Measurement of the Volume Flow and Hydraulic Conductivity Across the Isolated Dog Retinal Pigment Epithelium Shunji Tsuboi The isolated dog retinal pigment epithelium (RPE)-choroid was gently stretched on the inner surface of a spherical stainless mesh, retinal side upward, and clamped between half-chambers made of Kel-F. The volume flow across the tissue was monitored by the movement of water in capillary tubes connected to both chambers. With zero pressure difference across the RPE-choroid, retina-to-choroid fluid flow was determined to be 6.4 jul/hr/cm2 (absorption). Removal of HCO3~ from the solution did not affect the fluid flow. However, the flow was reduced 88% in Cl"-free medium, indicating a coupling between water and Cl~ absorption. The flow was also inhibited by ouabain ( 1 0 s M) and furosemide (10~4 M). Hydraulic conductivity (Lp) of the RPE-choroid was determined to be 0.0126 ^l/min/cm 2 / mm Hg which places the dog RPE-choroid in the category of a "leaky" epithelium. Invest Ophthalmol Vis Sci 28:1776-1782, 1987 Evidence is accumulating in vivo that the retinal The present study was undertaken to determine the pigment epithelium (RPE) absorbs fluid from the ret- volume flow across the isolated dog RPE-choroid. inal to the choroidal surface.1"3 This volume flow The effect of HCO3~, Cl~, ouabain, and furosemide may assist the sensory retina in attachment to the on the volume flow was then examined. Finally, the RPE,1'4 and is one of the unconventional routes for hydraulic conductivity (Lp) was determined. aqueous humor drainage in eyes with rhegmato- genous retinal detachments (postiridial flow).2"5 Di- Materials and Methods rect measurement of the flow has also been made Tissue Preparation using the isolated frog RPE-choroid, where the vol- ume flow is 4.8-7.8 nl/hr/cm2, from retina to cho- The eyes were obtained from adult dogs of both roid, without hydrostatic or osmotic pressure differ- sexes weighing 15-30 kg shortly after sacrifice. Prepa- ence across the membrane, indicating an isotonic ration of the dog RPE-choroid was described in detail electrolyte-linkedfluidflow.6-7 previously. 810 All procedures conformed to the ARVO Resolution on the Use of Animals in Re- Hughes, Miller, and Machen reported that HCO3~ search. is the major ion that drives water from retina to cho- roid in the frog RPE-choroid, based on the observa- Bathing Solution tion that removal of HCO3~ from the bathing solu- tion both inhibited the fluid movement and de- The standard bathing solution was composed of (in creased the short-circuit current (SCC).6 However, in mM) NaCl, 102; KC1, 0.8; NaHCO3, 24; KH2PO4, the isolated RPE-choroid of dogs8 and chickens,9 1.2; Na2SO4, 4; CaCl2, 2.5; MgSO4, 1.2; HEPES-Na, SCC is not decreased by the elimination of ambient 8.0; HEPES, 9.0; and glucose, 5.5. In addition, a so- HCO3~. It is thus likely that a different mechanism is lution where either HCO3" or Cl~ was replaced with involved in the water transport of warm-blooded ani- equivalent SO42~ was prepared. The RPE-choroid mals. was always immersed in the identical solution on both sides. The pH of the solutions was 7.4 at 37°C. From the Department of Ophthalmology, University of Minne- Osmolality was measured by a freezing point osmom- sota, Minneapolis, Minnesota. eter (micro osmette; Precision Systems, Inc., Natick, Supported by NIH grant EY-03277. MA) and adjusted, if necessary, to 300 mosmol/kg Dr. Tsuboi is Visiting Research Fellow, Department of Ophthal- using mannitol. mology, Osaka University Medical School, Osaka, Japan. Submitted for publication: June 9, 1986. During initial volumetric experiments, it was Reprint requests: Shunji Tsuboi, MD, University of Minnesota, readily noticed that expansion of the solution due to Box 493 Mayo, Minneapolis, MN 55455. gas bubbles resulted in a substantial error. The solu- 1776 No. 11 FLUID ADSORPTION IN DOG RPE / Tsuboi 1777 tions were thus slightly degassed using a vacuum pump, so that air bubbles did not appear when the temperature was raised to 37°C. This caused negligi- ble change in the pH and pO2. Tissue Mounting The RPE-choroid was gently placed, retinal side upward, on the inner surface of a spherical stainless mesh (radius = 3.54 mm, depth = 3.0 mm, Fig. 1). Fig. 1. Cross section of the tissue-holding ring. The RPE-choroid The area of the RPE-choroid exposed to the solution (1) is held between the silicone disk (2) and stainless mesh (3). The was 0.67 cm2. The mesh with the tissue was mounted mesh is spherical inside the ring, so that the area exposed to the in a tissue-holding ring. An elastic doughnut-shaped solution is enlarged (0.67 cm2). silicone plate (Dow Corning 500-9; Midlands, MI) was placed on the retinal side of the tissue. The tis- sue-holding ring, mesh, and silicone plate were tern was 30 nl. To prevent evaporation from the coated with high-vacuum grease. These procedures water surface, the ends of capillaries were covered were done under an operating microscope. with wet gauze. Water movement was always monitored at both sides of the half-chambers, ie, retinal and choroidal Chambers sides of the RPE-choroid. When the RPE-choroid Two pairs of Ussing-type chambers were used in transports water from retinal to choroidal surface, the each experiment. The shape of each half-chamber volume of the bathing solution on the retinal side was symmetrical, with a volume of 12.5 ml, and be- should decrease, while that on the choroidal side tween each half-chamber fit the tissue-holding ring should increase over time. When other factors such as (Fig. 2). The volume fluctuation originating from the temperature change, gas bubbles appearing in the so- system was occasionally tested by changing the orien- lution, and evaporation are also involved, simulta- tation of the tissue ring in the chamber. One of the neous increase or decrease at both ends may occur. chambers, constructed especially for the volume flow Experiments where simultaneous increase or de- measurement, was made of Kel-F, an extremely hy- crease occurred throughout the experiment were dis- drophobic material, instead of Lucite, which absorbs carded. a considerable amount of water.6 Moreover, the Piston-like movement of the tissue may cause a Kel-F chamber did not contain electrode holes in substantial error, since a rigid support was placed order to avoid a possible leak. only on the choroidal side. Due to water surface ten- The mounted RPE-choroid was first clamped be- sion in the capillaries, a pressure was required to initi- tween Lucite half-chambers, which were connected ate meniscus movement. In the present system, the to an automatic voltage clamp device through two frictional pressure was around 1 cm H2O, the pres- pairs of 3% agar-3 M KC1 bridges and calomel elec- sure capable of dislocating RPE-choroid from the net. trodes as described in detail previously.810 The trans- Initial experiments revealed that transepithelial water epithelial potential difference (Et) and SCC were then movement sometimes resulted in movement of the determined. The resistance (Rt) was calculated from tissue rather than meniscus. Thus, the following pro- Et and SCC by Ohm's law. Ten to 30 min later, when cession was followed in each experiment. Et became constant, the mounted RPE-choroid was moved to the Kel-F chamber. After the measurement of volumeflowas described below, electrical parame- ters were again examined in the Lucite chamber. Measurement of the Volume Flow Each half of the Kel-F chamber was connected to a glass capillary tube, with inner diameter of 0.66 mm, via polyethylene tubing. The capillaries were set hor- izontally at the same height. Volume flow across the Fig. 2. Kel-F chambers and tissue holding ring. 1. Water jacket. tissue was determined from the meniscus movement 2. Stopper and hole for air release and drug administration. 3. in the capillary." Since the movement of 0.1 mm was Connector to the capillary tube through polyethylene tubing. 4. detectable using a magnifier, the accuracy of the sys- Gasket holding temperature probe. 5. Tissue-holding ring. 1778 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1987 Vol. 28 7.3±0.4(mV) _ -^ 3.6 ±0.7 bathing solution was preheated at 37°C and added to 378±47 (O cm2) 330126 0.15 the solutions on both sides of the tissue chamber Sham using a micropipette (final concentrations, 10~5 M ouabain and 10~4 M furosemide). Symmetrical ad- 0.10 H-h- ministration of the drug precluded any change in os- motic pressure difference across the membrane. The solutions were stirred for 1 to 5 min using magnetic E stirring bars. A preliminary experiment showed that ^ 0.05 this mixing procedure was enough to cause a typical decrease in Et and SCC by these drugs in the isolated dog RPE-choroid. Control experiments with the L 0 same procedure without drugs were also performed. 30 60 90 120 Hydraulic Conductivity Time (min) After the volume flow measurement, where only Fig. 3. Time course of the volumeflow(Jv) in the control exper- compensatory and transient pressure was applied, iment. Each point represents means ± SE of seven experiments. At volume flow was also determined with a continuous t = 60 min, "sham" procedure is performed. An almost linear decline is noted regardless of the procedure. Et and Rt before and hydrostatic pressure on the retinal side. Three or 8 cm after volume flow measurement are also shown. H2O was applied. First, 3 cm H2O static pressure was applied on the Results retinal side for 10 min by lowering the capillary tube Control Experiments connected to the choroidal side. This produced a rapid movement of the meniscus, due to retina-to- Figure 3 summarizes seven control experiments. choroid tissue movement, but in seconds the menis- After the tissue-holding ring was placed in the Kel-F cus movement stopped, indicating that the tissue was chamber, less than 30 min was required for the tem- entirely attached to the stainless steel net. Experi- perature to become stabilized. Thereafter, fluid trans- ments with continuous meniscus movement result- port was observed from retina to choroid (absorption) ing from a leak were discarded. Capillaries were then for more than 2 hr. Mean Et before the volume flow set at the same height. Every 10 min, 3 cm H2O static measurement, 7.3 mV, retinal side positive, de- pressure was placed on the retinal side for 15 sec, and creased 50% after the measurement. Mean Rt, 378 readings were made. This transient pressure compen- ohm-cm2, decreased 9%. An almost linear decline in sated tissue dislocation. The 3 cm H2O pressure was Jv was noted regardless of the sham procedure. The shown to have negligible effect on the volume flow decay of Jv was, however, small enough to examine determination (see Results). Hydrostatic pressure was drug effects within the 2 hr time period. calibrated using a pressure transducer (Hewlett Pack- ard, 7754 B; Waltham, MA). HCO3~- and CP-Free Solutions Temperature Control It was virtually impossible in the present study to determine the volume flow of the RPE-choroid in The Kel-F chamber including tissue was placed in- two consecutive solutions, because of the spontane- side an oven whose temperature was kept around ous decay of Jv and relatively large time loss until the 32°C. In addition, the temperature of the circulating temperature of the substituted solution became stabi- water around the chamber (Fig. 2) was controlled by lized. Therefore, each preparation was used for only a proportional controller (Yellow Springs Instrument one of the bathing solutions. Co., Yellow Springs, OH; Model 72), so that the Table 1 summarizes Jv, Et, Rt, and SCC of the dog bathing solution, monitored by a tele-thermometer RPE-choroid determined in the standard, HCO3~- (Yellow Springs Model 43 TA) and temperature free, and Cr-free solutions. Jv values in this Table probes (Yellow Springs Model 423), was kept con- were taken one hour after the beginning of volume stant at 36-37 ± 0.05°C. flow measurement. Et, Rt, and SCC were measured in the Lucite chamber just prior to (standard and Effect of Ouabain and Furosemide HCO3"-free) or after (Cr-free) the volume flow mea- After the volume flow became constant, 125 /A of surement. Retina-to-choroid Jv in the HCO3"-free 10~3 M ouabain or 10~2 M furosemide in standard HEPES was 0.093 /il/min/cm 2 (5.6 2 No. 11 FLUID ADSORPTION IN DOG RPE / Tsuboi 1779 Table 1. Jv (retina-to-choroid), Et, Rt, and SCC Jv Et Rt SCC Solution (nl/min/cm2) (mV) (ohm-cm2) (nEq/hr/cm2) Standard 0.106 ±0.009 7.0 ± 1.1 365 ± 19 0.71 ±0.11 (n) (10) (14) (14) (14) HCOj-free 0.093 ±0.010 6.1 ± 1.0 365 ±31 0.60 ± 0.06 (n) (10) (10) (10) (10) Cr-free 0.012 ±0.008 1.7 ±0.3 289 ± 12 0.21 ±0.04 (n) (6) (5) (5) (5) Values are mean ± SE. which was significantly larger than zero (P < 0.001) bain was added into both half-chambers 1 hr after the but not significantly different from Jv in the HCO3~- beginning of volume flow measurement. HCO3"-free rich standard solution. Et, Rt, and SCC of these two HEPES solution was used in these experiments. After sets of experiments were not significantly different. the ouabain application, Jv decreased markedly. The In the Cl~-free medium Jv reduced markedly, and difference from control was obvious 20 min after the in 60 min it became not statistically different from application. zero (Fig. 4). Jv at t = 60 min, 0.012 ± 0.008 (SE) Reduction rates of Jv, Et, and Rt are summarized /il/min/cm2, was markedly lower than the control ex- in Table 2. Jv values used in this Table were taken periment superimposed in the Figure (0.099 ± 0.010, right before and 60 min after the drug administration P < 0.001). Et reduced 77% from the value measured (or sham) procedure. Et and Rt were taken right be- in the standard HEPES prior to the volume flow mea- fore and after the volume flow measurement. The surement (Fig. 4). The reduction rate was signifi- reduction of Jv by ouabain application was 83%, sig- cantly higher than control (25%/hr, P < 0.001, see nificantly larger than control (P < 0.002). At the end Table 2). Change in Rt was not significant, however. of the experiment Et was reduced 91%, whereas Rt decreased insignificantly. 10~5 M Ouabain 10~4 M Furosemide Addition of preheated 125 n\ aliquots including ouabain or furosemide into the 12.5 ml half- The effect of 10~4 M furosemide applied to both chambers had little effect on the temperature, so that half-chambers is summarized in Figure 6, which con- volume flow measurement was disturbed only for sists of four experiments with HCO3~-free HEPES several minutes from the drug administration. Figure solution and three with HCO3~-rich solution. Sixty 5 summarizes five experiments where 10~5 M oua- 6.6 + 0.9 (mV) . 0.6 10.2 328116 (Ocm2) 296 ± 15 7.5±1.9 (mV) _ 1.7±0.3 360 ±21 (O cm2) 289±12 0.15 r n-5 10"° M Ouabain 0.15 r Cr-free ST 0.10 E o.io o c E E | 0.05 | 0.05 L 0 L 0 30 60 90 120 30 60 90 120 Time (min) Time (min) Fig. 5. Effect of 10"5 M ouabain on the volume flow (Jv, solid Fig. 4. Volume flow is not maintained in the Cl"-free medium line). Each point represents mean ± SE of five experiments. After (solid line). Each point repcesents mean ± SE of six experiments. ouabain application Jv decreases markedly, and in 60 min it be- Note the difference from the control experiment (dotted line). Et comes statistically not different from zero. Note the difference from reduces 77% in the Cl~-free medium. the control experiment (dotted line). Et decreases 91%. 1780 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1987 Vol. 28 Table 2. Reduction rate(%)ofJv, Et, and Rt O.b Experiment (n) Jv Et Rt n4 Sham (control) (7) 39 ± 8 50 ±4 9± 6 Ouabain (5) 83 ± 8* 91 ±5f 14± 12 CVJ Furosemide (7) 84 ± 11$ 76 ±2* 18± 4 E 0.3 o Jv (jul/min/ and Values are mean ± SE. */>< 0.002, -fP< 0.001, tP < 0.01 with respect to "control" values. 0.2 0.1 minutes after the furosemide application, mean Jv decreased 84%. This rate of decline was significantly -8cm H o 0- larger than the control experiments (P < 0.01, Table 2). Et decreased 76% at the end of the experiment. 30 60 90 120 Time (min) Hydraulic Conductivity Fig. 7. A representative experiment for Lp measurement. After Application of continuous 3 cm H2O hydrostatic the volume flow (Jv) measurement with zero pressure (only tran- pressure on the retinal side did not significantly in- sient 3 cm H 2 O pressure, see Materials and Methods), 8 cm H 2 O crease the Jv, affirming the negligible effect of the pressure is applied continuously on the retinal side. Note that the transient 3 cm H2O pressure on the volume flow leveled Lp value is slightly higher during the 8 cm H 2 O pressure. measurement (n = 9). However, by 8 cm H2O pres- sure, Jv increased markedly, followed by a gradual decrease, and in about 20 min leveled off at a value roid, confirms that the dog RPE-choroid transports slightly larger than the initial value (Fig. 7). Using the fluid from retinal to choroidal surface (absorption), constant post-peak values, the Lp of six tissues was since fluid absorption occurs without any hydrostatic determined to be 0.0126 ± 0.0018 (SE) /il/min/cm2/ or osmotic pressure difference across the membrane. mm Hg in the HCO3~-free HEPES solution at 37°C. The amount of fluid transport, 6.36 Atl/hr/cm2 in the The mean (±SE) Et and Rt of these tissues measured HCO3"-rich HEPES, is comparable to the frog RPE- before Lp determination were 7.7 ± 0.6 mV and 329 choroid,6'7 rabbit corneal endothelium,12 and mam- ± 15 ohm-cm2, respectively. malian proximal tubules.13 Negi and Marmor re- ported that in the rabbit, RPE metabolic water trans- Discussion port accounts for 70% of the in vivo resorption rate of the subretinal fluid, 8.4 /ul/hr/cm2,14 which is close to The present study, the first direct determination of the present in vitro value. the volume flow across the mammalian RPE-cho- In a wide variety of epithelia, fluid transport is linked to electrolyte transport.15 Assuming isotonic 6.9 + 0.9 (mV) _ 1.7 + 0.3 volumeflowacross the dog RPE-choroid, Js/Jv = 300 334 + 21 (Ocm2) 276+25 mosmol/kg, where Js is the sum of net ion fluxes in 0.15 r -4 the open-circuit condition. Using the Jv value of ex- 10 M Furosemide periments with HCO3"-rich HEPES, 6.36 /il/hr/cm2, Js is calculated to be 1.91 /u,osmol/hr/cm2. In contrast ~ 0.10 to the frog RPE,6 HCO3~ is not the primary driving o force for Jv, since Jv is not inhibited by the elimina- c tion of ambient HCO3~. However, Cl" contributes E ^ 0.05 substantially to the Js, since Jv is inhibited 88% in the Cl~-free medium. In the short-circuited dog RPE, net Cl~ flux is determined to be 0.67 /ueq/hr/cm2 from L retina to choroid,8 about one-third as large as the Js. 0 Therefore, participation of the ions other than Cl~ 30 60 90 120 and HCO3~ is also suggested. Time (min) Since K+ is transported from retina to choroid in the frog RPE-choroid,16 K+ is another likely constitu- Fig. 6. Effect of 10 4 M furosemide on the volume flow (Jv, solid line). Each point represents mean ± SE of seven experiments. After ent. However, the net K+ flux is only a small fraction furosemide application Jv decreases significantly. Note the differ- of Js.6 Frambach and Misfeldt reported that in the ence from the control experiment (dotted line). Et decreases 76%. chicken RPE-choroid-sclera, net Na+ movement is No. 11 FLUID ADSORPTION IN DOG RPE / Tsuboi 1781 Fig. 8. A hypothetical scheme of the Retina RPE Choroid electrolyte-linked water transport (apical) (Basal) across the dog retinal pigment epithe- lium. Ouabain-sensitive Na-K-ATP- Et(+) Et(-) ase is distributed in the apical cell membrane, and produces the electro- Water chemical potential difference of Na+ across the membrane. Backflux of Na+ down the electro-chemical potential difference is linked with Cl~ via a furo- Water semide-sensitive neutral carrier. Cl" diffuses across the basolateral mem- brane into the paracellular space. Dif- Furosemide fusional movement of Na+ is absorp- tive because of the retina-positive Et. Ouabain Absorption of NaCl allows water to be absorbed isotonically. K from retina to choroid under the open-circuit condi- This is in good agreement with a previous report tion.9 They also noted that this movement of Na + is a where the diffusion of carboxyfluorescein (MW passive diffusion resulting from the potential differ- = 376) across the isolated dog RPE-choroid was ence, retinal side positive, across the RPE. Therefore, shown to occur through a paracellular pathway.22 Al- passive Na+ absorption may account for a part of Js, though it is debatable whether water also moves even if in the dog, net Na+ flux is from choroid to through the "tight" junctions of the "leaky" epithe- retina in the short-circuited condition.8 The ions con- lium,23 there is some evidence that in the cynomolgus stituting Js are, of course, only concluded by flux monkey, inward (choroid-to-retina) diffusion of car- studies under the open-circuit condition. boxyfluorescein interacts with outward fluid flow Ouabain is an inhibitor of Na-K ATPase located in across the RPE. 24 Metabolic (electrolyte-linked) the RPE apical membrane.17 In a previous study water transport across the dog RPE is summarized in using dogs, it was shown that 10~5 M ouabain inhibits Figure 8. active transport of both Na+ and Cl".8 In the present study, 10~5 M ouabain inhibits Jv in the HCO3~-free Key words: volume flow, hydraulic conductivity, retinal HEPES solution, suggesting that ouabain-induced Jv pigment epithelium, ouabain, furosemide, chloride trans- port, dog reduction is independent of ambient HCO3~. Thus, ouabain-induced Jv reduction may result from re- Acknowledgment duced Cl~ transport. In addition, abolition of Et by ouabain would cause a reduced passive diffusion of I thank Jonathan E. Pederson, MD, for his encourage- ment and helpful suggestions. Na+ in the retina-to-choroid direction. Ouabain-sen- sitive Jv is also encountered in the isolated frog RPE.6 References Furosemide at 10~4 M is an inhibitor of Cl~ trans- 1. Marmor MF, Abdul-Rahim AS, and Cohen DS: The effect of port, but not Na+ transport, in the dog RPE.9 There- metabolic inhibitors on retinal adhesion and subretinal fluid fore, inhibition of Jv by the 10~4 M furosemide, inde- resorption. Invest Ophthalmol Vis Sci 19:893, 1980. pendent of the ambient HCO3~, further supports the 2. Pederson JE and Cantrill HL: Experimental retinal detach- coupling between water and Cl~ transport. Furose- ment: V. Fluid movement through the retinal hole. Arch Oph- thalmo! 102:136, 1984. mide also inhibits Cl~ transport in the isolated RPE 3. Tsuboi S, Taki-Noie J, Emi K, and Manabe R: Fluid dynamics of frog18 and chicken.9 Furthermore, in the cyno- in eyes with rhegmatogenous retinal detachments. Am J Oph- molgus monkey eye with retinal detachment, retina- thalmol 99:673, 1985. to-choroid RPE permeability to fluorescein is de- 4. Tsuboi S and Pederson JE: Permeability of the blood-retinal creased by vitreous 10~4 M furosemide, suggesting barrier to carboxyfluorescein in eyes with rhegmatogenous ret- inal detachment. Invest Ophthalmol Vis Sci 28:96, 1987. furosemide-sensitive fluid transport in vivo.19 5. Brubaker RF and Pederson JE: Ciliochoroidal detachment. In the present study Lp of the dog RPE-choroid Surv Ophthalmol 27:281, 1983. was determined to be 0.0126 /il/min/cm2/mm Hg. 6. Hughes BA, Miller SS, and Machen TE: Effects of cyclic AMP Although this vdlue may possibly be an overestimate, on fluid absorption and ion transport across frog retinal pig- ment epithelium. J Gen Physiol 83:875, 1984. because of the edge damage, it is about one-third as 7. Frambach DA, Weiter JJ, and Adler AJ: A photogrammetric large as the bullfrog RPE-choroid,20 placing the RPE- method to measure fluid movement across isolated frog retinal choroid in the category of a "leaky" epithelium.21 pigment epithelium. Biophys J 47:547, 1985. 1782 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / November 1987 Vol. 28 8. Tsuboi S, Manabe R, and Iizuka S: Aspects of electrolyte trans- 16. Miller SS and Steinberg RH: Active transport of ions across port across isolated dog retinal pigment epithelium. Am J frog retinal pigment epithelium. Exp Eye Res 25:235, 1977. Physiol 250 (Renal Fluid Electrolyte Physiol 19):F781, 1986. 17. Ostwald TJ and Steinberg RH: Localization of frog retinal 9. Frambach DA and Misfeldt DS: Furosemide-sensitive Cl pigment epithelium Na+-K+ ATPase. Exp Eye Res 31:351, transport in embryonic chicken retinal pigment epithelium. 1980. Am J Physiol 244 (Renal Fluid Electrolyte Physiol 13):F679, 18. DiMattio JK, Degnan J, and Zadunaisky JA: A model for 1983. transepithelial ion transport across the isolated retinal pigment 10. Tsuboi S, Fujimoto T, Uchihori Y, Emi K, Iizuka S, Kishida epithelium of the frog. Exp Eye Res 37:409, 1983. K, and Manabe R: Measurement of retinal permeability to 19. Tsuboi S and Pederson JE: Experimental retinal detachment: sodium fluorescein in vitro. Invest Ophthalmol Vis Sci XI. Furosemide-inhibitable fluid absorption across the retinal 25:1146, 1984. pigment epithelium in vivo. Arch Ophthalmol 104:602, 1986. 11. Reid EW: Transport of fluid by certain epithelia. J Physiol 20. Brown JA and Zadunaisky JA: Ion coupled fluid movements 26:436, 1900-1901. across the bullfrog retinal pigment epithelium. ARVO Ab- 12. Fischbarg J, Lim JJ, and Bourguet J: Adenosine stimulation of stracts. Invest Ophthalmol Vis Sci 22(Suppl):102, 1982. fluid transport across rabbit corneal endothelium. J Membr 21. Fromter E and Diamond JM: Route of passive ion permeation Biol 35:95, 1977. in epithelia. Nature (London) 235:9, 1972. 13. Schafer JA, Troutman SL, and Andreoli TE: Volume reab- 22. Tsuboi S and Pederson JE: Permeability of the isolated dog sorption, transepithelial potential differences, and ionic per- retinal pigment epithelium to carboxyfluorescein. Invest Oph- meability properties in mammalian proximal straight tubules. thalmol Vis Sci 27:1767, 1986. JGen Physiol 64:582, 1974. 23. Levitt DG: Routes of membrane water transport. Comparative 14. Negi A and Marmor MF: Quantitative estimation of metabolic Physiology. In Water Transport Across Epithelia, Ussing HH, transport of subretinal fluid. Invest Ophthalmol Vis Sci Bindslev N, Lassen NA, and Sen-Knudsen O, editors. Copen- 27:1564, 1986. hagen, Munksgaard, 1981, pp. 248-257. 15. Diamond JM and Bossert WH: Standing-gradient osmotic 24. Tsuboi S and Pederson JE: Acetazolamide effect on the inward flow: A mechanism for coupling of water and solute transport permeability of the blood-retinal barrier to carboxyfluorescein. in epithelia. J Gen Physiol 50:2061, 1967. Invest Ophthalmol Vis Sci 28:92, 1987.
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