Rabbit conjunctival epithelium transports fluid, and
P2Y22 receptor agonists stimulate Cl
and
fluid secretion
Yansui
Li1,
Kunyan
Kuang1,
Benjamin
Yerxa2,
Quan
Wen1,
Heinz
Rosskothen1, and
Jorge
Fischbarg1,3
Departments of 1 Ophthalmology and 3 Physiology and
Cellular Biophysics, College of Physicians and Surgeons, Columbia
University, New York, New York 10032; and 2 Inspire
Pharmaceuticals, Incorporated, Durham, North Carolina 27703
 |
ABSTRACT |
Rabbit conjunctival epithelium exhibits UTP-dependent
Cl
secretion into the tears. We investigated whether
fluid secretion also takes place. Short-circuit current
(Isc) was 14.9 ± 1.4 µA/cm2
(n = 16). Four P2Y2 purinergic receptor
agonists [UTP and the novel compounds INS365, INS306, and INS440
(Inspire Pharmaceuticals)] added apically (10 µM) resulted in
temporary (~30 min) Isc increases (88%, 66%,
57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of
6.5 ± 0.7 µl · h
1 · cm
2 (range
2.1-15.3, n = 20). Fluid transport was stimulated
by mucosal additions of 10 µM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 µl · h
1 · cm
2,
n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 µl · h
1 · cm
2,
n = 5. Fluid transport was abolished by 1 mM
ouabain (n = 5) and was drastically inhibited by 300 µM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 µl · h
1 · cm
2,
n = 4). We conclude that this epithelium secretes fluid
actively and that P2Y2 agonists stimulate both
Cl
and fluid secretions.
dry eye; purinergic; adenosine 5'-triphosphate; uridine
5'-triphosphate; INS365; short-circuit current
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INTRODUCTION |
THE SECRETION OF TEAR
FLUID appears to have two main components: 1) baseline
secretion, and 2) secretion after a stimulus. It has been
proposed that the stimulated secretion arises from the main and
accessory (palpebral) lacrimal glands (18), but other
sources cannot be excluded. The nature and origin of the baseline
secretion is less clear. Some accounts (18) attribute it
to the accessory lacrimal glands of Krause and Wolfring, but others
even doubt it exists as such (19). These unsolved
questions lead naturally to the consideration of all conceivable routes of tear fluid secretion.
A possible source that had not been tested until now is the
conjunctival epithelium. Some evidence has appeared to implicate it,
since it has been reported (13) that, in the absence of or
at low levels of stimulation, the secretion of the cannulated rabbit
lacrimal gland is hypertonic (334 mosM) while the tears are isotonic.
This led to the suggestion that fluid from the conjunctiva and cornea
could dilute the lacrimal gland secretion (13). In keeping
with this possibility, it was recently found that the rabbit
conjunctival epithelium transports electrolytes (25, 26, 42,
44), expressing both secretory and absorptive electrolyte transport mechanisms (42). In addition, AQP3 water
channels have been located to conjunctival epithelium
(11). Water channels are conduits for transcellular and
translayer water flow in the kidney collecting duct, and they are
present in all fluid-transporting epithelia known. Fittingly, the
conjunctival apical surface is highly permeable to water, with a
transepithelial diffusional water permeability (1.3 µm/s) largely
exceeding paracellular water permeability (10
2 µm/s)
(5).
Given this background, we have investigated whether the rabbit
conjunctival epithelium can transport fluid, and we have found that it
does. The direction of fluid transport is from the stromal (basolateral) to the mucosal (apical) side, which is the same direction
in which Cl
is secreted by this layer (25,
42). Furthermore, we found that fluid and Cl
transports are stimulated by UTP and by a novel purinergic receptor agonist, P1,P4-di(uridine 5'-)tetraphosphate,
or Up4U (INS365), which are, respectively, natural and
synthetic agonists of the P2Y2 receptor (34,
38).
While this paper was undergoing review, another paper appeared
(45), reporting net fluid secretion across pigmented
rabbit conjunctiva in the serosal-to-mucosal direction. Such secretion was increased by mucosal addition of 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and 10 µM UTP and was abolished by 0.5 mM
ouabain, which is similar to the results we present. The two laboratories had previously reported some of these findings in abstract
form (29, 43).
 |
MATERIALS AND METHODS |
Solutions.
The physiological medium used to bathe the conjunctiva for
short-circuit current (Isc) measurements was
Tyrode solution (42), containing (in mM) 113 NaCl, 30 NaHCO3, 4 KCl, 0.8 NaH2PO4, 1.2 MgCl2, 1.8 calcium gluconate, and 5.6 D-glucose, pH 7.4-7.5 (maintained by gassing with
air-5% CO2), osmolarity 280 mosM. For fluid transport experiments, since gassing was impractical, we used modified Tyrode solution by adding HEPES (20 mM) while leaving the rest of the components unchanged; pH remained the same but the osmolarity was 300 mosM.
Rabbit conjunctival preparation.
New Zealand albino rabbits, each weighing 2.0-2.5 kg, were
euthanized with pentobarbital sodium solution injected into the marginal ear vein. The eyes including eyelids were enucleated immediately along the superior and inferior orbit. Only freshly dissected preparations were used, and that entailed that only one of
the eyes of a given rabbit was used for each experiment. The eyeball
was placed on a semispherical Lucite holder, where it was secured in
place by vacuum. The eyelids were then pulled up, which resulted in
extending the conjunctiva, forming a cylinder. At that point, the
eyelids were sutured to a circular holder held on a manipulator. The
holder was adjusted to maintain the conjunctiva in cylindrical shape,
with the epithelium covering the internal surface. The inside of the
conjunctival cylinder was filled with prewarmed (37°C) Tyrode
solution, and the subconjunctival tissues and extraocular muscles were
trimmed off the outside. The conjunctival cylinder was then cut open
from one of the canthi to the corneal limbus vertically, followed by
cutting all along the limbus. At that point, the entire conjunctiva
thus obtained, including the free bulbar and fornix sections plus the
palpebral conjunctiva still attached to the eyelids, was placed into a
dish with Tyrode solution at 37°C and gassed with air-5%
CO2. With the help of a dissection microscope, the
palpebral conjunctiva was dissected off the eyelids, which yielded the
whole conjunctiva isolated in one piece.
Isc.
These measurements were done with an Ussing system [World Precision
Instruments (WPI), Sarasota, FL]. The isolated rabbit conjunctival epithelium preparation was clamped in a model CHM2 WPI chamber. The solutions were bubbled with air-5% CO2
and maintained at 37°C, using the thermally jacketed glass lifts of
the system. The Isc across the tissue was
measured using a four-electrode WPI DVC 1000 voltage-current clamp.
Fluid transport.
Isolated rabbit conjunctival epithelial preparations were mounted in an
insert made of two flat Lucite rings, one of them having a stainless
steel mesh (Fig. 1) to support the
tissue. The insert was clamped between two thermally jacketed
fluid-filled chambers (37°C). The mucosal side (top) chamber was
stoppered and was in contact with the outside through a narrow piece of tubing (to minimize evaporation); the serosal (bottom) chamber was
closed except for the detector, also enclosed to minimize evaporation.
The nanoinjector-driven volume-clamp instrument (see Fig. 1) has been
described previously (10, 35). The rate of fluid
traversing the preparation was determined by keeping constant the
volume of the stromal side chamber. The microelectrode contact detector
used is triggered by volume variations of 1-3 nl; to avoid its
blockage, voltage to it was limited to ~100 mV. The nanoinjector
voltage output was proportional to the volume injected or (withdrawn)
by the syringe in each cycle; such output went to a pen-chart recorder
and to a computer in oscillograph mode. To function, the method
requires the tissue to be applied against its support by a pressure
head. In addition, if that pressure head is <1.5 cmH2O,
capillarity artifacts at the detector are possible. Because the tissue
does not normally have a pressure difference across it, for practical
considerations we chose a compromise value. The relative positions of
chamber and detector were therefore such that the hydrostatic pressure
difference (mucosa minus stroma) across the conjunctival preparation
was 3.0 cmH2O.

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Fig. 1.
Scheme for conjunctival fluid transport measurements. A
conjunctival preparation was placed on the insert, which in turn was
clamped between two thermally jacketed fluid-filled hemichambers. For
clarity, jackets are shown on left side only. The height of the chamber
assembly can be adjusted to obtain a hydrostatic pressure difference
( P) of 3 cmH2O between the two sides of the tissue;
bott, bottom.
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 |
RESULTS |
Isc.
Unless specified, after mounting, the Isc was
allowed to stabilize for 30 min, at which point the average
Isc across the rabbit conjunctival epithelium
was 14.9 ± 1.4 µA/cm2 (range 5-24
µA/cm2, n = 16). At that time, typically
one of four different P2Y2 purinergic receptor agonists
utilized was added to the apical side funnel. Each agonist at each
given concentration was tested in an individual preparation and only
once during a given experiment. After such addition, the
Isc initially increased rapidly in all cases.
Given that all experiments were done with the same protocol, the
behavior of Isc can be expressed as a percent of
its value immediately before the solution exchange. With 10 µM (final
concentration) of UTP, INS365, INS306, or INS440, the respective
increases above the control levels were 88%, 66%, 57%, and 28%
(n = 4 in all cases, Fig.
2). The stimulated
Isc gradually decreased to its control value
except for INS365 and INS440, for which Isc
remained 24% and 13% higher than the control level after 30 min,
respectively. The effects of these four compounds were concentration
dependent (Figs. 3 and
4). Maximal increases in
Isc (Emax) for UTP,
INS365, INS306, and INS440 at 10
3 M were 104 ± 19, 91 ± 2, 78 ± 14, and 58 ± 2% of the control level,
respectively (Fig. 3). The EC50 values calculated from these data are also given in Fig. 4; they were 2.5 ± 1.4, 3.6 ± 1.1, 3.5 ± 2.1, and 9.1 ± 1.5 µM,
respectively. Comparing the four compounds, UTP gave the highest change
in Isc but could keep Isc
stimulated for >30 min only at the highest concentration tested (10
3 M). INS365 not only stimulated the
Isc to reach a similar level, but also could
keep the Isc stimulated for a longer time at
10
3 and 10
5 M (Fig. 3B).
INS440 had the least effect on Isc.

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Fig. 2.
Transconjunctival short-circuit current
(Isc) and its stimulation by adding 4 different
P2Y2 purinergic receptor agonists at a concentration of 10 µM to the apical side after Isc being stable
for 30 min. Points represent means ± SE of 4 preparations. Here
and elsewhere, as mentioned in MATERIALS AND METHODS, each
preparation corresponds to a different eye and a different rabbit.
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Fig. 3.
Stimulation of Isc by adding 4 different
P2Y2 purinergic receptor agonists at 4 different
concentrations to the apical side of the rabbit conjunctiva after
Isc was stable for 30 min. Points represent
means ± SE of 4-5 preparations; n = 4.
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Fig. 4.
Effects of 4 different P2Y2 purinergic receptor
agonists at 4 different concentrations on the peak change (%) of
Isc. The curves shown were obtained with a
dose-response nonlinear fitting algorithm contained in the program
Origin (OriginLab, Northampton, MA). Emax,
maximum Isc.
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Fluid transport.
Rabbit conjunctival preparations were mounted in the insert with their
mucosal (apical) side facing the top chamber. We found that the
conjunctival epithelial preparations transported fluid from the bottom
to the top chamber (from stroma to mucosa) against a hydrostatic
pressure difference of 3 cmH2O. We calculated average rates
of fluid transport 30 min before and after adding agonists or
inhibitors. The baseline rate of fluid transport observed was 6.5 ± 0.7 µl · h
1 · cm
2
(range 2.1-15.3, n = 20). Figure
5 shows a typical experiment in which the
conjunctival preparation transports fluid spontaneously and
continuously at an average rate of 8.6 µl · h
1 · cm
2 for a
period of 90 min after mounting. We also calculated average rates of
fluid transport every 15 min within this entire 90-min period (bar
diagram, Fig. 5). As can be seen, fluid transport was reasonably stable
except for the first 15 min after mounting. Because the current
technique does not allow gassing of the serosal side, we only ran
relatively short experiments, all of them in freshly isolated
preparations. Because 10 µM UTP and INS365 had significant effects on
Isc, we also explored their effects on fluid
transport. With our procedure, to keep the volume of the bottom chamber
constant, it meant that the bottom chamber was inaccessible during an
experiment, and therefore test agents could only be added to the top
chamber (mucosal side). We waited for the conjunctiva to transport
fluid in stable fashion for about 0.5 h, and then we added UTP or
INS365 (10 µM each, final concentration). Both analogs induced a
significant increase in fluid transport that could last up to 60 min.
Figure 6 shows representative
experiments. Agonists induced changes in rates of fluid transport as
follows: for UTP, from 7.4 ± 2.3 µl · h
1 · cm
2 (range
2.8-15.3) to 10.7 ± 3.3 µl · h
1 · cm
2 (range
3.1-18.4; n = 5), and for INS365 from 6.3 ± 1.0 µl · h
1 · cm
2 (range
3.2-8.4) to 9.8 ± 2.5 µl · h
1 · cm
2 (range
3.7-20.3; n = 5). Pairing the control and
stimulated flow rates within each experiment yielded increases of
45.6 ± 29.5% with UTP and of 50.2 ± 16.2% with INS365.

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Fig. 5.
Typical fluid transport by an excised rabbit conjunctival
preparation from its serosal to its mucosal side against a hydrostatic
pressure difference ( P) of 3 cmH2O. Vertical deflections
represent volume accumulated during 5-s recording intervals, as
detailed at a magnified time scale in the inset. Bars denote
rates of fluid transport averaged for 15-min periods.
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Fig. 6.
Addition of 10 5 M INS365 (A) and UTP
(B) to the top chamber (mucosal side) stimulates rabbit
conjunctival fluid transport in 2 representative experiments. After the
fluid transport was stable for ~30 min, UTP or INS365 were added
(each 10 µM final concentration). Both agonists induced a significant
increase in fluid transport that could last up to 60 min. A
and B, top: accumulation (integral) of the volume
of fluid transported, and the slopes summarize the average rates of
fluid transport before and after stimulation. A and
B, bottom: reproduction of original chart
recordings of fluid transport measurements. A and
B, middle: bar diagrams denote rates averaged for
15-min periods; fl. tr., fluid transport.
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Furthermore, we characterized the effects of inhibitors of electrolyte
transporters and channels on transconjunctival fluid movements. We
added ouabain or quinidine to the apical side after fluid secretion was
stable for ~0.5 h. Fluid transport was abolished by 1 mM ouabain
after 30 min of exposure (n = 5) and was drastically inhibited by 300 µM quinidine [from 6.4 ± 1.2 µl · h
1 · cm
2 (range
3.0-8.6) to 3.6 ± 1.0 µl · h
1 · cm
2 (range
1.7-6.4)] during the first 30 min after exposure
(n = 4). Figure 7
shows representative experiments for the effects of ouabain and
quinidine.

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Fig. 7.
Addition of 1 mM ouabain
(A) and 300 µM quinidine (B) to the top chamber
(mucosal side) inhibits rabbit conjunctival fluid transport in 2 representative experiments. After fluid transport was stable for ~30
min, ouabain or quinidine were added at the times denoted by the arrows
at the final concentrations shown.
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DISCUSSION |
Isc.
From prior Isc measurements, active
Cl
secretion at the apical surface of the freshly excised
rabbit conjunctiva accounted for 75% (25) and 60%
(42) of the Isc, respectively. The
Isc values determined in our study are in
agreement with the values found previously (25, 42, 44).
As we expected, the mucosal application of UTP increased
Isc in a dose-dependent fashion, confirming that
UTP stimulates Cl
secretion (26). Our
results also show that the other three P2Y2 purinergic
receptor agonists we used (INS365, INS306, and INS440) could also
stimulate Isc in a concentration-dependent manner. As mentioned in the results, they induced maximal stimulation of Isc rapidly after application, and their
effects decreased gradually within 30 min. UTP (10 µM) simulated
Isc more than INS365, but the effects of the
latter remained much longer.
UTP and ATP have previously been demonstrated to be potent mediators of
goblet cell mucin secretion and Cl
secretion in airway
epithelia (22, 24, 27, 31), as well as in the conjunctiva
(20, 26). They act via the P2Y2 nucleotide receptors in the epithelial cells of these tissues and regulate ion
transport and secretory functions. The physiological importance of the
P2Y2 receptor, a G protein-coupled receptor, has been well established. Signal transduction by P2 receptors has been thoroughly reviewed (1, 16, 39). Studies have indicated that ATP, acting on P2 receptors in the cell membrane, plays a pivotal role in
various tissues as an extracellular transmitter (4, 23); ATP is known to affect intracellular Ca2+ concentration in
various tissues, including ocular tissues such as corneal epithelium
(23), endothelium (7, 46), and lacrimal acinar cells (15). The P2Y2 receptor found on
the apical surface of airway epithelia is believed to be the major
coordinator of mucociliary clearance mechanisms in the lung.
P2Y2 agonists (UTP, ATP) lead to increases in mucin release
(22, 27), protein secretion (32),
Cl
and ion transport (24, 31), water
transport into the airway surface (2, 17), and surfactant
release (14). Their role in regulation of mucosal
hydration and defense may provide clinical benefit for lung diseases
such as chronic bronchitis in cystic fibrosis. It also appears that a
whole class of uridine nucleotide-responsive receptors exists in a
broad range of tissues, since different receptors that are activated by
uridine nucleotides have been identified (36). This poses
the possibility of selective drug development for each of these receptors.
Fluid transport.
The main finding of this paper is that the conjunctival epithelium
transports fluid from its blood side outward into the tear side. Fluid
transport takes place against a pressure head of 3 cmH2O,
is abolished by 1 mM ouabain (a Na+ pump inhibitor), and is
drastically inhibited by 300 µM quinidine (a K+ channel
blocker), all of which suggest that the fluid movements observed arise
from active transport.
The average Isc we report (14.5 µA/cm2) corresponds to a monovalent ion flux of solute
flux (Js) = Isc/zF = 0.54 µeq · h
1 · cm
2 (where
F = Faraday's constant and z is signed
charge), which in turn would correspond to an isotonic fluid
flow of Jv = Js/Ciso = 3.6 µl · h
1 · cm
2
(Ciso = 150 mM where Ciso is the
concentration of an isotonic solution). This is less than the average
rate of fluid transport we determined (6.5 µl · h
1 · cm
2). However,
the ranges for Isc and Jv
measurements are relatively large (5-29 µA/cm2 and
2.1-15.3
µl · h
1 · cm
2).
Therefore, it is unclear whether the relative current deficit is
meaningful or is simply determined by the large relative deviations. As
mentioned above, the average Isc values
determined by us are consistent with values earlier determined by other
laboratories (25, 42, 44), although Kompella et al.
(25) report a suggestively wide range (5-44
µA/cm2). It may be that these preparations are especially
susceptible to edge damage; however, to determine that will require
further work.
One question the findings pose is whether such secretion can account
for part or all of the basal rate of tear fluid secretion. It would
appear that a large conjunctival contribution is entirely possible; the
total area of rabbit conjunctiva is ~7.2 cm2, and, at the
average fluid transport rate we determined (6.6 µl · h
1 · cm
2), the total
addition to tear fluid of conjunctival origin would amount to 0.79 µl/min or 47.5 µl/h. For comparison, the physiological basal (or
minimal) flow rate for rabbit conjunctival tear fluid is reportedly
(3) 0.72 µl/min or 43.2 µl/h. All other prior reports
summarized in Fig. 8A
(3, 6, 8, 9, 13, 19, 33, 37, 40, 41) are also consistent
with the possibility that the basal tear secretion may arise in good
part (if not mostly) from the conjunctiva. Some supportive indications
can be obtained from the data in Fig. 8B. The basal
secretion observed in the absence of the main lacrimal gland of
squirrel monkeys (~0.4 µl/min in our estimate, Fig. 8B)
might correspond to secretion by accessory glands, as interpreted
before (30), but might also include a component of basal
conjunctival secretion (~0.6 µl/min in the rabbit; cf. Fig.
8A).

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Fig. 8.
A: comparison of our present data with those in reports
of tear and lacrimal gland basal rates of secretion. B:
secretion of tears in eyes with and without lacimal
glands.
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We also report that, importantly, the rabbit conjunctival secretion is
subject to purinergic stimulus (Figs. 6 and 8A and see
discussion below). Whether the conjunctival secretion is also subject
to neural stimulus (as is tear secretion, Fig. 8B) remains to be determined. We find that fluid transport across the rabbit conjunctiva is stimulated by mucosal additions of the P2Y2
receptor agonists UTP and INS365. Figure 6 depicts experiments in which the stimulation caused by these agonists at 10
5 M is
clearly visible. However, on average, the extent of stimulation was
somewhat less than shown there, and amounted to ~50% (see RESULTS and Fig. 8A). A similar trend is visible
in the stimulation of Isc by 10
5 M
UTP and INS365 (Fig. 2): integrating the area under the respective curves yields stimulations of 26% and 35%, respectively. Within experimental error, the secretion of fluid is stimulated about the same
as Isc; whether electrolyte movements other than
apical Cl
secretion are involved in fluid transport
remains to be explored.
These results might conceivably account for a role of conjunctival
fluid in hydrating the surface of the cornea. In this connection, it
has been recently reported that topical instillation of INS365 in vivo
consistently increased tear secretion compared with untreated or
saline-treated rabbit eyes (47). The increase in tear
secretion was statistically significant at 5-15 min after the
application, and, consistent with the in vitro results reported here,
an 8.5% solution of INS365 caused an approximately twofold increase in tear secretion. It is known that P2Y2 receptors play an
important role in regulation of conjunctival mucin secretion. In
addition, UTP and ATP induce an increase in mucin release on topical
application on the conjunctiva (20), so activation by UTP
or its agonists is believed to trigger the coordinated release of mucin
as well as fluid by the conjunctiva.
Given these and prior observations, under some conditions, the
conjunctival epithelium might serve as a conduit for osmotic flow and
contribute to dilute tear gland secretion. In addition, since we have
observed that the conjunctival secretion can be modulated, it is also
possible that the conjunctival epithelium might serve as part of a
mechanism to sense and correct the osmolarity of the fluid bathing it.
As we mentioned above, both ATP and UTP are not only highly effective
Cl
secretagogues when applied to the apical surface of
the airway epithelia but also can induce fluid secretion across airway
epithelia (2, 17). A similar role of P2Y2
receptor mediated by UTP and ATP has been explored in the conjunctiva.
The results indicate that the rabbit and human conjunctival cells
contain functional P2Y2 nucleotide receptors
(20), which have been confirmed in human conjunctiva by
RT-PCR techniques (21).
A common, vexing condition in ocular pathology known as "dry eye
syndrome" is produced by abnormalities in precorneal tear film
components (aqueous, mucin, or lipid), which may lead to loss of tear
film stability (12). The resulting dry spots on the
corneal and conjunctival epithelia have adverse repercussions on the
ocular epithelial surface cells. The results from this and other
laboratories discussed above indicate that, by stimulating secretions
of fluid and mucin, P2Y2 agonists may prove useful to treat
dry eye disease.
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ACKNOWLEDGEMENTS |
This work was supported by Inspire Pharmaceuticals (Grant CU512848)
and in part by Research to Prevent Blindness.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. Fischbarg, Dept. of Physiology, College of Physicians and Surgeons, Columbia Univ., 630 West 168th St., New York, NY 10032 (E-mail: jf20{at}columbia.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 28 March 2000; accepted in final form 27 March 2001.
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