Agonist‐dependent cannabinoid receptor signalling in human trabecular meshwork cells

Background and purpose: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB 1 receptors (CB 1 ) are present in TM and cannabinoid administration decreases IOP. CB 1 signalling was investigated in a cell line derived from human TM (hTM). Experimental approach: CB 1 signalling was investigated using ratiometric Ca 2+ imaging, western blotting and infrared In‐Cell Western analysis. Key results: WIN55212‐2, a synthetic aminoalkylindole cannabinoid receptor agonist (10–100 μ M ) increased intracellular Ca 2+ in hTM cells. WIN55,212‐2‐mediated Ca 2+ increases were blocked by AM251, a CB 1 antagonist, but were unaffected by the CB 2 antagonist, AM630. The WIN55,212‐2‐mediated increase in [Ca 2+ ] i was pertussis toxin (PTX)‐insensitive, therefore, independent of G i/o coupling, but was attenuated by a dominant negative Gα q/11 subunit, implicating a G q/11 signalling pathway. The increase in [Ca 2+ ] i was dependent upon PLC activation and mobilization of intracellular Ca 2+ stores. A PTX‐sensitive increase in extracellular signal‐regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212‐2, indicative of a G i/o signalling pathway. CB 1 ‐G q/11 coupling to activate PLC‐dependent increases in Ca 2+ appeared to be specific to WIN55,212‐2 and were not observed with other CB 1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX‐sensitive increases in [Ca 2+ ] i at concentrations ≥15 μ M , and PTX‐sensitive increases in ERK1/2 phosphorylation. Conclusions and implications: This study demonstrates that endogenous CB 1 couples to both G q/11 and G i/o in hTM cells in an agonist‐dependent manner. Cannabinoid activation of multiple CB 1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP. British Journal of Pharmacology (2007) 152 , 1111–1120; doi: 10.1038/sj.bjp.0707495 ; published online 8 October 2007