Cannabinoid CB1 Receptor–Mediated Inhibition of Calcium Signaling in Rat Retinal Ganglion Cells

Abstract

Abstract: : Purpose: The cannabinoid CB1 receptor has been identified in the mammalian retina but the role of cannabinoids in visual processing has yet to be fully elucidated. The CB1 receptor agonist, WIN55,212–2, has differential effects on voltage–dependent Ca channels in salamander and goldfish photoreceptors. The present study investigates the actions of CB1 receptor ligands on voltage–dependent Ca channel currents and glutamate–induced Ca²⁺ influx in rat RGC cultures. Methods: The presence of CB1 receptors in the rat retina was shown via PCR analysis, and immunohistochemistry demonstrated labelling in the RGC layer. Purified RGCs, isolated from neonatal rats using a two step immuno–panning procedure, were used for Ca²⁺ imaging and whole–cell patch clamping experiments. RGCs were loaded with the ratiometric Ca²⁺–indicator dye fura–2, and the effect of WIN55,212–2 on [Ca²⁺]_i during exposure to glutamate (100 µM; with 10 µM glycine) was determined. The voltage–clamp technique was used to record high voltage–activated (HVA) Ca channel currents (using barium as charge carrier), and the effect of WIN55,212–2 alone and in the presence of CB1 receptor antagonists (AM 281 and SR141716) was measured. Results: Ca²⁺–imaging demonstrated that 5µM WIN55,212–2 significantly inhibited glutamate–induced Ca²⁺ influx in isolated RGCs (62% ± 5% mean influx of initial response, n=16) and that the inhibition was reversible. HVA Ca channel currents recorded with the patch–clamp technique were also significantly reduced by WIN55,212–2 (0.5 µM, 20% ± 6%, n=5; 1 µM, 25% ± 6%, n=4; 5 µM, 51% ± 9%, n=5). Ca channel current reduction produced by 5 µM WIN55,212–2 was inhibited in the presence of CB1 receptor antagonists, AM 281 (0.1 µM, 83% ± 7% remaining, n=4) and SR141716 (0.1 µM, 95% ± 9% remaining, n=5). Conclusions: The CB1 agonist WIN55,212–2 decreased the amplitude of HVA Ca channel current in a purified rat RGC culture. This decrease was blocked by CB1 receptor antagonists AM 281 and SR141716, confirming a selective CB1 receptor–mediated inhibition of Ca channel currents. These results support a role for endogenous cannabinoids in modulating glutamatergic Ca²⁺ signaling pathways.

Keywords: calcium • ganglion cells • ion channels