The Role of the Endogenous Cannabinoid Anandamide on Human Airway Epithelial Cell Permeability

VCM Shang, DA Kendall, RE Roberts. University of Nottingham, Nottingham, UK

In the past decades, the endocannabinoid system has been associated with the regulation of inflammation, including asthma. Studies in humans have demonstrated that the release of anandamide is up-regulated as a response to an inhaled allergen (Zoerner et al., 2011). Recent studies have reported that airway inflammation is manifested from bronchial epithelial dysfunction which functions as a physical barrier against environmental insults (Holgate, 2007). Interestingly, similar histological changes were also observed in childhood asthma hence hinted that epithelium desquamation is not exclusively a consequence to chronic local inflammation, but also implicated to asthma pathogenesis (Fedorov et al., 2005). Therefore, the aim of this investigation is to elucidate the roles of endocannabinoids in influencing bronchial epithelial permeability using Calu-3 cell line as an in vitro model.

The human epithelial cell line, Calu-3 cells were grown in 12-transwell plates at air-liquid interface for 21 to 28 days to allow development of tight junctions. Changes in epithelial permeability were assessed by measuring transepithelial electrical resistance (TEER) following treatment with anandamide (30 µM) or TNFα (10 ng/mL) for up to 48 hours. Methanandamide (100 nM) and URB597 (1 µM), a selective inhibitor of anandamide catabolic enzyme, fatty acid amide hydrolase (FAAH), were also used to identify whether the effect on TEER was caused by anandamide itself or its metabolites respectively. Stimulation of the cannabinoid receptors by anandamide was also investigated using CB1 antagonist AM251 (100 nM) and CB2 antagonist SR144528 (1 µM). Data were presented as %TEER by calculating the relative change in resistance at various time points from basal reading. Each number of experiments, n, represents a set of TEER readings that were measured in triplicates from a 12-transwell plate. A consecutive n number is only assigned when experiments were repeated using cells grown at different passages from separate stock cultures.

Treatment with anandamide at 30 µM (85±5%, n=5) produced a significant reduction in TEER starting at 2 hours post drug addition, which persisted for 48 hours (p<0.01, two-way ANOVA compared to vehicle control (0.3 % ethanol)). A similar effect was observed with TNFα (73±8%, n=5, p<0.01, two-way ANOVA). Unlike anandamide, methanandamide (100 nM) did not cause any significant changes in TEER values throughout the duration of the experiment. Pre-incubation with URB597 (1 µM) for an hour prevented anandamide-induced TEER reduction (%TEER with anandamide alone = 81±9%, t = 2.5 hours, compared to 91±7% in the presence of URB597 1 µM n=5, p<0.01 two-way ANOVA). Pre-treatment with cannabinoid receptor antagonists had no effect on the anandamide-induced reduction in TEER.

These data demonstrate that anandamide causes a reduction in transepithelial resistance, indicative of an increase in epithelial permeability. This effect does not appear to be mediated through cannabinoid receptors. The fact that inhibition of anandamide metabolism with URB597 prevented the effects of anandamide suggests that the reduction in TEER is mediated by a metabolite of anandamide. Studies to investigate the potential downstream roles of cyclooxygenase (COX) or lipooxygenase (LOX) metabolites are currently underway.

Fedorov IA et al, Thorax 60:389, 2005

Holgate ST, Journal of Allergy and Clinical Immunology 120:1233, 2007

Zoerner AA et al, Clinical Pharmacology & Therapeutics 90:388, 2011