INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE TRABECULAR MESHWORK (TM) is the primary site through which aqueous humor exits the eye. Through regulating resistance to aqueous humor outflow, the TM also regulates intraocular pressure (IOP) (15). In the normal eye, IOP is maintained within narrow limits through finely tuned coupling of aqueous production and outflow. However, in the most common form of glaucoma, primary open-angle glaucoma (POAG), an improperly functioning TM is thought to cause increased resistance to outflow and, consequently, increased IOP, which in turn can cause damage to the optic nerve and ultimately lead to blindness (19, 26). Despite the well-recognized functional importance of TM cells in regulating aqueous outflow, cellular mechanisms underlying these functions are not well understood. We and others have hypothesized that modulation of TM cell volume is one means by which aqueous outflow across the TM is regulated (1, 2, 10, 11, 30, 38). Previous studies in our laboratory have demonstrated that, in cultured TM cells, the Na-K-Cl cotransporter functions to maintain steady-state cell volume under basal, isotonic conditions (35), most likely by offsetting ion efflux pathways such as K and Cl channels and/or K-Cl cotransport (32). The cotransporter also mediates volume recovery following hypertonicity-induced TM cell shrinkage, and it causes changes in cell volume following TM cell exposure to hormones, drugs, and neurotransmitters (30). We have also shown that the permeability of cultured TM cell monolayers to [¹⁴C]sucrose is modulated by changes in Na-K-Cl cotransport activity and intracellular volume (30). These findings led us to hypothesize that the Na-K-Cl cotransporter contributes to regulation of TM barrier function through modulating TM cell volume and thereby changing the extracellular space available for bulk flow of aqueous humor through the tissue in vivo. In support of this are studies using an in vitro anterior chamber organ perfusion model to evaluate the contribution of TM cell volume to outflow facility across the intact TM. These studies found that, when bovine (1, 11) or human (1) anterior segments are perfused with hypotonic medium (which swells TM cells), aqueous outflow is decreased, whereas perfusion with hypertonic medium (which transiently shrinks TM cells) increases outflow facility (1, 11). Furthermore, perfusion with bumetanide (which inhibits the Na-K-Cl cotransporter and causes sustained shrinkage of TM cells) was also found to increase outflow facility (2).

Recent studies from our laboratory have revealed that there are differences in regulation of the Na-K-Cl cotransporter as well as in steady-state cell volume in normal vs. glaucomatous human TM cells (35). These studies demonstrated that Na-K-Cl cotransport activity and cotransporter protein expression are reduced in cultured glaucomatous TM cells compared with cultured normal TM cells. In addition, the Na-K-Cl cotransporter activity remaining in glaucomatous TM cells is insensitive to inhibition by cAMP, whereas cotransport activity of normal TM cells is inhibited by cAMP. Furthermore, our studies showed that glaucomatous TM cell volume is elevated compared with normal TM cell volume, suggesting that TM cells of glaucomatous eyes exhibit a higher volume set point than those of nonglaucomatous eyes. The reason for the increased volume and decreased cotransport activity of glaucomatous TM cells has yet to be determined. However, one possibility is that the activity of ionic efflux pathway(s), such as K and Cl channels and/or K-Cl cotransport, is reduced in glaucomatous TM cells, causing elevated intracellular concentrations of these ions and elevated cell volume. The reduced cotransport activity, in turn, could be a consequence of a sustained elevation of cell volume and/or elevated intracellular Cl concentration ([Cl]_i) and/or intracellular K concentration (24, 37).

[Cl]_i has been shown to be a potent regulator of Na-K-Cl cotransport in secretory epithelia (9, 14, 25, 37), squid giant axon (5), and avian erythrocytes (23). Reduction of [Cl]_i appears to stimulate Na-K-Cl cotransport activity in these cells by direct phosphorylation of the cotransporter protein (13, 23-25), whereas elevation of [Cl]_i has been shown to inhibit Na-K-Cl cotransport activity of secretory epithelial cells (24, 37). Regulation of the Na-K-Cl cotransporter by [Cl]_i has been hypothesized to play a role in mediating cross talk between the basolaterally located Na-K-Cl cotransporter and apical Cl channels in secretory epithelia (12, 24, 37). In this case, [Cl]_i may act as the signal coupling Cl entry (through the Na-K-Cl cotransporter) with Cl exit (through Cl channels). [Cl]_i has also been hypothesized to act as the signal regulating Cl influx and efflux in nonepithelial cells and thereby to play a role in determining cell volume. As an initial approach to clarify the relationship between ion efflux pathways and Na-K-Cl cotransport in normal TM cells, the present study was conducted to investigate the possibility that activity of the TM cell cotransporter is regulated by [Cl]_i. Our findings indicate that [Cl]_i is a potent regulator of TM cell cotransporter activity and thus may contribute to the regulation of aqueous outflow across the TM in vivo through modulation and regulation of TM cell volume.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TM cell isolation and culture. Human TM cells (HTMC) were isolated from TM explants excised from eye bank donor rims as described previously (30, 34). The explants were obtained from fresh donor rims immediately following corneal transplantation. Postmortem donor screening by eye bank personnel excluded any donors with a known history of glaucoma. HTMC were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 5% bovine calf serum, essential and nonessential amino acids, penicillin/streptomycin and L-glutamine. Two separate cell cultures were used in these studies; HTMC-5 was isolated from a 5-yr-old male and was used to generate data (see Figs. 1, 3, and 4), whereas HTMC-22 was isolated from a 12-yr-old male and was used to generate data (see Figs. 2 and 5-8). Previous studies in our laboratory have demonstrated no significant difference in basal Na-K-Cl cotransport values in cultured HMTC isolated from young donors (2-38 yr) compared with older donors (48-68 yr) (35). Cells were maintained in collagen-coated 75-cm² tissue culture flasks and were used between passages 3 and 8.

For experiments, cells were removed from flasks by brief trypsinization and were subcultured onto collagen-coated 24-well plates. Cells were used 4-7 days later as confluent monolayers, and growth medium was replaced every 3-4 days. For culturing in stock flasks and 24-well plates, all cells were maintained in a 95% air-5% CO₂ atmosphere.

K influx determination. Na-K-Cl cotransport was measured as ouabain-insensitive, bumetanide-sensitive K influx, using ⁸⁶Rb as a tracer for K. Details of this method have been published previously (28). TM cell monolayers on 24-well cluster plates were preequilibrated for 30 min at 37°C in an isotonic, HEPES-buffered minimal essential medium (MEM) containing (in mM) 143 Na, 136 Cl, 5.8 K, 1.2 Ca, 4.2 HCO₃, 0.33 HPO₄, 0.4 H₂PO₄, 0.81 Mg, 0.81 SO₄, 5.6 dextrose, and 20 HEPES. In some experiments, the cells were then preincubated for 25 min at 37°C in MEM containing the above but with extracellular Cl concentrations ([Cl]_o) ranging from 0 to 136 mM. MEM containing <136 mM [Cl]_o (reduced [Cl]_o media) was prepared using the anion substitute methane sulfonic acid (MSA) (33). In other experiments, the cells were preincubated for 25 min at 37°C in MEM containing extracellular K concentrations ([K]_o) ranging from 5.8 to 80 mM. Media containing >5.8 mM [K]_o (elevated [K]_o media) were prepared by substituting K for Na. In these experiments, MEM extracellular Na ranged from 58 mM (in the 80 mM [K]_o MEM) to 122 mM (in the 20 mM [K]_o MEM). The cells were then pretreated and assayed (5 min each) in either isotonic (290 mosM) or hypertonic (390 mosM by addition of sucrose) MEM containing 1 mM ouabain, 10 or 0 µM bumetanide, normal [Cl]_o (136 mM) or reduced [Cl]_o (0-122 mM), and normal [K]_o (5.8 mM) or elevated [K]_o (20-80 mM). Assay medium also contained ⁸⁶Rb (1 µCi/ml). For experiments testing the effects of Cl channel blockers, the pretreatment and assay media also contained niflumic acid (NA; 0.3 µM to 1 mM), DIDS (1 or 3 mM), SITS (1 or 3 mM), or diphenylamine-2-carboxylic acid (DPC; 1 or 3 mM). Solutions containing DIDS and SITS were prepared in MEM immediately before the start of each experiment and protected from light. NA and DPC were dissolved in DMSO and bumetanide was dissolved in ethanol before they were added to the MEM, while ouabain was dissolved directly in MEM. The maximum final concentration of either DMSO or ethanol in MEM was 0.1%. The assay was terminated by rinsing the cluster plate wells with ice-cold isotonic 0.1 M MgCl₂ and then the contents were extracted with 1% SDS, and the amount of radioactivity present was determined by liquid scintillation (Tri-Carb model 2500 TR; Packard Instruments, Downers Grove, IL). K influx was determined by using the specific activity (counts · min¹ · µmol¹) of the assay medium. For each experiment, specific activities were calculated for each assay condition. Samples of SDS extracts were also used to determine the individual protein content of each well using the bicinchoninic acid (BCA) method (40). Osmolarities of all preincubation, pretreatment, and assay media were verified by osmometry (model 3W2; Advanced Instruments).

Cell volume assessment. Intracellular volume of HTMC was determined by radioisotopic evaluation of TM cell monolayer intracellular water space using [¹⁴C]urea and [¹⁴C]sucrose as markers of total and extracellular space, respectively. Details of this method have been described previously by O'Donnell (29). HTMC monolayers on 24-well plates were preequilibrated for 30 min in isotonic MEM at 37°C in an air atmosphere and then preincubated for 20 min in MEM containing either normal [Cl]_o (136 mM) or reduced [Cl]_o (27, 68, or 82 mM), either normal [K]_o (5.8 mM) or elevated [K]_o (20, 40, or 80 mM), either 100 or 0 µM NA, and either 10 or 0 µM bumetanide. The cells were then incubated for 10 additional min in the same medium containing either [¹⁴C]urea or [¹⁴C]sucrose (both at 1 µCi/ml). We have found that [¹⁴C]urea and [¹⁴C]sucrose are fully equilibrated by 5 min of incubation with the cell monolayers (data not shown). To terminate the assay, monolayers were rinsed with isotonic ice-cold 0.1 M MgCl₂ then extracted with SDS. Radioactivity of the SDS extracts was determined by liquid scintillation, and the protein content of each extract was assessed by the BCA method (40). The amounts of radioactivity in assay media containing [¹⁴C]urea and [¹⁴C]sucrose (in counts · min¹ · ml¹) were used to calculate water space associated with the radioactive markers (expressed as µl/mg protein). Intracellular volume was calculated as the difference between water space determined for [¹⁴C]urea (a marker for intracellular plus trapped extracellular space) and [¹⁴C]sucrose (a marker for trapped extracellular space).

Cell Cl content determination. For determination of cell Cl content, the ³⁶Cl equilibration method was used, as has been described previously (18, 36, 39). For ³⁶Cl equilibration time courses, HTMC monolayers on 24-well cluster plates were preequilibrated for 30 min in MEM at 37°C in an air atmosphere and then incubated for a total of 30 min in MEM containing either normal [Cl]_o (136 mM) or reduced [Cl]_o (27 or 82 mM) with ³⁶Cl (0.8 mCi/ml) also present during the final 2, 6, 10, 20, or 30 min of the incubation. In other experiments, TM cell monolayers were simply incubated for 30 min in MEM containing either normal [K]_o (5.8 mM) or elevated [K]_o (20, 40, or 80 mM), either 100 or 0 µM NA, either 10 or 0 µM bumetanide, and ³⁶Cl (0.8 mCi/ml). To terminate the assay, cell monolayers were rinsed with isotonic ice-cold 0.1 M MgCl₂, and then SDS extracts were prepared to determine radioactivity and protein content of each well (40). In each experiment, specific activities (counts · min¹ · µmol¹) of ³⁶Cl were determined for each assay condition and used to calculate intracellular Cl content (expressed as µmol/mg protein).

Materials. Bumetanide was purchased from ICN Pharmaceuticals (Costa Mesa, CA) and ouabain from Boehringer Mannheim Biochemicals (Indianapolis, IN). MSA, NA, DIDS, and SITS were obtained from Sigma Chemical (St. Louis, MO). DPC was obtained from Aldrich Chemical (Milwaukee, WI), and ⁸⁶Rb, [¹⁴C]urea, [¹⁴C]sucrose, and ³⁶Cl were from Dupont New England Nuclear (Boston, MA). Eagle's minimal essential medium was purchased from JRH Biosciences (Lenexa, KS), fetal bovine serum and FCS were from Hyclone Laboratories (Logan, UT), and collagen (type I) was from Collaborative Research (Bedford, MA).

Statistical analysis. Experimental results were analyzed by the unpaired Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of reduced [Cl]_i on Na-K-Cl cotransport in HTMC. Evaluation of bumetanide-sensitive K influx in HTMC following 30 min of preincubation with media of varying reduced [Cl]_o (0 to 136 mM) resulted in concentration-dependent stimulation of Na-K-Cl cotransport, as seen in Fig. 1. HTMC exhibited a bumetanide-sensitive K influx of 7.95 ± 0.21 µmol · g protein¹ · min¹ under control conditions (136 mM [Cl]_o preincubation) that was increased in a concentration-dependent manner by reduced [Cl]_o preincubation from 8.58 ± 0.32 µmol · g protein¹ · min¹ after preincubation with 122 mM [Cl]_o medium to 15.06 ± 0.66 µmol · g protein¹ · min¹ after preincubation with 0 mM [Cl]_o medium, an ~89% stimulation over control.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of preincubation with media containing reduced extracellular Cl concentration ([Cl]o) on Na-K-Cl cotransport activity in human trabecular meshwork cells (HTMC). Na-K-Cl cotransport activity of HTMC monolayers was assessed as bumetanide-sensitive K influx, as described in MATERIALS AND METHODS. Cells were preincubated with HEPES-buffered media of varying [Cl]o (0-136 mM) for 25 min then pretreated in the same media containing 1 mM ouabain and 10 or 0 µM bumetanide for 5 min. Cells were then assayed for 5 min in media containing normal [Cl]o (136 mM), 1 mM ouabain, 10 or 0 µM bumetanide, and 86Rb (1 µCi/ml). Reduced [Cl]o media (media containing <136 mM Cl) were prepared by using methane sulfonic acid as an anion substitute for Cl. Data are means ± SE of quadruplicate determinations from 5 experiments; prot, protein.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   A: cell Cl content of HTMC after incubation with reduced [Cl]o media. Cell Cl content of HTMC was assessed by 36Cl equilibration as described in MATERIALS AND METHODS. Cells were incubated for a total of 30 min in HEPES-buffered media containing either normal [Cl]o (136 mM) or reduced [Cl]o (82 or 27 mM) plus 36Cl (0.8 µCi/ml) during last 2, 6, 10, 20, or 30 min of incubation. Data are means ± SE of quadruplicate determinations from a representative experiment. Three other experiments gave similar results. B: effect of reduced [Cl]o media on HTMC volume. Cell volume was evaluated by radioisotopic determination of intracellular water space as described in MATERIALS AND METHODS. Confluent HTMC monolayers were incubated in HEPES-buffered media containing either normal [Cl]o (136 mM) or reduced [Cl]o (82 or 27 mM; each prepared using methane sulfonic acid as the anion substitute) for 20 min then incubated for 10 min in same media containing either [14C]urea or [14C]sucrose (1 µCi/ml). Amounts of [14C]urea and [14C]sucrose in assay medium (counts · min1 · ml1) were used to calculate total and extracellular water space, respectively. Data are means ± SE of quadruplicate determinations from 2 experiments.

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View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of reduced [Cl]o preincubation time on Na-K-Cl cotransport activity in HTMC. HTMC monolayers were preincubated for a total of 0-36 min in HEPES-buffered medium containing 27 mM Cl. During last 5 min of each preincubation, cells were pretreated in same medium containing 1 mM ouabain and 10 or 0 µM bumetanide. In the case of the 2- and 4-min time points, the cells were still pretreated with ouabain plus or minus bumetanide for a total of 5 min before the assay but were only exposed to reduced [Cl]o medium for 2 and 4 min of this time. Cells were then assayed for bumetanide-sensitive K influx for 5 min in medium containing normal Cl (136 mM), 1 mM ouabain, 10 or 0 µM bumetanide, and 86Rb (1 µCi/ml). Data are means ± SE of quadruplicate determinations from 4 experiments.

Additive effects of cell shrinkage and reduced [Cl]_i on stimulation of HTMC Na-K-Cl cotransport activity. To further investigate the effect of reduced [Cl]_i on HTMC Na-K-Cl cotransport activity, we tested whether lowering [Cl]_i would stimulate the cotransporter in osmotically shrunken cells. We have found that the HTMC Na-K-Cl cotransporter is stimulated by cell shrinkage and that maximal cotransport activity is obtained with 350-400 mosM hypertonic media (Putney, unpublished observations). Figure 4 shows the results of experiments in which we examined the combined effects of reduced [Cl]_o preincubation and maximally stimulatory hypertonic medium on cotransporter activity. For these studies, HTMC were preincubated for 25 min in either normal or reduced [Cl]_o medium (136 and 68 mM, respectively) and then assayed for bumetanide-sensitive K influx in normal [Cl]_o media that were either isotonic (290 mosM) or hypertonic (390 mosM). As described in MATERIALS AND METHODS, cells were given the appropriate 5-min pretreatment in isotonic or hypertonic media containing normal or reduced [Cl]_o, 1 mM ouabain, and 10 or 0 µM bumetanide. We found that reducing [Cl]_i caused a stimulation of Na-K-Cl cotransporter activity whether the cells were assayed in isotonic or hypertonic medium (Fig. 4A). Na-K-Cl cotransport activity was increased from a control value of 5.86 ± 0.31 µmol K · g protein¹ · min¹ (measured under isotonic/normal [Cl]_o incubation conditions) to 9.04 ± 0.28 and 8.36 ± 0.34 µmol K · g protein¹ · min¹ after exposure to reduced [Cl]_o and hypertonic media, respectively, whereas exposure of HTMC to both stimulators increased cotransport activity to 12.03 ± 0.44 µmol K · g protein¹ · min¹. In related experiments, we also evaluated the effect of varying assay media tonicity on Na-K-Cl cotransport activity in HTMC preincubated with normal [Cl]_o vs. reduced [Cl]_o. We found that cells with reduced [Cl]_i do not exhibit an altered response to varying media tonicity compared with HTMC with normal [Cl]_i. Although cotransport activity is higher in cells with reduced [Cl]_i compared with normal [Cl]_i, at every tonicity examined (between 290 and 590 mosM), we found that 390 mosM media provide maximal cell-shrinkage-induced stimulation of cotransport activity in both normal and reduced [Cl]_i cells (data not shown). Thus the effects of reduced [Cl]_i and maximally stimulatory hypertonicity appear to be truly additive.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   A: additive effects of hypertonicity (Hyper) and reduced intracellular Cl concentration ([Cl]i) on Na-K-Cl cotransport activity in HTMC. Cell monolayers were preincubated in isotonic HEPES-buffered media containing either normal [Cl]o (136 mM) or reduced [Cl]o (68 mM) for 25 min and then were pretreated in either isotonic or hypertonic media containing normal or reduced [Cl]o, 1 mM ouabain, and 10 or 0 µM bumetanide for 5 min. Cells were then assayed for 5 min in either isotonic or hypertonic media containing normal [Cl]o (136 mM), 1 mM ouabain, 10 or 0 µM bumetanide, and 86Rb (1 µCi/ml). Level of hypertonicity used was 390 mosM (by addition of sucrose), which was previously found to be maximally stimulatory for Na-K-Cl cotransport activity in TM cells (data not shown). Data are means ± SE of quadruplicate determinations from three experiments. B: effects of hypertonicity and reduced [Cl]i on HTMC volume. Confluent HTMC monolayers were incubated in HEPES-buffered media containing either normal [Cl]o (136 mM) or reduced [Cl]o (68 mM) for 20 min then assayed for 10 min in either isotonic or hypertonic media containing either normal [Cl]o (136 mM) or reduced [Cl]o (68 mM) and [14C]urea or [14C]sucrose (1 µCi/ml). Amounts of [14C]urea and [14C]sucrose in assay medium (counts · min1 · ml1) were used to calculate water associated with total and extracellular space, respectively. Data are means ± SE of quadruplicate determinations from 3 experiments.

Effect of elevated [K]_o on Na-K-Cl cotransport activity in HTMC. To examine the effect of increasing [Cl]_i on HTMC Na-K-Cl cotransport activity, we preincubated cells with media containing elevated [K]_o as a means of diminishing K efflux and thus decreasing electrically coupled Cl efflux (24). For these studies, bumetanide-sensitive K influx of cultured HTMC was evaluated after 30 min of preincubation with media of varying elevated [K]_o (5.8, 20, 40, 60, or 80 mM). Preincubation with high [K]_o media resulted in inhibition of Na-K-Cl cotransport activity and also a concomitant increase in [Cl]_i, as shown in Fig. 5. HTMC exhibited a bumetanide-sensitive K influx of 6.65 ± 0.25 µmol · g protein¹ · min¹ under control conditions (5.8 mM [K]_o preincubation) that was decreased in a concentration-dependent manner by elevated [K]_o preincubation, from 5.08 ± 0.21 µmol · g protein¹ · min¹ after preincubation with 20 mM [K]_o medium to 1.00 ± 0.16 µmol · g protein¹ · min¹ after preincubation with 80 mM [K]_o medium (Fig. 5A). Incubation of HTMC with elevated [K]_o media also caused a concentration-dependent increase in cell Cl content, assessed by ³⁶Cl equilibration (Fig. 5B). Cell Cl content rose from a control value of 0.328 ± 0.024 µmol/mg protein after incubation with 5.8 mM [K]_o medium for 30 min to 0.415 ± 0.031, 0.461 ± 0.038, and 0.585 ± 0.023 µmol/mg protein after incubation with elevated [K]_o media containing 20, 40, and 80 mM K, respectively, for the same duration. Figure 5C shows the results of experiments in which we evaluated the effect of elevated [K]_o media on TM cell volume. Intracellular volumes of HTMC exposed for 30 min to media containing 20 or 40 mM [K]_o were not significantly different from the volume assessed under control conditions (5.8 mM K). Exposure to media containing 80 mM [K]_o did, however, cause an increase in cell volume. The possible reasons for this are considered in the DISCUSSION. For HTMC incubated with control media (5.8 mM K), [Cl]_i was calculated to be 58.7 ± 2.6 mM, whereas, for cells incubated with 80 mM [K]_o media, the calculated [Cl]_i was 87.4 ± 2.4 mM (calculations based on data shown in Fig. 5, B and C). Thus incubation of HTMC with elevated [K]_o media of 20 or 40 mM caused significant reductions in cotransport activity and also significant increases in [Cl]_i, with no change in intracellular volume. This indicates that elevated [K]_o-induced increases in [Cl]_i cause inhibition of cotransport activity by a mechanism independent of cell volume changes.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   A: effect of elevated [K]o preincubation on HTMC Na-K-Cl cotransport activity. Cells were preincubated in HEPES-buffered media containing either normal [K]o (5.8 mM) or elevated [K]o (20, 40, 60, or 80 mM) for 25 min and then were pretreated in same media containing 1 mM ouabain and 10 or 0 µM bumetanide for 5 min. Cells were then assayed for bumetanide-sensitive K influx for 5 min in media containing normal [K]o (5.8 mM), 1 mM ouabain, 10 or 0 µM bumetanide, and 86Rb (1 µCi/ml). Data are means ± SE of quadruplicate determinations from 4 experiments. B: cell Cl content of HTMC after incubation with elevated [K]o media. HTMC monolayers were incubated in HEPES-buffered media containing either normal [K]o (5.8 mM) or elevated [K]o (20, 40, or 80 mM) plus 36Cl (0.8 µCi/ml) for 30 min. Specific activities (counts · min1 · µmol1) of 36Cl in assay media were determined to calculate intracellular Cl content. Data are means ± SE of quadruplicate determinations from 3 experiments. C: effect of elevated [K]o media on HTMC volume. Confluent HTMC monolayers were incubated in HEPES-buffered media containing either normal [K]o (5.8 mM) or elevated [K]o (20, 40, or 80 mM) for 20 min then assayed for 10 min in same media containing either [14C]urea or [14C]sucrose (1 µCi/ml). Amounts of [14C]urea and [14C]sucrose in assay medium (counts · min1 · ml1) were used to calculate total and extracellular water space, respectively. Data are means ± SE of quadruplicate determinations from 3 experiments.

View larger version (45K): [in this window] [in a new window]   Fig. 6.   Alteration of HTMC Na-K-Cl cotransport activity by Cl channel modulators. Cells were equilibrated in HEPES-buffered media for 30 min and then pretreated for 5 min in same media containing 1 or 3 mM DIDS, 1 or 3 mM SITS, 0.1 or 1 mM diphenylamine-2-carboxylic acid (DPC), 0.1 or 1 mM niflumic acid (NA), 1 mM ouabain, and 10 or 0 µM bumetanide. Cells were then assayed for bumetanide-sensitive K influx for 5 min in same media plus 86Rb (1 µCi/ml). Data are means ± SE of at least 3 determinations from 3-6 experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effect of low-concentration NA on Na-K-Cl cotransport in HTMC. Cells were equilibrated in HEPES-buffered media for 30 min and then pretreated for 5 min in media containing 1 mM ouabain, 10 or 0 µM bumetanide, and 0.3 µM to 1 mM NA. Cells were then assayed for bumetanide-sensitive K influx for 5 min in media identical to pretreatment media but also containing 86Rb (1 µCi/ml). K influx value for 0 mM NA control (not shown) was not significantly different from that at 0.1 µM (107 M). Dashed line indicates control level of Na-K-Cl cotransport. Data are means ± SE of at least 4 determinations from 3 experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   A: additive reduction of cell Cl in HTMC by bumetanide and low-concentration NA. Confluent HTMC monolayers were incubated and assayed in HEPES-buffered media containing 10 or 0 µM bumetanide (Bumet), 100 or 0 µM NA, and 36Cl (0.8 µCi/ml) for 30 min. Specific activities (counts · min1 · µmol1) of 36Cl in assay media were determined to calculate intracellular Cl content. Significant differences: * P < 0.05 and ** P < 0.001. Data are means ± SE of quadruplicate determinations from 2 experiments. B: additive reduction of HTMC volume by bumetanide and low-concentration NA. Confluent HTMC monolayers were incubated for 20 min in HEPES-buffered media containing 10 or 0 µM bumetanide and 100 or 0 µM NA and then assayed for 10 min in same media containing either [14C]urea or [14C]sucrose (1 µCi/ml). Amounts of [14C]urea and [14C]sucrose in assay medium (counts · min1 · ml1) were used to calculate total and extracellular water space, respectively. Significant differences: * P < 0.05 and ** P < 0.001. Data are means ± SE of quadruplicate determinations from 5 experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results of the present study demonstrate that the HTMC Na-K-Cl cotransport system is highly sensitive to changes in [Cl]_i. We have found that reducing [Cl]_i in HTMC stimulates Na-K-Cl cotransport activity and that elevating [Cl]_i inhibits cotransport activity in a manner independent of cell volume changes. Our studies also demonstrate that several known Cl channel inhibitors cause a reduction of Na-K-Cl cotransport activity in HTMC. These studies further show that NA at 100 µM lowers HTMC Cl content, stimulates activity of the Na-K-Cl cotransporter, and decreases intracellular volume in a manner additive with bumetanide. Together, the results of this investigation suggest that [Cl]_i is an important regulator of HTMC Na-K-Cl cotransporter activity and thus may influence cell volume of TM cells and, consequently, aqueous outflow across the TM in vivo.

The Na-K-Cl cotransporter has been shown to be activated by reduced [Cl]_i in secretory epithelial cells, squid axon, and avian erythrocytes (5, 14, 23, 25, 37). In the present study, we found that the HTMC Na-K-Cl cotransporter is also stimulated by reduced [Cl]_i, with a maximum stimulation of about twofold occurring after preincubation of cells with 0 mM [Cl]_o medium for 30 min. These findings are consistent with those reported by others. For example, Breitwieser et al. (5) showed that dialyzing the squid giant axon with 0 mM [Cl]_o medium for 150 min resulted in an approximately threefold stimulation of the Na-K-Cl cotransporter. Haas et al. (12) demonstrated that incubation of nystatin-treated dog tracheal epithelial cells with 32 mM [Cl]_o medium for 40 min caused an approximately twofold stimulation of Na-K-Cl cotransporter activity and, similarly, Isenring et al. (17) demonstrated a twofold stimulation of cotransporter activity in response to reduced [Cl]_i in HEK cells transfected with the human Na-K-Cl cotransporter protein. In a study of Na-K-Cl cotransport in shark rectal gland tubules, Lytle and Forbush (24) found a 20-fold increase in cotransporter activity after incubation with 15 mM [Cl]_o medium. It should be noted that these comparisons are of relative magnitudes of stimulation and that absolute levels of cotransport activity vary with cell and tissue type. In our studies, the half time (t_1/2) for reduced [Cl]_o (27 mM) activation of HTMC Na-K-Cl cotransport was ~5 min, a parameter that may also be species and/or cell type specific. In shark rectal gland tubules, exposure to reduced [Cl]_o (15 mM) resulted in a t_1/2 of 2.5-5 min (24), whereas the transfected HEK cells exhibited a t_1/2 of 12 min for reduced [Cl]_o (2.5 mM) activation of the cotransporter (17).

The stimulation of HTMC Na-K-Cl cotransport by reduced [Cl]_i cannot be explained by cell-shrinkage-induced activation of the cotransporter. We found no decrease in intracellular volume of HTMC after incubation of the cells with varying reduced [Cl]_o media for 30 min. Furthermore, we observed that the stimulatory effect of reduced [Cl]_o preincubation on HTMC cotransport activity is additive with that observed following exposure of the cells to a maximally stimulatory hypertonic medium, a finding inconsistent with reduced [Cl]_o media acting via cell shrinkage. Because acute cell shrinkage increases [Cl]_i, it is possible that, in cells treated to reduce [Cl]_i, a tonic inhibition of cotransport activity is removed, allowing a greater stimulation with cell shrinkage. We have found that HTMC with reduced [Cl]_i exhibit increased cotransport activity at all tonicities examined between 300 and 600 mosM and that the shape of the dose-response relationship between tonicity and cotransporter activity is not different in reduced [Cl]_i cells compared with normal [Cl]_i cells. Thus it is unlikely that stimulation of HTMC cotransporter activity by hypertonicity and by reduced [Cl]_i occur by the same signaling pathway. Our observations are consistent with previous findings in secretory epithelial cells and squid axon, which suggest that hypertonicity and reduced [Cl]_i act independently to stimulate Na-K-Cl cotransport (5, 12, 24, 37).

Our findings also reveal that the HTMC Na-K-Cl cotransporter is inhibited in a concentration-dependent manner by elevated [Cl]_i. Preincubation of HMTC with elevated [K]_o medium (80 mM), a maneuver that significantly diminishes K and Cl loss from the cells and elevates [Cl]_i, results in an ~85% reduction in Na-K-Cl cotransport activity. Our findings are consistent with those reported by others that cotransport activity is inhibited in shark rectal gland by elevated [K]_o media (24) and in secretory acinar cells in response to direct elevation of [Cl]_i (37). Previous studies in our laboratory have shown that hypotonic medium-induced cell swelling of bovine TM cells inhibits Na-K-Cl cotransport activity (30). In the present study we demonstrate that Na-K-Cl cotransport inhibition by elevated [Cl]_i (via incubation with elevated [K]_o media) is not simply due to cell swelling, because incubation of the cells with [K]_o media varying from 5.8 to 40 mM did not alter cell volume. However, preincubation of the cells with 80 mM [K]_o medium did cause an elevation of intracellular volume (~29%) and thus the inhibition of cotransport activity observed with 80 mM [K]_o preincubation could be due, at least in part, to cell swelling. Nevertheless, the magnitude of cell swelling observed under these conditions is not likely to account for the entire cotransport inhibition (~85%), since our previous studies in bovine TM cells indicate that a hypotonic-medium-induced cell swelling of 30% causes only 45% inhibition of cotransporter activity.

In the present study we also found that exposing TM cells to agents known to inhibit Cl channels causes reduction of Na-K-Cl cotransport activity. Thus DPC, well recognized for its ability to block Cl channels (3, 41), inhibits the HTMC Na-K-Cl cotransporter. NA, reported to inhibit Cl channels in a variety of cell types (16, 20, 41), also inhibits the HTMC cotransporter, but only at concentrations >100 µM. Furthermore, our studies also show that DIDS and SITS, blockers of Cl channels and Cl/HCO₃ exchange, also inhibited the HTMC cotransporter. DIDS was more potent than SITS in inhibiting HTMC Na-K-Cl cotransport activity, consistent with DIDS having a higher potency for Cl channel inhibition than SITS (7, 8). The finding that the Cl channel inhibitors lose their ability to inhibit HTMC Na-K-Cl cotransport when [Cl]_i is reduced suggests that inhibition of cotransport activity by these agents may indeed depend on the elevation of [Cl]_i. In the presence of reduced [Cl]_i, NA (1 mM) even stimulated cotransporter activity. Although the reason for this stimulatory effect on cells with reduced [Cl]_i remains to be determined, NA has been reported to either increase or decrease Cl channel conductance, depending on the concentration. Furthermore, we have found that NA stimulates HTMC cotransporter activity at concentrations <1 mM (while inhibiting at 1 mM). Thus it is possible that reduction of [Cl]_i may influence the concentration dependence of NA effects on Cl channels. The observation that DIDS, SITS, DPC, and NA fail to reduce cotransport activity in the presence of reduced [Cl]_i also suggests that these agents do not inhibit HTMC Na-K-Cl cotransport activity by binding to and inhibiting the cotransporter protein directly.

Our findings are consistent with previous reports that DIDS and DPC (each at 1 mM) inhibit Na-K-Cl cotransport activity (by 57% and 15%, respectively) in nonpigmented ciliary epithelial cells of the eye (6). If the net direction of Cl movement through Cl channels in HTMC is outward, then it is predicted that these inhibitors will reduce Na-K-Cl cotransport activity in HTMC by inhibiting channel-mediated Cl efflux and elevating [Cl]_i. Our findings that NA (0.1 mM) stimulates HTMC cotransporter activity and that DIDS, DPC, and NA (all at 1 mM) inhibit the cotransporter are consistent with previous reports that the Na-K-Cl cotransporter is regulated indirectly by changes in [Cl]_i brought about by activation of Cl channels in airway epithelial cells (12, 14), ciliary epithelial cells (6), and shark rectal gland tubules (24).

NA is a nonsteroidal anti-inflammatory drug that has been shown to inhibit Cl/HCO₃ exchange in human erythrocytes (21) and Ca-activated Cl channels in rabbit (16) and rat (20) portal vein in the concentration range of 1-100 µM, as well as to stimulate ATP-gated K channels in a similar concentration range (16, 20). However, in two independent studies using retinal pigmented epithelial cells, NA at 100 µM was shown to open Cl channels (4, 27). Consistent with this, our studies show that NA, at concentrations of 0.3-100 µM, stimulates rather than inhibits HTMC Na-K-Cl cotransport and, furthermore, that 100 µM NA significantly reduces HTMC cell Cl content. Thus our findings support the possibility that NA opens a Cl channel in these cells, thereby reducing cell Cl and stimulating Na-K-Cl cotransporter activity. Alternatively, it is possible that NA at this concentration activates ATP-gated K channels in HTMC, resulting in K and Cl loss, reduction of cell Cl, and stimulation of Na-K-Cl cotransport (16, 20).

There is strong evidence that modulation of Na-K-Cl cotransport by [Cl]_i plays an important role in regulating cell volume (22, 37). In vascular endothelial cells, we have found that [Cl]_i levels increase as volume is restored after hypertonic cell shrinkage (regulatory volume increase; RVI) and that elevation of [Cl]_i reduces Na-K-Cl cotransport activity in these cells (O'Donnell, unpublished observations). This regulation may provide a mechanism to prevent overshoot of intracellular volume during the RVI. In addition, we find that the RVI in HTMC is markedly augmented when cell Cl has first been reduced by exposing cells to hypotonic media [i.e., the RVI following a regulatory volume decrease (RVD) is significantly faster than a normal RVI (Putney, unpublished observations)]. Recent studies in our laboratory have also demonstrated that the RVD in HTMC is diminished when the cells are exposed simultaneously to hypotonicity and to the Cl channel inhibitors DIDS, DPC, or NA (1 mM each) (31). This suggests that the RVD in HTMC is mediated at least in part by channel-mediated Cl efflux. In this regard, it is possible that [Cl]_i is the signal that coordinates Cl efflux (through Cl channels) and influx (through the Na-K-Cl cotransporter) in HTMC and thus regulates intracellular volume recovery as well as resting cell volume. Furthermore, it is important to note that [Cl]_i and cell volume are intimately linked in vivo, since Cl is the major intracellular permeant anion.

Previous studies in our laboratory and those of others have shown that the Na-K-Cl cotransporter and intracellular volume appear to be determinants of TM barrier function (1, 2, 11, 30). Our recent findings that regulation of the Na-K-Cl cotransporter in glaucomatous human TM cells compared with normal TM cells is aberrant while cell volume is elevated and cotransport activity is reduced prompt the speculation that Cl efflux pathways and/or regulation of the Na-K-Cl cotransporter by [Cl]_i may be impaired in the glaucomatous TM cells (35). Whether these processes are, in fact, altered in the glaucomatous TM cells will require further study. In any case, our finding that NA (100 µM) decreases cell Cl content and causes cell shrinkage in HTMC suggests that agents that open Cl channels may be of therapeutic value in promoting aqueous humor outflow across the TM in vivo and in lowering IOP in patients with POAG, particularly if coupled with the cell-volume-reducing effect of Na-K-Cl cotransport inhibition.