Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium.

The authors used a modified capacitive probe technique to simultaneously assess the effect of apical epinephrine on fluid transport rate (Jv), transepithelial potential (TEP), and transepithelial resistance (Rt) across bovine retinal pigment epithelium (RPE). In control Ringer, the RPE absorbed fluid at a rate of 1.42 +/- 0.34 microliters/cm2.hr (mean +/- SEM; 22 tissues). Tissues with the highest TEP (8-9 mV) and Rt (160-220 omega.cm2) had maximum fluid absorption rates (3-4 microliters/cm2.hr). Apical epinephrine (100 nM) stimulated Jv by a factor of 3, from 0.70 +/- 0.18 microliter/cm2.hr to 2.17 +/- 0.24 microliters/cm2.hr and TEP from 4.6 +/- 0.4 mV to 7.0 +/- 0.6 mV (n = 6). The epinephrine-induced transport changes were inhibited by apical bumetanide (0.1 mM). The alpha-1 adrenergic antagonist prazosin (1 microM) completely blocked the epinephrine-induced stimulation of Jv and TEP. In contrast, the beta adrenergic antagonist propranolol (1 microM) had no effect on epinephrine-induced transport changes. These results, coupled with previous studies on bovine RPE, suggest that the mechanisms underlying the epinephrine-induced stimulation of fluid absorption include an apical membrane alpha-1 adrenergic receptor, a bumetanide-inhibitable apical membrane Na-K-2Cl cotransporter, and a basolateral membrane Cl conductance.     

Response properties of the toad retinal pigment epithelium

Electrophysiologic responses were recorded in vitro from the retina of the toad, Bufo marinus. In the isolated retinal pigment epithelium (RPE)-choroid preparation, a decrease in the K+ concentration outside the apical membrane evoked a sequence of trans-epithelial (TEP) and membrane potential changes. A hyperpolarization originating at the RPE apical membrane that increased the TEP was followed by a delayed hyperpolarization originating at the basal membrane that decreased the TEP. This response sequence differed from the well-studied bullfrog RPE in exhibiting potentials generated at the basal membrane; it more closely resembled responses from the reptilian and avian RPE. The toad direct-current electroretinogram recorded from a neural retina-RPE-choroid preparation also differed from other amphibians and was more similar to responses recorded in reptiles, birds, and mammals. In this initial study, the electrophysiologic characteristics of the RPE's Na+/K+ pump were examined. Contrary to a previous report, ouabain, a specific inhibitor of the pump, depolarized the apical membrane and decreased the TEP. Ba2+ was used to block apical K+ conductance and unmasked a modulation of the Na+/K+ pump by subretinal K+. Decreasing the K+ concentration in the presence of Ba2+ depolarized the apical membrane and decreased the TEP. These responses were blocked by ouabain, indicating that the K+ concentration decrease slowed the Na+/K+ pump.     

Na-dependent pHi regulatory mechanisms in native human retinal pigment epithelium.

This study provides the first information about pHi regulatory mechanisms in human retinal pigment epithelium (RPE). The experiments were carried out on fresh explant tissues from adult donor and fetal eyes, and pHi was measured using fluorescence microscopy techniques and the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In adult donor RPE, the resting pHi is 7.30 +/- 0.14 (mean +/- standard deviation; n = 6) in HCO3 Ringer's solution. In HCO3 Ringer's solution, apical Na removal caused rapid cell acidification with an initial rate of 0.40 +/- 0.10 pH U/min (n = 4). This Na-dependent acidification was partially inhibited by apical amiloride (n = 1) and DIDS (n = 1). In HCO3 Ringer's solution, pHi recovery from an acid load (NH4 prepulse) also was blocked by apical Na removal. In nominally HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells. These results suggest that the apical membrane of adult human RPE contains an Na/H exchanger and possibly a Na-dependent, DIDS-inhibitable pH regulatory mechanism, perhaps a NaHCO3 cotransporter. For the fetal RPE, the resting pHi was 7.16 +/- 0.10 (n = 9) and 7.19 +/- 0.10 (n = 20) in HCO3 and HCO3-free Ringer's solution, respectively. In HCO3 and HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells and the removal of apical Na caused cell acidification with an initial rate of 0.30 +/- 0.08 (n = 32) and 0.58 +/- 0.29 (n = 6) pH U/min, respectively. The pHi recovery from an acid load also was blocked by apical amiloride and apical Na removal. These results suggest that the apical membrane Na/H exchanger is the dominant acid extrusion mechanism in human fetal RPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue

PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.     

Effect of taurine and apical potassium concentration on electrophysiologic parameters of bovine retinal pigment epithelium

The purpose of this study was to assess the effect of taurine and apical potassium concentration modelling in vivo light evoked changes on the transepithelial potential (TEP) and the transepithelial resistance (TER) of isolated bovine retinal pigment epithelium (RPE). Isolated specimens of bovine non-tapetal RPE-Bruch's-choroid (RPE-BC) were mounted in modified Ussing chambers. The apical and the basolateral side of the preparations were exposed to 10 mm and 10 microm concentrations of taurine in Krebs' medium with either 6.04 or 2.2 mm potassium in the apical compartment. TEP and TER were recorded over 140 min. TEP and TER decreased with exposure to taurine over the course of 1 hr followed by a stabilisation. The degree of this response did not depend on the concentration of taurine but was more pronounced when taurine was added to the apical compartment. Lowering apical potassium from 6.04 to 2.2 mm further pronounced the decrease of TEP and TER. The data show that light-induced release of taurine from the outer retina and light-induced decrease of the potassium concentration in the subretinal space synergistically lead to a temporary decrease in TEP and TER. Thereby, taurine uptake into the RPE is reduced probably by a reduction of the activity of the electrogenic Na+/taurine co-transporter of the apical RPE cell membrane. The findings suggest a mechanism whereby the sustained presence of taurine in the interphotoreceptor matrix following exposure to light may protect photoreceptor outer segments from light-induced oxidative stress.     

Retinal pigment epithelial function: a role for CFTR?

In the vertebrate eye, the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE) are separated by a small extracellular (subretinal) space whose volume and chemical composition varies in the light and dark. Light onset triggers relatively fast (ms) retinal responses and much slower voltage and resistance changes (s to min) at the apical and basolateral membranes of the RPE. Two of these slow RPE responses, the fast oscillation (FO) and the light peak, are measured clinically as part of the electrooculogram (EOG). Both EOG responses are mediated in part by apical and basolateral membranes proteins that form a pathway for the movement of salt and osmotically obliged fluid across the RPE, from retina to choroid. This transport pathway serves to control the volume and chemical composition of the subretinal and choroidal extracellular spaces. In human fetal RPE, we have identified one of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) by RT-PCR, immunolocalization, and electrophysiological techniques. Evidence is presented to suggest that the FO component of the EOG is mediated directly or indirectly by CFTR.     

The distribution of Na+,K(+)-ATPase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture

The polarity of retinal pigmented epithelia (RPE) from chicken embryos was studied in primary cell culture. Since cultured RPE approximates the morphological polarity of RPE in vivo, we investigated whether this polarity extends to the distribution of plasma membrane proteins that are peculiar to RPE. In contrast to other epithelia, the Na+,K(+)-ATPase of RPE is located in the apical rather than basolateral plasma membrane. To examine this property, we cultured RPE on extracellular matrix-coated filters. Primary cultures were compared to embryonic RPE in situ using electron microscopy and indirect immunofluorescence of frozen sections. The viability and morphology of RPE was improved by using a serum-free medium containing a bovine pituitary extract in conjunction with an extracellular matrix coating derived from Engelbreth-Holm-Swarm tumors. Cultured RPE mimicked the morphology of RPE in vivo with microvilli and junctional complexes on the apical pole and infoldings along the basolateral plasma membrane. Functional tight junctions formed as demonstrated by an EDTA-sensitive, transepithelial electrical resistance, and by the retention of [3H]inulin added to the apical chamber. In 2 hr, only 4-6% of the [3H]inulin crossed the monolayer, compared to 24% in control filters. Despite these features of polarity, the Na+,K(+)-ATPase was detected in both apical and basolateral membranes by immunofluorescence. In embryonic eyes in which the neural retina was removed, the Na+,K(+)-ATPase was confined to the apical membrane. In addition, the polarity of cultured RPE was probed with vesicular stomatitis virus. In contrast to other epithelia, budding virus particles were observed emerging from the apical, as well as basolateral, domain further suggesting the cultured cells were only partially polarized. These data indicate that structural criteria are inadequate to determine if cultured RPE have become polarized in the same manner as the epithelium in vivo.     

Effects of dopamine on the chick retinal pigment epithelium. Membrane potentials and light-evoked responses

Dopamine, a retinal neurotransmitter, is known to affect electrical measures of retinal pigment epithelial (RPE) function: the standing potential and the DC ERG. To locate the origin of these effects, studies were performed on in vitro preparations of chick retina-RPE-choroid, which were separately perfused on the retinal and choroidal tissue surfaces. Dopamine (250 micrograms) in the retinal bath depolarized the RPE basal membrane, decreased the apparent basal membrane resistance (Rba) and increased the ERG c-wave. At concentrations less than or equal to 100 microM, retinal dopamine often caused a transient basal membrane hyperpolarization, accompanied by an apparent increase in Rba and decrease in c-wave. Surprisingly, 20-100 microM choroidal dopamine induced similar changes in basal membrane potential, resistance and c-wave amplitude, and the transient hyperpolarization and increase in Rba were often more pronounced than at comparable concentrations of retinal dopamine. Experiments in RPE-choroid preparations suggested that the effects of retinal dopamine were not secondary to effects on the neural retina. The effects of retinal and choroidal dopamine in the same tissue often were distinct, suggesting separate receptor populations on the apical and basolateral membranes of the RPE. The c-wave changes could be explained by the changes in Rba, and not by an effect on the light-evoked decrease in subretinal [K+]0. Choroidal perfusion with 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), which appears to block a Cl- conductance in chick RPE, blocked the effects of dopamine perfusion on either side of the tissue. These results suggest that perfusion with either retinal or choroidal dopamine leads to electrical effects on the RPE basal membrane, possibly via a second-messenger system affecting a basal membrane Cl- conductance. Dopamine could suppress the "light-peak" depolarization of the RPE basal membrane. When either retinal or choroidal dopamine induced a large net change in trans-tissue potential (originating as a change in basal membrane potential), the light peak was severely depressed, while smaller changes produced correspondingly smaller decreases in light-peak amplitude. We found, however, that light-peak amplitude was not significantly reduced when there was little net change in the trans-tissue potential, even though dopamine may have produced sizable transient effects. Thus, despite apparent occupation of dopamine receptors on the RPE, the light peak persisted under these conditions. Similar relations between light-peak amplitude and net change in trans-tissue potential have been observed for a variety of different conditions, suggesting that the effect of dopamine on the light peak is nonspecific.     

Fresh and cultured human lens epithelial cells: an electrophysiological study of cell coupling and membrane properties

Microelectrode studies of fresh human and rabbit lens epithelia revealed stable membrane potentials [VR (human) = -36 mV; VR (rabbit) = -45 mV] and low input resistances [Ri (human) = 10 M omega; Ri (rabbit) = 20 M omega]. Coupling studies, using two voltage microelectrodes, demonstrated that the low input resistance of the fresh epithelial tissue was due to electrotonic coupling, which was found to be extremely labile and sensitive to perfusion of the apical (fibrefacing) surface of the epithelium. The intercellular coupling could be stabilized by raising the calcium concentration of the perfusate. Studies performed on confluent monolayers of cultured human lens epithelial (HLE) cells demonstrated a membrane potential (VR = -33 mV) and input resistance (Ri = 29 M omega) similar to their fresh counterparts. The intercellular coupling of these cells was found to be much more robust. Ultrastructural studies revealed that the apical junction of cultured HLE cells was less complex than that found in fresh tissue, the latter exhibiting multiple interdigitations and folds. The cultured monolayer was dissociated into single cells by a variety of methods and the membrane properties of individual cells were studied. Single cells were found to have a lower membrane potential (-20 to -25 mV) and an input resistance in the range 110-170 M omega, depending on the method of dissociation. Channel blocking and ion replacement studies revealed significant conductance pathways for potassium, sodium and chloride and a cell-attached patch clamp investigation revealed three distinct channel types. Of the two channels with inward currents at the resting potential, one, with a conductance of 25 pS, is identified as a non-selective cation channel, and the other, with a conductance of 14 pS and reversal potential of - 14 mV, is a possible candidate for a chloride channel but has yet to be characterized. A third channel with an outward current at the resting potential is identified as a potassium channel with a conductance of 49 pS. A link between epithelial uncoupling and certain types of cataract is proposed.     

Effects of bicarbonate ion on chick retinal pigment epithelium: membrane potentials and light-evoked responses

The purpose of this study was to determine how changes in [HCO3-] alter the electrical properties of the retinal pigment epithelium (RPE). Experiments were conducted on the isolated chick retina-RPE-choroid preparation. The chamber holding the preparation allowed independent perfusion of the retinal and the choroidal surfaces. The light-evoked trans-tissue potential (TTP), the trans-epithelial potential (TEP), the trans-retinal potentials, and the intracellularly-recorded apical and basal membrane potentials were studied. Increasing the [HCO3-]0 in the choroidal bath from 25 to 40 mEq/1 led to an increase in the TTP and TEP. The same change in the retinal bath decreased the TTP because of a biphasic change of the RPE membrane potentials. There was also an increase in the amplitudes of the TEP, the c-wave and the slow PIII. The light-evoked subretinal K+ decrease was greater which is consistent with an increase in the photoreceptor light response. These observations indicated that the decrease of TTP resulted from a basal membrane hyperpolarization followed by an apical membrane depolarization induced by an increase in retinal [HCO3-]0. The relationship of these potential changes to the human bicarbonate responses is discussed.     

Initial observations of rabbit retinal pigment epithelium-choroid-sclera preparations

Retinal pigment epithelium (RPE)-choroid-sclera preparations from black dutch-belted rabbits were sealed in an Ussing chamber maintained at 37-39 degrees C. Typical preparations produced a spontaneous voltage (Ve) of 12.5 mV (retina side positive) and possessed an electrical resistance (R) of 350 ohm-cm2. Both of these values can be attributed to the RPE. Ouabain and amiloride diminished the Ve without affecting R. Ouabain was effective when applied to the apical but not to the basolateral side of the preparation, suggesting the presence of a Na-K ATPase on rabbit RPE apical membrane similar to that found in bullfrogs, embryonic chickens, cats and dogs. Dinitrophenol also reduced Ve. Digoxin, furosemide, bumetanide, ethacrynic acid and chlorothiazide had no apparent effect upon Ve and R. The lack of response to furosemide, bumetanide and ethacrynic acid strongly suggests that, unlike RPE from other species, rabbit RPE does not possess Na-dependent Cl transport and/or does not possess furosemide receptors on its apical membrane.     

Electrical membrane properties of a cell clone derived from human nonpigmented ciliary epithelium

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.     

Anion channels in the apical membrane of mammalian corneal epithelium primary cultures.

Chloride ion (Cl-) secretion by the rabbit corneal epithelium involves a catecholamine-stimulated Cl- conductance at the apical membrane, but the characteristics of the ion channel(s) responsible are unknown. A primary cell culture system was developed that induces stratified epithelial differentiation of rat and rabbit corneal epithelial cells, as detected by differential interference contrast light and transmission electron microscopy. In patch clamp studies, gigaOhm seal, were obtained easily during the first 1-4 days in vitro, and patches contained a voltage-dependent outwardly rectifying channel. Single-channel conductance at 24 degrees C was about 10, 29, and 72 pS in symmetric 150 mM NaCl at membrane potentials of -60, 0, and +60 mV, respectively. The current-voltage relationship measured with 75 mM NaCl and sucrose in the bath indicated anion selectivity. Increasing the temperature to 37 degrees C and replacing HEPES buffer with tricine, increased channel conductance and decreased rectification. Characteristics of this channel and a low conductance Cl- channel are compared with those of anion channels of other vertebrate epithelia.     

Distribution of chloride ion in intercellular space of retinal pigment epithelium--effects of various agents

The distribution of chloride ion (Cl-) in the frog retinal pigment epithelium (RPE) was investigated histologically to determine the effects of various agents on the Cl- concentration in the intracellular spaces of the RPE. Fixation with osmium tetroxide containing silver acetate resulted in the deposit of silver chloride (AgCl) in the RPE, and this was confirmed by X-ray microanalysis. The Cl- concentration in the intercellular space decreased significantly after the fixation and washing with sucrose solution before Cl- deposition procedures, as well as after the 2,4-dinitrophenol treatment, which inhibits the active transport mechanism of the RPE. The intercellular Cl- concentration decreased after the cyclic AMP or cyclic GMP treatment which, respectively, decreases or might decrease Cl- transport from the apical to the basolateral side of the RPE. The Cl- concentration in the intercellular spaces did not change after the ouabain treatment. The results of the present study suggest that the changes in the Cl- concentration in the intercellular spaces of the RPE are related to the status of Cl- transport in the RPE.     

A model for transepithelial ion transport across the isolated retinal pigment epithelium of the frog

The contribution of chloride ion movement and sodium and bicarbonate concentrations to the net current across the isolated choroid-retinal pigment epithelium (RPE) of the bullfrog were studied. The presence of a ouabain-sensitive Na+/K+-pump on the retinal side was confirmed and complete inhibition of this pump with Na+ removal and ouabain treatment abolished nearly all the RPE transepithelial transport and SCC suggesting that all ionic transport was dependent on sodium. It was found that apical to basal (AB) chloride flux accounted for 26 +/- 2% (mean +/- S.E.M.) of the short circuit (SCC). Results suggest that AB bicarbonate and/or basal to apical (BA) hydrogen ion net transport accounts for 38 +/- 2% of the SCC while BA sodium is presumably responsible for the remaining 34% of the SCC. Transport was inhibited by apical administration of known chloride inhibitors. Trans-RPE 36Cl flux measurements indicate that furosemide (10(-4) M) and SITS (10(-3) ) decrease the retinal-choroid flux. Results suggest that net transport of chloride and bicarbonate are independent of each other and additive. It was found that a bicarbonate-free preparation was relatively unaffected by changes in pH (5.5-8.5) indicating that pH has little, if any, effect on sodium or chloride current in this range. A model is presented which is compatible with the various data. It is suggested that along with the apical Na+/K+-ATPase pump, there exists an apical Na+/Cl- -co-transport system which is driven by the established sodium gradient. Moreover, this pump established sodium gradient is postulated to drive a Na+/HCO3- -co-transport system tentatively placed on the retinal side of the RPE.     

Roles of cyclic AMP and Ca in epithelial ion transport across corneal epithelium: a review

The messenger roles of cyclic AMP and the calcium ion in stimulus-secretion coupling are considered in the frog and bovine corneal epithelium, respectively. In the frog cornea, epinephrine stimulates net C1 transport by increasing cyclic AMP content. This stimulation is associated with a larger apical membrane C1 conductance and basolateral membrane ionic conductance. The response of the apical membrane conductance is thought to result from an increase in cyclic AMP content whereas the basolateral membrane ionic conductance increase is unrelated based on measurements of the effects of the calcium channel antagonist, diltiazem, and the beta agonist, isoproterenol, on the electrical parameters and cyclic AMP content. The basolateral membrane is essentially K permselective since the K channel blocker, Ba, depolarized the intracellular potential difference and increased the basolateral membrane resistance. Diltiazem had even larger effects on these parameters suggesting that this compound is a more effective inhibitor of K channel activity than barium. In broken cell preparations of bovine corneal epithelium, a high affinity form of Ca + Mg activated ATPase is present (Km = .06 microM for Ca) and is essentially of plasma membrane origin. This ATPase activation is at a Ca activity similar to the expected intracellular value and suggests that this activity is the enzymatic basis for net Ca transport.     

High levels of E-/P-cadherin: correlation with decreased apical polarity of Na/K ATPase in bovine RPE cells in situ

PURPOSE: The adherens junction protein E-cadherin induces a basolateral polarity of Na/K ATPase in most epithelial cells that express it, whereas in retinal pigment epithelium (RPE) cells, Na/K ATPase is largely apical. The purpose of this study was to determine whether the distribution of Na/K ATPase differs in RPE cells in situ, that differ in levels of junctional E-cadherin. METHODS: Bovine RPE cells in situ were immunostained with an E-cadherin antibody (which has some cross-reactivity with the closely related epithelial cadherin P-cadherin), and RPE cells with different levels of junctional stain were identified. RPE cells with low and high E-/P-cadherin were costained in various combinations with Na/K ATPase and interacting proteins of the membrane cytoskeleton (ankyrin, fodrin, and actin) and analyzed by confocal imaging. RESULTS: Individual RPE cells within the same monolayer differed in amount of Na/K ATPase, with a lower frequency of high expressing cells in the area centralis. High expressing Na/K ATPase cells were found among cells with both low and high E-/P-cadherin levels. In cells with low E/P-cadherin, Na/K ATPase localized to apical microvilli, whereas in high E-/P-cadherin cells, Na/K ATPase was on basolateral surfaces in addition to microvilli. Actin staining showed that microvillar domains were smaller and that lateral membrane domains were taller in high E-/P-cadherin cells. In high but not low E-/P-cadherin cells, ankyrin and fodrin levels varied among cells, with a subset of cells showing distinctly higher expression. Both ankyrin and fodrin had complex subcellular distribution patterns, although they tended to be enriched basal to rather than apical to the adherens junction. Cells with high Na/K ATPase did not necessarily have commensurately higher levels of ankyrin or fodrin. Where both Na/K ATPase and ankyrin were high, they codistributed weakly in apical microvilli but more prominently on the basal cell surface. CONCLUSIONS: Within the same RPE monolayer, the polarity of Na/K ATPase differs among cells, with a more basal polarity found in cells with high levels of junctional E-/P-cadherin. The increased basal Na/K ATPase was due to a combination of a smaller microvillar domain, a taller lateral domain, and more basolateral staining for Na/K ATPase, perhaps because of an enrichment of a basal ankyrinfodrin membrane cytoskeleton with which Na/K ATPase is known to associate.     

Initial observations on the isolated retinal pigment epithelium-choroid of the cat.

An in vitro preparation of a mammalian retinal pigment epithelium (RPE)-choroid was developed, that of the cat, in order to study the RPE in isolation from the neural retina. The purpose of these initial experiments was to evaluate the electrical characteristics of the tissue and the ionic properties of the RPE apical membrane. They were designed so that results would be directly comparable to those previously obtained on a more extensively studied cold-blooded preparation, the bullfrog. The electrical characteristics of the best cat tissues were similar to those routinely obtained in bullfrog, whereas the average cat apical membrane potential was 20 mV more depolarized than that of frog. The apical membrane of cat resembled the frog's in having a large sensitivity to potassium, but it had a relatively smaller sensitivity to bicarbonate and a relatively larger sensitivity to sodium. The cat, like the frog, also had a ouabain-sensitive mechanism on its apical membrane that directly contributed to membrane potential. Two factors contributing to the lower apical membrane potential in cat were the higher potassium concentration of the mammalian salt solution and the sodium conductance of the apical membrane.     

Intracellular voltage recordings in bovine non-pigmented ciliary epithelial cells in primary culture

Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na+ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3 +/- 0.5 mV (n = 36). Relative K+ conductance was increased by extracellular alkalinization. Removing extracellular K+ led to a depolarization and readdition of K+ to K+ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na+/K+ ATPase is inhibited by K+ replacement, and that there is overshoot-activation of the pump when K+ is readded. Extracellular Cl- replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stilbene-sensitive Cl(-)-conductance. Removing HCO3- led to a Na+ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na+ was not influenced by DIDS or the extracellular HCO3(-)-concentration.