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 immumoglobulin-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 [S-35]GTP gammaS binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [S-35]GTP gammaS binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 muM 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. (C) 2001 Elsevier Science B.V. All rights reserved.