1 Laboratory of Ocular Physiology and Biochemistry, Department of Optometry and Radiography, Hong Kong Polytechnic University, Hung Hom, Hong Kong; and Departments of 2 Ophthalmology and 3 Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029
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ABSTRACT |
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The possible existence
of transepithelial bicarbonate transport across the isolated bovine
ciliary body was investigated by employing a chamber that allows for
the measurement of unidirectional, radiolabeled fluxes of
CO2 + HCO was used, a net Cl
flux of
1.12 µeq · h
1 · cm
2 (30 µA/cm2) in the blood-to-aqueous direction was detected.
Acetazolamide, as well as removal of HCO
flux and
Isc. Because such removal should increase
HCO
channels. The acetazolamide effect on Cl
fluxes can be
explained by a reduction of cellular H+ and
HCO
via Na+/H+ and
Cl
/HCO
transport. The fact that the net Cl
flux is about three times larger than the Isc is
explained with a vectorial model in which there is a secretion of
Na+ and K+ into the aqueous humor that
partially subtracts from the net Cl
flux. These transport
characteristics of the bovine ciliary epithelium suggest how
acetazolamide reduces intraocular pressure in the absence of
HCO
aqueous humor secretion; chloride fluxes; bicarbonate fluxes; Ussing chamber; short-circuit current
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INTRODUCTION |
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IT IS WIDELY ACCEPTED that the two-cell-layered epithelium of the ciliary body is responsible for the production of the aqueous humor that circulates through the chambers of the anterior segment of the eye and maintains intraocular pressure (IOP). Most investigators believe that a large fraction, if not all, of the aqueous humor is produced by a mechanism of secretion secondary to active ionic transport of the epithelial layer (12, 14, 21, 24). Thus, to reduce IOP in glaucoma, one common strategy is to target transport mechanisms to bring the secretion down to levels comparable to the reduced aqueous outflow.
To better understand how this can be accomplished, during the past 15 years several laboratories have developed techniques to study the
transport properties of the isolated ciliary body to identify
transporters, exchangers, and channels involved in this secretory
process (3, 8, 16, 23, 26, 28, 29, 34, 35). Despite
intensive research and the identification of several of the transport
elements present in the ciliary epithelium, a satisfying model
consistent with all the available experimental data is still elusive.
There are, in our opinion, mainly three reasons for this situation: the
complexity of this epithelium with two juxtaposed apical membranes, the
contribution of the capillary pressure to aqueous humor secretion by
ultrafiltration, and the possible variability in transport elements
among the studied species. Despite a similar anatomic configuration and
the presence of common transport elements in the rabbit and bovine
ciliary epithelium, differences in transport properties were already
apparent. While HCO transport in the same direction seems to account for
the short-circuit current (Isc) in the bovine
tissue (14, 30). Very recently, Crook et al.
(12) also described a net Cl
transport as a
component of the Isc of the isolated rabbit
ciliary epithelium. Because acetazolamide, an inhibitor of the carbonic anhydrase enzyme (CA), reduces the Isc in the
bovine preparation (unpublished observations), we thought that a
transepithelial transport of HCO
Another previous puzzling finding with the bovine ciliary epithelium
was a net Cl flux that was consistently two to three
times larger than the Isc, without any other net
ionic transport to account for the discrepancy. Here we report that we
were unable detect any net flux of HCO
flux by
acetazolamide, by reducing the substrates available to parallel
Na+/H+ and
Cl
/HCO
flux may be larger than the Isc and why
HCO
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METHODS |
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Freshly enucleated bovine eyes were obtained from a local
abattoir and used in our experiment. The detailed procedure of tissue preparation has been described elsewhere (14, 30).
Briefly, a sector of intact bovine iris ciliary body was dissected and mounted in either of two types of modified Ussing chambers. Only the
ciliary body was exposed to the chamber cavity with a cross-sectional area of 0.30 cm2 in both chambers. The standard
HEPES-buffered Ringer solution comprised (in mM) 113.0 NaCl, 4.6 KCl,
21.0 NaHCO3, 0.6 MgSO4, 7.5 D-glucose, 1.0 glutathione (reduced form), 1.0 Na2HPO4, 10.0 HEPES, and 1.4 CaCl2.
To obtain HCO fluxes, a
previously described chamber was used (30). Essentially, it consisted of two potential-sensing tubes that were connected to each
other by a bypass arm through three-way stopcocks. At the beginning of
the experiment, both the potential-sensing tubes and the bypass arm
were filled with normal Ringer solution. The potential-sensing tubes
were then fitted into the PD-sensing arms of the chamber. By switching
the three-way stopcocks to a neutral position (A), any junction
potentials between the 0.9% NaCl and Ringer solutions or associated
with the Ag-AgCl electrodes could be nullified. The stopcocks were then
switched back to a measuring position (B) to allow for the measurement
of the electrical parameters across the preparation in the chamber. At
that point, the fluid junction was exactly the same as in
position A (0.9% NaCl and Ringer solutions). The offset
potential was checked frequently with the bypass arm and stopcocks
throughout the experiment.
Measurement of radiolabeled Cl flux.
In the initial experiments, after a brief stabilization period, Ringer
solution with acetazolamide was perfused to the preparation until its
full effect on the Isc was demonstrated. Later,
a radiolabeled (hot) solution with that drug was perfused and allowed
to equilibrate before the flux measurements, as described earlier
(30), were performed. In the subsequent experiments, we
loaded the drug together with the hot solution and skipped the
drug-only treatment. The results were not different from those before,
and the electrical parameters remained stable throughout the experiments.
Measurements of unidirectional fluxes of
CO2 and
HCO
Radiolabeled isotopes and pharmacological agents.
The radioactivity was measured with a liquid scintillation counter
(Wallac 1414 Winspectral DSA). The radiolabeled isotopes [14C]sodium bicarbonate and [36Cl]sodium
chloride were purchased from Amersham Radiochemicals. The
HCO
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RESULTS |
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Fluxes of labeled CO2 + HCO
Table 1 shows the results of nine
experiments in which the effects of acetazolamide (104 M
in both bathing solutions) on the unidirectional fluxes of labeled
CO2 + HCO
1 · cm
2 net flux
is statistically not significant and is also opposite to the expected
direction to drive fluid into the posterior chamber. In all but one
experiment, acetazolamide produced a small (~6%) increase, which was
statistically significant as paired data but still inconsequential. The
possible reasons for this increase will be discussed later.
Acetazolamide, however, had a clear inhibitory effect on the
Isc (except for 1 experiment) amounting to an
average decrease of 38%. In our existing database of 21 experiments,
acetazolamide inhibits the Isc from 11.7 ± 1.29 to 7.6 ± 0.83 µA/cm2, a 35% decrease.
Thus, by an undetermined mechanism, acetazolamide must be inhibiting
the Cl
transport, which is the main component of the
Isc. Further confirmation that the bovine
ciliary body lacks a HCO
4 M)
had no effect on the control unidirectional fluxes of ~0.20 µeq · h
1 · cm
2 (data not
shown).
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In three other experiments, after control values were established in
HCO1 · cm
2,
indicating that ~75% of the label moves as HCO
Because we could not detect an effect of acetazolamide on
HCO
fluxes, which seem to support the Isc. These
results are shown in Table 2.
Acetazolamide in either bathing solution clearly decreases the
blood-to-aqueous Cl
flux (Jba)
while having a smaller, not significant, inhibitory effect on the
opposite flux. The decrease in Jba by
acetazolamide on the aqueous side was statistically significant. The
reduction in the net flux amounted to 42% but had a borderline
significance of P = 0.12. The effect of acetazolamide
on the blood side was less pronounced, and a significant net flux was
detected.
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In the rabbit ciliary body, where transport of HCO transport. Indeed, in 10 experiments in which we tested the effect of
CO2/HCO
fluxes, Jab
increased from the control 5.20 ± 0.30 to 6.20 ± 0.24 µeq · h
1 · cm
2 with
little effect on Jba and reducing the net
Cl
flux by 58%.
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Because changes from a HCO exchangers that supply part
of the Cl
uptake that subsequently exits the NPE and
contributes to the Isc. However, as shown in
Table 4, changing the bubbling from 5%
CO2 to air (which increases the solution pH to 8.7) did not reduce the Isc; on the contrary, a small
increase was observed. The effect was reversible, because the
electrical parameters returned to control values when 5%
CO2 bubbling was reintroduced. Thus CO2 removal
was not the cause of the Isc reduction. When
CO2 and HCO
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It is possible that the effects of the absence of
HCO
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DISCUSSION |
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The ciliary epithelium that lines the posterior chamber side of
the ciliary body is a complex epithelium with two cell layers juxtaposed by their apical side. Many of its transport characteristics and components including channels and transporters have been identified (1, 12, 15, 19, 20, 31). It is widely known that the
composition of the aqueous humor of different species varies, particularly with regard to its Cl and
HCO
concentration is higher and
HCO
transport toward
the aqueous humor (14, 30), which is consistent with a
higher-than-plasma concentration of Cl
in the aqueous
humor, as found in humans. Despite these differences, inhibitors of CA
reduce aqueous Cl
concentration in humans and
HCO
In this work we have shown that there is no noticeable transport of
HCO fluxes and
Isc, and the reasons for the discrepancy between
net Cl
transport and Isc.
We have previously shown the reliability of our system for the
measurement of CO2 + HCO (6).
In the present experiments, baseline fluxes were very stable, equal in both directions, and unaffected by acetazolamide and DIDS. These findings support the notion that there is no bicarbonate transporter and channels working in unison to produce a net transepithelial flux
and that the inhibitory effect of acetazolamide must be on Cl
fluxes. The small increase in both unidirectional
CO2 and HCO
/HCO
/HCO
Regardless of the possibility that H+ and
HCO
Because an HCO transport, which
accounts for more than the measured Isc. Indeed, Table 2 shows a net Cl
flux from blood to aqueous humor
as well as its inhibition by acetazolamide. Even though the net
Cl
flux persists in the two experimental conditions, it
is smaller by 42 and 21%, respectively. More importantly, the decrease
in Jba is statistically significant for
acetazolamide on the aqueous side, whereas the decrease in
Jab is not. A logical conclusion is that
acetazolamide reduces Isc by inhibiting the
Jba Cl
flux.
Also, the inhibition of the Isc produced by
HCO flux. Indeed, we have found a
58% net Cl
flux reduction in HCO
flux was due to an
increase in the back flux Jab with little effect
on Jba, the paracellular pathway should be
involved. A decrease in transcellular Jba
simultaneous with an increase in its paracellular component will leave
this unidirectional flux unchanged, while increasing the opposite
Jab flux and thus reducing the net. If so, this
would also preclude the detection of resistance changes. The
possibility that the effect of aqueous HCO
flux was confirmed with
experiments in which electrical parameters were measured. As shown in
Table 3, the effect of HCO
/HCO
We tried to establish whether there was a direct relationship between
the inhibitory effects of acetazolamide and the removal of
HCO/HCO
exit at the basolateral side of the NPE. As
indicated before, this interpretation cannot be applied to the
HCO
and K+
permeability with a larger effect on Cl
, thus reducing
the Isc. In addition, cell acidification, which is known to close gap junctions, will reduce the
Isc across this tissue (34).
We propose a model (shown in Fig. 1) of
the ciliary epithelium that includes channels and transporters
well-characterized in bovine and rabbit tissues. This model is similar
to those previously proposed by several investigators (14, 24,
32). We should note, however, that our previously proposed model
(14) has been modified. The interpretation of new data in
the present study compels us to include the parallel
Cl/HCO
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The main improvements in this model are the quantification of all vectorial fluxes and its dynamic nature that allows for the changes in one vector to be transmitted to all others so that a new steady state is attained. This is accomplished by the use of spreadsheet software (e.g., QPRO or Excel) in which two basic principles are preserved: 1) the net ionic current is the same at each limiting membrane and is equal to the Isc; and 2) the total flux of an ion leaving the cell compartment is equal to the total flux entering the cell compartment for each ion. Descriptions of the equations in the spreadsheet cells are provided in APPENDIX A.
Figure 1 shows the control condition with the average values of the
Isc and vectorial fluxes compiled from many
experiments during the past five years. Notice that the net
Cl flux emerging from the NPE (27.0 µA/cm2)
is 3.23 times larger than the Isc. This was a
consistent finding difficult to explain up to the present time. The
model shows that the difference is due (at the aqueous side) to the
pump-originated Na+ flux and the net K+ efflux.
For simplicity we have chosen two vectors to be variable inputs to the
system: the Cl
entering the cell from the blood side via
the two transporters. The relative value of these two vectors was
selected on the basis of previous results indicating the inhibition of
DIDS and furosemide on Cl
fluxes. Obviously, it could be
modified. All the other vectors are interconnected in such a way that
the two enunciated principles are respected. At the bottom of the
model, the relative activity of the two oppositely directed
Na+-K+ pumps, the relative permeability of the
NPE and PE K+ channels, and the relative contribution of
the aqueous-side Cl
/HCO
permeability of the basolateral
Cl
channel of the NPE is considered to be 1 in the
control condition but can be changed.
The "0.70" value means that 70% of the Na+ entering
the epithelium from either side will leave the cell via the NPE pump;
likewise for the K+ flux (72%). These values were chosen
on the basis of the effects of ouabain and K+ channel
inhibitors (26). It was found that the effects were larger
at the aqueous side. These values also can be changed; this will not
affect the Cl fluxes but will change the
Isc and some of the other vectors. Also shown in
the model inside the cell is a nonvectorial production of
H+ and HCO
, they must be equal to each other at the basolateral
membranes. Although we have chosen the Cl
flux exchanged
for HCO
fluxes at the PE basolateral membrane, 21.0 and 6.0 µA/cm2. All the other values are results of
well-established relations necessary to satisfy the defined conditions
and steady state. To test this model, we can analyze the effect of
acetazolamide on the Isc and Cl
fluxes. In the model shown in Fig. 2, we
have reduced the production of H+ and
HCO
entering the cell via the
Cl
/HCO
entry is coupled to available HCO
flux to 21 and
the Isc to 5.95 µA/cm2,
which is consistent with the observed experimental results. In this
example, we reduced the H+ and HCO
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Going back to Fig. 1, we can see that under short-circuit conditions,
there is a net secretion of NaCl and KCl with a small HCO
In summary, we have presented results indicating that
HCO fluxes and Isc by reducing the
available HCO
,
which contributes in large measure to the Isc.
We chose a model in which the ratio of Cl
entering the PE
via the 2Cl
-K+-Na+ cotransporter
to the that entering via the Cl
/HCO
We think that the bovine ciliary epithelium may be a good model to study the mechanisms of aqueous secretion and to understand the effects of pharmacological agents used to reduce IOP in humans.
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APPENDIX A |
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For those interested in reproducing the model to try different
variations, the equations for the relevant spreadsheet cells are
indicated below.
A3 = (A4 ![]() ![]() ![]() ![]() ![]() |
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A4 = (D11) | |
A12 = (1 ![]() |
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A15 = (1 ![]() |
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A18 = (C17) | |
C5 = (A4) | |
C7 = (0.5*C8) | |
C8 = (A8*F22) | |
C9 = (C7) | |
C13 = (0.6666*A12) | |
C17 = (A17*F22) | |
D10 = (D11) | |
D11 = (A18 + H18) | |
F13 = (0.6666*H12) | |
F17 = (H18) | |
H3 = (H8 + F13 + H18 ![]() ![]() ![]() |
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H8 = (C8 + C17 + F17) | |
H12 = (B21*(C9 + C5)) | |
H15 = (B22*(F13 + C7 + C13)) | |
H18 = (F21*A18*F22) | |
J5 = (H8 ![]() |
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J6 = (H18) | |
J7 = (H12) | |
J8 = (H15 ![]() |
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APPENDIX B |
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We give three examples for the utilization of the spreadsheet model presented in the DISCUSSION with cells identically defined as in the legend to Fig. 1 and in APPENDIX A.
We can observe what happens if the relative K+ permeability
of one of the sides is changed, for example, by Ba2+
blockade of the PE or NPE side. In Fig.
3, a blockade of the NPE K+
channels is simulated, which results in an increase of the
Isc without a change in the Cl
fluxes. A similar effect of less magnitude is obtained by ouabain on
the NPE side. On the contrary, Ba2+ or ouabain on the PE
side will decrease the Isc. These effects have
been observed experimentally (26).
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In another example (Fig. 4), we can
simulate the effect of reducing the permeability of the
Cl channel on the aqueous side of the NPE by changing the
control value of 1 to 0.6. By simply restricting the output of
Cl
, the model gets unbalanced unless the Cl
influx across the transporters in the PE is also restricted. This is
accomplished by making the Cl
fluxes across the
transporters (cells C8 and C17) equal to their respective inputs (cells
A8 and A17) multiplied by the Cl
permeability. Figure 4
then simulates the effect of HCO
flux and Isc.
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We can also explain our previously reported results that reducing
Cl concentration in the bathing solutions to 60 mM
abolished the Isc (14). Further
reduction produced a reversal of the Isc. We can
speculate that at this Cl
concentration, the
Na+-K+-2Cl
cotransporter is
dominated by the large K+ gradient from cell to bath and
reverses direction [Cl
(60:15)2 + Na+(136:15) < K+(140:4.6)], where 15, 15, and 140 are assumed millimolar cellular concentrations of
Cl
, Na+, and K+,
respectively. We have represented this situation in Fig.
5, in which we reduced the activity of
the Cl
/HCO
flux across the
Na+-K+-2Cl
cotransporter of
3.00. This reduced the Isc to near zero and produced an absorption of KCl across the NPE. One must realize that the
values in the model are only approximations because possible changes in
electrical potentials are not considered. Nevertheless, the model is
self-consistent and is a useful aid in interpreting and predicting
experimental results.
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ACKNOWLEDGEMENTS |
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This work was supported by Hong Kong Polytechnic University Research Grants V423, S151, P190, and P197 and by National Eye Institute Grants EY-11631, EY-00160, and EY-01867, as well as an unrestricted grant from Research to Prevent Blindness, Inc. (New York).
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FOOTNOTES |
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Address for reprint requests and other correspondence: O. A. Candia, Dept. of Ophthalmology, Mount Sinai Medical Center, One Levy Place, New York, NY 10029-8574 (E-mail: oscar.candia{at}mssm.edu).
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. Section 1734 solely to indicate this fact.
Received 14 August 2000; accepted in final form 20 December 2000.
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