Evidence that the alpha and alpha (+)isoforms of the catalytic subunit of (Na+, K+)-ATPase reside in distinct ciliary epithelial cells of the mammalian eye

Polyclonal antibodies against the canine kidney (Na+,K+)-ATPase were used to examine the localization and distribution of this protein in intact ciliary processes (CP) from bovine eyes by indirect immunofluorescence. The basolateral surface of non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells was found to be stained specifically for the (Na+,K+)-ATPase. Immunoblot analysis of intact CP, separated PE and NPE cells by density gradients and cultured ciliary epithelial cells, revealed two forms of the catalytic subunit of the (Na+,K+)-ATPase: the alpha and alpha (+). The alpha (+) form was enriched in NPE cells while alpha was in PE cells.     

Ciliary epithelia of the mammalian eye in cultured explants

The ultrastructural characteristics of ciliary epithelium from bovine, pigmented rabbit, and fetal albino rabbit were studied in cultured explants. The tips of ciliary processes were cultured in plastic dishes with Dulbecco Modified Eagle Medium (DMEM) containing 5% fetal bovine serum. More than half of the explants adhered to the plastic culture dish, and epithelial cells spread as monolayers within a few days. Initially the explant contains two layers, the outer (nonpigmented cells) and the inner (pigmented cells). Later the explant exhibits three layers: 1) outermost lightly pigmented flattened cells, 2) an outer layer of non-pigmented cells, and 3) an inner layer of densely pigmented cuboidal cells. The cells of the outermost layer are continuous with the cells of the inner layer. A narrow space lies between the outermost layer and the outer layer. The columnar cells in the outer layer contain well developed organelles but no pigment granules; they possess a basement membrane, lateral interdigitations, and junctional complexes near their apices. Numerous focal junctions and some ciliary channel-like structures were detected between the columnar cells of the outer layer and the cuboidal cells of the inner layer. The cuboidal cells of the inner layer are filled with pigment granules. These observations suggest that the columnar cells of the outer layer are nonpigmented epithelium, the cuboidal cells of the inner layer are pigmented epithelium, and the flattened cells in the outermost layer are derived from pigmented epithelium.     

Connexin 43 and the glucose transporter,GLUT1, in the ciliary body of the rat

To investigate the relationship between the gap junction protein connexin 43 and the glucose transporter GLUT1, their localization was visualized by double-immunofluorescence microscopy using frozen sections as well as immunogold staining of ultrathin frozen sections. In pigmented epithelial cells, most of the GLUT1 was localized along the plasma membrane facing the blood vessels, whereas in non-pigmented epithelial cells. it was present along the plasma membrane facing the aqueous humor. Connexin 43 was abundant in the ciliary body and localized mainly in the gap junctions connecting the pigmented and non-pigmented epithelial cells. Localization of GLUT1 and connexin 43 in the blood-aqueous barrier suggests that GLUT1, connexin 43, and GLUT1 disposed in this order could be a machinery responsible for the transport of glucose across the blood-aqueous barrier.     

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye^1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment⁴; however the cells are still capable of forming the different cell types found within the neural retina^1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury⁵, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent^6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation^9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.     

Colocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye

 The facilitative glucose transporter GLUT1 is abundant in cells of the blood-ocular barrier and serves as a glucose transport mechanism in the barrier. To see the relationship between the glucose transfer function and junctional proteins in the barrier, we examined the localization of GLUT1 and the tight junction proteins, occludin and ZO-1, in the mouse eye. Their localization in the retina, ciliary body, and iris was visualized by double-immunofluorescence microscopy and immunogold electron microscopy. Occludin and ZO-1 were colocalized at tight junctions of the cells of the barrier: retinal pigment epithelial cells, non-pigmented epithelial cells of the ciliary body, and endothelial cells of GLUT1-positive blood vessels. Occludin was restricted to these cells of the barrier. ZO-1 was found, in addition, in sites not functioning as a barrier: the outer limiting membrane in the retina, in the cell border between pigmented and non-pigmented epithelial cells in the ciliary body, and GLUT1-negative blood vessels. These observations show that localization of occludin is restricted to tight junctions of cells of the barrier, whereas ZO-1 is more widely distributed. Accepted: 7 September 1998     

Electron microprobe analysis of ouabain-exposed ciliary epithelium: PE-NPE cell couplets form the functional units

Aqueous humor is secreted by the bilayered ciliary epithelium. Solutes and water enter the pigmented ciliary epithelial (PE) cell layer, cross gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, and are released into the aqueous humor. Electrical measurements suggest that heptanol reduces transepithelial ion movement by interrupting PE-NPE communication and that gap junctions may be a regulatory site of aqueous humor formation. Several lines of evidence also suggest that net ciliary epithelial transport is strongly region dependent. Divided rabbit iris-ciliary bodies were incubated in chambers under control and experimental conditions, quick-frozen, cryosectioned, and freeze-dried. Elemental intracellular contents of NPE and PE cells were determined by electron probe X-ray microanalysis. With or without heptanol, ouabain produced concentration- and time-dependent changes more markedly in anterior than in posterior epithelium. Without heptanol, there were considerable cell-to-cell variations in Na gain and K loss. However, contiguous NPE and PE cells displayed similar changes, even when nearby cell pairs were little changed by ouabain in aqueous, stromal, or both reservoirs. In contrast, with heptanol present, ouabain added to aqueous or both reservoirs produced much larger changes in NPE than in PE cells. The results indicate that 1) heptanol indeed interrupts PE-NPE junctions, providing an opportunity for electron microprobe analysis of the sidedness of modification of ciliary epithelial secretion; 2) Na and K undergo faster turnover in anterior than in posterior epithelium; and 3) PE-NPE gap junctions differ from PE-PE and NPE-NPE junctions in permitting ionic equilibration between adjoining ouabain-stressed cells. pigmented ciliary epithelial cells; nonpigmented ciliary epithelial cells; gap junctions; aqueous humor; Na⁺/K⁺ exchange pump; rabbit iris-ciliary body     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Ion transport in an immortalized rat submandibular cell line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.     

Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.     

Conductive properties of the proximal tubule in Necturus kidney

The electrical properties of the proximal tubule of the in vivo Necturus kidney were investigated by injecting current (as rectangular waves) into the lumen or into the epithelium of single tubules and by studying the resulting changes of transepithelial (VL) and/or cell membrane potential (VC) at various distances from the source. In some experiments paired measurements of VL and VC were performed at two abscissas x and x'. The luminal length constant of about 1,030 micrometer was shown to provide a good estimate of the transepithelial resistance, specific resistance (RTE = 420 omega.cm2) and/or per unit length (rTE = 1.3 x 10(4) omega.cm). The apparent intraepithelial length constant was subject to distortions arising from concomitant current spread in the lumen. The resistances of luminal membrane (rL), basolateral membrane (rB), and shunt pathway (rS) were estimated by two independent methods at 3.5 x 10(4), 1.2 x 10(4), and 1.7 x 10(4) omega.cm, respectively. The corresponding specific resistances were close to 1,200, 600, and 600 omega.cm2. There are two main conclusions of this study. (a) The resistances of cell membranes and shunt pathway are of the same order of magnitude. The figure of the shunt resistance is at variance with the notion that the proximal tubule of Necturus is a leaky epithelium. (b) A rigorous assessment of the conductive properties of concentric cylindrical double cables (such as renal tubules) requires that electrical interactions arising from one cable to another be taken into account. Appropriate equations were developed to deal with this problem.     

Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.     

A primary culture of guinea pig gallbladder epithelial cells that is responsive to secretagogues

We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (R(t)) of approximately 500 Omega. cm(2). The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (I(sc)) and decreased R(t). The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl(-) and/or HCO(3)(-). Mucosal addition of the anion transport inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the response to ionomycin. The response to forskolin was blocked by DPC but not by DIDS. Ionomycin, but not forskolin, increased intracellular Ca(2+) concentration in fura 2-loaded cells. PGE(2), histamine, vasoactive intestinal polypeptide, and secretin elicited a sustained increase in I(sc). Responses to ATP and CCK were transient. Thus cultured guinea pig gallbladder epithelia display the range of responses observed in the native tissue and are an appropriate model for studies of ion transport in gallbladder and intestinal epithelia.     

12(R)-hydroxyeicosatetraenoic acid synthesis by 3-methylcholanthrene- and clofibrate-inducible cytochrome P450 in porcine ciliary epithelium.

Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family.     

Electrical properties of monolayers cultured from cells of human tracheal mucosa

Dispersed isolated cells were obtained from human tracheal mucosa by digestion with collagenase. Up to 1.5 X 10(8) cells were obtained per trachea and showed up to 95% viability, as judged by trypan blue exclusion. When grown in culture, the cells formed monolayers after approximately 4 days. Electron microscopy of the monolayers revealed a polarized structure. An apical membrane, containing microvilli and a pronounced glycocalyx, was separated from a relatively unspecialized basolateral membrane by typical tight junctions. Monolayers grown on nucleopore filters showed resistances of 44-1,800 omega. cm2 and transepithelial potential differences of 0.1-7.6 mV. Short-circuit current (Isc) was increased by isoproterenol, prostaglandins E2 and F2 alpha, and bradykinin. The loop diuretic, bumetanide, reduced Isc when added to the basolateral (serosal) side but had no effect from the apical (mucosal) side of the monolayers. Furosemide and MK-196 also inhibited Isc. Mucosal amiloride inhibited Isc. Serosal amiloride or mucosal ouabain had no effect on Isc. Serosal ouabain brought Isc to zero after approximately 15 min.     

Induction of pigmentation by continuous X-irradiation of amelanotic tumors of B16-XI mouse melanoma and induced change in chromosomes of amelanotic cells

Non-pigmented tumor cells of B16-XI mouse melanoma were found to contain a diploid number of chromosomes similarly to those of melanotic tumors and the parental cells in tissue culture. A major difference between pigmented and non-pigmented cells was in the number of biarmed chromosomes per cell. There was no difference in growth rate between non-pigmented and pigmented tumors, but growth usually begins about 2 days earlier in the former. Pigmentation lost in the course of serial transplantation was restored by irradiating the non-pigmented tumor continuously with 2,500-3,000 rads/passage of X-rays during six transfer generations. In the course of repeated irradiations, the chromosomes changed structurally and numerically as the pigmentation of the tumor was gradually restored. The observations of tumor growth and chromosomal changes are discussed in relation to the pigmentation of B16-XI melanoma cells.     

Type I cell-like morphology in tight alveolar epithelial monolayers

The pulmonary alveolar epithelium separates air spaces from a fluid-filled interstitium and might be expected to exhibit high resistance to fluid and solute movement. Previous studies of alveolar epithelial barrier properties have been limited due to the complex anatomy of adult mammalian lung. In this study, we characterized a model of isolated alveolar epithelium with respect to barrier transport properties and cell morphology. Alveolar epithelial cells were isolated from rat lungs and grown as monolayers on tissue culture-treated Nuclepore filters. On Days 2-6 in primary culture, monolayers were analyzed for transepithelial resistance (Rt) and processed for electron microscopy. Mean cell surface area and arithmetic mean thickness (AMT) were determined using morphometric techniques. By Day 5, alveolar epithelial cells in vitro exhibited morphologic characteristics of type I alveolar pneumocytes, with thin cytoplasmic extensions and protruding nuclei. Morphometric data demonstrated that alveolar pneumocytes in vitro develop increased surface area and decreased cytoplasmic AMT similar to young type I cells in vivo. Concurrent with the appearance of type I cell-like morphology, monolayers exhibited high Rt (greater than 1000 omega.cm2), consistent with the development of tight barrier properties. These monolayers of isolated alveolar epithelial cells may reflect the physiological and morphological properties of the alveolar epithelium in vivo.     

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was -6.8 mV (apical side), transepithelial resistance was 1,050 Omega.cm(2), and short-circuit current (I(sc)) was 8.1 microA/cm(2). Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kOmega.cm(2), respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of alpha-epithelial Na(+) channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na(+)-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na(+) flux (19 nmol.min(-1).cm(-2)) was two times as large as the reverse flux, and net transepithelial Na(+) flux was nearly double the amiloride-sensitive I(sc). In plain Ringer solution, the amiloride-sensitive I(sc) went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl(-)-dependent I(sc) consistent with the stimulation of transepithelial Cl(-) secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na(+) absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl(-) secretion activated by cAMP.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

Molecular identification and cellular localisation of GSH synthesis, uptake, efflux and degradation pathways in the rat ciliary body

The aim of this study is to determine the contribution of the ciliary epithelium to glutathione (GSH) levels in the aqueous by mapping GSH metabolism and transport pathways in the rat ciliary body. Using a combination of molecular and immunohistochemical techniques, we screened and localised enzymes and transporters involved in GSH synthesis, uptake, efflux and degradation. Our findings indicate that both the pigmented epithelial (PE) and the non-pigmented epithelial (NPE) cell layers are capable of accumulating precursor amino acids for GSH synthesis, but only the NPE cells appear to be involved in the direct uptake of precursor amino acids from the stroma. The localisation of GSH efflux transporters to the PE cell and PE–NPE interface indicates that GSH and potentially GSH-S conjugates can be removed from the ciliary epithelium into the stroma, while the location of GSH efflux transporters to the basolateral membrane of the NPE indicates that these cells can mediate GSH secretion into the aqueous. GSH secreted by the ciliary into the aqueous would remain largely intact due to the absence of the GSH degradation enzymes γ-glutamyltranspeptidase (γ-GGT) labelling at the basolateral membrane of the NPE. Therefore, it appears that the ciliary epithelium contains the molecular machinery to mediate GSH secretion into the aqueous.     

Passive ionic properties of frog retinal pigment epithelium.

The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was --88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8--20 mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductance Ti of each membrane. For the apical membrane: TK approximately equal to 0.52, THCO3 approximately equal to 0.39 and TNa approximately equal to 0.05, and its specific resistance was estimated to be 6000--7000 omega cm2. For the basal membrane: TK approximately equal to 0.90 and its specific resistance was estimated to be 400--1200 omega cm2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110 mM. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered "tight". The shunt pathway did not distinguish between anions (HCO--3, Cl--, methylsulfate, isethionate) but did distinguish between Na+ and K+.