Effects of HCO3 on Cell Composition of Rabbit Ciliary
Epithelium: A New Model for Aqueous Humor Secretion
Charles W. Mclaughlin,1 David Peart,2 Robert D. Purves,5 David A. Carre,4
Anthony D. C Macknight,1 and Mortimer M. Civan4'5
whether the Na+-K+-2CF symport or the parallel Na + /H + and CF/HCO3~
antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways
are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells,
after which Na+ and Cl~ diffuse across the gap junctions into the nonpigmented ciliary epithelial
(NPE) cells and are released into the aqueous humor.
PURPOSE. TO determine
Rabbit iris ciliary bodies were preincubated in HCO3~/CO2-containing or HCO3~/CO2free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis.
METHODS.
The NPE and the PE cells contained more K and Cl when incubated with bicarbonate.
Inhibition of carbonic anhydrase with 0.5 raM acetazolamide had little effect in HCO3~-free
medium but prevented the increase in Cl in both cell types in HCO3~/CO2 solution. Inhibition of
the Na+-K+-2C1~ symport with 10 to 500 /LLM bumetanide caused Cl loss from both cell types in
HCO3~-free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3~/CO2
solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3~-free
solution and prevented the gains of Cl and Na in HCO3~/CO2 solution.
RESULTS.
The present results indicate that the dominant entry pathway of NaCl from the stroma
into the ciliary epithelial syncytium is through an acetazolamide-inhibitable C1~/HCO3~ and a
parallel Na + /H + antiport. The dominant release pathways into the aqueous humor appear to be a
Na+-K+-2C1~ symport, which can be outwardly directed under physiological conditions, together
with the Na+/K+-exchange pumps and Cl~ channels. (Invest Ophthalmol Vis Sci. 1998;39:
1631-1641)
CONCLUSIONS.
he ciliary epithelium secretes the aqueous humor.1 Inhibition of this secretion, with consequent lowering of
the intraocular pressure, is a major strategy in treating
open-angle glaucoma. Many transport mechanisms underlying
secretion are known, but it is unclear how these components
are integrated to form the aqueous humor.1"6
The ciliary epithelium is unique, comprising two cell layers whose apical membranes are juxtaposed. Secretion involves primary solute transfer with accompanying water movement from the blood or stromal side to the aqueous humor.
Transcellular transfer requires solutes and water to cross the
basolateral membranes of the pigmented epithelial (PE) cells,
pass through gap junctions7"12 linking PE cells to the nonpigmented (NPE) cells, and then cross the basolateral membranes
T
From the Departments of 'Physiology, 2 Ophthalmology, and
Pharmacology, University of Otago Medical School, Dunedin, New
Zealand; and the Departments of 4Physiology and 5Medicine, University of Pennsylvania, School of Medicine, Philadelphia.
Supported in part by a Project Grant from the Health Research
Council of New Zealand and Lottery Health and by research grants
EY08343 and EY01583 (for core facilities) from the National Institutes
of Health, Bethesda, Maryland.
Submitted for publication October 29, 1997; revised March 5,
1998; accepted April 28, 1998.
Proprietary interest category: N.
Reprint requests: Mortimer M. Civan, Department of Physiology,
University of Pennsylvania, Richards Building, Philadelphia, PA 191046085.
3
Investigative Ophthalmology & Visual Science, August 1998, Vol. 39, No. 9
Copyright © Association for Research in Vision and Ophthalmology
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of the NPE cells to reach the aqueous humor. Fluid can also be
secreted between the cells through the tight junctions between the NPE cells.8 Secretion is largely transcellular, because
inhibitors of metabolism and the Na+ pump markedly slow
aqueous humor formation.13"14
One major regulator of aqueous humor formation is
carbonic anhydrase,15 but the underlying mechanism remains to be established. In a previous article from our
laboratory,12 we established that the technique of x-ray
microanalysis could be used to detect changes in cell ion
contents in both PE and NPE cells under different conditions
of incubation. As part of that study, we found that the
presence of solution HCO3~/CO2 resulted in elevated cell Cl
in both cell types. The present study examines this finding
in more detail. We show that this elevation is prevented by
the carbonic anhydrase inhibitor, acetazolamide. Furthermore, although bumetanide, an inhibitor of Cl~ cotransport,
results in a decrease in cell Cl~ in a bicarbonate-free medium, it paradoxically increases cell Cl~ in a bicarbonate
medium. The explanation for this finding suggests a new
model for aqueous humor secretion.
MATERIALS AND METHODS
The methods used in this study have been described in detail
elsewhere.12
1631
1632
McLaughlln et al.
Cellular Model
Dutch-belted or New Zealand White rabbits of either sex, and
older than 6 weeks were obtained from the Department of
Laboratory Animal Sciences, University of Otago Medical
School, and were treated in accordance with the ARVO Resolution on the Use of Animals in Research. The animals were
anesthetized with 25 to 40 mg/kg sodium pentobarbital and
killed by injecting air into the marginal ear vein. After the eye
was enucleated, the iris-ciliary body was excised16 and cut into
quarters, and each quarter was bonded at its edge to plastic
frames with cyanoacrylate. Dissected tissue was then incubated for at least 2 hours in either bicarbonate or bicarbonatefree medium. Pairs of quadrants (one from each eye) were then
incubated separately for at least 30 minutes in a beaker under
the different experimental conditions. The temperature for
incubation was chosen to be room temperature (18-22°C) for
several reasons. First, we wanted to develop as complete a
database as possible with the ciliary epithelial cells under
similar conditions. Conducting the microprobe analyses at
room temperature complements the measurements of shortcircuit current, cell volume, and single-channel and whole-cell
currents conducted in Philadelphia at a comparable temperature. Second, conducting the experiments at the higher temperature is expected to introduce additional complexities,
both in delivering sufficient oxygen to meet the enhanced
metabolic demands and in making unstirred layers more important. These concerns were supported by our initial comparison of ionic compositions measured at room temperature
and 37°C, which indicated that the ciliary cells were less able
to maintain a high intracellular K+ concentration and low
intracellular Na+ concentration at the higher temperature under our experimental conditions (Bowler JM, Peart D, MacKnight ADC, Civan MM, unpublished data, March 1991). Third,
qualitatively similar responses in short-circuit current are
evoked by adding cardiotonic steroids or forskolin to the aqueous solution bathing rabbit iris-ciliary body at 37°c l6 - 17 anc j a t
room temperature.18
Depending on whether or not HCO3~ was included, the
gas bubbled through the solution throughout incubation consisted of either 95%O2-5%CO2 or pure O2, respectively. After
incubation, the tissues were blotted and a 30% albumin solution was applied briefly to the epithelial surface of the NPE
cells (i.e., to the basement membrane supporting the NPE
cells). Excess albumin was removed by blotting, and the tissue
segment was then plunged into liquid propane at — 180°C to
freeze the preparation quickly before ions and water could
undergo redistribution. Sections were then cut 0.2 to 0.4 jam in
thickness at -80 to -90°C with a cryoultramicrotome, freezedried at 10~4 Pa (equivalent to 7.5 X 10~7 Torr), and transferred for analysis to a scanning electron microscope QSM 840;
JEOL, Tokyo, Japan) equipped with an energy-dispersive spectrometer.
Solutions and Chemicals
The bicarbonate medium contained (in millimolar): 145 Na+,
5.9 K+, 122.1 CF, 15.0 HEPES [4-(2 hydroxyethyl)-l-piperazineethanesulfonic acid], 1.2 Mg2+, 2.5 Ca2+, 1.2 H2PO4~, 30
HCO3~, and 10 glucose at pH 7.30 to 7.45 and 305 to 315
mOsm. Bicarbonate-free solution was prepared by isosmolar
replacement of HCO3~ by Cl~. All chemicals were reagent
grade. Bovine albumin (RIA grade; Immuno Chemical Prod-
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IOVS, August 1998, Vol. 39, No. 9
ucts, Auckland, New Zealand) was dialyzed for 48 to 60 hours,
freeze-dried at -70°C, and stored at 4°C. A 30% (wt/vol)
solution was prepared by dissolving the albumin in the same
medium in which the tissue was incubated. Both acetazolamide and bumetanide were added from stock solutions in
dimethylformamide. In each case, the same concentration of
solvent vehicle (0.1% vol/vol) was applied to the parallel control preparations.
Principle of Electron Probe X-Ray Microanalysis
Both small intracellular areas and extracellular samples were
irradiated with an electron beam. Incident electrons with sufficient energy can knock electrons out of the inner shell of an
atom within the irradiated area. When an electron from an
outer orbit relaxes into the vacated orbit, a quantum of x-ray
energy is released that is characteristic of the atom that is
bombarded. In addition to these "characteristic x-rays," deceleration of the incident electrons by electrostatic interaction
with the positive atomic nucleus produces an x-ray continuum.
The intensity of this x-ray continuum or "white radiation"
depends on the mass of the irradiated region.
Data Acquisition and Reduction
The dried sections were imaged with a transmitted electron
detector. Measurements were collected with an x-ray detector
(30 mm2; Tracor Northern, Mountain View, CA), using a probe
current of 140 pA to 200 pA for 100 seconds at an accelerating
voltage of 20 kV. When peripheral standards were analyzed, at
least two large areas (^10 X 20 /xm) of albumin were examined, and the resulting data were pooled to obtain an average
value. The intracellular data were obtained by electron beam
scanning of a rectangular area within the nucleus of each
selected NPE or PE cell, which varied from ~ 0.9/Am X 1.2 /xm
to ~ 2.4ju,m X 3.0 jLtm, depending on the size of the nucleus
analyzed. It must be emphasized that a great strength of the
electron probe is the capability of directly visualizing the same
cells and subcellular targets that are analyzed, thereby ensuring
that only the analyzed epithelial cells and not the extracellular
compartment or other cellular components contribute data on
elemental composition.
The elemental peaks were quantified by filtered leastsquare fitting to a library of monoelemental peaks.19 The library spectra for Na, Mg, Si, P, S, Cl, K, and Ca were derived
from microcrystals sprayed onto a Formvar film. White counts
were summed over the range 4.6 keV to 6.0 keV and corrected
for the nontissue contributions arising from the Al specimen
holder and Ni grid.
Although we attempted to calculate ion contents and
concentrations using an external albumin standard,12 we have
found that we cannot obtain consistent data by relying on this
approach in the ciliary epithelium. The reasons for this include
problems posed by albumin's failing to adhere to the epithelial
surface, the dilution of albumin in tissue infoldings, and variations in section thickness. Therefore, as in our previous
study,'2 for purposes of data reduction, the elemental peaks
were routinely normalized to the phosphorus signal obtained
in the same scanned area of each cell. The values that we
report for Na/P, Cl/P, and K/P are the measured estimates of
the intracellular Na, Cl, and K contents, respectively. Although,
it is impossible to estimate ion concentrations in millimolar
from this data, the intracellular contents of (Na + K) or of (Na
Effects of Bicarbonate on Cell Composition
IOVS, August 1998, Vol. 39, No. 9
1633
TABLE 1. Comparisons of Bicarbonate and Bicarbonate-Free Solutions: All Available Results
Na
P
Cl
P
K
P
(Na + K)
(Na + K + Cl)
(Na + K - Cl)
P
P
P
0.145
±0.007
0.118
±0.006
0.026
±0.009
<0.05
0.259
±0.006
0.216
0.004
0.043
±0.007
<0.001
0.932
±0.010
0.902
±0.008
0.029
±0.013
1.335
±0.013
1.237
±0.010
0.099
±0.016
<0.001
0.817
±0.009
0.804
±0.009
-0.013
±0.013
NS
1.076
±0.010
1.020
±0.008
0.056
±0.013
<0.05
0.167
±0.008
0.125
±0.006
0.042
±0.010
<0.01
0.329
±0.006
0.255
±0.004
0.074
±0.007
<0.001
1.014
±0.010
0.966
±0.008
0.049
±0.013
<0.01
1.181
±0.012
1.090
±0.011
0.091
±0.016
<0.001
1.510
±0.016
1.346
±0.014
0.164
±0.021
<0.001
0.852
±0.010
0.835
±0.009
0.017
±0.013
C. Differences between incubation in HCO3 ~ and HCO3 "-free solutions
NPE cells
0.022
0.070
0.083
±0.011
±0.008
±0.014
NS
P
<0.001
<0.001
0.121
±0.016
<0.001
0.194
±0.021
<0.001
0.049
±0.013
<0.01
0.070
±0.014
<0.001
0.109
±0.017
<0.001
0.031
±0.013
A. Incubation in HCO3 -free solution!
NPE cells
302 (56)
PE cells
280 (43)
Differences
P
B. Incubation in HCO3~ solution:):
NPE cells
300 (52)
PE cells
262 (34)
Differences
P
PE cells
P
0.007
±0.008
NS
0.039
±0.006
<0.001
0.063
±0.011
<0.001
NS
NS
NS
NPE, nonpigmented epithelial; NS, not significant; PE, pigmented epithelial.
* The numbers in parentheses are the number of observations included in our previous article.1'
t Data from 43 sections from eyes of 11 animals.
! Data from 44 sections from eyes of 10 animals.
+ K 4- Cl) have been taken by many investigators to be
approximate indices of intracellular water content.20 For this
reason, the measured values of (Na + K)/P and of (Na + K +
Cl)/P are included in Table 1 and are referred to as appropriate
in the text.
Values are presented as means ± 1 SE, with n as the
number of cells analyzed. The differences between groups
have been analyzed by ANOVA using nonparametric (KruskalWallis) testing, and the probabilities (P) of the null hypothesis
have been calculated with the Dunn Multiple Comparisons
post-test.
RESULTS
Effects of Incubation in Bicarbonate Solution
Table 1 presents all the data obtained in this laboratory over
several years in which segments of ciliary body from the same
eyes have been incubated in either bicarbonate-free or bicarbonate-containing solution. Some of these data were included
in our previous article, as indicated in Table 1.
Comparison of the NPE and PE cell compositions in the
two media indicates that both cell types contained significantly
more Cl and K in the bicarbonate solution, but similar amounts
of Na. Also, the (Na + K + Cl)/P, which reflects total cell ions,
and therefore water, is significantly higher for both cell types
in a bicarbonate medium. Finally, incubation in a bicarbonate
medium has increased (Na + K — Cl)/P, which is a reflection
of the total cellular anionic charge other than that carried by
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Cl . This increase presumably results from increased cell bicarbonate under these conditions.
In both solutions, the ratios of Na/P and Cl/P were greater
in NPE cells than in PE cells. The K/P ratio was also higher in
the NPE cells but the difference only reached statistical significance for cells in bicarbonate medium. It is important to note
that these differences in content do not necessarily imply
differences in ion concentrations between the two cell types.
In part, the differences in content may reflect a lower P/dry
weight content and/or a higher intracellular water/P content of
the NPE cells. Indeed, our two measures of cell water
content([Na+K]/P and [Na+K+Cl]/P) were both significantly
higher in the NPE than in the PE cells. Possibly of greater
importance is the fact that the cell content of the measured
diffusible ions, Na, K, and Cl, must also balance charges on
multivalent organic solutes. Almost all the P signal from the
cells comes from P incorporated into nondiffusible P-containing organic compounds (phosphoproteins, phospholipids, organic phosphates), many of which carry multivalent charges.
Thus, variations in the ion/P ratios between the cell types
reflect variations in the fractions of these ions balancing
charges on nondiffusible cell macromolecules as well as alterations in the amounts of Na, K, and Cl entering and leaving the
cells. Differences between the cell types in the the nature and
amount of these nondiffusible P-containing organic compounds may well underlie the differences in die ion ratios
found between the NPE and PE cells. Since the patterns of
change were the same in the two cell types in all the experi-
1634
McLaughlin et al.
1.8
IOVS, August 1998, Vol. 39, No. 9
HCO3 -free medium
HCO3 medium
1.6
1.4
1.2
.8
.6
.4 '
.2 •
o •
-.2
Na
Cl
K
Na
Cl
K
1. Effects of acetazolamide (0.5 mM) on ciliary epithelial Na/P, Cl/P, or K/P ratios in
HCO3~ or HCO3~-free media. The medians are indicated by the central horizontal lines, the
lower and upper lines include all data between the 25th and 75th percentiles, and the
"whiskers" display the data range between the 10th and 90th percentiles. Circles are individual
data points that lie outside this range. The open and filled symbols present control and
experimental results, respectively. *Significant differences from controls (**P < 0.001). Data
were obtained from experiments using eyes from four animals: HCO3~ solution, 247 cells from
23 sections; HCO3~ solution + acetazolamide, 225 cells from 18 sections; HCO3~-free
solution, 215 cells from 19 sections; and HCO3~-free solution + acetazolamide, 225 cells from
18 sections.
FIGURE
mental protocols examined in the present study, we have
combined the data in the remaining text, figures, and the
tables.
Effects of Inhibition of Carbonic Anhydrase
Activity on Composition in Bicarbonate and
Bicarbonate-Free Solutions
Figure 1 illustrates the effects of the carbonic anhydrase inhibitor acetazolamide2' on cell composition in the presence and
absence of HCO3~/CO2. In tissues incubated in HCO3~-containing solution, acetazolamide significantly decreased the total
amount of measured diffusible solute (Na+K+Cl)/P (A =
-0.133, P < 0.001). It also significantly decreased Na/P (A =
-0.054, P < 0.001) and Cl/P (A = -0.063, P < 0.001) but not
K/P (A = -0.015, P > 0.05). In HCO3"-free medium, acetazolamide had no significant effects on the cell ion contents.
Effects of Inhibition of Na+-K+-2C1~ Cotransport
Activity on Composition in Bicarbonate and
Bicarbonate-Free Solutions
Bumetanide is a 5-sulfamoylbenzoic acid loop diuretic which
blocks the Na+-K+-2C1~ symport.22 Its effects on cell compo-
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sition in the presence and absence of HCO3 /CO2 are illustrated in Figure 2. In tissues incubated in HCO3~-free solution,
bumetanide significantly decreased the Cl/P ratio (A = —0.045,
P < 0.001) without significant changes in Na/P or K/P. In these
experiments, a 500-jaM concentration was applied to completely abolish symport activity and to avoid the theoretical
concern of adsorption of the drug onto nonepithelial tissues.
However, we have observed similar results with lower concentrations of bumetanide, as well. In an additional experiment
conducted in HCO3~-free solution, the mean Cl/P ratio for the
NPE and PE cells was measured as follows: 0.175 ± 0.005
(control, n = 48), 0.125 ± 0.006 (10 yM bumetanide, n = 50),
0.101 ± 0.004 (100 JU-M bumetanide, n = 50), and 0.114 ±
0.004 (500 jitM bumetanide, n = 50). Evidently, a maximal
effect was reached at 100 to 500 /xM bumetanide, with a mean
Cl/P value of 0.108 ± 0.006. Thus, 80% to 90% maximal
inhibition of the symport was achieved by adding 10 /xM
bumetanide.
In contrast to its effects in HCO3~/CO2-free solution, bumetanide significantly increased the Cl/P in tissues incubated
with HCO3 (A = +0.099, P < 0.001, Fig. 2). This increased
Cl was associated with increased Na/P (A = +0.144, P <
Effects of Bicarbonate on Cell Composition
IOVS, August 1998, Vol. 39, No. 9
1.8
HCO3 medium
1635
HCO3 -free medium
1.6
1.4
1.2
c_
o
1
U
Z .8
o
-.2
Na
Cl
K
Na
Cl
K
2. Effects of bumetanide (0.5 mM) on ciliary epithelial Na/P, Cl/P, or K/P ratios in
HCO3~ or HCO3~-free media. The medians are indicated by the central horizontal lines, the
lower and upper lines include all data between the 25th and 75th percentiles, and the
"whiskers" display the data range between the 10th and 90th percentiles. Circles are individual
data points that lie outside this range. The open and filled symbols present control and
experimental results, respectively. *Significant differences from controls (**P < 0.001). Data
were obtained from experiments using eyes from three animals: HCO3~ solution, 158 cells
from 13 sections; HCO3~ solution + bumetanide, l6l cells from 14 sections; HCO3~-free
solution, 172 cells from 15 sections; and HCO3~-free solution + bumetanide, 148 cells from
12 sections.
FIGURE
0.001) and some decreases in K/P which did not reach significance (A = -0.027, P > 0.05). The net effect of these changes
in a HCO3~ solution was that measured cell solute
[(Na+K+Cl)/P] rose significantly (A = +0.216, P < 0.001)
following exposure to bumetanide.
The presence of both bumetanide and the carbonic anhydrase inhibitor acetazolamide, in a bicarbonate-free medium,
resulted in greater loss of Cl (AC1/P = -0.091, P < 0.001),
together with K (AK/P = -0.071), than occurred with either
inhibitor alone (Fig. 3 compared with Figs. 1 and 2). That is,
the combination of inhibitors resulted in a loss of —44% of cell
Cl over 30 minutes compared with the loss of —20% with
bumetanide alone. In HCO3~/CO2 solution, the combination
of inhibitors prevented the gains in Cl and Na seen with
bumetanide alone. Indeed, cells lost some Na (ANa/P =
-0.054, P < 0.001) and gained K (AK/P = -0.079, P <
0.001).
DISCUSSION
Both bicarbonate and carbonic anhydrase are known to modulate aqueous humor formation,1 but the underlying mecha-
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nisms have been uncertain. Recent reports have stressed the
potential importance of the membrane-bound form (CA-IV) of
carbonic anhydrase.23'24 In particular, a membrane-impermeant polymeric form of carbonic anhydrase inhibitor has
been reported to increase the short-circuit current across the
isolated rabbit ciliary epithelium only when applied to the
aqueous (NPE) surface.23 Studies of cultured rabbit NPE cells
have indicated that CA-IV likely contributes 20% to 30% of the
carbonic anhydrase activity displayed by these cells.24 These
recent results and earlier intracellular recordings of the intact
rabbit ciliary epithelium3 have led to a focus on the possible
role of carbonic anhydrase and bicarbonate at the aqueous
surface of the ciliary epithelium. However, a substantial fraction of the total solute secreted by the rabbit ciliary epithelium
may proceed by electroneutral transport both across the stromal-PE and NPE-aqueous interfaces.25 The results of the
present study bear on this possibility. Wiederholt et al.2 have
stressed the potential importance of bicarbonate-mediated
stimulation of Cl~ uptake at the stromal surface through a
C1~7HCO3~ antiport. The relative quantitative contribution of
this mechanism has been unclear, given the operation of a
Na+-K+-2CF symport at the same membrane.4 The Na+-K+-
1636
McLaughlin et al.
1.8
IOVS, August 1998, Vol. 39, No. 9
HCO3 medium
0
HCO3 -free medium
1.6
1.2
o 1
U
re"
z ,s
o
.4
0 '
Na
Cl
K
Na
Cl
K
3. Effects of bumetanide (0.5 mM) and acetazolamide (0.5 mM) on ciliary epithelial
Na/P, Cl/P, or K/P ratios in HCO3~ or HCO3~-free media. The medians are indicated by the
central horizontal lines, the lower and upper lines include all data between the 25th and 75th
percentiles, and the "whiskers" display the data range between the 10th and 90th percentiles.
Circles are individual data points that lie outside this range. The open and filled symbols
present control and experimental results, respectively. *Significant differences from controls
C*P < 0.001). Data were obtained from experiments using eyes from two animals: HCO3~
solution, 99 cells from 8 sections; HCO3~ solution + acetazolamide + bumetanide, 96 cells
from 8 sections; HCO3~-free solution, 96 cells from 8 sections; and HCO3~-free solution +
acetazolamide + bumetanide, 97 cells from 8 sections.
FIGURE
2C1 symport has been functionally detected in the ciliary
epithelium26 and in isolated PE427 and NPE cells.28"32
In the present study, exposure to HCO3~/CO2 increased PE and NPE cell Cl, K, and water (taking Na+K+Cl
as an index of volume) (Table 1). Paradoxically, inhibiting
the Na+-K+-2C1~ symport with bumetanide in the presence
of HCO3~/CO2 increased cell Cl. These results strongly
indicate that C1~/HCO3~ exchange is the dominant pathway
for Cl~ uptake from the stroma by the PE cells (Fig. 4). Na+
can accompany Cl~ uptake into the PE cells through the
Na + /H + antiport and be subsequently exchanged for K+ by
the Na+/K+-exchange pump (Fig. 5), leading to a net gain of
cell KC1 (Table 1).
The present data also suggest the likely major site of action
of the clinically important carbonic anhydrase inhibitors. In the
presence of HCO3~/CO2, acetazolamide decreased the rate of
formation of HCO3~ and H + , thereby slowing uptake of NaCl
through the paired Cr/HCO 3 ~ and Na + /H + antiports (Table
1, Fig. 4). As anticipated, the carbonic anhydrase inhibitor had
little effect on Cl~ uptake in the absence of HCO3~/CO2. It
must be emphasized that the contribution of the C1~/HCO3~
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antiport to Cl uptake at the stromal-PE cell interface cannot
be precisely quantified from the current data because of a
possible contribution to Cl~ uptake from C1~/HCO3~ antiport
activity at the NPE cell-aqueous interface, as well.
Exposure to bumetanide, an inhibitor of the Na+-K+2C1~ symport, resulted in the expected significant loss of Cl
in a bicarbonate-free medium. Cell Na and K were not
altered significantly, presumably because other ion transport
pathways were able to compensate for the decreased rate of
entry of Na and K on the transporter. However, with
HCO3~/CO2 present, bumetanide produced a paradoxical
increase in the cell Cl content of both PE and NPE cells. This
increase was associated with a large gain of Na, partly in
association with the gain in Cl and partly to replace K lost
from the cells. This unexpected effect of bumetanide can
be explained if, in the presence of HCO3~/CO2, the net
driving force for the Na+-K+-2C1~ cotransporter is oriented from cell to extracellular fluid (Fig. 4). Thus, under
physiological conditions (with HCO3~ present), the transporter removes Cl"" brought into the cells by the Cl~/
HCO3~ exchanger; inhibition by bumetanide now reduces
Effects of Bicarbonate on Cell Composition
IOVS, August 1998, Vol. 39, No. 9
1637
CO, / HCO " present
PE
Stroma
Na+
Na+
Cl"
cr
CO 2 / HCO3" absent
B
Stroma
PE
Na+
Na+
K+
Cl
2CI"
Na+
cr
FIGURE 4. Model of aqueous humor secretion: uptake of NaCl from the stroma into the
pigmented ciliary epithelial (PE) cells. For purposes of clarity, only a few components among
the full array of pumps, symports, antiports, and channels considered elsewhere3 are included
in Figures 4 and 5. In the presence of HCO3~/CO2 (A), Na+ and Cl~ are largely taken up by
the PE cells at the stromal surface by the parallel Na + /H + and C1~/HCO3~ antiports, whose
turnover rates exceed that of the oppositely directed Na+-K+-2C1~ symport. Carbonic
anhydrase (CA) ensures the adequate delivery of HCO3~ for C1~/HCO3~ exchange. In the
absence of HCO3~/CO2 (B) or when acetazolamide inhibits carbonic anhydrase, continued
NaCl secretion into the aqueous humor depletes the epithelial cells of NaCl, so that the
thermodynamic driving force now favors uptake of Na+-K+-2C1~ into the PE cells, albeit at a
reduced rate.
this Cl removal, thereby increasing the cell content of
this ion.
As illustrated in Figure 6, calculations using a range of cell
ion concentrations, indicate that the driving force for the
Na+-K+-2C1~ symport is such that relatively small changes in
cell Cl, consistent with the differences in Cl that we have
observed between bicarbonate-free and bicarbonate media,
would alter the direction of net ion movement through the
symport.
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The continued entry of Na+ in exchange for H + in the
situation where inhibition of the cotransporter limits Na+
extrusion, results in increased cell Na. The decrease in cell K is
less easily explained but may result from increased cell volume,
possibly together with change in cell pH, opening basolateral
membrane K+ channels.33
It will be appreciated that bumetanide and other loop
diuretics, like most pharmacologic inhibitors, have been reported to exert multiple effects on membrane transport at high
1638
McLaughlin et al.
IOVS, August 1998, Vol. 39, No. 9
CO, / HCO " present
NPE
Aqueous
Na'
cr
B
CO 2 / HCO3" absent
NPE
2K
+
Aqueous
Na+
Na+
cr
cr
FIGURE 5- Model of aqueous humor secretion: Release of NaCl from the nonpigmented ciliary
epithelial (NPE) cells into the aqueous humor. After uptake at the stromal surface (Fig. 4), Na+
and Cl~ cross gap junctions to enter the NPE cells from the PE cells. In the presence of
HCO3~/CO2 (A), uptake of NaCl by the PE cells is sufficiently active to establish a thermodynamic driving force favoring Na+-K+-2C1~ symport release into the aqueous humor. Under
these conditions, Cl~ is released into the aqueous humor through the outwardly directed
Na+-K+-2CP symport and Cl~ channels. Na+ is secreted through the Na+/K+-exchange
pumps and the symport. In the absence of HCO3~/CO2 (B) or when acetazolamide inhibits
carbonic anhydrase, uptake of NaCl by the PE cells is slowed, reducing the NaCl concentration
within the ciliary epithelial syncytium and favoring Na+-K+-2C1~ uptake from the aqueous
humor into the NPE cells. Under these conditions, Cl~ is released only through Cl~ channels,
and Na+ is secreted only through the pump.
concentrations in some preparations, including inhibition of
Cl~ channels, carbonic anhydrase, and cell metabolism.34
However, in HCO3~/CO2-free solution, bumetanide exerted
80% to 90% of the maximal effect at 10 /uM, an order of
magnitude lower than the concentration at which nonspecific
effects have been reported.34 Furthermore, none of these nonspecific effects could have produced the observed results: (i)
Blockage of Cl~ channels in HCO3~/CO2-free solution would
have increased the Cl content, contrary to observation, (ii)
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Inhibition of carbonic anhydrase should have produced effects
similar to those resulting from acetazolamide; actually, bumetanide reduced Cl/P only in HCO3~/CO2-free solution, whereas
acetazolamide reduced Cl/P only in HC03~/C02-containing
solution, (iii) Had bumetanide been acting as a nonspecific
metabolic inhibitor, similar results should have been exerted in
HCO3~/CO2-free and HCO3~/CO2-containing solutions,
whereas diametrically opposite results were observed. Thus,
nonspecific effects are unlikely to have significantly influenced
Effects of Bicarbonate on Cell Composition
IOVS, August 1998, Vol. 39, No. 9
1639
2.5-,
2-
1.5-
-9—
changing [Cl] & [K]
O
changing [Cl] & [Na]
O -
changing [Cl] & [Na+K]
0.5-
30
40
60
50
70
80
cell [Cl]
6. The sensitivity of the net direction of ion movement through the symport to cell
ion concentrations in a HCO3~/CO2 medium. Because the Na+-K+-2C1~ symport is electroneutral, the thermodynamic driving force favoring outward movement is proportional to the
logarithm of the term {[Na+]C[K+]C[C1~]C2}, where the subscript c refers to the fluid phase
within the ciliary epithelial syncytium. The thermodynamic driving force favoring inward
movement is proportional to the logarithm of the term {[Na+]e[K+]e[Cl~]e2}, where the
subscript e refers to the extracellular fluid outside the ciliary epithelial syncytium. The ratio
{[Na+]JK+]e[Cl]e2}/{[Na+]c[K+]c[Cr]c2} is plotted on the j-axis. Values greater than 1 indicate net movement from medium to cell, values less than 1 indicate net movement out of cell.
It is assumed that electroneutrality is maintained by matching the increase in [Cl] to increases
in the diffusible cations (Na and K). To illustrate the relative sensitivity of the ratio to changes
in the ion concentrations, three lines are shown. In one, it is assumed that the increase in [Cl]
is matched by a similar increase in [K], with [Na] remaining constant. In a second, it is assumed
that the increase in [Cl] is matched by a similar increase in [Na], with [K] remaining constant,
and in the third, [Na] and [K] increase equally with the increase in [Cl].
FIGURE
the observed responses to bumetanide. The same conclusion
holds for acetazolamide. Were the observed changes in composition produced by nonspecific actions of the carbonicanhydrase inhibitors, we would have expected acetazolamide
to exert similar effects on the Cl content whether or not
HCO3~/CO2 was present, contrary to observation. The precise
mechanism for the acetazolamide-triggered inhibition of C\~/
HCO3 exchange cannot be identified from the data obtained.
It is reasonable that the inhibition reflects a reduction in the
rate of delivery of HCO3~ to the exchanger. However, secondary changes involving inter alia, intracellular pH and Ca2+
activity, could also play a role.
The hypothesis illustrated in Figures 4 to 5 is supported by
the results obtained when both bumetanide and acetazolamide
are present in the media (Fig. 3). In a HCO3~-free solution, this
combination results in a significantly greater loss of Cl (accompanied by K) than occurs with either acetazolamide or bumetanide alone. In a HCO3~ medium, the inclusion of acetazolamide with bumetanide prevented Cl accumulation. Instead,
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cells lost Cl and Na and gained K. These findings can be
explained by a combination of decreased HCO3~ (and H + )
formation and extrusion, together with inhibition of K"1" extrusion on the cotransporter.
These results suggest a new model for aqueous humor
formation (Figs. 4, 5). Secretion is fundamentally based on the
Na+/K+-exchange pumps thought to be in higher concentration at the aqueous than at the stromal surface. However, the
current data now indicate that Cl and Na+ uptake at the
stromal surface proceeds by C1"~/HCO3~ and Na + /H + exchange, at turnover rates higher than that of the Na+-K+-2C1~
cotransporter whose net driving force is directed oppositely,
back into the intersititial fluid. We now suggest that Cl~ is
released at the contralateral surface from the NPE cells into the
aqueous humor by the Na+-K+-2C1~ cotransporter (whose
thermodynamic driving force favors secretion), as well as by
passage through Cl~ channels (Fig. 5). The Na+/K+-exchange
pumps recycle the K+ extruded by the cotransporter and
contribute to net secretion by moving additional Na+ into the
1640
McLaughlin et al.
aqueous humor. The net movement of ions through the cells
drives osmotic water flow through both aquaporin-1 water
channels of the NPE cells35 and the paracellular pathway with
resultant isosmotic secretion.
It must be emphasized that the same Na+-K+-2C1~ cotransporter can provide net ion transport in either direction,
depending on the net thermodynamic force (Fig. 6). In this
respect, the hypothesis is entirely consistent with previous
data obtained by other techniques. For example, Dong and
Delamere29 detected bumetanide-sensitive 86Rb efflux from
ouabain-treated, but not from control, cultured rabbit NPE
cells. This agrees with our observation that ouabain increases cell Na by about fivefold while reducing cell K by
only about half, with possibly some decrease in Cl content.12 The cell ion concentrations must broadly reflect
these changes and the calculated thermodynamic driving
force for ion efflux through the symport is reversed so that
there is now net ion efflux through this pathway in a
ouabain-containing medium. The novel concept of this study
is that the Na+-K+-2C1~ symport can support net movement
out of the ciliary syncytium into the aqueous humor under
our baseline conditions (with CO2 and bicarbonate present).
The net driving force on the symport can well be oppositely
directed in preparations of freshly isolated and cultured
cells,4'29 given its dependence on the square of the intracellular Cl~ concentration. It is of interest that data obtained by
Keep et al.36 led them to propose a model of cerebrospinal
fluid secretion very similar to the present hypothesis of
aqueous humor secretion.
The current model is based on analyses of rabbit tissues
studied in vitro, so the extent of its applicability to the secretion of aqueous humor in vivo in humans is uncertain. The
hypothesis does, however, accommodate several otherwise
puzzling observations: the effectiveness of carbonic anhydrase
inhibitors in reducing aqueous humor formation,15 the basis
for the large fraction of solute secretion through an electroneutral pathway,25 and the inability of bumetanide alone to
suppress secretion.37 The current model predicts that bumetanide together with a Cl "-channel blocker would be necessary
to fully suppress secretion by the NPE cells and that a supplemental carbonic anhydrase inhibitor would be necessary to
also suppress uptake of NaCl by the PE cells at the stromal
surface.
Acknowledgment
The authors thank Sylvia Zellhuber-McMillan for superb technical
support in the x-ray microanalysis studies.
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Erratum in: "Essential Iris Atrophy, Pigment Dispersion, and Glaucoma in DBA/2J Mice," by
John et al. (Invest Ophthalmol Vis Sci. 1998;39:951-962).
Figure 2 on page 957 and Figure 3 on page 958 were cropped erroneously, and as a result
the scale bars were shortened in the middle and right columns of the figures. The scale bars
should be the same as those shown in the left column of each figure. Corrected reprints can
be obtained from the author. The Journal regrets the errors.
Erratum in: "The Role of Eosinophils and Neutrophils in Helminth-Induced Keratitis," by
Pearlman et al. (Invest Ophthalmol Vis Sci. 1998;39:1176-1182).
Figure 3 on page 1179 should have been printed in color so that eosinophils can be
distinguished from neutrophils. The color version of the figure is printed below. Corrected
reprints can be obtained from the author (e-mail: [email protected]). The Journal regrets the
error.
C57BI/6
Eosinoohils
Neutraohils
IL-5 ko
FIGURE 3. Clinical and histologic appearance of corneas of interleukin (IL)-5 gene knockout (IL-5 ' ) mice after intrastromal
injection of helminth antigens. C57B1/6 and IL-5-/~ mice w^ere immunized and injected intrastromally with Onchocerca volvulus
antigens as described in the Materials and Methods section. Seven days later, corneal opacification and neovascularization had
developed in both mouse strains (leftpanels). Eyes werefixedin formalin, and adjacent sections were immunostained with antisera
to eosinophil major basic protein (MBP: center panels) or anti-neutrophil monoclonal antibody 7/4 (right panels). Note the
presence of eosinophils in the epithelial layer and extracellular MBP (upper center panel) and the presence of neutrophils in the
anterior chamber (lower right panel)- Original magnifications: left panels X25; all others X400. Sections are representative of two
experiments conducted in groups of 15 mice each.
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