Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

Luanna K. Putney, Cecile Rose T. Vibat, and Martha E. O'Donnell

Department of Human Physiology, School of Medicine, University of California, Davis, California 95616-8644


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.

glaucoma; aqueous outflow; intracellular volume; chloride channel; niflumic acid


    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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 · min-1 · µmol-1) 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 · min-1 · ml-1) 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 · min-1 · µmol-1) 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
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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 protein-1 · min-1 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 protein-1 · min-1 after preincubation with 122 mM [Cl]o medium to 15.06 ± 0.66 µmol · g protein-1 · min-1 after preincubation with 0 mM [Cl]o medium, an ~89% stimulation over control.


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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.

To determine the extent to which preincubation of HTMC with reduced [Cl]o media also reduces [Cl]i, we measured cell Cl content and cell volume following incubation of HTMC with media of varying [Cl]o, as shown in Fig. 2. For these experiments, Cl content was assessed by 36Cl equilibration. Cells were exposed to varying [Cl]o media for a total of 30 min with 36Cl present during the last 2, 6, 10, or 20 min or during the entire 30-min incubation (Fig. 2A). We found a time-dependent uptake of 36Cl that reached a plateau (isotopic equilibrium) by ~20 min in cells incubated with normal [Cl]o (136 mM) or reduced [Cl]o (82 or 27 mM). In some experiments, we also measured cell Cl content after 60 min exposure to 36Cl, where cells were first exposed to media containing 136 mM [Cl]o for 30 min and then to media containing either normal [Cl]o (136 mM) or reduced [Cl]o (27 or 82 mM) plus 36Cl (0.8 mCi/ml) for an additional 30 min (data not shown). We found no significant differences between the cell Cl contents measured after 30 min of 36Cl exposure and those measured after 60 min of exposure for any of the varying [Cl]o incubations, indicating that 36Cl is truly equilibrated by 30 min. The experiments depicted in Fig. 2A also demonstrate that the cell Cl content of HTMC at each of the plateaus (after 30 min of 36Cl exposure) correlates with the [Cl]o of the incubation media, i.e., incubation of cells with 136 mM [Cl]o medium (control) for 30 min resulted in a cell Cl content of 0.355 ± 0.018 µmol/mg protein, whereas incubation of cells with 82 and 27 mM [Cl]o media for the same amount of time resulted in cell Cl contents of 0.262 ± 0.004 and 0.096 ± 0.002 µmol/mg protein, respectively. If the anion used to substitute for Cl in these studies (MSA) is sufficiently less permeable than Cl, incubating the cells with the reduced [Cl]o media could decrease HTMC cell volume, causing shrinkage-induced stimulation of cotransporter activity. To test for this possibility, as well as to assess changes in [Cl]i, we also evaluated intracellular volume of the cells after incubation with media of varying [Cl]o. We found no change in cell volume after 30 min of incubation with reduced [Cl]o media (27 or 82 mM) compared with control (136 mM Cl; Fig. 2B). HTMC volume in control media was 4.34 ± 0.10 µl/mg protein, a value not significantly different from that measured after incubation with 27 or 82 mM [Cl]o media (4.41 ± 0.25 and 4.59 ± 0.18 µl/mg protein, respectively). Furthermore, [Cl]i for HTMC after incubation with control media (136 mM), 82 mM [Cl]o media, or 27 mM [Cl]o media values were calculated to be 58.7 ± 2.6, 48.3 ± 2.4, and 23.3 ± 0.9 mM, respectively (quadruplicate determinations from 3 experiments; representative experiments shown in Fig. 2, A and B). These findings indicate that incubation of HTMC with reduced [Cl]o media reduces [Cl]i as well as cell Cl content.



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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 · min-1 · ml-1) were used to calculate total and extracellular water space, respectively. Data are means ± SE of quadruplicate determinations from 2 experiments.

The time course of Na-K-Cl cotransport stimulation by preincubation of HTMC with 27 mM [Cl]o medium is shown in Fig. 3. Significant elevation of cotransport activity was observed after 2 min of preincubation with the reduced [Cl]o medium, from a control value of 8.64 ± 0.28 µmol K · g protein-1 · min-1 (normal [Cl]o preincubation) to 11.57 ± 0.38 µmol K · g protein-1 · min-1 after 2 min of preincubation with reduced [Cl]o medium. Maximum stimulation was observed by 10 min incubation with reduced [Cl]o medium, with cotransport activity of 15.87 ± 0.50 µmol K · g protein-1 · min-1.


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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 protein-1 · min-1 (measured under isotonic/normal [Cl]o incubation conditions) to 9.04 ± 0.28 and 8.36 ± 0.34 µmol K · g protein-1 · min-1 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 protein-1 · min-1. 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.



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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 · min-1 · ml-1) were used to calculate water associated with total and extracellular space, respectively. Data are means ± SE of quadruplicate determinations from 3 experiments.

Experiments conducted in parallel with those shown in Fig. 4 revealed that when HMTC were preincubated for 20 min with normal isotonic [Cl]o medium (136 mM) and then exposed for 10 min to isotonic or hypertonic medium (both with 136 mM Cl), the resulting [Cl]i values were calculated to be 67.1 ± 3.1 and 127.2 ± 5.6 mM, respectively. When HTMC were preincubated for 20 min with isotonic reduced [Cl]o medium (68 mM) and then exposed for 10 min to isotonic or hypertonic media (both with 68 mM Cl), [Cl]i values were 48.9 ± 2.0 and 57.3 ± 4.1, respectively (data not shown; quadruplicate determinations from 3 experiments). These findings, together with the data of Fig. 4, indicate that hypertonic medium stimulates the Na-K-Cl cotransporter to a degree similar to that observed with reduction of [Cl]i, even though HTMC in hypertonic medium exhibit greatly elevated [Cl]i. This observation is addressed further in the DISCUSSION.

In these experiments, we also evaluated the intracellular volume of HTMC occurring in response to reduced [Cl]o (68 mM) incubation and hypertonic medium (390 mosM; Fig. 4B). Here we found that the hypertonicity-induced cell shrinkage was the same whether HTMC had been incubated with normal or reduced [Cl]o media. Exposure of HTMC to hypertonic medium for 10 min resulted in shrinkage of cells from a control value of 5.06 ± 0.19 µl/mg protein (measured under isotonic/normal [Cl]o incubation conditions) to 2.98 ± 0.15 µl/mg protein, ~59% of control volume. Similarly, incubation of HTMC with reduced [Cl]o medium for 20 min followed by a 10-min assay for cell volume in hypertonic medium containing reduced [Cl]o resulted in a cell volume decrease to 3.35 ± 0.18 µl/mg protein, a value not significantly different from the volume measured in hypertonic medium containing normal [Cl]o.

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 protein-1 · min-1 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 protein-1 · min-1 after preincubation with 20 mM [K]o medium to 1.00 ± 0.16 µmol · g protein-1 · min-1 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.




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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 · min-1 · µmol-1) 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 · min-1 · ml-1) were used to calculate total and extracellular water space, respectively. Data are means ± SE of quadruplicate determinations from 3 experiments.

Effect of Cl channel inhibitors on HTMC Na-K-Cl cotransport. Our previous studies have shown that the HTMC Na-K-Cl cotransporter mediates a net ion uptake under basal, isotonic conditions and that bumetanide inhibition of cotransport activity causes the cells to shrink (30). This suggests that, under steady-state conditions, the cotransporter mediates a net ion influx that offsets efflux pathways such as Cl and K channels and/or K-Cl cotransport. If [Cl]i is an important regulator of HTMC Na-K-Cl cotransport, then it is possible that changes in the activity of Cl efflux pathways will modulate Na-K-Cl cotransport activity. To test this, we evaluated the effects of Cl channel inhibitors on HTMC Na-K-Cl cotransport activity. We found that a 10-min exposure of HTMC to DIDS (1 or 3 mM), SITS (3 mM), DPC (1 mM), or NA (1 mM) resulted in significant inhibition of bumetanide-sensitive K influx, as shown in Fig. 6. HTMC Na-K-Cl cotransport activity was reduced by ~33% and 70% after exposure of cells to 1 and 3 mM DIDS, respectively. SITS, a stilbene derivative structurally similar to DIDS but with lower potency for Cl channel inhibition, was without effect on HTMC Na-K-Cl cotransport activity at 1 mM but caused an ~22% inhibition of cotransport activity at 3 mM. Exposure of the cells to DPC at 1 mM resulted in an ~43% inhibition of bumetanide-sensitive K influx, whereas 0.1 mM DPC had no effect. At 1 mM, NA also inhibited HTMC Na-K-Cl cotransport activity by ~22%. However, exposure of the cells to a lower dose of NA (0.1 mM) caused a stimulation of cotransport activity. We found no significant changes in the intracellular volume of HTMC after exposure of the cells to either 3 mM DIDS, 3 mM SITS, or 1 mM DPC for 10 min compared with control data; however, 1 mM NA caused an ~22% elevation of cell volume compared with control (data not shown). Thus alteration of Na-K-Cl cotransport in HTMC in response to Cl channel inhibitors does not appear to be due to signaling associated with cell volume changes.


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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.

In a separate set of experiments, we reduced [Cl]i in HTMC by 30 min of preincubation with medium containing 0 mM Cl and then assessed the effects of the Cl channel modulators DIDS, SITS, DPC, and NA on bumetanide-sensitive Na-K-Cl cotransport activity in the presence of normal flux medium containing 136 mM Cl. We found that, when HTMC were preincubated with medium containing normal Cl, Na-K-Cl cotransport was inhibited 33.9 ± 3.8% by 1 mM DIDS, 27.2 ± 3.2% by 3 mM SITS, 42.6 ± 3.8% by 1 mM DPC, and 37.8 ± 4.9% by 1 mM NA relative to control (quadruplicate determinations from 4 experiments). These findings are similar to the results shown in Fig. 6. However, when HTMC were preincubated with Cl-free medium, the Cl channel blockers lost their ability to reduce Na-K-Cl cotransport activity. Specifically, under these conditions, DIDS, SITS, and DPC reduced cotransport activity 8.5 ± 5.5%, 2.7 ± 6.0%, and 2.8 ± 9.6%, respectively. Exposure of the cells to 1 mM NA even caused a 59.1 ± 18.1% stimulation of cotransport activity in cells with reduced [Cl]i (data not shown; quadruplicate determinations from 4 experiments for all four agents). The possible reasons for NA stimulation of cotransport activity under these conditions are considered in the DISCUSSION. In any case, our findings suggest that the observed inhibitory effects of DIDS, SITS, DPC, and NA on HTMC cotransport activity are dependent on elevation of [Cl]i.

Effects of low-NA concentrations on HTMC Na-K-Cl cotransport and intracellular volume. There is much evidence that NA inhibits various Cl channels at a concentration as low as 1 µM in a variety of cell types (16, 20, 41). However, NA (100 µM) has also been shown to have stimulatory effects on Cl channels in ocular cells (4, 27). Because the initial findings of the present study (Fig. 6) suggested that there might be a concentration-dependent effect of NA on the activity of the Na-K-Cl cotransporter in HTMC, we examined cotransport activity over a range of NA concentrations, as shown in Fig. 7. We found that, whereas NA at 1 mM inhibited cotransport activity of HTMC, it stimulated the cotransporter over a range of lower concentrations (0.3-100 µM). Bumetanide-sensitive K influx increased in a concentration-dependent manner to a maximum of ~35% above control after exposure of cells to 10 µM NA but then fell by ~23% compared with control after exposure of cells to 1 mM NA.


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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 (10-7 M). Dashed line indicates control level of Na-K-Cl cotransport. Data are means ± SE of at least 4 determinations from 3 experiments.

If low-concentration NA stimulates HTMC Na-K-Cl cotransport activity by positively regulating a Cl channel and thus promoting Cl efflux, we would predict a concomitant reduction in cell Cl content. To test this, we measured HMTC Cl content after exposure of the cells to 100 µM NA for 30 min (Fig. 8). We also measured cell Cl content after exposure of HTMC to either 10 µM bumetanide alone or 10 µM bumetanide plus 100 µM NA for 30 min, as a means of determining whether the predicted reduction of cell Cl by 100 µM NA was additive, with the expected reduction of cell Cl after Na-K-Cl cotransport inhibition. Figure 8A shows that exposure of HTMC to either 10 µM bumetanide or 100 µM NA for 30 min caused a significant reduction in cell Cl content, 14% and 11%, respectively. When HTMC were exposed to both 10 µM bumetanide and 100 µM NA for 30 min, cell Cl content was reduced in an additive manner, by ~34%. These findings support the hypothesis that NA opens a Cl channel in HTMC, causing Cl efflux and reduction of cell Cl content. We also investigated the effects of bumetanide and NA on HTMC volume and found that changes in cell volume reflect changes in cell Cl content occurring after exposure of the cells to these agents (Fig. 8B). Here bumetanide (10 µM) and NA (100 µM) separately reduced cell volume by 12% and 11%, respectively, whereas together they produced an additive cell shrinkage of ~21%. These data support the hypothesis that NA, at concentrations of 100 µM and lower, open a Cl channel in HTMC, promoting Cl efflux and reducing cell Cl content and cell volume.



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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 · min-1 · µmol-1) 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 · min-1 · ml-1) 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 (t1/2) 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 t1/2 of 2.5-5 min (24), whereas the transfected HEK cells exhibited a t1/2 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.


    ACKNOWLEDGEMENTS

This work was supported in part by grants from the Glaucoma Research Foundation (to M. E. O'Donnell), Merck Research Laboratories (to M. E. O'Donnell), Fight for Sight, Inc., New York City, a division of Prevent Blindness America (to L. K. Putney), and the National Academy of Sciences through Sigma Xi, the Scientific Research Society (to L. K. Putney).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. E. O'Donnell, Dept. of Human Physiology, One Shields Ave., Univ. of California, Davis, CA 95616-8644 (E-mail: meodonnell{at}ucdavis.edu).

Received 8 January 1999; accepted in final form 29 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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