Epinephrine-Induced Increases in [Ca2+]in and KCl-Coupled Fluid Absorption in Bovine RPE

Jodi Rymer; Sheldon S. Miller; Jeffrey L. Edelman

Abstract

purpose. To define the ionic basis for the apical epinephrine-induced increase of fluid absorption (J _V) 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 μCi) 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 α₁-adrenoreceptor antagonist prazosin (1 μM) and blocked by CPA(5 μM), 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 (J _V), 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 μM).

conclusions. Epinephrine increased fluid absorption across bovine RPE by activating apical membrane α₁-adrenergic receptors, increasing[ Ca²⁺]_in, and stimulating bumetanide-sensitive Na,K,2Cl uptake at the apical membrane and KCl efflux at the basolateral membrane.

In 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 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 solute-linked fluid transport across the RPE.⁵ ⁸ ⁹ ¹⁰ ¹¹ 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 ¹⁴ ¹⁵ ¹⁶ 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 α₁-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 (J _V) 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 Ca²⁺ 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 μM 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 photomultiplier 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 μM 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 = K _d (F _min/F _max)[(R − R _min)/(R _max− R)], where K _d is the dissociation constant for fura-2 AM (220 nM) and F _min and F _max 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 (R _T) 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-μA current pulse across the tissue, and the R _T was calculated from the current-induced change in TEP.

Ion Flux Experiments

The method for determining the transepithelial unidirectional fluxes of ³⁶Cl, 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 stainless-steel 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 R _T. R _T was calculated from the change in TEP induced when a 5-μA pulse was passed across the tissue.

Radioisotope (5–7 μCi) 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. L-shaped 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 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). R _T was obtained by passing a 6-μA 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 (J _V) and tissue electrical parameters (TEP and R _T) 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). ²²Na 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 [Ca²⁺]_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 R _T, as previously observed in microelectrode studies.¹¹

Figure 2 illustrates the effect of prazosin, anα 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 R _T (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 epinephrine-induced 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 μM). CPA is an inhibitor of endoplasmic reticulum (ER) Ca²⁺-ATPase,²⁹ which depletes ER Ca²⁺ stores by blocking uptake through the Ca²⁺ pump 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.

[HCO₃]_o Dependence of Epinephrine-Induced Changes in J _V

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 J _V. This possibility was examined in the experiments summarized in Figure 4 J _V (Fig. 4A 4B ; top) and TEP/R _T (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; pH_o 7.4), J _V decreased from 2.5μ l/cm² per hour to 0.8μ 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 R _T increased from approximately 220 to 230 Ω · cm². Very similar changes in J _V, TEP, and R _T were previously observed in this preparation (n = 22).¹² At t = 50 minutes, 100 nM epinephrine was added apically and increased J _V by a factor of 3, to 2.5 μl/cm² per hour. The TEP increased transiently to 10 mV (from a baseline of 5 mV), and R _T decreased by approximately 25Ω · cm². At t = 125 minutes, 100 μM bumetanide was added to the apical bath and decreased J _V by 1.3μ l/cm² per hour and concomitantly decreased TEP to 0 and increased R _T by 10 Ω · cm².

Figure 4B shows a similar result obtained from a different tissue measured in HCO₃-free Ringer (10 mM HEPES, pH_o 7.4). The J _V (top) is shown along with the simultaneous measurement of TEP and R _T. After a temperature equilibration period of approximately 45 minutes, J _V was approximately 1μ l/cm² per hour. At 116 minutes, 100 nM epinephrine was added to the apical bath, and J _V increased by a factor of three, to approximately 3 μl/cm² per hour. The TEP transiently increased by approximately 5 mV and then decreased toward baseline. At 200 minutes, 100 μM bumetanide was added to the apical bath and decreased J _V by approximately 2.1μ 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 J _V from 0.31 ± 0.54 to 2.03 ± 0.95 μl/cm² per hour, and TEP from 5.7 ± 1.8 to 10.4 ± 1.2 mV. R _T was not significantly changed (183 ± 43 to 172 ± 34 Ω · cm²). In two of these experiments, the epinephrine-induced stimulation of J _V was inhibited by 1.4 and 2.1μ l/cm² per hour with bumetanide (100 μM). These results indicate that bath HCO₃ is not required for the epinephrine-induced stimulation of J _V.

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 μeq/cm² per hour, respectively, and there was a small but significant net K absorption of 0.05 μeq/cm² per hour. TEP ranged from approximately 6.5 to 10 mV and R _T from approximately 155 to 165 Ω · cm². There was no significant difference in TEP and R _T between groups of paired tissues.

Figure 5 illustrates the effect of epinephrine on the simultaneous measurement of ³⁶Cl transport, TEP, and R _T, across paired tissues from the same eye cup. The solid circles represent the unidirectional apical-to-basal Cl flux rate (J _Cl ^a-b), and the open circles depict the unidirectional basal-to-apical flux rate (J _Cl ^b-a). In the top panel, the solid lines represent the mean J _Cl ^a-b and the dashed lines the mean J _Cl ^b-a. Net Cl flux is the difference between the mean unidirectional fluxes. In control Ringer, net Cl absorption rate was approximately 0.20μ eq/cm² per hour. TEP and R _T 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 μeq/cm² per hour, primarily by stimulating J _Cl ^a-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 R _T (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 R _T. At 120 minutes, 100 μM 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 R _T by 15 to 20Ω · 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 μeq/cm² per hour, TEP was increased by approximately 3 mV. In three of these experiments, 100 μM 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 μeq/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 R _T across paired tissues. The solid circles represent data from the tissue used to measure the unidirectional J _K ^a-b flux rate, and the open circles depict J _K ^b-a. The unidirectional fluxes are shown in the top panel and TEP and R _T in the middle and bottom panels, respectively. In control Ringer, there was a small but significant net absorption of K of approximately 0.05 μeq/cm² per hour. At 75 minutes, 100 nM epinephrine was added to the apical bath, and net K absorption was increased by stimulating J _K ^a-b by a factor of 6, to approximately 0.30 μeq/cm² per hour. It also caused a transient increase in TEP, as expected. At 180 minutes, 100 μM bumetanide was added to the apical bath and decreased net K transport to approximately 0 by reducing J _K ^a-b. TEP decreased and R _T increased in both tissues by approximately 7 mV and approximately 30Ω · cm², respectively.

Table 2 summarizes the effects of epinephrine on the simultaneous measurement of net K (⁸⁶Rb) absorption, TEP, and R _T. In four experiments, 100 nM epinephrine stimulated net K absorption from 0.038 ± 0.015 to 0.207 ± 0. μeq/cm² per hour by increasing J _K ^a-b. The TEP was increased by approximately 3 mV, but the R _T was not significantly changed. In two of these experiments, 100 μM bumetanide decreased the epinephrine-stimulated net K absorption from 0.26 to 0.04μ eq/cm² per hour and 0.31 to 0.08μ 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 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 containα 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 signal-transduction 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

In many systems it has been shown that activation of G-protein–coupled receptors (e.g.,α 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 thatα -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, anα 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 α and β 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 cAMP-dependent 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 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 cAMP-mediated 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 (J _s) and J _V are isotonically coupled,³⁸ ³⁹ so that J _V= J _s/C, where J _s = J _Cl + J _Na + J _K, and C is the solute concentration of the bathing medium.¹ ²⁸ The results summarized in Table 1 show that J _s = 1.40 μeq/cm² per hour, which predicts a fluid absorption rate of 4.7 μl/cm² per hour in control Ringer. This value is in relatively good agreement with the maximum J _V rate (4μ l/cm² per hour) measured in tissues of similarly high TEP and R _T.¹²

The data summarized in Table 2 show that epinephrine stimulated J _s (J _Cl + J _K) by approximately 0.5μ eq/cm² per hour, which predicts an epinephrine-induced increase in J _V of 1.7 μeq/cm² per hour. This prediction is in excellent agreement with previous measurements showing that 100 nM epinephrine increased J _V by 1.4μ l/cm² per hour.¹² It indicates that Cl and K are the ions responsible for the epinephrine-induced 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.

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 ⁴⁴ ⁴⁵ ⁴⁶ ⁴⁷ ⁴⁸ The 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.

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. [email protected]

Figure 1.

Effect of epinephrine on [Ca2+]in (top), TEP, and R T (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 R T decreases of 7.5, 8.5, and 5Ω
· cm2. All Ca2+ responses were obtained
from the same set of approximately 10 cells within the RPE.

View Original Download Slide

Effect of epinephrine on [Ca²⁺]_in (top), TEP, and R _T (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 R _T decreases of 7.5, 8.5, and 5Ω · cm². All Ca²⁺ responses were obtained from the same set of approximately 10 cells within the RPE.

Figure 2.

Effect of prazosin on the epinephrine-induced[
Ca2+]in increase, TEP, and R T. 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 R T decrease. The simultaneous application of theα 1-adrenoreceptor antagonist prazosin (1 μM) with
epinephrine (100 nM) fully prevented the ratio increase and R T decrease and diminished the TEP change by approximately 50% (B). The
effects of prazosin were not fully reversible (C).

View Original Download Slide

Effect of prazosin on the epinephrine-induced[ Ca²⁺]_in increase, TEP, and R _T. 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 R _T decrease. The simultaneous application of theα ₁-adrenoreceptor antagonist prazosin (1 μM) with epinephrine (100 nM) fully prevented the ratio increase and R _T decrease and diminished the TEP change by approximately 50% (B). The effects of prazosin were not fully reversible (C).

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 μM), 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.

View Original Download Slide

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 μM), 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.

Figure 4.

Effect of epinephrine on J V rate
(top), TEP, and R T (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). J V and TEP increased and R T decreased. At approximately 125
minutes, 100 μM bumetanide was added to the apical bath and reversed
the epinephrine-induced changes. (B) A similar experiment on
a different tissue measured in nominally
HCO3-free Ringer buffered with 10 mM HEPES and
equilibrated with room air (pH 7.4). The stimulation of J V and TEP with 100
nM apical epinephrine and the inhibition with 100 μM bumetanide
produced effects very similar to those measured in
HCO3 Ringer.

View Original Download Slide

Effect of epinephrine on J _V rate (top), TEP, and R _T (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). J _V and TEP increased and R _T decreased. At approximately 125 minutes, 100 μM bumetanide was added to the apical bath and reversed the epinephrine-induced changes. (B) A similar experiment on a different tissue measured in nominally HCO₃-free Ringer buffered with 10 mM HEPES and equilibrated with room air (pH 7.4). The stimulation of J _V and TEP with 100 nM apical epinephrine and the inhibition with 100 μM bumetanide produced effects very similar to those measured in HCO₃ Ringer.

Table 1.

View Table

Open Circuit Net Fluxes of ³⁶Cl, ⁸⁶Rb, and ²²Na across Bovine RPE

Table 1.

Open Circuit Net Fluxes of ³⁶Cl, ⁸⁶Rb, and ²²Na across Bovine RPE

Tracer	³⁶Cl	⁸⁶Rb	²²Na
Apical-to-Basal (n = 6)
J^a−b (μeq/cm² · hr)	4.08 ± 0.27	0.16 ± 0.01	3.21 ± 0.32
TEP (mV)	8.1 ± 1.4	9.3 ± 1.1	6.5 ± 1.2
R_T (Ω · cm²)	159 ± 11	154 ± 14	160 ± 7
Basal-to-Apical (n = 6)
J^b−a (μeq/cm² · hr)	3.41 ± 0.24	0.11 ± 0.01	2.53 ± 0.21
TEP (mV)	9.1 ± 1.4	9.9 ± 1.4	6.3 ± 1.6
R_T (Ω · cm²)	169 ± 10	160 ± 16	165.9 ± 9
Net flux (μeq/cm² · hr)	0.67	0.05	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.

Figure 5.

Simultaneous measurement of unidirectional 36Cl flux, TEP, and R T across paired
tissues from the same eye. (•) J Cl a-b; (○) J Cl b-a. Top, solid
lines: mean J Cl a-b; dashed lines: mean J Cl b-a. The difference between
these two values at every time point is the net flux. In control
Ringer, net Cl absorption rate was approximately 0.20μ
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 0.60μ
eq/cm2 per hour. Epinephrine transiently increased TEP, which was followed by a sustained increase, but did
not alter the R T. At 120 minutes, 100 μM
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 R T in both tissues.

View Original Download Slide

Simultaneous measurement of unidirectional ³⁶Cl flux, TEP, and R _T across paired tissues from the same eye. (•) J _Cl ^a-b; (○) J _Cl ^b-a. Top, solid lines: mean J _Cl ^a-b; dashed lines: mean J _Cl ^b-a. The difference between these two values at every time point is the net flux. In control Ringer, net Cl absorption rate was approximately 0.20μ 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 0.60μ eq/cm² per hour. Epinephrine transiently increased TEP, which was followed by a sustained increase, but did not alter the R _T. At 120 minutes, 100 μM 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 R _T in both tissues.

Table 2.

View Table

Stimulation of ³⁶Cl, ⁸⁶Rb (K), and ²²Na Absorption by Apical Epinephrine

Table 2.

Stimulation of ³⁶Cl, ⁸⁶Rb (K), and ²²Na Absorption by Apical Epinephrine

Treatment	J ^a−b	J ^b−a	J ^net (μeq/cm² · hr)	TEP (mV)	R _T (Ω · cm²)
³⁶Cl
Control	3.85 ± 0.19	3.57 ± 0.14	0.28 ± 0.12	7.9 ± 1.0	155 ± 7
Epinephrine	3.92 ± 0.18	3.32 ± 0.10	0.60 ± 0.17^*	11.0 ± 1.2	154 ± 6
⁸⁶Rb (K)
Control	0.142 ± 0.007	0.104 ± 0.014	0.038 ± 0.015	10.7 ± 1.1	175 ± 10
Epinephrine	0.310 ± 0.042^*	0.103 ± 0.012	0.207 ± 0.044^*	13.6 ± 1.0^*	170 ± 9
²²Na
Control	3.74 ± 0.56	2.74 ± 0.37	1.00 ± 0.63	7.6 ± 1.6	152 ± 7
Epinephrine	4.01 ± 0.65	2.96 ± 0.20	1.15 ± 0.61	10.0 ± 1.0^*	145 ± 9

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.

Figure 6.

Simultaneous measurement of unidirectional 86Rb fluxes (K
substitute), TEP, and R T across paired tissues from the same eye. (•) J Cl a-b; (○) J Cl b-a. Top, solid
lines: mean J Cl a-b; dashed lines: mean J Cl b-a. In control Ringer, net 86Rb (K) absorption rate was approximately 0.05μ
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 μeq/cm2 per hour, and TEP was transiently increased, followed by a sustained
increase. R T was decreased in both tissues
by 5 to 10 Ω · cm2. At 180 minutes, 100 μM 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 R T by
20 to 30 Ω · cm2 in both tissues.

View Original Download Slide

Simultaneous measurement of unidirectional ⁸⁶Rb fluxes (K substitute), TEP, and R _T across paired tissues from the same eye. (•) J _Cl ^a-b; (○) J _Cl ^b-a. Top, solid lines: mean J _Cl ^a-b; dashed lines: mean J _Cl ^b-a. In control Ringer, net ⁸⁶Rb (K) absorption rate was approximately 0.05μ 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 μeq/cm² per hour, and TEP was transiently increased, followed by a sustained increase. R _T was decreased in both tissues by 5 to 10 Ω · cm². At 180 minutes, 100 μM 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 R _T by 20 to 30 Ω · cm² in both tissues.

Figure 7.

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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.

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