Epinephrine-Induced Increases in [Ca2ⴙ]in and
KCl-Coupled Fluid Absorption in Bovine RPE
Jodi Rymer,1 Sheldon S. Miller,1,2 and Jeffrey L. Edelman2,3
PURPOSE. To define the ionic basis for the apical epinephrineinduced increase of fluid absorption (JV) across isolated bovine
RPE– choroid.
METHODS. Epinephrine-induced changes in RPE [Ca2⫹]in levels
were monitored with the ratioing dye fura-2. Transepithelial
potential, resistance, and unidirectional fluxes of 36Cl, 86Rb (K
substitute), and 22Na were simultaneously determined in
paired tissues from the same eye mounted in modified Üssing
flux chambers. Radioisotopes (5–7 ␮Ci) were added to the
apical bath of one tissue and the basal bath of the other, and
the appearance of label in the opposite bath was measured.
RESULTS. Apical epinephrine (100 nM) transiently increased
[Ca2⫹]in by 153 ⫾ 78 nM. This increase was inhibited by the
␣1-adrenoreceptor antagonist prazosin (1 ␮M) and blocked by
CPA(5 ␮M), an inhibitor of endoplasmic reticulum Ca2⫹-adenosine triphosphatases (ATPases). Apical epinephrine (100 nM)
more than doubled the net Cl absorption rate, increased net K
(86Rb) absorption by fivefold, and tripled net fluid absorption
( JV), as predicted by isotonic coupling between ion and fluid
transport. The epinephrine-induced increases in ion and fluid
transport were completely inhibited by apical bumetanide
(100 ␮M).
CONCLUSIONS. Epinephrine increased fluid absorption across
bovine RPE by activating apical membrane ␣1-adrenergic receptors, increasing [Ca2⫹]in, and stimulating bumetanide-sensitive Na,K,2Cl uptake at the apical membrane and KCl efflux
at the basolateral membrane. (Invest Ophthalmol Vis Sci. 2001;
42:1921–1929)
I
n the back of the eye, fluid is driven across the retinal
pigment epithelium (RPE) by hydrostatic and oncotic pressure and by active solute-linked fluid transport. The RPE contains a variety of plasma membrane proteins and intracellular
signaling molecules that maintain the chemical composition
and volume of the RPE and its extracellular environment on
either side of the cell.1,2 Active ion-linked fluid transport across
the RPE has been demonstrated both in vitro and in vivo.1,3
Although most epithelia can absorb or secrete fluid, they are
commonly categorized as fluid absorbing or secreting based on
the polarity of their Na,K,2Cl cotransporters and Cl channels.4
In secreting epithelia, the cotransporters are normally located
at the basolateral membrane and the Cl channels at the apical
From the 2School of Optometry and the 1Department of Molecular
and Cell Biology, University of California, Berkeley.
3
Present affiliation: Biological Sciences, Allergan, Inc., Irvine, California.
Supported by National Eye Institute Grant EY02205 (SSM) and
Core Grant EY03176.
Submitted for publication January 3, 2001; revised March 5, 2001;
accepted April 6, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Sheldon S. Miller, University of California,
Berkeley, 360 Minor Hall, Berkeley, CA 94720-2020.
smiller@socrates.berkeley.edu
Investigative Ophthalmology & Visual Science, July 2001, Vol. 42, No. 8
Copyright © Association for Research in Vision and Ophthalmology
membrane. In epithelia, such as the RPE, that normally absorb
fluid, the converse is true: The cotransporters are located in
the apical membrane and the Cl channels at the basolateral
membrane.5,6 Of course, there are many interesting exceptions—for example, the choroid plexus, which normally secretes fluid but contains both Na,K,2Cl cotransporters and Cl
channels on the apical membrane.7
In vivo there are many paracrine and hormonal signals that
impinge on the RPE during light and dark. The RPE responds to
the integrated activity of these input signals, for example,
adenosine triphosphate (ATP), dopamine, neuropeptide Y,
5-HT, or epinephrine through a variety of plasma membrane
receptors and second-messenger pathways that activate solutelinked fluid transport across the RPE.5,8 –12 In vitro it has been
shown that many of these receptors are metabotropically coupled to cell Ca2⫹ and cAMP in mammalian and human
RPE.8,13–17 Physiological analysis of the intact epithelium in
vitro, in native rabbit, bovine, or human RPE5,8,9,11,12,18,19 or in
vivo 3 have focused mainly on adrenergic or purinergic receptors. In particular, it has been shown that activation of apical
membrane ␣1-adrenergic or P2Y2 purinergic receptors activated apical membrane Na,K,2Cl cotransporters, and basolateral and apical membrane K and Cl channels, respectively.8,11,20 In both cases, an electrophysiological analysis suggested the possibility that increased KCl transport across the
RPE could provide the driving force for increased fluid absorption.8,11,12
In the present experiments, we have provided the ionic
basis for the epinephrine-induced stimulation of fluid absorption ( JV) using radiolabeled ion tracers and capacitive probe
fluid transport measurements. In addition, using the Ca2⫹sensitive dye fura-2, we have identified intracellular Ca2⫹ as the
second messenger involved in epinephrine signaling in bovine
RPE.
MATERIALS
AND
METHODS
The bovine eyes used in these experiments were obtained from a local
slaughterhouse, 15 to 40 minutes after death. They were placed in
ice-cold Ringer and transported to the laboratory. Techniques for
isolating bovine RPE– choroid have been previously described.21
Intracellular Ca2ⴙ Measurements
Intracellular Ca2⫹ levels were monitored with the fluorometric ratioing
dye fura-2 AM (Molecular Probes, Eugene, OR) in a modified Üssing
chamber. The chamber and setup have been described previously.22,23
Briefly, melanotic RPE– choroid preparations were bathed in Ringer
solution containing 12.5 to 25 ␮M fura-2 AM (dissolved in dimethyl
sulfoxide [DMSO]⫹20% pluronic acid) for 2 hours (8% CO2, room
temperature) to load them with dye. In addition, 1 mM probenecid was
included in the loading solution and all subsequent apically perfused
Ringer solutions to inhibit dye extrusion by the organic anion transporter located in the apical membrane.23 Tissues were mounted between the two halves of the perfusion chamber, and the perfusion
solutions were maintained at 34°C. Photic excitation was achieved
using a xenon light source filtered at 340 and 380 nm every 0.5
seconds. Approximately 10 cells were present in each field. The emission fluorescence was measured at 510 nm or more with a photomul-
1921
1922
Rymer et al.
tiplier tube (Thorn, EMI) and the ratio of the fluorescence intensities at
340/380 nm (R) was determined every second. The technique and
computer software for data acquisition have been described previously.24 Solution changes were made at a distance of 30 cm from the
chamber, causing an approximate 30-second delay in the arrival of a
new solution to the apical bath.
Fura-2 calibration was performed in situ by first perfusing both
membranes with Ca2⫹-free Ringer solution containing 5 to 10 mM
EGTA, which chelates any residual free Ca2⫹, and 10 ␮M ionomycin, a
Ca2⫹ ionophore that facilitates the equilibration of [Ca2⫹]in and external Ca2⫹ ([Ca2⫹]o). The tissue was then exposed to a saturating (1.8
mM) concentration of Ca2⫹. [Ca2⫹]in was determined according to the
equation [Ca2⫹]in ⫽ Kd (Fmin/Fmax)[(R ⫺ Rmin)/(Rmax ⫺ R)], where Kd
is the dissociation constant for fura-2 AM (220 nM) and Fmin and Fmax
are the fluorescence intensities at 380 nm in the absence and presence,
respectively, of saturating Ca2⫹.25 For technical reasons, calibrations
could not be obtained in all experiments. Transepithelial potential
(TEP) and transepithelial resistance (RT) were determined concurrently with Ca2⫹ measurements. This was achieved using calomel
electrodes in series with Ringer–agar bridges (4%) to make contact
with the apical and basolateral baths. A voltage– current clamp
(VCC600; Physiologic Instruments, San Diego, CA) was used to pass a
1-␮A current pulse across the tissue, and the RT was calculated from
the current-induced change in TEP.
Ion Flux Experiments
The method for determining the transepithelial unidirectional fluxes of
36
Cl, 86Rb (K substitute), and 22Na was similar to that described
earlier.26,27 Briefly, paired pieces of RPE– choroid were obtained from
the same eye and mounted in identical clamping chips and flux chambers with an exposed surface area of 0.3 cm2. The apical and basolateral baths (1.8 ml volume) were maintained at approximately 37°C by
warmed circulated water flowing through the water-jacketed chambers. Gas was injected into both bathing solutions through stainlesssteel 30-gauge needles, which mixed the baths and maintained the pH
at approximately 7.4. Ringer–agar bridges located on either side of the
tissues were used to measure TEP and RT. RT was calculated from the
change in TEP induced when a 5-␮A pulse was passed across the
tissue.
Radioisotope (5–7 ␮Ci) was added to the apical bath of one tissue
and basal bath of the other, and the appearance of label was measured
from the opposite bath at either 10- or 15-minute intervals. Samples
were counted on a scintillation counter (LS 7500; Beckman, Berkeley,
CA) and ion flux calculated as microequivalents per square centimeter
per hour.
Fluid Transport Measurements
The capacitive probe technique used to determine transepithelial fluid
transport has been described previously.8,12,28 Isolated bovine RPE–
choroid was placed in a clamping chip with an exposed surface area of
0.71 cm2. The clamping chip was placed between two water-jacketed
chamber halves, each with 12 ml total bath volume. Heated-circulated
water kept the bathing solution temperature at 35°C ⫾ 0.05°C. Lshaped canals, approximately 1 mm in diameter, connected the baths
with Teflon cups located at the top of each half chamber. The chamber
was placed in an incubator kept at a constant 31.5°C with open water
reservoirs that kept the relative humidity at 80% ⫾ 5%. The bathing
solutions were preheated to 38°C and perfused into the chambers
using a push–pull system equipped with 50-ml gas-tight feed-and-drain
syringes (Hamilton Co., Reno, NV). The measurement of fluid transport
was initiated after a 30- to 45-minute temperature equilibration period.
Stainless-steel capacitive probes (Accumeasure System 1000; MT
Instruments, Latham, NY) were lowered into the Teflon cup of each
half chamber. The capacitance of the air gap between probe and fluid
meniscus was measured and the system calibrated so that the voltage
output from each probe reflected the loss of fluid from one chamber
and the concomitant increase of fluid from the opposite chamber. The
IOVS, July 2001, Vol. 42, No. 8
difference signal was converted into fluid transport rate (microliters
per square centimeter per hour) and plotted as a function of time. The
TEP was measured using Ringer–agar bridges in contact with Ag-AgCl
electrodes and monitored with a voltage– current clamp (VCC600;
Physiologic Instruments). RT was obtained by passing a 6-␮A current
pulse across the tissue through Ag-AgCl electrodes for a duration of 1
second at 1-minute intervals and monitoring the change in TEP. Signals
from the capacitive probes ( JV) and tissue electrical parameters (TEP
and RT) were digitized, stored, and plotted online by microcomputer
(IBM, Armonk, NY).
Bathing Solutions and Materials
The control Ringer solution consisted of (in mM): 120 NaCl, 23
NaHCO3, 5 KCl, 1 MgCl2, 1.8 CaCl2, and 10 glucose gassed with 85%
N2, 10% O2, and 5% CO2 (to keep pH constant at 7.4 ⫾ 0.05).
Nominally HCO3-free Ringer was prepared by substitution of 23 mM
NaHCO3 with 12 mM sodium cyclamate and 10 mM HEPES and equilibrated with room air (pH 7.4). All solutions had a final osmolarity of
295 ⫾ 5 mOsm. To increase tissue longevity for flux experiments, 1
mM glutathione was added to the Ringer.21,27 For Ca2⫹ experiments, 2
mM glutathione was added at the beginning of each experiment.
Epinephrine (bitartrate salt), prazosin, cyclopiazonic acid (CPA), and
bumetanide were obtained from Sigma Chemical Co. (St. Louis, MO).
22
Na and 86Rb were obtained from Amersham Corp. (Arlington
Heights, IL) and 36Cl from ICN (Irvine, CA). CPA was prepared as a
stock solution in DMSO, and diluted to a final concentration of less
than 0.02% (vol/vol). DMSO alone does not alter bovine RPE [Ca2⫹]in
at this concentration.
All data are summarized as mean ⫾ SEM, unless stated otherwise.
Statistical comparisons were made using Student’s t-test. Differences
were considered significant at P ⬍ 0.05.
RESULTS
Effect of Epinephrine on [Ca2ⴙ]in
The ratiometric Ca2⫹-sensitive dye fura-2 was used to determine the effects of apical bath epinephrine on intracellular
Ca2⫹ concentrations ([Ca2⫹]in) in bovine RPE. In each experiment, approximately 10 to 15 cells were examined within the
RPE cell sheet, and the same cells were monitored for the
duration of the experiment. Mean (⫾SD) baseline [Ca2⫹]in was
96 ⫾ 8 nM in four RPE– choroid preparations. One-minute
applications of epinephrine (100 nM) in the apical bathing
solutions caused rapid, transient [Ca2⫹]in increases of 153 ⫾ 78
nM. Typical responses are illustrated in Figure 1. Repeated
applications of epinephrine in a single preparation led to repeated [Ca2⫹]in increases. Ten separate epinephrine applications in four RPE– choroid preparations caused a range of
[Ca2⫹]in increases between 53 and 292 nM. Epinephrine also
transiently increased TEP and decreased RT, as previously observed in microelectrode studies.11
Figure 2 illustrates the effect of prazosin, an ␣1-adrenoreceptor antagonist, on 100 nM epinephrine-induced changes in
[Ca2⫹]in. In this experiment, prazosin completely inhibited the
transient [Ca2⫹]in increase caused by epinephrine treatment,
and in two other experiments, prazosin decreased the Ca2⫹
response by 90%. In all preparations, prazosin inhibited TEP
responses by approximately 50% and completely prevented
the changes in RT (n ⫽ 3). It has been shown that prazosin
inhibits epinephrine-induced changes in basolateral and apical
membrane voltage and resistance.11
Figure 3 summarizes the effect of CPA on the epinephrineinduced Ca2⫹ increase. RPE monolayers were treated with
epinephrine (100 nM) in the absence (Fig. 3, left trace) and
presence (Fig 3, right trace) of CPA (5 ␮M). CPA is an inhibitor
of endoplasmic reticulum (ER) Ca2⫹-ATPase,29 which depletes
ER Ca2⫹ stores by blocking uptake through the Ca2⫹ pump
IOVS, July 2001, Vol. 42, No. 8
FIGURE 1. Effect of epinephrine on [Ca2⫹]in (top), TEP, and RT (bottom). Epinephrine (100 nM) was applied apically for approximately
1-minute intervals, indicated by the filled boxes. Epinephrine caused
transient increases of 225, 112, and 292 nM in [Ca2⫹]in. Epinephrine
also led to TEP increases of 0.6, 0.8, and 0.95 mV, and RT decreases of
7.5, 8.5, and 5 ⍀ 䡠 cm2. All Ca2⫹ responses were obtained from the
same set of approximately 10 cells within the RPE.
without affecting efflux through leakage pathways. If the epinephrine-induced increase in cytosolic Ca2⫹ levels is due to
release of Ca2⫹ from the ER, CPA should abrogate the epinephrine-induced Ca2⫹ change. This result is illustrated in Figure 3.
In five other preparations, epinephrine (100 nM) had no effect
on [Ca2⫹]in in the presence of CPA. In Figure 3, the Ca2⫹
response was reduced by 96% in the presence of CPA.
KCl-Coupled Fluid Absorption across RPE
1923
FIGURE 2. Effect of prazosin on the epinephrine-induced [Ca2⫹]in
increase, TEP, and RT. Ca2⫹ data are represented as fura-2 ratio, which
increases with [Ca2⫹]in. Apically applied epinephrine (100 nM) led to
a transient ratio increase (A), TEP increase, and RT decrease. The
simultaneous application of the ␣1-adrenoreceptor antagonist prazosin
(1 ␮M) with epinephrine (100 nM) fully prevented the ratio increase
and RT decrease and diminished the TEP change by approximately 50%
(B). The effects of prazosin were not fully reversible (C).
JV by 1.3 ␮l/cm2 per hour and concomitantly decreased TEP to
0 and increased RT by 10 ⍀ 䡠 cm2.
Figure 4B shows a similar result obtained from a different
tissue measured in HCO3-free Ringer (10 mM HEPES, pHo 7.4).
The JV (top) is shown along with the simultaneous measure-
[HCO3]o Dependence of Epinephrine-Induced
Changes in JV
NaHCO3 cotransporters have been identified at the apical and
basolateral membranes of bovine RPE.30 Fluid transport across
frog RPE has been shown to be mediated by an apical membrane NaHCO3 cotransporter.1 An epinephrine-induced alteration in the activities of one or more of these transporters
could contribute to the observed change in JV. This possibility
was examined in the experiments summarized in Figure 4. JV
(Fig. 4A, 4B; top) and TEP/RT (Fig. 4A, 4B; bottom) were
measured in the presence (Fig. 4A) and absence (Fig. 4B) of
exogenous HCO3. In control Ringer (23 mM HCO3/5% CO2;
pHo 7.4), JV decreased from 2.5 ␮l/cm2 per hour to 0.8 ␮l/cm2
per hour during the first 50 minutes of the experiment shown
in Figure 4A. During this time TEP decreased from approximately 8 to 5mV and RT increased from approximately 220 to
230 ⍀ 䡠 cm2. Very similar changes in JV, TEP, and RT were
previously observed in this preparation (n ⫽ 22).12 At t ⫽ 50
minutes, 100 nM epinephrine was added apically and increased
JV by a factor of 3, to 2.5 ␮l/cm2 per hour. The TEP increased
transiently to 10 mV (from a baseline of 5 mV), and RT decreased by approximately 25 ⍀ 䡠 cm2. At t ⫽ 125 minutes, 100
␮M bumetanide was added to the apical bath and decreased
FIGURE 3. Effect of CPA on the epinephrine-induced [Ca2⫹]in increase. Left: apical epinephrine alone (100 nM, dashed line) caused a
transient [Ca2⫹]in increase of 225 nM. Right: apical CPA (5 ␮M), an ER
Ca2⫹-ATPase blocker, caused a transient increase in Ca2⫹ followed by
a much smaller steady state elevation above baseline. In the presence
of CPA, epinephrine (dashed line) did not significantly increase
[Ca2⫹]in.
1924
Rymer et al.
IOVS, July 2001, Vol. 42, No. 8
TABLE 1. Open Circuit Net Fluxes of
Bovine RPE
Tracer
Apical-to-Basal (n ⫽ 6)
J a⫺b (␮eq/cm2 䡠 hr)
TEP (mV)
R T (⍀ 䡠 cm2)
Basal-to-Apical (n ⫽ 6)
J b⫺a (␮eq/cm2 䡠 hr)
TEP (mV)
R T (⍀ 䡠 cm2)
Net flux
(␮eq/cm2 䡠 hr)
36
Cl,
36
Cl
86
Rb, and
22
Na across
86
Rb
22
Na
4.08 ⫾ 0.27
8.1 ⫾ 1.4
159 ⫾ 11
0.16 ⫾ 0.01
9.3 ⫾ 1.1
154 ⫾ 14
3.41 ⫾ 0.24
9.1 ⫾ 1.4
169 ⫾ 10
0.11 ⫾ 0.01 2.53 ⫾ 0.21
9.9 ⫾ 1.4
6.3 ⫾ 1.6
160 ⫾ 16
165.9 ⫾ 9
0.67
0.05
3.21 ⫾ 0.32
6.5 ⫾ 1.2
160 ⫾ 7
0.68
Data are mean ⫾ SEM for a minimum of 1 hour of open-circuit
measurement across paired tissues from the same eye cup. There was
a significant net absorption (retina-to-choroid) for the three ions (P ⬍
0.05), but TEP and R T were not significantly different between the
paired groups.
ment of TEP and RT. After a temperature equilibration period of
approximately 45 minutes, JV was approximately 1 ␮l/cm2 per
hour. At 116 minutes, 100 nM epinephrine was added to the
apical bath, and JV increased by a factor of three, to approximately 3 ␮l/cm2 per hour. The TEP transiently increased by
approximately 5 mV and then decreased toward baseline. At
200 minutes, 100 ␮M bumetanide was added to the apical bath
and decreased JV by approximately 2.1 ␮l/cm2 per hour. This
was accompanied by a rapid decrease in TEP of approximately
6 mV. All these electrical responses have been previously
documented, by using intracellular recording techniques.6,11,31
In three experiments, measured in HCO3-free Ringer (three
eyes), epinephrine increased JV from 0.31 ⫾ 0.54 to 2.03 ⫾
0.95 ␮l/cm2 per hour, and TEP from 5.7 ⫾ 1.8 to 10.4 ⫾ 1.2
mV. RT was not significantly changed (183 ⫾ 43 to 172 ⫾ 34
⍀ 䡠 cm2). In two of these experiments, the epinephrine-induced stimulation of JV was inhibited by 1.4 and 2.1 ␮l/cm2
per hour with bumetanide (100 ␮M). These results indicate
that bath HCO3 is not required for the epinephrine-induced
stimulation of JV.
Effect of Epinephrine on the Net Fluxes
of Cl, Na, and K
Table 1 summarizes the results from a series of radioactive
tracer experiments using 36Cl, 22Na, and 86Rb (K substitute).
The value shown for net flux is the difference between the
unidirectional fluxes obtained across six paired tissues per
isotope. The direction of net flux was from apical-to-basal
(absorption) for all three ions (P ⬍ 0.05). The net Cl and Na
absorption rates were 0.67 and 0.68 ␮eq/cm2 per hour, respectively, and there was a small but significant net K absorption of
0.05 ␮eq/cm2 per hour. TEP ranged from approximately 6.5 to
10 mV and RT from approximately 155 to 165 ⍀ 䡠 cm2. There
Š
FIGURE 4. Effect of epinephrine on JV rate (top), TEP, and RT (bottom) in the presence and absence of bath HCO3. (A) In control Ringer
(23 mM HCO3-5% CO2, pH 7.4), 100 nM epinephrine was added to the
apical bath (50 minutes). JV and TEP increased and RT decreased. At
approximately 125 minutes, 100 ␮M bumetanide was added to the
apical bath and reversed the epinephrine-induced changes. (B) A
similar experiment on a different tissue measured in nominally HCO3free Ringer buffered with 10 mM HEPES and equilibrated with room air
(pH 7.4). The stimulation of JV and TEP with 100 nM apical epinephrine and the inhibition with 100 ␮M bumetanide produced effects very
similar to those measured in HCO3 Ringer.
KCl-Coupled Fluid Absorption across RPE
IOVS, July 2001, Vol. 42, No. 8
FIGURE 5. Simultaneous measurement of unidirectional 36Cl flux,
TEP, and RT across paired tissues from the same eye. (F) JCla-b; (E)
JClb-a. Top, solid lines: mean JCla-b; dashed lines: mean JClb-a. The
difference between these two values at every time point is the net flux.
In control Ringer, net Cl absorption rate was approximately 0.20
␮eq/cm2 per hour. At 60 minutes, 100 nM epinephrine was added to
the apical side of both tissues as a concentrated stock solution. It
increased net 36Cl flux to approximately 0.60 ␮eq/cm2 per hour.
Epinephrine transiently increased TEP, which was followed by a sustained increase, but did not alter the RT. At 120 minutes, 100 ␮M
bumetanide was added to the apical bath of both tissues. It inhibited
both unidirectional 36Cl fluxes, decreased the net flux to approximately 0, decreased TEP, and increased RT in both tissues.
was no significant difference in TEP and RT between groups of
paired tissues.
Figure 5 illustrates the effect of epinephrine on the simulTABLE 2. Stimulation of
Treatment
36
Cl,
86
Rb (K), and
1925
taneous measurement of 36Cl transport, TEP, and RT, across
paired tissues from the same eye cup. The solid circles represent the unidirectional apical-to-basal Cl flux rate ( JCla-b), and
the open circles depict the unidirectional basal-to-apical flux
rate ( JClb-a). In the top panel, the solid lines represent the
mean JCla-b and the dashed lines the mean JClb-a. Net Cl flux is
the difference between the mean unidirectional fluxes. In
control Ringer, net Cl absorption rate was approximately 0.20
␮eq/cm2 per hour. TEP and RT were similar in both tissues
(middle and bottom panels). At 60 minutes, 100 nM epinephrine was added to the apical bath. It more than tripled the rate
of Cl absorption to 0.64 ␮eq/cm2 per hour, primarily by stimulating JCla-b. The TEP was rapidly increased in both tissues by
4 to 5 mV, followed by a rapid decrease to near baseline and a
slower sustained increase. The reduction in RT (Fig. 1) could
not be seen in the tracer flux experiments, because the frequency of the current pulses (one every 8 minutes) was too
low to capture the relatively rapid Ca2⫹-induced changes in RT.
At 120 minutes, 100 ␮M bumetanide was added to the apical
bath. It decreased both unidirectional fluxes and reduced the
net flux to 0, decreased the TEP by approximately 7 mV and
increased the RT by 15 to 20 ⍀ 䡠 cm2 in both tissues.
The stimulation of Cl absorption by epinephrine is summarized in Table 2. Data from five paired tissues (five eyes)
indicate that epinephrine (100 nM) more than doubled the rate
of net Cl absorption from 0.28 ⫾ 0.12 to 0.60 ⫾ 0.17 ␮eq/cm2
per hour, TEP was increased by approximately 3 mV. In three
of these experiments, 100 ␮M apical bumetanide decreased
the epinephrine-stimulated net Cl absorption rate by a factor of
20, from 0.66 ⫾ 0.21 to 0.03 ⫾ 0.10 ␮eq/cm2 per hour. These
results support the notion that epinephrine stimulates the Cl
absorption pathway, which includes the apical membrane
Na,K,2Cl cotransporter.
Figure 6 shows the effect of epinephrine on the simultaneous measurement of unidirectional K (86Rb) flux, TEP, and
RT across paired tissues. The solid circles represent data from
the tissue used to measure the unidirectional JKa-b flux rate,
and the open circles depict JKb-a. The unidirectional fluxes are
shown in the top panel and TEP and RT in the middle and
bottom panels, respectively. In control Ringer, there was a
small but significant net absorption of K of approximately 0.05
␮eq/cm2 per hour. At 75 minutes, 100 nM epinephrine was
added to the apical bath, and net K absorption was increased
by stimulating JKa-b by a factor of 6, to approximately 0.30
␮eq/cm2 per hour. It also caused a transient increase in TEP, as
expected. At 180 minutes, 100 ␮M bumetanide was added to
the apical bath and decreased net K transport to approximately
0 by reducing JKa-b. TEP decreased and RT increased in both
22
Na Absorption by Apical Epinephrine
Jaⴚb
Jbⴚa
Jnet
(␮eq/cm2 䡠 hr)
TEP (mV)
RT
(⍀ 䡠 cm2)
3.85 ⫾ 0.19
3.92 ⫾ 0.18
3.57 ⫾ 0.14
3.32 ⫾ 0.10
0.28 ⫾ 0.12
0.60 ⫾ 0.17*
7.9 ⫾ 1.0
11.0 ⫾ 1.2
155 ⫾ 7
154 ⫾ 6
0.142 ⫾ 0.007
0.310 ⫾ 0.042*
0.104 ⫾ 0.014
0.103 ⫾ 0.012
0.038 ⫾ 0.015
0.207 ⫾ 0.044*
10.7 ⫾ 1.1
13.6 ⫾ 1.0*
175 ⫾ 10
170 ⫾ 9
3.74 ⫾ 0.56
4.01 ⫾ 0.65
2.74 ⫾ 0.37
2.96 ⫾ 0.20
1.00 ⫾ 0.63
1.15 ⫾ 0.61
7.6 ⫾ 1.6
10.0 ⫾ 1.0*
152 ⫾ 7
145 ⫾ 9
36
Cl
Control
Epinephrine
86
Rb (K)
Control
Epinephrine
22
Na
Control
Epinephrine
Flux data (columns 2– 4) are mean ⫾ SEM for 1 hour or more of steady state measurement taken from five sets of paired tissues (Cl), four sets
of paired tissues (K), and three sets of paired tissues (Na) before and after the addition of 100 nM epinephrine. Data for TEP and R T were measured
just before and approximately 15 minute after drug addition (peak change) and include the combined data from all tissues (n ⫽ 12).
* P ⬍ 0.05; Student’s t-test.
1926
Rymer et al.
IOVS, July 2001, Vol. 42, No. 8
TEP (2.4 mV). These results suggest that the Na,K pump rate is
not measurably altered by epinephrine and therefore strengthens our conclusion that the epinephrine-induced stimulation of
KCl absorption is mainly determined by the bumetanide-sensitive Na,K,2Cl cotransporter.
DISCUSSION
The apical membrane of both bovine and native human RPE
contain ␣1-adrenergic receptors that can be activated by nanomolar amounts of epinephrine.5,11 In the present experiments
in bovine RPE, we showed that apical epinephrine elevated
cell calcium and increased the rate of net Cl (36Cl) and K (86Rb)
absorption, without affecting net Na (22Na) transport. Net fluid
and KCl absorption were stimulated by apical epinephrine and
inhibited by apical bumetanide. The epinephrine-induced
[Ca2⫹]in changes were blocked at two points in the signaltransduction pathway: at the apical membrane, by prazosin,
and intracellularly by the ER Ca2⫹-ATPase inhibitor CPA. These
[Ca2⫹]in changes coincided in time with the epinephrine-induced increase in basolateral membrane Cl conductance and
decrease in apical membrane K conductance11 that ultimately
produced the increase in KCl-coupled fluid absorption. The
model summarized in Figure 7 can be used to understand the
plasma membrane and intracellular signaling pathways that
mediate fluid absorption across the RPE.
Receptor Activation and Signal Transduction
FIGURE 6. Simultaneous measurement of unidirectional 86Rb fluxes
(K substitute), TEP, and RT across paired tissues from the same eye. (F)
JCla-b; (E) JClb-a. Top, solid lines: mean JCla-b; dashed lines: mean JClb-a.
In control Ringer, net 86Rb (K) absorption rate was approximately 0.05
␮eq/cm2 per hour. At 75 minutes, 100 nM epinephrine was added to
the apical bath. Net 86Rb (K) absorption rate was stimulated to approximately 0.27 ␮eq/cm2 per hour, and TEP was transiently increased,
followed by a sustained increase. RT was decreased in both tissues by
5 to 10 ⍀ 䡠 cm2. At 180 minutes, 100 ␮M bumetanide was added to the
apical bath and decreased net 86Rb (K) transport to approximately 0,
decreased the TEP by approximately 8 mV, and increased the RT by 20
to 30 ⍀ 䡠 cm2 in both tissues.
tissues by approximately 7 mV and approximately 30 ⍀ 䡠 cm2,
respectively.
Table 2 summarizes the effects of epinephrine on the simultaneous measurement of net K (86Rb) absorption, TEP, and RT.
In four experiments, 100 nM epinephrine stimulated net K
absorption from 0.038 ⫾ 0.015 to 0.207 ⫾ 0. ␮eq/cm2 per
hour by increasing JKa-b. The TEP was increased by approximately 3 mV, but the RT was not significantly changed. In two
of these experiments, 100 ␮M bumetanide decreased the epinephrine-stimulated net K absorption from 0.26 to 0.04 ␮eq/
cm2 per hour and 0.31 to 0.08 ␮eq/cm2 per hour.
These experiments, taken together, show that net KCl absorption is stimulated by epinephrine and strongly suggest that
the bumetanide-sensitive Na,K,2Cl cotransporter mediated this
response. However, K also enters the cell through the apical
membrane Na,K-ATPase.21,27,32 If the Na,K pump was stimulated by epinephrine, then net Na transport may have been
altered.28,33
The effect of 100 nM apical epinephrine on net Na (22Na)
transport is summarized in Table 2. In three experiments
(three eyes), both unidirectional 22Na fluxes were increased
slightly, but there was no significant change in unidirectional
or net flux (P ⬎ 0.50). Epinephrine significantly increased the
In many systems it has been shown that activation of Gprotein– coupled receptors (e.g., ␣1-adrenoreceptors) leads to
the phospholipase C (PLC)–mediated production of inositol
triphosphate (IP3), and IP3 then interacts with its receptors on
the ER membrane to cause a transient increase in cell Ca2⫹.34
Previous work in cultured human and rat RPE cells has indicated that ␣-adrenergic receptor activation is coupled to PLC
activation.14,35 The RPE contains several other metabotropic
receptors that could activate this signal-transduction pathway
to increase basolateral membrane Cl channel activity.5,8,10,13,14
In bovine RPE, we have shown that A23187, a Ca2⫹ ionophore,
mimics the epinephrine-induced electrical responses, and that
BAPTA, a membrane-permeable Ca2⫹ buffer, prevents the
epinephrine-induced membrane potential and resistance
changes,11 suggesting that Ca2⫹ was the second messenger
mainly responsible for mediating the RPE responses to epinephrine. The present experiments strengthened this conclusion in two ways: by showing that prazosin, an ␣1-adrenergic
receptor antagonist, inhibited the epinephrine-induced increase in [Ca2⫹]in and by implicating the ER stores (Fig. 3) as
the source of Ca2⫹ that caused the epinephrine-induced
changes in membrane voltage and resistance.
In native fetal human RPE, epinephrine activates ␣ and ␤
adrenergic receptors at the apical membrane and two intracellular second messengers, Ca2⫹ and cAMP, seem to determine
the physiological responses.5 In bovine RPE,11 we previously
showed that epinephrine also increases intracellular cAMP
levels, and this suggests the possibility of cross talk between
the Ca2⫹ and cAMP transduction pathways. Figure 7 indicates
a possible locus of interaction for these two second messengers at the ER Ca2⫹-ATPase regulatory protein phospholamban
(PLB). It has been shown that this protein is present in bovine
and human RPE.9 In its unphosphorylated state, PLB inhibits ER
Ca2⫹-ATPase activity.36 This inhibition is removed by cAMPdependent protein kinase A (PKA) phosphorylation of PLB,
which stimulates Ca2⫹ uptake into ER stores and reduces
[Ca2⫹]in.37
The physiological effects of cAMP on bovine RPE physiology were studied by elevating cell cAMP using a cocktail of
IOVS, July 2001, Vol. 42, No. 8
KCl-Coupled Fluid Absorption across RPE
1927
FIGURE 7. Model of RPE apical
membrane receptor–mediated transepithelial fluid transport. In control
Ringer, ion-linked fluid absorption
(arrow) is mediated by apical membrane cotransporters and basolateral
membrane Cl channels. Potassium
(K) enters the cell through apical
membrane Na,K,2Cl cotransporters
or the Na,K pumps and is recycled
through apical membrane K channels. Na enters the cell through apical membrane Na,K,2Cl cotransporters and is recycled through Na,K
pumps. The apical membrane contains at least three G-protein– coupled receptors activated by ATP
(UTP), or epinephrine (EP). Cross
talk between the Ca2⫹ and cAMP signaling pathways is possibly mediated
by PLB, an ER-associated protein.9 At
the basolateral membrane, extracellular Cl is exchanged for intracellular
HCO3, Cl channel efflux is recycled
through this exchanger, and HCO3
is cotransported with Na. ADP, adenosine diphosphate. Lactate (Lac)
transporters are at the basolateral
membrane and a transmembrane adenylate cyclase (tmAC) is located at
the apical membrane.
cAMP-elevating drugs. In striking contrast to the epinephrine
results,11,12 elevation of cell cAMP per se decreased basolateral
membrane Cl conductance, increased cell Cl, and reversed the
direction of fluid transport from absorption to secretion. All
these changes were blocked by CPA, consistent with a cAMPmediated alteration in PLB phosphorylation.9 In the model
shown in Figure 7, we hypothesize that elevation of cell cAMP
acts on PLB to lower cell Ca2⫹ and, in effect, provides a
negative regulation of Ca2⫹-activated Cl conductance (and
fluid) increase that opposes the dominant effect of epinephrine.
Ionic Basis of Fluid Absorption across
Bovine RPE
In many epithelia, including frog RPE,28 net solute ( Js) and JV
are isotonically coupled,38,39 so that JV ⫽ Js/C, where Js ⫽ JCl
⫹ JNa ⫹ JK, and C is the solute concentration of the bathing
medium.1,28 The results summarized in Table 1 show that Js ⫽
1.40 ␮eq/cm2 per hour, which predicts a fluid absorption rate
of 4.7 ␮l/cm2 per hour in control Ringer. This value is in
relatively good agreement with the maximum JV rate (4 ␮l/cm2
per hour) measured in tissues of similarly high TEP and RT.12
The data summarized in Table 2 show that epinephrine
stimulated Js ( JCl ⫹ JK) by approximately 0.5 ␮eq/cm2 per
hour, which predicts an epinephrine-induced increase in JV of
1.7 ␮eq/cm2 per hour. This prediction is in excellent agreement with previous measurements showing that 100 nM epinephrine increased JV by 1.4 ␮l/cm2 per hour.12 It indicates
that Cl and K are the ions responsible for the epinephrineinduced stimulation of fluid absorption and suggests that ion
and fluid transport are isotonically coupled across bovine RPE.
KCl Absorption and RPE Physiology and Function
In combination with previous electrophysiological and fluid
transport studies,6,11,12,31 the present results demonstrate that
the rate of Na,K,2Cl cotransporter and conductive Cl efflux
were both increased by epinephrine. Activation of the cotransporter could have been a direct result of epinephrine-induced
increases in [Ca2⫹]in or cAMP, because the cotransport protein
has consensus protein kinase C (PKC) and PKA phosphorylation sites, and protein phosphorylation activates the cotransporter. Another possibility, based on work in a wide variety of
systems, is that Na,K,2Cl cotransporters are activated after a
decrease in cell Cl ([Cl]in), which then alters cotransporter
phosphorylation.40 Additional experiments would be required
to evaluate these possibilities.
In control Ringer, most of the K that enters the cell across
the apical membrane is recycled across that membrane (see
Fig. 7) through a large apical membrane K conductance.21,27
The small but significant net absorption of K measured in the
present study was probably driven through the paracellular
pathway by TEP, because no net K flux was measured under
short-circuited conditions.27 Epinephrine decreased the apical
membrane K conductance,11 which would reduce the recycling of K across the apical membrane and possibly stimulate
the transepithelial absorption of K, mediated by the cotransporter or pump27 (see Fig. 7).
The increase in net K (86Rb)Cl absorption stimulated by
epinephrine was completely inhibited by apical bumetanide
(Figs. 5, 6) indicating that the cotransporter mediated this
response. If net Na flux was altered by epinephrine, this may
indicate that the Na,K pumps were stimulated,33 but Table 2
shows that net Na flux was not significantly altered by epinephrine. One possibility is that that Na is recycled at the apical
membrane in the presence or absence of a secretagogue. Alternatively, if the cotransporter and the pump were both stimulated, KCl uptake might increase without an effect on Na
transport—a possibility that cannot be eliminated by the
present data.
1928
Rymer et al.
The basolateral membrane contains electrically coupled Cl
and K conductances that mediate KCl efflux.6,21,27,31 The epinephrine-induced K efflux could have been mediated by the
Ca2⫹ increase, by the change in membrane potential, or by
both. It is also possible that an epinephrine-induced increase in
cAMP increased K efflux at the basolateral membrane.11,41
Because epinephrine significantly depolarized the basolateral
membrane, it would increase the driving force for conductive
K efflux.6,21,42 Thus, in the steady state, K and Cl and osmotically obliged fluid exit the basolateral membrane, balanced by
the influx of K and Cl and osmotically coupled fluid at the
apical membrane.
In vitro and in vivo experiments have demonstrated that the
apical membrane cotransporters and apical and basolateral
membrane K and Cl channels are the main determinants of RPE
cell volume as well as subretinal space hydration and chemical
composition.32,43– 48The present experiments illustrate another level of regulation (Fig. 7) by showing that a putative
retinal paracrine signal such as epinephrine2,49,50 can activate
this pathway and perhaps act synergistically with other paracrine3 or hormonal signals to control the hydration and chemical composition of the extracellular spaces on either side of
the RPE.
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