Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells

Area of Science:

• Ocular Physiology
• Molecular Pharmacology
• Cell Signaling

Background:

• The trabecular meshwork (TM) regulates aqueous humor outflow and intraocular pressure (IOP).
• Cannabinoid CB1 receptors are present in the TM, and cannabinoid administration is known to decrease IOP.
• Understanding CB1 signaling in TM is crucial for potential therapeutic interventions.

Purpose of the Study:

• To investigate the signaling pathways activated by CB1 receptors in a human TM (hTM) cell line.
• To determine the G protein coupling and downstream effectors involved in CB1 receptor activation in hTM cells.

Main Methods:

• Ratiometric calcium (Ca2+) imaging was employed to measure intracellular Ca2+ levels.
• Western blotting and infrared In-Cell Western analysis were used to assess protein phosphorylation.
• Specific antagonists (AM251, AM630) and pertussis toxin (PTX) were utilized to dissect signaling pathways.

Main Results:

• WIN55,212-2, a CB1 agonist, increased intracellular Ca2+ in hTM cells via a Gq/11 pathway, involving PLC activation and intracellular Ca2+ store mobilization.
• This Ca2+ increase was PTX-insensitive, indicating independence from Gi/o coupling.
• WIN55,212-2 also induced a PTX-sensitive increase in ERK1/2 phosphorylation, suggesting concurrent Gi/o signaling.
• CB1 coupling to Gq/11 was agonist-specific, as other agonists like CP55,940 primarily activated Gi/o pathways.

Conclusions:

• Endogenous CB1 receptors in hTM cells exhibit agonist-dependent coupling to both Gq/11 and Gi/o signaling pathways.
• The activation of multiple CB1 signaling cascades in the TM could differentially modulate aqueous humor outflow and IOP.
• These findings provide insights into the complex role of cannabinoids in ocular physiology.