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B. Li, W. Shen; Regulation of Glycine Response by Na+–K+–2Cl– Cotransporter (NKCC) on Amphibian Rods . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3452.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Glycine is an inhibitory neurotransmitter in retinas. However, glycine can also produce excitatory effect on horizontal cells due to an high intracellular chloride concentration in the neurons. In many cases, increase of chloride equilibrium potential in cells depends on the expression of NKCC cotransporters that carry chloride ions into the cells. The action of NKCC cotransporters could lead to the result of shifting inhibitory effect of glycine to become excitatory. In amphibian rods, it has been found that the chloride equilibrium is around –35mV. It is possible that NKCC cotransporters express on the rods. Activating glycine receptors in the rods might provide a positive effect. This effect might be directly related to glycine feedback signal from glycinergic interplexiform cells in the retinal network. Thus, the regulation of glycine response by NKCC cotransporters is crucial for the first step synapse in retinal neural transduction. This study is carried out on investigating NKCC cotransporters and glycine receptors in the rods. Methods: Tiger salamander retinas were used in experiments. Whole–cell patch clamp, immunostaining and Western blotting techniques were used to identify the function and localization of glycine receptors and NKCC cotransporters in the rods. Results: Glycine receptors were identified by antibody against the bata subunits on the axon terminals of isolated rods. NKCC cotransporter positive staining was also observed on the rods. Whole cell recording experiments indicated that 50µM bumetanide, a NKCC cotransporter inhibitor, could hyperpolarize dark membrane potential and reduce light response in the rods in dark adapted retinal slices, suggesting that the expression of NKCC cotransporters on the rods helps to stabilize the excitatory membrane potentail at dark adapted condition. Voltage clamp experiment indicated that bumetanide could reduce glycine current when a rod was held at –60mV, below the chloride equilibium potential. Possibly, this is due to bumetanide lowering chloride equilibrium potential in the rod. The effect of NKCC cotransporters on chloride equilibium potentials was studied in the rods at dark and light adaptation, in order to evaluate the modulation of glycine response by NKCC cotransporters. The regulation of NKCC cotransporters by dark and light adaptation were also quantitatively analyzed in Western blotting and signal cell RT–PCR experiments. Conclusions: Both glycine receptors and NKCC cotransporters express in salamander rods. The function of NKCC cotransporters controls glycine feedback signal from glycinergic interplexiform cells on the rod photoreceptors.
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