| In the central nervous system endocannabinoids, acting on G-protein coupled cannabinoid receptor, regulats a variety of physiological functions, including memory, pain perception, synaptic transmission and synaptic plasticity. Two cannabinoid receptors have been cloned, CB1 and CB2 receptor (CB1R/CB2R). Cannabinoid CB1R signaling is also extensively distributed in the vertebrate retina. CB1Rs and endocannabinoids, anandamide and 2-Arachidonoylglycerol (2-AG), as well as an endocannabinoid degradative enzyme, fatty acid amide hydrolase (FAAH), were found in a variety of retinal cell populations and in the retinal inner plexiform (IPL). To date, our knowledge about the functions of cannabinoid CB1r signaling in retina is mainly from its modulation of Ca2+ and K+ channels in retinal neurons and transmitter release in retinal discs. Retinal ganglion cell (RGC), output neuron of retina, conveys visual signals to visual cortex. Modulation of RGC’s excitability would affect the visual signal transmission. Potassium channels are one of key factors in determining the membrane excitability of neurons. Modulation of potassium channels would change the excitability of RGCs. However, effects of cannabinoid CB1R signaling on the K+ channels in rat RGCs are poorly understood. In the present work, we first investigated the expression of CB1R on RGCs. We further investigated the effect of WIN55212-2 (WIN) on outward potassium currents of rat RGCs.Using immunohistochemistry, we confirmed the expression pattern of CB1R in rat retina. CB1R positive signals (red) were detected abundantly in the retinal cells, including cells in the outer nuclear layer (ONL), the inner nuclear layer (INL), the ganglion cell layer (GCL), and the synapse enriched outer plexiform layer (OPL) and IPL. Double immunofluorescence labeling experiments showed that CB1R is expressed in RGCs. Using patch clamp whole cell recording technique, we studied the effects of WIN on outward potassium currents in acute isolated rat RGCs. The cells were held at -70 mV and depolarized to +30 mV to induce outward potassium currents in the presence of 0.4 μM TTX and 100 μM CdCl2. Perfusion of 2 μM WIN significantly and reversibly suppressed outward potassium currents in the RGCs. At+30 mV test potential the average current amplitudes were inhibited by WIN to 69.6 ± 3.0% of control (n= 8, P< 0.001), and washout returned the currents to 96.2 ± 2.2% of control (n= 7, P> 0.05). Similar effect was also observed by using AEA, aonther CB1R/CB2R agonist. We then tested the time course of WIN affecting the K+ currents. Extracellular application of 2 pM WIN eventually decreased the current amplitudes and the maximal effect was appeared at 2 min after application of WIN. The average current amplitude was 78.2 ± 6.0% of that at 0 min (n= 7, P< 0.01). Washout of WIN, the average current amplitude was eventually returned to control level. WIN suppressed the outward K+ currents in a concentration-dependent manner (IC5o= 4.7 μM). Unexpectedly, the inhibition of outward potassium currents by WIN in the RGCs is not mediated by activating cannbinoid receptors. Selective CB1R antagonist AM251 (2 μM) or SR141716 (1 μM) alone did not change the K+ current amplitude in the cells. The average current amplitudes were 96.4 ± 2.0% of control (n= 8, P> 0.05) when 2 μM AM251 was applied alone, and the currents were reduced to 79.7 ± 2.2% of control (n= 8, P< 0.001) after co-application of AM251 and WIN, which was comparable to that of WIN alone. In the presence of 1 μM SR141716 the K+ current amplitude were 99.3 ± 0.5% of control (n= 5, P> 0.05), and the currents were decreased to 76.3 ± 2.4% of control (n= 5, P< 0.01) when SR141716 and WIN were co-applied to the cells. AM630 is selective CB2R antagonist. The average current amplitudes were 97.9 ± 0.7% of control (n= 5, P> 0.05) when 100 nM AM630 was applied alone, and the currents were reduced to 72.9 ± 2.9% of control (n= 5, P< 0.001) after co-application of AM630 and WIN. Similar results were obtained when the concentration of AM630 was increased to 1 μM (AM630:97.0 ± 1.3% of control, n= 6, P> 0.05; AM630+WIN:73.1 ± 0.7% of control, n= 6, P< 0.001).Under our experiment condition the outward potassium currents of RGCs contain at least four components, Ba2+-sensitive, TEA-sensitive,4-AP-sensitive, and glybenclamide (Gb)-sensitive ones. With the help of pharmacological analysis, we found that WIN selectively suppressed TEA-sensitive and Gb-sensitive components. Moreover, extracellular application of 2 μM WIN shifted the steady-state inactivation curve of the currents towards left. The Vh values obtained before and after WIN application were-45.1 ± 5.6 mV and-52.0 ± 5.6 mV, respectively (n= 7, P< 0.001). Intracellular dialysis of WIN had no effect on outward potassium currents. cAMP-protein kinase A (PKA) signaling pathway was unlikely involved in WIN-induced suppression of the K+ currents because PKA inhibitor H-89 and Rp-cAMP did not block the WIN effects.In conclusion, in the present work, we found that CB1Rs are expressed on rat RGCs. WIN receptor-independently suppressed the TEA-and glybenclamide-sensitive K+ current components in rat RGCs, thus regulating the cell excitability. |
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