Experimental Study On Expression And Related Functions Of Chloride Channels In The Trabecular Meshwork

Objective:Glaucoma is one of the main irreversible blinding eye diseases, the exact pathogenesis is currently not very clear. The increased resistance of aqeous humor outflow resulting in high intraocular pressure is a major risk factor for glaucoma occurred. Trabecular network is the main part of aqeous humor outflow and regulation of intra-ocular pressure. The mechanisms of the regulation of aqeous humor outflow resistance are multiple, including the trabecular meshwork contractility, cytoskeleton rearrangement, spreading, and interactions of extracellular matrix, changes of gene expression, and the changes of trabecular meshwork cell volume et al. Trabecular meshwork cell volume and contraction determines the tissue space, and plays a positive and important role in the regulation of resistance of aqeous humor outflow. The contraction of trabecular meshwork cells and the maintenance of volume may be related to different ion channels and pumps adjustments.Chloride ion is the most abundant negion, participating in a variety of biological functions through transmembrane transport and ion channels, there are diverse types of chloride channels. Volume-activated chloride channel,VACC (I C| voi),also called swelling-activated chloride channel(I c, svven)expresses in almost all eukaryotic cells, and plays an important role in the maintenance of the balance of cell volume and as well as participating in regulation of cell proliferation, differentiation and apoptosis. The molecular mechanism of I ci voi remains unclear currently because of lacking specific high-affinity blocker. It is very difficult to determine protein molecule structure, patch-clamp technique is used to study it. Srinivas etc reported that cell volume increased dramatically after giving hypotonic stimulation to meshwork cells in cultured bovine trabecular. Patch-clamp experiment showed that characteristic chloride current was activated and the current could be inhibited by NPPB and fluoxetine, furthermore, the permeability of I" was greater than that of C\-. All these consistent with the characteristics of volume-sensitive chloride current. Soto and his group reported that the regulation of bovine trabecular meshwork cell volume was related with chloride channels and potassium channel. Mitchell et al reported that the regulation of human trabecular meshwork cell volume was related with chloride channels. Voltage-gated chloride channel (C1C) is a a large family in chloride ion channel, widely distributed in the cell membrane and organelle membrane of the body, involved in cell electrical activity adjustment, volume adjustment, transendepidermis material transport, pH regulation in cell, cellular immune response, cell migration, cell proliferation and differentiation, apoptosis, and many other activities and functions. Comes and other reports C1C-2-C1C-7 were expressed in cultured human trabecular meshwork cells, but no expression of C1C-1. Srinivas etc reported that C1C-2, C1C-5 and P-glycoprotein expressed in cultured bovine trabecular meshwork cells, and C1C-3 did not express.It can be confirmed that the volume-sensitive chloride channels were involved in the regulation of trabecular meshwork cell volume and a variety of voltage-gated chloride ion channels are expressed in the trabecular meshwork. However, the research in this field is also limited, and the detailed role of two types of chloride channels in the trabecular meshwork is not entirely clear. The purpose of this study is to further explore the possible role of C1C and I ci^ voi in the trabecular meshwork and provide some theoretical basis for glaucoma pathogenesis.Content:Human immortalized trabecular meshwork cells were cultured in vitro, trabecular membrane chloride channel currents were recorded using whole-cell patch-clamp under isotonic and hypotonic conditions,and the current characteristics were observed. Chloride channel currents were recorded under hypotonic conditions after bathed by chloride channel blockers NPPB and tamoxifen, and ATP. The cultured trabecular meshwork cells were added by lCT'mol/L dexamethasone and then continued to be cultured for 1 day,3 days,7 days,the chloride currents were observed under hypotonic conditions. Sodium hyaluronate intracameral injection induced chronic ocular hyperten-sion model in rat, one time injection per week for ten weeks, and intraocular pres-sure was observed before injection to insure to form a stable model of chronic ocular hypertension.Using RT-PCR to detect mRNA levels of voltage-gated chloride channel C1C-1, C1C-2, C1C-3, C1C-4, C1C-5, C1C-K1, C1C-K2 of the trabecular meshwork in normal rat. Using immunohistochemical methods to detect protein levels of C1C-2 and C1C-3 expression of the trabecular meshwork tissue in rat. After 3 weeks and 10 weeks of sodium hyaluronate intracameral injection induced chronic ocular hypertension model, the above-mentioned work were repeated to observe the expression changes between the model eyes and normal eyes.Finding:Under isotonic condition, only weak and stable background currents were observed in cultured human trabecular meshwork cells; The currents were increased rapidly by perfusing the cells with hypotonic solution and outward currents were prominent, This current is sensitive to changes in cell volume, it was activated when cell swells, when the cell volume gradually reduced, the current gradually weakened. The current was not time-compliance, no significant current was activated, reversal potential was near to the Cl" ion equilibrium potential. In the hypotonic state, after the bath were added NPPB (100 fimol-L"1), tamoxifen (50 umol-L"1) and ATP (2 jiumol-L"1) for 20min,the current was significantly suppressed. The culture medium was added bylO"7mol/L dexamethasone and continued to be cultured for 1 day,3 days,7 days and we found that with the extension of dexamethasone treatment time, outward and inward current density of trabecular meshwork cells gradually reduced after hypotonic stimulation. After adding dexamethasone for 1 day, outward and inward current density value were lower than those of hypotonic stimulation, but no statistically significant difference (P>0.05), After adding dexamethasone for 3 days,7 days, outward and inward current density value were significantly lower than those of hypotonic stimulation, and the difference was significant (P<0.05).Sodium hyaluronate intracameral injection induced chronic ocular hyperten- sion model in rat. IOP values before operation and after operation for one week, three weeks, six weeks, ten weeks were 11.47±0.90 mmHg;13.20±0.85 mmHg; 21.79±1.47 mmHg; 22.96±1.04 mmHg; 23.27±1.42 mmHg respectively. We can see from this that intraocular pressure increased to a lesser extent after a single injection, but it continued to slowly rising after repeated several times injection to form a stable model of chronic ocular hypertension.We found that seven kinds of voltage-gated chloride ion channels (C1C-1, C1C-2, C1C-3, C1C-4, C1C-5, C1C-K1, C1C-K2) were expressed using RT-PCR in trabecular tissue of normal rat; We found C1C-2 and C1C-3 expression in rat trabecular meshwork tissue using immunohistochemical method from the protein level. After sodium hyaluronate intracameral injection induced chronic ocular hypertension model for 3 weeks, we found that seven C1C chloride channel expression at the mRNA transcription level and the C1C-2, C1C-3 at the protein level in trabecular tissue of model group were inhanced than those in normal rats. While after ten weeks of ocular hypertension model,all the above indicators were reduced compared with normal rats.Achievement and its position,significance and values in this field:Cell volume and contractility of trabecular meshwork play a positive and important role in the regulation of aquous humor outflow. Foreign scholars have confirmed the volume-sensitive chloride channels were involved in the regulation of trabecular meshwork cell volume,however, there was no domestic relevant reports.It is the first time in the country to study the physiological characteristics of the volume-sensitive chloride current in cultured human trabecular meshwork cells in this paper, and to observe the effects of dexamethasone on the curr-ent.We conclude that chloride currents formed after hypotension stimulation in cultured human trabecular meshwork cells was in line with characteristics of volume-sensitive chloride current(I cl. vol);the channel was involved in the regulation of trabecular meshwork cell volume, and dexamethasone may influence the trabecular meshwork cell volume-sensitive chloride channels,thus involved in the occurrence of steroid-induced glaucoma.Foreign scholars have confirmed the presence of voltage-gated chloride channel expression in cultured human and bovine trabecular meshwork cells, however there were not relevant reports about ClC chloride channels in rat trabecular meshwork at home and abroad. As is known that the structure and physiological characteristics of rat eyes are similar to those of human eyes in many ways, such as the trabecular meshwork, Schlemn tubes, ciliary body, retinal blood vessels, scleral vein,et al,and its aqueous drainage channels are also very similar. We confirmed the existence of voltage-gated chloride channel expression in rat trabecular tissue for the first time, and found that sodium hyaluronate intracameral injection induced chronic ocular hypertension model model affect there expression. Furthermore,we conclude that the CIC-type chloride channels are closely linked with trabeculae cell function.This study provided a theoretical basis for exploring the possible role of Icl, vol and ClC chloride channel in the pathogenesis of glaucoma, and offered new ideas for further study of the function of chloride channels in the pathogenesis of glaucoma and development of anti-glaucoma drug.