Modulation Of Kir Channel Proteins By Activation Of MGluR I In The Cultured Rat Retinal Mller Cells

Glaucoma, one of the most common diseases of Ophthalmology, is listed as the second blinding eye disease by the World Health Organization and the first leading cause of irreversible blindness. This disease is characterized by optic nerve degeneration, reduced number of retinal ganglion cells and vision loss. Elevated intraocular pressure and hemodynamic changes are commonly regarded as hallmark risk factors and apoptotic death of retinal ganglion cells is its basic pathogenesis. In regard to the pathogenesis of glaucoma, cell excitotoxicity induced by extracellular excessive accumulation of glutamate is recognized world widely. However, the exact mechanisms underlying retinal ganglion cell death following the onset of glaucoma are still poorly understood.In the vertebrate retina, Muller cells are principal glial cells that stretch across the whole thickness of the retina and contact with the somata and processes of all retinal neurons. In addition to supporting the retinal neurons, information exchange and interaction between Muller cells and retinal neurons may play a curious role in regulating and maintaining retinal neuronal functions. Growing evidence has demonstrated that reactivation (gliosis) of glial cells occurs in almost all kinds of central nervous system injury and disease. Muller cell gliosis is also a common response in a variety of pathological alternations of the retina. Muller cells gliosis, transformation of glial cells from matured stage to non-matured differentiation stage, may be involved in two contradictory functions:protecting retinal tissue from further damage and contributing to neurodegeneration. As an early response to cell injury, Muller cells release antioxidants and neurotrophic factors, and remove extracellular excess glutamate, thus promoting axonal regeneration and synaptic remodeling. In contrast, reactivated Muller cells produce and release various cytotoxic factors, such as nitric oxide, tumor necrosis factor-α, reactive oxygen species, and prostaglandin E2, which contribute to retinal ganglion cell apoptosis. Meanwhile, gliosis may also hamper axonal regeneration and neurite outgrowth by forming scar tissues. Therefore, to study the Muller cell reactivation is important for understanding the neuronal injury mechanisms, nerve regeneration and repair.The functional changes of Muller cells in retinal injuries and diseases, including glaucoma, have been widely studied. One of the common features is reactivation of Muller cells, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), a cellular marker of an early gliotic response. Meanwhile, the ion channels in Muller cell membrane, such as K+ and Ca2+channels, have been up-or down-regulated, in which downregulation of membrane K+conductance, especially Kir4.1-mediated membrane currents, is of great importance. However, the mechanisms underlying Muller cell gliosis and downregulation of K+currents in glaucoma and other retinal injuries and diseases are largely unknown. Our previous study has shown that elevated intraocular pressure in a rat experimental glaucoma model induced a reduction of Kir currents of Muller cells due to over-activated group I metabotropic glutamate receptors (mGluR I), thus contributing to Muller cell gliosis. However, there are still many issues remained to be explored. What are the molecular mechanisms of downregulation and/or trafficking of Kir proteins? Whether or not activation of mGluR I may affect the synthesis of Kir channel proteins? Apart from Kir4.1, what happened to Kir2.1? To answer these questions, in vitro purified cultured retinal Muller cells are ideal preparation.In the present work, we studied effects of DHPG, a selective mGluR I agonist, on GFAP and Kir4.1 protein expression in purified cultured rat retinal Muller cells by using primary cell culture, immunocytochemistry, Western blot and real-time PCR techniques. The results are showed below. (1) Purified rat retinal Muller cells were successfully obtained from 5-day-old Sprague Dawley rats. More than 90% of the cells were glutamine synthetase (GS), a Muller cell marker, positive in the third-generation cultured Muller cells identified by immunocytochemistry; (2) After the cells were treated with 10 μM DHPG for different times (0.5-24 h), Western blot experiments were performed to examine the expression of GFAP. The results showed that GFAP protein levels were increased significantly from 5 h to 9 h, suggesting the reactivation of Muller cells; (3) We found that total Kir4.1 protein levels were not significantly changed by DHPG treatment, as compared to that obtained from control. (4) However, Kir4.1 protein levels in cell membrane component of Muller cells were remarkably decreased with a peak at 7-9 h after the DHPG treatment. These results suggest that activation of mGluR I may induce Kir4.1 channel protein internalization from the cell membrane, thus resulting in downregulation of functional Kir4.1 channel proteins in Muller cells; (5) We performed double immunocytochemistry experiments to further confirm the changes of Kir4.1 proteins in cell membrane of Muller cells. The results showed that co-localization of caveolin-1, a membrane protein marker, and Kir4.1 membrane protein was gradually decreased at 3-9 h after DHPG treatment, consistent with the changes of Kir4.1 protein in membrane component of the cells by Western blotting. (6) Finally, we examined the DHPG treatment-induced changes of Kir4.1 (KCNJ10 gene) and Kir2.1 (KCNJ12 gene) mRNAs by using real-time PCR technique. Our results showed that Kir4.1 mRNA revealed a transient reduction at 0.5 h after DHPG treatment, and then returned to the control level. Similarly, DHPG treatment induced significant decrease of Kir2.1 mRNA at 0.5 h and 9 h. These results suggest that activation of mGluR I receptor may decrease the synthesis of Kir4.1 and Kir2.1 proteins in Muller cells.In conclusion, we successfully established a cell model in vitro for Muller cell gliosis by using DHPG to activate the mGluR I receptor in rat purified cultured retinal Muller cells. Activation of mGluR I in rat cultured retinal Muller cells induced membrane Kir4.1 protein trafficking, and attenuated Kir4.1 and Kir2.1 mRNA transcription, which may result in decrease of functional Kir channels in the membrane of Muller cells, thus contributing to Muller cell gliosis. All these results provide a foundation for us to further explore the molecular mechanisms underlying Muller cell gliosis and Kir channel down-regulation, as well as to search the pathways for inhibiting glial cell reactivation and protecting retinal neurons against damage.