| Glaucoma is a severe blind-causing disease in the world and the mechanism of glaucoma is still not very clearly yet. In the research about glaucoma neuroprotection, the function of glial cells, especially Müller cell, were got more and more attention. Some researches show that the loss of appropriate interaction with the extracellular matrix might be an important signal within the retina for initiating axon degeneration and apoptosis in RGCs.Müller cell, a principal glial cell of the mammalian retina, is specialized radial glial cells which span the entire thickness of the retina, and is closely related to structure and function of retinal blood vessels and neurons. It has a bipolar morphology characteristicits soma is located in the inner nuclear layer; the inwardly directed process terminates in an expanded endfoot at the surface of the retina inner limiting membrane, the outwardly directed process that ends in the photoreceptor layer. The surface-to-volume ratio of the Müller cell process is very high and it can contact almost all neuronal elements. There are abundant of different ions channel on Müller cell, such as ligand receptors, transmembraneous transporter molecules, and enzymes. The notable character of the Müller cell membrane is abundant of potassium channel-activity may be regulated by voltage, calcium and neurotransmitters, and high K+ conductance. Potassium channels are grouped into families based on their structural and functional properties. The 2TM domain family, which is known as an inward-rectifier K channel family, includes the strong inward-rectifier K channels(KIR2.x and(KIR4.x).Adenosine is widely exists in intracellular fluid and extracellular fluid. Adenosine produces biological effect through adenosine receptor(AR), which characterized as G-protein linked receptor, are grouped into four subtypes- A1、A2A、A2B and A3 receptor and all four adenosine receptors are located in retina. It was shown in some research that adenosine and adenosine receptor agonist or antagonist increased the potassium channel activity. As with the A2 A adenosine receptor, very early studies indicated that agonists or antagonist at A2 A AR can produce protection of the CNS against several insults and disease, including ischemia, Parkinson’s disease and excitotoxins such as kainite.Purpose To investigate the effect of A2 A adenosine receptor antagonist SCH442416 on potassium currents and the expression of Kir2.1, Kir4.1, TASK-1, glutamine synthetase(GS) and L-Glutamate/L-Aspartate Transporter(GLAST) in rat retinal Müller cells under the chronic ocular hypertension rat model.Method Animals and rat glaucoma models. Rat chronic ocular hypertension(COH) model was induced in the right eye of each Male Sprague Dawley rat, however, the left eye was not considered as a control eye. We choice another rat right eye shammed operation as a control eye in order to avoid influence from operate eye and intravitreal injection medicine.Drugs and Intravitreal injection. The pupil was dilated with tropicamide drops, and 10μM adenosine, 10μM(8-cyclopentyl-1,3-dipropylxanthine(DPCPX)+10μM adenosine, 100 n M 5-amino-2-(2-furyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine(SCH442416) +10μM adenosine, 10μM(3-ethyl 5-benzyl2-methyl-6-phenyl-4-phenyl-ethynyl-1, 4-(+)-dihydropyridine-3,5-dicarboxylate, MRS 1191) +10μM adenosine. Eyes that received only an injection of saline in the same manner served as a vehicle control. m RNA and protein expression were tested by RT-PCR, Western-blot and Immunohistochemistry. Potassium currents of dissociated Müller cells tested by Whole-cell patch-clamp recordings.Results We established the chronic ocular hypertension model by blocking three episcleral veins. Kir2.1, Kir4.1, TASK-1 and GLAST(EAAT1) expression in rat glaucoma models Kir2.1, Kir4.1, TASK-1 and GLAST(EAAT1) protein expression and m RNA expression were evaluated by Western blotting, Realtime PCR and immunofluorescence double stain. In rat glaucoma model retina Müller cell, Kir4.1 m RNA and protein expression was strongly downregulated detected by R-T PCR, Western blot and immunohistochemistry compared with shamed-operation rat Müller cell. The protein expression of Kir4.1 remained at a low level in rat glaucoma model, downregulated 35.7%、51.8% and 40.6%from 2 weeks to 8 weeks(n=6, P<0.05). Kir2.1 was mild downregulated compared to Kir4.1, the protein expression of Kir2.1 downregulated 13.5%、22.8% and 22.0%from 2 weeks to 8 weeks(n=6, P<0.05). The protein expression of TASK-1 downregulated 21.6%、22.7% and 37.0% from 2 weeks to 8 weeks(n=6, P<0.05). The protein expression of GS downregulated 20.0%、23.6% and17.9% from 2 weeks to 8 weeks(n=6, P<0.05).The protein expression of GLAST downregulated 35.0%、42.1% and 38.6% from 2 weeks to 8 weeks(n=6, P<0.05). Consistently, Kir2.1 protein, Kir4.1 protein, TASK-1 protein and GLAST protein expression show a similar trend with m RNA expression.After 2 weeks, 4 weeks and 8 weeks ocular hypertension, immunostaining data of the GLAST expression reduced significantly while the Kir2.1 protein expression was a weakly downregulation.The protein and m RNA of Kir4.1, TASK-1 GS and GLAST in Müller cells expression in the group of adenosine+SCH442416 imcreased significantly compared control.The Müller cells potassium currents induced in by a series of hyperpolarized voltage pulses from a holding potential of-160 m V in increments of 20 m V. There is a significant downregulation of potassium currents in the rat glaucoma models. Müller cells of glaucoma model rat retina strongly downregulate transmembrane potassium currents. The peak current amplitudes were decreased to approximately 47% of control even on G1d(45.4 ±9.8% control, n=6, p<0.05), and remained at this level for next 2 weeks.SCH442416 was significantly increased the potassium currents in isolated Müller cells. The peak of Kir currents increased 35.8 ±8.5% compared control Kir currents(n=6, p<0.05).Conclusions 1.We established the chronic ocular hypertension model successfully.2. Retina Müller cell potassium channel Kir currents significantly decreased in the chronic ocular hypertension models. Consistently, the m RNA and protein expression decreased in a similar extent. That suggested the protein expression decrease mainly because of protein synthesis decreased but not transfer or metabolism increased. 3. A2 A adenosine receptor antagonist SCH442416 increased the potassium channel expression and Kir currents of retina Müller cells, and therefore increase the GLAST and GS expression and function, which maybe accelerate the clearance extracellular glutamate. |
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