Differential expression of the human chloride channel genes in the trabecular meshwork under stress conditions Nu ´ria Comes a , Xavier Gasull b , Arcadi Gual b , Teresa Borra ´s a, * a Department of Ophthalmology, University of North Carolina School of Medicine, 6109 Neuroscience Research Building CB 7041, 103 Mason Farm Road, Chapel Hill, NC 27599-7041, USA b Department of Physiological Sciences I-IDIBAPS, Faculty of Medicine, University of Barcelona, Barcelona, Spain Received 8 July 2004; accepted in revised form 13 December 2004 Available online 2 February 2005 Abstract Among other channels, voltage-gated chloride channels (ClC) regulate cell volume, membrane potential and cellular transport. Because changes in trabecular meshwork (TM) cell volume influence outflow facility and because the relative abundance of a gene’s transcript is an indication of the relevance of the gene’s function, we investigated the presence and relative expression of seven members of the CLCN gene family in the human TM. To elucidate the role of ClC-2 and ClC-3 in cell swelling, we studied changes in their mRNA levels after hypotonic shock. In addition, to examine the potential involvement of these two channels in conditions associated with glaucoma, we determined their transcripts levels in response to elevated intraocular pressure (IOP) and dexamethasone (DEX). For our evaluations, we used non- transformed human TM cells and perfused human anterior segments from post-mortem donors. For hypotonic shock, cells were exposed to 260 mOsm kg K1 medium for 15 and 30 min. For DEX, cells were treated with 0$1 mM DEX for 1, 4 and 10 days. For elevated IOP, one eye of each pair of perfused human anterior segments was subjected to DP 38G4 mmHg for 1 hr, 4 and 7 days while the contralateral remained at baseline pressure as a control. ClCs transcripts were determined by relative quantitative RT-PCR. Our results showed that all transcripts but ClC-1 were detected in HTM cells. ClC-2 and ClC-3 were the most abundant and comprised about twice the amount of ClC-6 and ClC-7 and four times that of ClC-4 and ClC-5. Hypotonic conditions consistently up regulated CLCN2 and slightly up regulated CLCN3. After short periods of elevated pressure, ClC-2 and ClC-3 transcripts were increased but ClC-2 induction was significantly higher than that of ClC-3. In contrast, after long pressure insults (7 days), ClC-3 mRNA was significantly increased while CLCN2 was not changed. DEX treatment markedly down regulated CLCN3 and little, if any, reduced ClC-2. The extent of response of the CLCN2 and CLCN3 to these conditions was markedly affected by individual traits but at all times maintained the relative expression pattern of both genes. CLCN2 gene expression was predominantly influenced by cell volume regulation while that of CLCN3 was preferentially affected by conditions associated with TM pathology. q 2005 Elsevier Ltd. All rights reserved. Keywords: human chloride channels; trabecular meshwork; perfused anterior segments; osmotic shock; elevated intraocular pressure; differential gene expression 1. Introduction The maintenance of physiological IOP is essential for proper ocular function. In the eye, IOP is determined by both the rate of secretion of aqueous humour and by the resistance offered to this fluid by the outflow tissues (Nilsson, 1994; Lu ¨tjen-Drecoll and Rohen, 1996; Lu ¨tjen- Drecoll et al., 2001). In humans, the main route of aqueous humour outflow is the conventional outflow pathway, which consists of the trabecular meshwork (TM) and Schlemm’s canal (SC). Far from being a passive tissue, the TM has an active role in modulating aqueous humour outflow. It is well established that disruptions of TM function lead to an increase in resistance to flow and to the generation of elevated IOP. In a yet unknown manner, this elevated IOP exerts a mechanical insult to the posterior region of the eye 0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.12.009 Experimental Eye Research 80 (2005) 801–813 www.elsevier.com/locate/yexer * Corresponding author. Dr Teresa Borra ´s, Department of Ophthal- mology, University of North Carolina School of Medicine, 6109 Neuroscience Research Building CB 7041, 103 Mason Farm Road, Chapel Hill, NC 27599-7041, USA. E-mail address: tborras@med.unc.edu (T. Borra ´s).