Lack of effect of cannabinoids on nitric oxide production in two rodent microglial cell lines

Khalil Eldeeb, Stephen Alexander, David Pritchard, David Kendall

School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK

Microglial cells are key players in a variety of CNS disorders such as stroke and multiple sclerosis. Activated microglial cells release nitric oxide (NO), an excess of which causes neuronal damage, while inhibition of NO over-production would be expected to increase cell survival. Cannabinoid compounds have well known immunomodulatory properties, via their actions predominantly on CB2 cannabinoid receptors, which are regarded as promising pharmaceutical targets (Di Marzo, 2008). The aim of the present study was to investigate whether cannabinoids could modify NO responses in microglial cells.

Two microglial cell lines were employed; mouse brain-derived BV2 cells (Walter et al., 2003) and HAPI cells (rat highly aggressively proliferating immortalized microglial cells, Cheepsunthron et al., 2001). HAPI and BV2 cells (4 × 105 cells/ml in flat-bottomed 24-well plates) were cultured overnight, then stimulated with bacterial lipopolysaccharide (LPS 4 μg/ml) in the presence and absence of the cannabinoid CB1/CB2 receptor agonist CP55940 or the selective CB2 agonist JWH133 (100 pM to 10 μM) for 24 hours. Generation of NO was determined by measuring accumulation of nitrite in culture supernatant using the Griess reagent (absorbance at 540 nm). Standard curves were constructed using sodium nitrite (0.1 -100 μM) in culture medium. Data from three or more separate experiments were analysed using one-way ANOVA with post hoc Dunnett’s multiple comparison test.

Basal NO formation was lower in HAPI (1.5 ± 0.2 μM nitrite mean±SEM) compared with BV2 cells (10.9 ± 0.9 μM nitrite) but the maximum effect of LPS was nearly identical (100 ± 12 and 99 ± 5 μM nitrite, respectively). JWH133 at 10 μM failed to alter significantly LPS-stimulated NO formation in HAPI or in BV2 cells (111 ± 18 and 105 ± 4 μM nitrite, respectively). A high concentration of CP55940 (10 μM) significantly attenuated NO formation in both HAPI cells and in BV2 cells (62 ±6 and 66 ± 5 μM nitrite, P<0.01). A number of endocannabinoids (oleamide and N-oleoyl-, N-palmitoyl-,N-stearoyl- and N-arachidonoyl-ethanolamine, all at 1 μM) failed to alter significantly LPS-evoked increases in NO formation. No treatment significantly affected cell viability (resazurin test). The CB1 and CB2 receptor antagonists rimonabant and SR144528 (both 1 μM) were also without effect.

In conclusion, although cannabinoids have been reported to modulate some microglial functions e.g. migration (Walter et al., 2003), in our hands, we have been unable to show that LPS-elevated NO formation in microglia is modified by cannabinoid receptor activation, at least in the BV2 and HAPI cell lines.

This work was made possible by a scholarship to KE-D from the Egyptian Government

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Di Marzo V (2008). Nat Rev Drug Discovery 7: 438-455.

Walter L et al. (2003). J Neurosci 23: 1398-405.