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 μM) 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 μM, 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.