Cannabinoid Receptor Agonists Modulate Calcium Channels On Rat Retinal Müller Cells

Muller cells are the major glia cells in the retina. Different types of calcium channels have been found in Muller cells of toads, rabbits, human and rats. Calcium channels play important roles in cellular functions. Modulation of calcium channels may affect the excitability of Muller cells, thus regulating neuronal functions in the retina. Cannabinoid receptors are widely distributed in retina and modulate the ion channels in a variety of cells, such as bipolar cells, ganglion cells and photoreceptors. Previous studies have demonstrated that activation of cannabinoid receptors modulates T-and N-type calcium channels. However, there are not any reports about the modulation of calcium channels on rat Muller cells. Therefore, we first detected expression of T-and L-type calcium channel subtypes, as well as cannabinoid receptors on rat retinal Muller cells using immunohistochemistry. And then we examined the modulation of cannabinoid receptor agonists on calcium channel currents on rat Muller cells.Our results showed that three subtypes of T-type calcium channel (Cav3.1, Cav3.2 and Cav3.3), and one subtype of L-type calcium channel (Cav1.2) were expressed on rat Muller cells. Cav1.3, a subtype of L-type calcium channel, was not detected. We further identified the functional expression of calcium channels by patch-clamp technique. In a divalent cation-free bath solution, Na+-carried currents through calcium channels in acutely isolated Muller cells were induced by a serial depolarization pulses from-80 to+10 mV after a-120 mV prepulse. The currents were sensitive to Nimodipine, an L-type calcium channel blocker, and Mibefradil, a T-type calcium channel blocker, but insensitive to TTX, a sodium channel blocker. All these results demonstrated that the inward currents we recorded were mediated by T-and L-type calcium channels. We then tested whether cannabinoid receptor agonist WIN55212-2 (WIN), and two endocannabinoids, Arachidonoylethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG), may modulate the calcium channel currents of Muller cells. Extracellular application of WIN (5 μM) reversibly suppressed the peak current amplitudes of calcium channels. The suppression was dose-dependent with an IC50 value of 4.0 μM. Further experiments showed that WIN mainly suppressed the T-type calcium channels. WIN is a CB1 receptor antagonist. We tested whether the suppression was mediated by CB1 receptor. The results showed that AM251 (2 μM) and SR141716 (SR; 1 μM), CB1 receptor antagonists, could not block the WIN-induced suppression on calcium channel currents. In addition, we examined the effects of AEA and 2-AG on calcium channel currents. Similar to the WIN effect, these two endocannabinoids suppressed the calcium currents in a CB1 receptor-independent manner.Whilst WIN has a highly selectivity for CB1 receptor, WIN also exhibits a modest degree of selectivity for CB2 receptor. Therefore, we investigated whether WIN, AEA and 2-AG may suppress the calcium currents through CB2 receptor. Double immunohistochemical labeling showed that CB2 receptor was expressed on rat Muller cells. Electrophysiologically, AM630 (100 nM), a CB2 receptor selective antagonist, failed to block the WIN-or 2-AG-induced suppression on calcium channel currents, but partially reduced the AEA effect.In summary, our results demonstrate that functional T-and L-type calcium channels are expressed on rat retinal Muller cells. WIN and 2-AG suppress the calcium channel currents on Muller cells in a CB1 and CB2 receptor-independent manner, while AEA suppresses the calcium channels partially mediated by CB2 receptors. The existence of receptor-dependent and-independent mechanisms suggests that cannabinoids may modulate Muller cell functions through multiple pathways.