Role of the GPR55 receptor in microglial cell function

Samia Hassan, David Kendal, Steve Alexander. University of Nottingham/Biomedical School, NG7 2UH Nottingham, United Kingdom.

Background Microglial cells could be regarded as the macrophages of the central nervous system. Upon activation they produce a number of pro-inflammatory mediators including nitric oxide (NO), which normally play an immune protective role, but excessive activity might be damaging, as seen in stroke, Alzheimer’s disease and neuropathic pain. The orphan G protein-coupled receptor GPR55 (1) is suggested to be related to the cannabinoid receptor family and is thought to be involved in pain signalling and to produce anti-inflammatory effects in microglia. Aim of this study; to examine the potential involvement of GPR55 receptors in the control of NO formation in the BV-2 murine microglial cell line.

Methods: Generation of NO was assessed by measuring accumulation the stable end-product nitrite in BV-2 culture supernatants by the Griess assay (2) using an optical density endpoint (540nm) determined spectophotometrically. Cell viability was assessed using the Neutral Red assay. Data were analysed using ANOVA and post-hoc Dunnett’s tests. Results represent the means of at least 3 separate experiments, each performed in duplicate and results represent as % of LPS. Western immunoblotting; Membrane of BV-2 fractions were probed with an iNOS anti-mouse antibody (1:1000) and proteins visualised using a secondary polyclonal antibody (goat ant-rabbit conjugated with peroxidase, 1:2500) using an Odyssey imaging system (Li-Cor Bioscience).

Result: BV-2 cells produced time- and concentration-dependent increases in NO in the presence of LPS. CP55-940 (cannabinoid CB1/2 receptor agonist and putative GPR55 antagonist) (72±3.3%), AM251 (CB1 antagonist and putative GPR55 antagonist) (75±1%) and the phytocannabinoid cannabidiol CBD (65±0.03%) (also a putative GPR55 antagonist) all at 10µm, produced significant (p<0.05) inhibitions of LPS (100ng/ml)-stimulated NO. The GPR55 receptor agonists VSN19 (100±3.4%) and O1916 (100±4.5%) were without effect, as was the endogenous GPR55 agonist lysophosphatidylinositol (LPI) (84±8.6%) number of other cannabinoid receptor agonists and antagonists were also without effect on LPS-stimulated NO formation. The inhibitory effects of CBD did not appear to be due to a reduction in cell viability. LPS (100ng/ml) (11.4±1.5, n = 3) increased iNOS protein expression and this was inhibited by CBD (10 µM) (1.5±0.9).

Conclusion The data presented demonstrate that a number of structurally different GPR55 receptor antagonists reduced LPS-stimulated NO formation which appears to be mediated by a reduced expression iNOS protein. On the other hand, GPR55 agonists were without effect. Whether this indicates that some part of the LPS effect is mediated by an endogenous GPR55 agonist (e.g. LPI) or by a more direct effect of the drugs on NO formation remains to be investigated.

(1) Ross R.A (2009) The enigmatic pharmacology of GPR55. Trends in pharmacological Sciences 30(3); 156-163.

(2) Green LC., Wagner DA Glogowski J, Skipper PL, Wishnok JS and Tannenbaum SR (1982) Analysis of nitrate, nitrite and [15N] in biological fluids. Anal Biochem. 126(1): 131-8.

Thanks to: Liague Latif, Paul Millns for technical help and The Libyan Cultural Bureau for financial support.