Departments of 1 Ophthalmology and Visual Sciences and 2 Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; and Departments of 4 Physiology and Cellular Biophysics and 3 Ophthalmology, Columbia University, New York, New York 10032
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ABSTRACT |
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We report for the
first time that cultured nonpigmented human ciliary epithelial (NPE)
cell layers transport fluid. Cells were grown to confluence on
permeable membrane inserts, and fluid transport across the resulting
cell layers was determined by volume clamp at 37°C. These cell layers
translocated fluid from the apical to the basal side at a steady rate
of 3.6 µl · h1 · cm
2
(n = 4) for 8 h. This fluid movement was
independent of hydrostatic pressure and was completely inhibited by 1 mM ouabain, suggesting it arose from fluid transport. Mercuric
chloride, a nonspecific but potent blocker of
Hg2+-sensitive aquaporins, and aquaporin-1 antisense
oligonucleotides both partially inhibited fluid transport across the
cell layers, which suggests that water channels have a role in NPE cell
homeostasis. In addition, these results suggest that of the two ciliary
epithelial layers in tandem, the NPE layer by itself can transport
fluid. This cultured layer, therefore, constitutes an interesting model that may be useful for physiological and pharmacological
characterization of ciliary epithelial fluid secretion.
aquaporins; ciliary epithelium; aqueous humor
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INTRODUCTION |
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CILIARY EPITHELIUM IS RESPONSIBLE for the secretion of aqueous humor into the posterior chamber of the eye. Most of the aqueous humor secretion is driven by the active transport of electrolytes from the plasma into the posterior chamber followed by rapid movement of water and solutes (17). The ciliary epithelium is composed of two layers juxtaposed, the proximal (or outer) pigmented epithelium (PE) and the distal (or inner) nonpigmented epithelium (NPE). These two layers exhibit characteristics found in transporting epithelia (16, 43). Ciliary epithelial cells have been cultured for several years and have been characterized for the presence of several ion transporters and receptors (24, 29, 41). In addition, ex vivo preparation of rabbit ciliary epithelium has been established and utilized for studying the ion transport across this bilayer (7, 22, 36). For the NPE layer (both in vivo and in cultured cells), the Na+-K+-ATPase, which is located on its basolateral membranes, provides the gradient required by a host of Na+-dependent cotransporters (2, 12, 13, 28).
In contrast to the rich information on electrolyte movements, very little is known about water movements across ciliary epithelial cell membranes. Presumably, part of the reason for that is the lack of convenient in vitro models for studying fluid transport across ciliary epithelial layers. We now report that the NPE cells, when grown on permeable supports, can be advantageously utilized to study translayer fluid movements. It is not clear whether or not ciliary fluid secretion in vivo requires the simultaneous presence of both the PE and NPE layers transporting in tandem. Our findings now strongly suggest that the cultured NPE layer alone transports fluid actively. The implications of these findings for the mechanism of fluid secretion in vivo remain to be determined.
Last, using molecular, immunological, and biochemical techniques, we have recently demonstrated that functional water channels (aquaporin-1 or AQP1) are expressed in simian virus 40 (SV40)-transformed human NPE cells (15). Our present results suggest that AQP1 contributes to the homeostasis of these cells.
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MATERIALS AND METHODS |
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Cultured human NPE cell layers.
The SV40-transformed and well characterized clone (ODM2) of
human NPE cells (3, 6, 41) was used in this study. NPE cells (passage 8) were grown on 24.5-mm-diameter
transparent, permeable, collagen-treated supports (Transwell,
Costar) at a density of 1 × 106 per support in
Dulbecco's modified Eagle's medium (DMEM), supplemented with 10%
fetal bovine serum (FBS), in a 5% CO2 atmosphere at
37°C. Experiments were performed within 1-7 days after
confluence. Confluence was determined by microscopy and was also
ascertained by determining the resistance of each insert before the
experiment. Electrical resistance of NPE layers was determined using an
Endohm chamber and a resistance meter (WPI, Sarasota, FL). The
resistance determined with a control insert was subtracted from that of
an insert with the cell layer. Specific resistance was calculated by
multiplication by the area of the insert (4.7 cm2); the
usual value for a control insert was 56.4 · cm2. For a set of five NPE cultured layers
representative of those utilized, the (corrected) specific resistance
was 73.32 ± 15.6
· cm2. These values are
of the order of those seen for other cell layers grown on the same
support by us (
TN4 cells: 130 ± 7.3
· cm2; bovine lens epithelium: 85.4 ± 5.9
· cm2) (8). For 2 h before
beginning and throughout the experiments, the cell layers were bathed
by DMEM containing FBS without antibiotics. The basal side of the
confluent NPE layer grown in this fashion is closer to the collagen
support, and the apical side is facing away from the support.
Antisense oligonucleotides.
A phosphorothioated antisense oligonucleotide targeted toward the
region encompassing the ATG start codon of AQP1 mRNA (from base 7 to
+11) (39) was synthesized. The oligonucleotide had natural
nucleotides flanked by four phosphorothioate-modified deoxynucleic
acids at the 5' and 3' ends
(TCGCTGGCCATGCTGGCA). Bold letters in
the oligonucleotide refer to phosphorothioated bases, and the
underlined region corresponds to the location of the start codon.
Phosphorothioated oligos are stable (exonuclease resistant) and are
transported into cells more efficiently than unmodified oligos
(35). A second oligonucleotide, with the same base
composition but with a scrambled (nonsense) sequence
(TGGGCTCACACCGTCGGT), was used as control for nonspecific or potential
toxic effects of the oligonucleotide. Scrambling was done using DNA
analysis software (MacVector). Cells were grown without
oligonucleotides and incubated with antisense or nonsense
oligonucleotide (5 µM) for 16-18 h in serum-free media before measurements.
Fluid flow measurements. The chamber utilized has been described in prior publications (8). Briefly, it consists of two Lucite halves with water jackets for temperature control that are kept saturated with water when not in use. The top half of the chamber holds the Costar Transwell insert, and a rubber gasket separates the plastic bottom of the insert from a seat in the bottom chamber. The clamping force to seal the chamber is exerted on the plastic surfaces and not on the cells. The bottom chamber seat for the insert accommodates a stainless steel wire mesh to prevent sagging of the flexible permeable support. The bottom chamber is sealed by a plug and an O-ring and is pierced by a 16-gauge stainless steel tube. The top chamber is also sealed by a plug and an O-ring and pierced by several tubes; it was gassed continuously with moist 95% air and 5% CO2. The steel tube coming from the bottom plug is connected to the automatic fluid level detector. The current nanoinjector method uses the signal from the detector to provide negative feedback and to keep the volume of the bottom compartment constant. Volume flow recordings correspond to the rate of injection (or withdrawal) of fluid from the bottom chamber. The relative positions of the chamber and the detector were such that the hydrostatic pressure difference applied to the apical side of the cell layer (top chamber) was 3 cmH2O. To be noted, the configuration of the setup precludes gassing of the bottom compartment.
Immunoblotting of AQP1 in NPE cell membranes. Immunoblot analysis was carried out on membrane preparations of the cultured cells. Cells were grown without oligonucleotides and were incubated for 18 h with sense and antisense AQP1 oligonucleotides (5 µM). For membrane preparations, cells were first lysed in the lysis buffer (2 mM HEPES and 2 mM EDTA, pH 7.4) on ice for 15 min, vortexed briefly, and centrifuged at 37,000 g for 30 min. The membrane pellet was resuspended in Tris · HCl (50 mM, pH 7.5) buffer, solubilized in the homogenizer, 25 µg of membrane protein was mixed with the sample buffer and resolved by 13% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. The membrane was blocked with 1% bovine serum albumin in Tris-buffered saline (TBS; 20 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature and was incubated with AQP1-specific polyclonal antibody (Alomone Labs, Jerusalem, Israel). The membranes were washed four times with TBS, incubated with an anti-rabbit IgG-alkaline phosphatase conjugate antibody (Promega, Madison, WI), and immunoreactive bands were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Northern blotting of AQP1 in NPE cells.
Cells were grown without oligonucleotides and were incubated for
18 h with sense and antisense AQP1 oligonucleotides (5 µM) for
18 h. Northern blot analysis was performed with the
poly(A)+ RNA isolated using a poly(A)+ RNA
isolation kit from Qiagen (Chatsworth, CA). Denatured
poly(A)+ RNA (1.0 µg) was separated on 1.5% agarose gel
containing formaldehyde. After electrophoresis, the RNA was
electrotransferred to a nitrocellulose membrane using the Bio-Rad
Transblot apparatus. RNA was cross-linked to the nitrocellulose
membrane by an ultraviolet crosslinker. The blot was prehybridized at
42°C for 2 h with 100 µg/ml of denatured salmon sperm DNA in
50% formaldehyde, 0.04% polyvinylpyrrolidone, 0.04% bovine serum
albumin, 0.04% Ficoll, and 1% SDS in 5× saline sodium citrate (SSC)
buffer. PCR-amplified DNA labeled with 32P was used as a
probe. Hybridization was carried out at 42°C for 16 h. Filters
were washed twice in 2× SSC at room temperature for 15 min and once in
1× SSC for 15 min containing 1% SDS. The blots were exposed to Kodak
X-Omat AR film with intensifying screen at 80°C for 3 days to
detect the AQP1 mRNA expressed in NPE cells.
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RESULTS |
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Fluid transport by NPE cell layers.
The introduction of permeable tissue culture supports has made it
possible to measure the ability of cells to transport fluid in
monolayer cultures as well as to measure the electrical resistance and
potential differences across the monolayers. We used a modified Bourguet-Jard technique (10, 30) to measure fluid
transport across cultured monolayers of NPE cells. This method has been successfully used to measure water and fluid transport across lens
epithelium and corneal limiting layers (8, 9, 11, 26, 45),
gives resolutions as high as 1-3 nl, and can be applied to any
layer of cultured cells. From the direction of fluid transport across
the NPE cell layer in vivo, if the cultured cells would behave
likewise, we would expect fluid movement from the top chamber to the
bottom chamber. Figure 1 shows that fluid
movement across an NPE cell layer indeed occurs in the expected
direction, from the apical to the basal surface, corresponding to the
activity of an absorptive epithelium. This movement of fluid took place spontaneously and continuously for several hours; the longest time
monitored was 8 h. In other experiments, varying pressure heads
were applied to the NPE cell layer. Hydrostatic pressure differences
(up to 7 cmH2O) had no effect on fluid movement, as exemplified in Fig. 2. Because such high
pressure differences are unlikely across an in vivo layer, we chose a
pressure head of 3 cmH2O as a standard. This pressure is
required to keep the cells and their support relatively immobile; at
the same time, it lessens capillarity artifacts in the sensor tube.
After ~8 h in the chamber, the rate of fluid movement increased
progressively, which is consistent with an increasing leak across the
deteriorating cell layers (driven by the existing pressure head). The
average rate of fluid movement was 3.6 + 0.3 µl · h1 · cm
2
(n = 4).
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DISCUSSION |
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In prior experimental work that examined fluid secretion by
ciliary epithelium in vivo (4, 18, 32), it has not been possible to ascertain whether the simultaneous presence of both layers
is required for fluid secretion because its two layers (PE and NPE) are
always together. In addition, there is no prior instance in which fluid
movements have been determined across an isolated or cultured
preparation of this important tissue. Here we report for the first time
that the NPE layer by itself (albeit cultured) is capable of
translocating fluid in a direction (from apical to basolateral; Fig. 1)
and at a rate (3.6 ± 0.3 µl · h1 · cm
2; cf. Fig.
1) consistent with fluid transport across it. The direction of fluid
movement observed is the same as that for fluid transport across this
absorptive layer in vivo. In addition, the blockage of fluid movement
by ouabain further suggests that fluid movement across NPE cells indeed
represents solute-coupled fluid transport.
Whether these observations across cultured cells are totally applicable to the ciliary epithelium in vivo remains to be determined. Still, there is that presumption a priori, since the cultures transport fluid actively, and they do so in the same direction as the in vivo process. Anatomically, it is well documented in the literature that NPE cells in vivo establish zonular structures at the intersections of their apical-lateral membrane domains (27, 42). Correspondingly, NPE cells in culture also form zonulae in primary culture (1, 38).
As mentioned earlier in this paper, varying hydrostatic pressures progressively between 3 and 7 cm of H2O had no effect on the rate of fluid movement (Fig. 2). This suggests that the fluid movement observed is due to fluid transport and not to a leak or an artifact. To further characterize this finding, we chose to study the possible role of the AQP1 water channel in fluid transport. The presence of AQP1 has been demonstrated by others and ourselves in human and rat NPE (14, 31, 37), and, more recently (15), in the same cultured human NPE cells with which this study was done. As detailed in RESULTS, we found that fluid movement across the NPE layer was significantly reduced in cells treated with AQP1 antisense oligonucleotide (Fig. 5). Nonsense oligonucleotide that had the same base composition as the antisense oligonucleotide, but scrambled, had no effect on fluid movement. This, again, suggests that the fluid movement observed is due to an active cellular mechanism (rather than a leak) and that AQP1 plays a role in the cellular processes, resulting in fluid transport by NPE cells.
The observed partial inhibition of fluid movement by HgCl2 is telling in at least two senses. It also militates against the existence of a fluid leak or an artifact, since under the applied hydrostatic pressure, a nonspecific or toxic effect of HgCl2 would have presumably increased the leak, and, therefore, increased the rate of fluid movement. Instead, the opposite happened. However, an explanation for this effect must also take into account the fact that the inhibition was partial (~50%). If the water flow observed traversed membrane water channels, one might argue that perhaps half of the flow would traverse other water-permeable membrane routes. Against this, however, is the observation that upon mounting, cells treated with antisense AQP1 oligonucleotide displayed an initial rate of fluid transport quite comparable to those of untreated cells. As mentioned earlier in this paper, the AQP1 protein content was already much decreased in the antisense oligonucleotide-treated cells, which points away from the fluid movement traversing AQP1 water channels.
An alternative explanation for the inhibitions seen with HgCl2 and antisense oligonucleotide is that AQP1 could have a central role in maintaining cell homeostasis. To be noted, as mentioned previously, despite downregulation of AQP1, the initial rate of transepithelial water transport is not reduced but the later water flow is lowered. This suggests that the downregulation may have a primary role on transepithelial solute flux in some unidentified way and is not limited to a unique effect on the water conduits. Consistent with this, clearly, cells in which AQP1 is diminished or inhibited show a functional deficit some time after mounting. In going from the incubator to the chamber, the surrounding fluid is the same but the lower chamber cannot be gassed with air-CO2, and its pH increases with time. There are reports that AQP1 serves as a conduit for CO2 gas (5, 25, 33), although there is also evidence against that possibility (44). Hence, although the precise reason remains unclear, lack of proper water or gas permeation might conceivably underlie the functional deficit seen in our case when AQP1 channels are inhibited or diminished in number.
As mentioned earlier in this paper, the rate of fluid movement observed
was 3.6 ± 0.3 µl · h1 · cm
2, which is
of the order of those in other fluid-transporting layers (8,
26). The ciliary epithelium comprises ~70 major ciliary processes that are ~2 mm long and 0.5 mm wide (23),
which results in, roughly, a 7-cm2 surface area for the
folded ciliary epithelium. If a factor of 6.7 is used, as described by
Krupin et al. (20, 21), to relate the real area to the
projected area for unfolded rabbit ciliary epithelium, the
7-cm2 surface area will result in a 46.9-cm2
surface area. Because the height of the ciliary processes is not
constant, the real area for unfolded ciliary epithelium is an
estimate. If such a surface area is used, the rate of fluid transport that we observe yields a putative rate of aqueous humor secretion of 2.8 µl/min
1, which is in agreement with
the in vivo values reported previously in the literature (19,
20).
Our observation of fluid transport by NPE alone leads naturally to a discussion of the presumed role of the gap junctions known to exist between PE and NPE. Their role and the modulation of their open and closed states is unclear, but they have been mentioned in the literature as a possible route for fluid movement from PE to NPE working in tandem (20). For example, Walker et al. (40) recently observed sequential cell regulatory volume changes first in PE and then in NPE, consistent with cyclic movement of fluid from PE to NPE. From their observations, one might argue that fluid could have crossed from PE to NPE across timely opening gap junctions. However, in our own results, NPE cells transport fluid without PE present. Our findings, therefore, pose the questions of whether the tandem model can explain the results in vivo or whether an alternative explanation needs to be sought. In this connection, we note that fluid production by the ciliary epithelium is considerable. Given a ciliary epithelial volume of 8 µl in vivo (20) and a rate of secretion of aqueous humor of 3-4 µl/min, the ciliary epithelium transports its own volume approximately every 2.5 to 3 min (20). Given our current observation that the NPE layer by itself transports fluid, it might be argued that transport by the PE and NPE layers juxtaposed could add up, as it would with two impellent pumps in series. This, of course, would require the PE to be able to transport fluid on its own, the capability of which is unknown.
In summary, we present evidence that a human cultured NPE layer actively transports a sizable amount of fluid from its apical to its basal side and that AQP1 has a role to allow these cells an optimal rate of transport. To be noted, there are aquaporins in all the tissues involved in the major route of aqueous humor flow. The possibility exists that NPE in vivo could transport fluid on its own, and improvements in our understanding of the role of aquaporins in connection with this transport could provide crucial new insights into physiological and disease mechanisms. Our results also indicate that cultures of the individual cell layers, like the one utilized here, could constitute useful in vitro models to study the mechanisms of aqueous humor secretion.
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ACKNOWLEDGEMENTS |
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We thank Drs. Kunyan Kuang and Quan Wen for technical expertise.
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FOOTNOTES |
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This work was supported by National Eye Institute Grants EY-10423 (to R. V. Patil), EY-06178 (to J. Fischbarg), Core Grant EY-02687, and, in part, by unrestricted grants from Research to Prevent Blindness, Inc. R. V. Patil is a Research to Prevent Blindness Olga Keith Wiess Scholar.
Address for reprint requests and other correspondence: R. Patil, Dept. of Ophthalmology and Visual Sciences, Washington Univ. School of Medicine, 660 S. Euclid, St. Louis, MO 63110 (E-mail: patil{at}vision.wustl.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 22 May 2000; accepted in final form 16 May 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Cilluffo, MC,
Fain MJ,
and
Fain GL.
Tissue culture of rabbit ciliary body epithelial cells on permeable supports.
Exp Eye Res
57:
513-526,
1993[ISI][Medline].
2.
Coca-Prados, M,
and
Lopez-Briones L.
Evidence that the and
(+) isoforms of the catalytic subunit of (Na+,K+)-ATPase reside in distinct ciliary epithelial cells of the mammalian eye.
Biochem Biophys Res Commun
145:
460-466,
1987[ISI][Medline].
3.
Coca-Prados, M,
and
Wax MB.
Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of 2-adrenergic receptors.
Proc Natl Acad Sci USA
83:
8754-8758,
1986[Abstract].
4.
Cole, DF.
Effects of some metabolic inhibitors on the formation of aqueous humor in rabbits.
Br J Ophthalmol
44:
739-750,
1960.
5.
Cooper, GJ,
and
Boron WF.
Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant.
Am J Physiol Cell Physiol
275:
C1481-C1486,
1998
6.
Cooper, HS,
Manning DR,
and
Wax MB.
G protein complement of SV40-transformed ciliary epithelial cells.
Curr Eye Res
9:
493-499,
1990[ISI][Medline].
7.
Crook, RB,
Takahashi K,
Mead A,
Dunn JJ,
and
Sears ML.
The role of NaKCl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium.
Invest Ophthalmol Vis Sci
41:
2574-2583,
2000
8.
Fischbarg, J,
Diecke FP,
Kuang K,
Yu B,
Kang F,
Iserovich P,
Li Y,
Rosskothen H,
and
Koniarek JP.
Transport of fluid by lens epithelium.
Am J Physiol Cell Physiol
276:
C548-C557,
1999
9.
Fischbarg, J,
Liebovitch LS,
and
Koniarek JP.
Inhibition of transepithelial osmotic water flow by blockers of the glucose transporter.
Biochim Biophys Acta
898:
266-274,
1987[ISI][Medline].
10.
Fischbarg, J,
Lim JJ,
and
Bourguet J.
Adenosine stimulation of fluid transport across rabbit corneal endothelium.
J Membr Biol
35:
95-112,
1977[ISI][Medline].
11.
Fischbarg, J,
Warshavsky CR,
and
Lim JJ.
Pathways for hydraulically and osmotically-induced water flows across epithelia.
Nature
266:
71-74,
1977[ISI][Medline].
12.
Flugel, C,
and
Lutjen-Drecoll E.
Presence and distribution of Na+/K+-ATPase in the ciliary epithelium of the rabbit.
Histochemistry
88:
613-621,
1988[ISI][Medline].
13.
Ghosh, S,
Freitag AC,
Martin-Vasallo P,
and
Coca-Prados M.
Cellular distribution and differential gene expression of the three subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.
J Biol Chem
265:
2935-2940,
1990
14.
Hamann, S,
Zeuthen T,
La Cour M,
Nagelhus EA,
Ottersen OP,
Agre P,
and
Nielsen S.
Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye.
Am J Physiol Cell Physiol
274:
C1332-C1345,
1998
15.
Han, Z,
Yang J,
Wax MB,
and
Patil RV.
Molecular identification of functional water channel aquaporin-1 in cultured human ciliary epithelial cells.
Curr Eye Res
20:
242-247,
2000[ISI][Medline].
16.
Helbig, H,
Korbmacher C,
Berweck S,
Kuhner D,
and
Wiederholt M.
Culture of ciliary body epithelia and studies of ion transport.
Fortschr Ophthalmol
85:
42-45,
1988[Medline].
17.
Jacob, TJ,
and
Civan MM.
Role of ion channels in aqueous humor formation.
Am J Physiol Cell Physiol
271:
C703-C720,
1996
18.
Jones, RF,
and
Maurice DM.
New methods of measuring the rate of aqueous flow in man with fluorescein.
Exp Eye Res
5:
208-220,
1966[ISI][Medline].
19.
Kinsey, VE,
and
Barany E.
The rate of flow of aqueous humor. II. Derivation of rate of flow and its physiological significance.
Am J Ophthalmol
32:
189-196,
1949[ISI].
20.
Krupin, T,
and
Civan MM.
Physiologic basis of aqueous humor formation.
In: The Glaucomas (2nd ed.), edited by Ritch R,
Shields MB,
and Krupin T.. Philadelphia, PA: Mosby, 1996, p. 251-280.
21.
Krupin, T,
Reinach PS,
Candia OA,
and
Podos SM.
Transepithelial electrical measurements on the isolated rabbit iris-ciliary body.
Exp Eye Res
38:
115-123,
1984[ISI][Medline].
22.
Mead, A,
Sears J,
and
Sears M.
Transepithelial transport of ascorbic acid by the isolated intact ciliary epithelial bilayer of the rabbit eye.
J Ocul Pharmacol Ther
12:
253-258,
1996[ISI][Medline].
23.
Morrison, JC,
and
Freddo TF.
Anatomy, microcirculation and ultrastructure of the ciliary body.
In: The Glaucomas (2nd ed.), edited by Ritch R,
Shields MB,
and Krupin T.. Philadelphia, PA: Mosby, 1996, p. 125-138.
24.
Mukhopadhyay, P,
Geoghegan TE,
Patil RV,
Bhattacherjee P,
and
Paterson CA.
Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells.
Biochem Pharmacol
53:
1249-1255,
1997[ISI][Medline].
25.
Nakhoul, NL,
Davis BA,
Romero MF,
and
Boron WF.
Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes.
Am J Physiol Cell Physiol
274:
C543-C548,
1998
26.
Narula, P,
Xu M,
Kuang KY,
Akiyama R,
and
Fischbarg J.
Fluid transport across cultured bovine corneal endothelial cell monolayers.
Am J Physiol Cell Physiol
262:
C98-C103,
1992
27.
Noske, W,
Stamm CC,
and
Hirsch M.
Tight junctions of the human ciliary epithelium: regional morphology and implications on transepithelial resistance.
Exp Eye Res
59:
141-149,
1994[ISI][Medline].
28.
Okami, T,
Yamamoto A,
Omori K,
Akayama M,
Uyama M,
and
Tashiro Y.
Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.
J Histochem Cytochem
37:
1353-1361,
1989[Abstract].
29.
Ortego, J,
and
Coca-Prados M.
Functional expression of components of the natriuretic peptide system in human ocular nonpigmented ciliary epithelial cells.
Biochem Biophys Res Commun
258:
21-28,
1999[ISI][Medline].
30.
Parisi, M,
Bourguet J,
Ripoche P,
and
Chevalier J.
Simultaneous minute by minute determination of unidirectional and net water fluxes in frog urinary bladder. A reexamination of the two barriers in series hypothesis.
Biochim Biophys Acta
556:
509-523,
1979[ISI][Medline].
31.
Patil, RV,
Saito I,
Yang X,
and
Wax MB.
Expression of aquaporins in the rat ocular tissue.
Exp Eye Res
64:
203-209,
1997[ISI][Medline].
32.
Pollack, IP,
Becker B,
and
Constant MA.
The effect of hypothermia on aqueous humor dynamics. I. Intraocular pressure and outflow facility of the rabbit eye.
Am J Ophthalmol
49:
1126-1131,
1960[ISI].
33.
Prasad, GV,
Coury LA,
Finn F,
and
Zeidel ML.
Reconstituted aquaporin 1 water channels transport CO2 across membranes.
J Biol Chem
273:
33123-33126,
1998
34.
Preston, GM,
Carroll TP,
Guggino WB,
and
Agre P.
Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein.
Science
256:
385-387,
1992[ISI][Medline].
35.
Robinson, ES,
Nutt DJ,
Jackson HC,
and
Hudson AL.
Antisense oligonucleotides in psychopharmacology and behaviour: promises and pitfalls.
J Psychopharmacol
11:
259-269,
1997[ISI][Medline].
36.
Sears, ML,
Yamada E,
Cummins D,
Mori N,
Mead A,
and
Murakami M.
The isolated ciliary bilayer is useful for studies of aqueous humor formation.
Trans Am Ophthalmol Soc
89:
131-152,
1991[Medline].
37.
Stamer, W,
Snyder R,
Smith B,
Agre P,
and
Regan J.
Localization of aquaporin CHIP in the human eye: implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance.
Invest Ophthalmol Vis Sci
35:
3867-3872,
1994[Abstract].
38.
Strauss, O,
and
Wiederholt M.
Transepithelial resistance of ciliary epithelial cells in culture: functional modification by protamine and extracellular calcium.
Comp Biochem Physiol A Physiol
100:
987-993,
1991[ISI].
39.
Umenishi, F,
and
Verkman AS.
Isolation of the human aquaporin-1 promoter and functional characterization in human erythroleukemia cell lines.
Genomics
47:
341-349,
1998[ISI][Medline].
40.
Walker, VE,
Stelling JW,
Miley HE,
and
Jacob TJ.
Effect of coupling on volume-regulatory response of ciliary epithelial cells suggests mechanism for secretion.
Am J Physiol Cell Physiol
276:
C1432-C1438,
1999
41.
Wax, MB,
and
Patil RV.
Immunoprecipitation of A1 adenosine receptor-GTP-binding protein complexes in ciliary epithelial cells.
Invest Ophthalmol Vis Sci
35:
3057-3063,
1994[Abstract].
42.
Wiederholt, M,
and
Zadunaisky JA.
Effects of ouabain and furosemide on transepithelial electrical parameters of the isolated shark ciliary epithelium.
Invest Ophthalmol Vis Sci
28:
1353-1356,
1987[Abstract].
43.
Wolosin, JM,
Chen M,
Gordon RE,
Stegman Z,
and
Butler GA.
Separation of the rabbit ciliary body epithelial layers in viable form: identification of differences in bicarbonate transport.
Exp Eye Res
56:
401-409,
1993[ISI][Medline].
44.
Yang, B,
Fukuda N,
van Hoek A,
Matthay MA,
Ma T,
and
Verkman AS.
Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in reconstituted proteoliposomes.
J Biol Chem
275:
2686-2692,
2000
45.
Yang, H,
Reinach PS,
Koniarek JP,
Wang Z,
Iserovich P,
and
Fischbarg J.
Fluid transport by cultured corneal epithelial cell layers.
Br J Ophthalmol
84:
199-204,
2000
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