Cannabinoid CB₁ receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB₁ class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB₁ receptor-specific oligodeoxynucleotide primers, cannabinoid CB₁ receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB₁ receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB₁ receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB₁ immunoglobulin-G in tissue sections localized cannabinoid CB₁ receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [³⁵S]GTPγS binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [³⁵S]GTPγS binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 μM digitonin (p < 0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB₁ receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB₁ receptors by CP-55,940 resulted in a 2.3 ± 0.3 and 1.7 ± 0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p < 0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB₁ mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB₁ receptors in the trabecular meshwork and increase aqueous outflow.