Department of Human Physiology, School of Medicine, University of California, Davis, California 95616-8644
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 · min1 · 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 · min1 · 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 · min1 · 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.
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RESULTS |
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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 · 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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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 · 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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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 · 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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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