIONSAEA can strongly suppress the proliferation of colorectal cancer CaCo-2 cells via the CB1 receptor and membrane cholesterol-LRs and induce apoptosis via lipid rafts. Anandamide plays a very important role in the carcinogenesis and development of colorectal cancer. MCD is a critical member in this system.

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

BACKGROUNDVascular hyporeactivity, which occurs in the terminal stage of hemorrhagic shock, is believed to be critical for treating hemorrhagic shock. The present study was designed to examine whether the CB1 cannabinoid receptor (CB1R) was involved in the development of vascular hyporeactivity in rats suffering from hemorrhagic shock.

METHODSSixteen animals were randomly divided into two groups (n = 8 in each group): sham-operated (Sham) and hemorrhagic shock (HS) groups. Hemorrhagic shock was induced by bleeding. The mean arterial pressure (MAP) was reduced to and stabilized at (25 +/- 5) mmHg for 2 hours. The vascular reactivity was determined by the response of MAP to norepinephrine (NE). In later experiments another twelve animals were used in which the changes of CB1R mRNA and protein in aorta and superior mesenteric artery (SMA) were analyzed by RT-PCR and Western blotting. In addition, we investigated the effects of a CB1R antagonist on the vascular hyporeactivity and survival rates in rats with hemorrhagic shock. Survival rates were analyzed by the Fisher's exact probability test. The MAP response was analyzed by one-way analysis of variance (ANOVA).

RESULTSVascular hyporeactivity developed in all animals suffering from hemorrhagic shock. The expression of CB1R mRNA and protein in aorta and 2 - 3 branches of the SMA were significantly increased in the HS group after the development of vascular hyporeactivity when compared to those in Sham group. When SR141716A or AM251 was administered, the MAP response to NE was (41.75 +/- 4.08) mmHg or (44.78 +/- 1.80) mmHg respectively, which was higher than that in saline groups with (4.31 +/- 0.36) mmHg (P < 0.01). We also showed an increased 4-hour survival rate in the SR141716A or AM251-treated group with 20% or 30%, but with a statistically significant difference present between the AM251-treated and saline groups (P < 0.05).

CONCLUSIONSCB1R is involved in vascular hyporeactivity resulting from hemorrhagic shock in rats, and CB1R antagonist may be useful in treating patients with traumatic, hemorrhagic shock who need field-rescue or initial treatment.

Animals ; Blotting, Western ; Gene Expression Regulation ; drug effects ; Hypotension ; metabolism ; Male ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Cannabinoid, CB1 ; antagonists & inhibitors ; genetics ; metabolism ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Shock, Hemorrhagic ; metabolism ; mortality ; Survival Rate

Being a great threaten for human health, obesity has become a pandemic chronic disease. There have been several therapeutic treatments for this social health issue, including diet and exercise therapy, medication and surgery, among which the diet is still the most common way. However, none of these therapeutic measures available is ideal, making it necessary to find an effective medical treatment. The endocannabinoid system, which is well known for its contributions in certain mental processes such as relaxation, amelioration of pain and anxiety, and sedation initiation, has been recently reported to play an essential role in regulating appetite and metabolism to maintain energy balance, leading to the belief that endocannabinoid system is closely related to obesity. This new discovery deepens our understanding of obesity, and provides us with a new direction for clinical obesity treatment. Rimonabant is an antagonist for CB1, and has entered the market in some countries. However, although effective as an anti-obesity drug, rimonabant also causes obviously adverse side-effects, thus is being doubted and denied for medical usage.
Animals ; Anti-Obesity Agents ; therapeutic use ; Cannabinoid Receptor Modulators ; antagonists & inhibitors ; metabolism ; Endocannabinoids ; Humans ; Obesity ; drug therapy ; metabolism ; Piperidines ; therapeutic use ; Pyrazoles ; therapeutic use ; Receptors, Cannabinoid ; metabolism

OBJECTIVEIn recent years, abnormal changes in the endocannabinoid system have been found in schizophrenia. The superior temporal gyrus (STG) is strongly implicated in the pathophysiology of schizophrenia, particularly with regards to auditory hallucinations. In this study, we investigated the binding density of cannabinoid CB1 receptors in the STG of schizophrenia patients compared to control subjects.

METHODSQuantitative autoradiography was used to investigate the binding densities of [(3)H]SR141716A (a selective antagonist) and [(3)H]CP-55940 (an agonist) to the CB1 receptors in the STG. Post-mortem brain tissue was obtained from the NSW Tissue Resource Centre (Australia).

RESULTSContrasting to previous findings in the alterations of CB1 receptor densities in the prefrontal, anterior and posterior cingulate cortex of schizophrenia, which were suggested to be associated to impairment of cognition function, no significant difference was found between the schizophrenia and control cases in both [(3)H]SR141716A and [(3)H]CP-55940 binding.

CONCLUSIONWe suggest that CB1 receptors in the STG are not involved in the pathology of schizophrenia and the auditory hallucination symptom of this disease.

Adult ; Aged ; Autoradiography ; Case-Control Studies ; Humans ; Middle Aged ; Receptor, Cannabinoid, CB1 ; agonists ; antagonists & inhibitors ; metabolism ; Reference Values ; Schizophrenia ; metabolism ; physiopathology ; Temporal Lobe ; metabolism