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). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM). Experimental approach: CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis. Key results: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 mu M) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+](i) was pertussis toxin (PTX)-insensitive, therefore, independent of G(i/o) coupling, but was attenuated by a dominant negative G alpha(q/11) subunit, implicating a G(q/11) signalling pathway. The increase in [Ca2+](i) was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTXsensitive 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. CB1-G(q/ 11) coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+](i) at concentrations >= 15 mu M, and PTX-sensitive increases in ERK1/2 phosphorylation. Conclusions and implications: This study demonstrates that endogenous CB1 couples to both G(q/11) and G(i/o) in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.