Abstract
Abstract: :
Purpose: Voltage–gated chloride channels (CLCN) regulate cell volume, membrane potential and cellular transport. Because changes in trabecular meshwork cell volume influences outflow facility, we investigated the presence of seven members of the CLCN family in the human TM (HTM). We determined changes in CLCN2 and CLCN3 expression after hypotonic shock (known to activate these channels), dexamethasone (DEX) and high intraocular pressure (HP) (factors associated with glaucoma pathology). Methods: Three HTM cell lines were derived from three different individuals with no history of glaucoma. For hypotonic shock, subconfluent cells were subjected to 260 mOsm/kg media for 15, 30 and 120 min. For DEX, confluent cells were treated with 100 nM DEX for 1, 4 and 10 days. For HP, paired anterior segments were perfused for 24 h at a constant flow of 3 µl/min. After reaching baseline values, the flow of one eye was raised to achieve an increase in pressure of 30 mmHg for a period of 1 h, 4 and 7 days while the contralateral eye was maintained at 3 µl/min as a control. Expression of CLCNs was determined by relative quantitative RT–PCR of total RNA using intron spanning primers and 18S primer:competimers as internal controls. Quantification was done by a gel dock optical density system. Results: Expression of CLCN2, 3, 4, 5, 6 and 7 was detected in three HTM cell lines, while CLCN1 was not present. Relative abundance studies showed each channel had a different expression level, with CLCN3 being the highest and CLCN2 the lowest. Hypotonic conditions upregulated CLCN2 and barely influenced expression of CLCN3. DEX treatment mostly downregulated both channels especially CLCN3. After 1 h HP, CLCN2 showed a higher upregulation than CLCN3 but the pattern was reversed at 4 and 7 days. Conclusions: The presence of six out seven CLCNs in the human TM indicates their relevance in the physiology of this tissue. CLCN3 and CLCN2 (the most and least abundant) respond differently to a HP pathological condition. Upregulation of CLCN2 by hypotonic shock suggests a role for this gene in volume cell regulation and in potential regulation of outflow facility. J.L.V. current affiliation: Inspire Pharmaceuticals, Inc., Durham, NC.
Keywords: trabecular meshwork • gene/expression • ion channels