A new cannabinoid CB2 receptor agonist HU-910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury.

BACKGROUND AND PURPOSE: Cannabinoid CB(2) receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. EXPERIMENTAL APPROACH: We have investigated the effects of a novel CB(2) receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. KEY RESULTS: Displacement of [(3) H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB(2) or CB(1) receptors (hCB(1/2) ) yielded K(i) values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB(2) CHO cells (EC(50) = 162 nM) and yielded EC(50) of 26.4 nM in [(35) S]GTPγS binding assays using hCB(2) expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB(1) antagonist tended to enhance them. CONCLUSION AND IMPLICATIONS: HU-910 is a potent CB(2) receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes.

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.

Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway.

Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.

Anandamide receptor signal transduction.

In the 15 years since its discovery anandamide has been implicated in many physiological processes. The signaling pathways mediating many of these processes are now coming to light, particularly in the CNS. The complexity of the cannabinoid system and the identification of many potential other receptors for anandamide have made conclusive evidence of molecular pathways stimulated by this molecule significantly more difficult to achieve. It is becoming obvious that anandamide receptor signal transduction is not a simple process and that many different cascades can be activated depending on a range of both experimental and physiological variables. This chapter explores the signaling pathways activated by anandamide both through the cannabinoid receptors and through other cellular targets.

Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!

The study of endogenous cannabinoid CB1 receptor proteins in neuronal tissues and cells relies on the availability of highly specific antibodies. We have tested the ability of a series of CB1 antibodies to detect endogenous receptors in brain as well as hemagglutinin (HA)-tagged receptors transfected into HEK-293 cells using a combination of immunological methods. An initial comparison of several CB1 antibodies in mouse brain revealed substantial differences in staining pattern to ligand binding by autoradiography. Antibodies were then tested immunocytochemically against HEK cells expressing HA-tagged rat and human CB1 receptors. None of the commercial antibodies tested were able to detect the receptor in this context. All antibodies were then screened by Western blotting using lysates from the HEK cells and rodent brain homogenates. Again, none of the commercially available antibodies detected proteins of the correct molecular weight in transfected cell lines or brain homogenates, although all recognized multiple proteins in brain tissues. We conclude that the commercially available antibodies we tested failed to detect CB1 receptors abundantly expressed in HEK cells or native receptors in brain slices or homogenates. As such, comprehensive validation of the specificity of these CB1 antibodies for a particular application is essential before use.