Cannabinoid Receptor-2 Activation in Keratinocytes Contributes to Elevated Peripheral ß-Endorphin Levels in Patients With Obstructive Jaundice.

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

Expression Analysis of CB2-GFP BAC Transgenic Mice.

The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

Endocannabinoid system activation may be associated with insulin resistance in women with polycystic ovary syndrome.

OBJECTIVE: To assess the levels of endocannabinoids and cannabinoid receptors (CB) 1 and 2 in women with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: University teaching hospital. PATIENT(S): In total, 20 women with PCOS and 20 healthy women in a control group, who were matched for body mass index and age, were enrolled in this study. INTERVENTION(S): The homeostasis model index was used to assess insulin resistance. MAIN OUTCOME MEASURE(S): Omental adipose tissue and human peripheral blood mononuclear cells (PBMCs) from PCOS and the controls were analyzed using real-time polymerase chain reactions for the expressions of CB1 and CB2. The levels of endocannabinoids were analyzed using high-performance liquid chromatography. RESULT(S): The levels of anandamide and 2-arachidonoylglycerol, and the expression of CB1 and CB2 mRNA (messenger ribonucleic acid) in the PBMCs were significantly higher in the women with PCOS than in the women serving as controls. We found that expression of CB1, but not CB2, in adipose tissue was significantly higher in the women with, vs. without, PCOS. The expressions of CB1 mRNA and endocannabinoids showed a significant positive correlation with 2-hour glucose and insulin levels 2 hours after glucose loading in the PBMCs and adipose tissue. CONCLUSION(S): Activation of endocannabinoids and overexpression of cannabinoid receptors, especially CB1, may be associated with insulin resistance in women with PCOS.

Detailed characterisation of CB2 receptor protein expression in peripheral blood immune cells from healthy human volunteers using flow cytometry.

It is commonly accepted from gene expression studies that the CB2 receptor is expressed by most cell types of the rodent and human immune system. However, the exact identity of cells expressing CB2 receptor protein in human blood or the abundance of receptors expressed by each immune subset is not well characterised. We conducted a detailed analysis of CB2 protein levels expressed by blood-derived immune cells from healthy human donors. Flow-cytometry was conducted using 4 commercially available anti-CB2 polyclonal antibodies in conjunction with a selection of immune cell specific markers. Across multiple healthy subjects we observed that NK cells, B-lymphocytes and monocytes expressed a higher level of CB2 receptor than CD4+ or CD8+ T-lymphocytes. Neutrophils also expressed a low level of CB2 receptor. NK cells had the greatest variation in CB2 expression levels, whereas for each of the other cell types CB2 levels were relatively similar between subjects. In contrast to other methods, the high sensitivity of flow-cytometry revealed that CB2 receptors are present on resting T-lymphocytes at low abundance in some healthy subjects. These data provide the first detailed analysis of CB2 protein levels in blood leukocyte subsets from healthy donors and identifies the cell types which could be targeted with CB-mimetic drugs in humans.

Substantially altered expression pattern of cannabinoid receptor 2 and activated endocannabinoid system in patients with severe heart failure.

Animal studies suggest that the endocannabinoid system (ECS) plays a role in the regulation of myocardial contractility and in the pathogenesis of heart failure. The current study aimed to proof the existence of endocannabinoid receptors on human ventricular myocardium and to determine whether human chronic heart failure (CHF) is associated with changes in endocannabinoid receptor expression and distribution. Expression of cannabinoid receptor 1 (CB1) and cannabinoid receptor (CB2) on human heart was assessed by means of real-time PCR and immunohistochemistry. On healthy human left ventricular myocardium, mRNA transcripts of CB1 and CB2 receptors were expressed in an almost equal proportion. In patients with CHF, mRNA expression of CB1 receptors was shown to be downregulated 0.7-fold (0.7.+/-0.15, n=12, p<0.01), whereas expression of CB2 receptors was upregulated more than 11-fold (11.6+/-4.5; n=12; p<0.005). Corresponding results were obtained by immunohistochemistry. Blood levels of endocannabinoids were significantly elevated (anandamide 3.5-fold (p<0.001); 2-AG 7-fold (p=0.02)) in patients with CHF, as compared to healthy volunteers. Both CB1 and CB2 receptors are present on healthy human left ventricular myocardium in a balanced distribution. Patients suffering from CHF exhibit a shift of the CB1-CB2 receptor ratio towards expression of CB2 receptors combined with significantly elevated peripheral blood levels of endocannabinoids indicating an activation of the ECS. These results might open up new perspectives regarding the role of endocannabinoid signalling in CHF and its potential as a target for pharmacological modulation.