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

Charles W. McLaughlin,1 Sylvia Zellhuber-McMillan,1 Anthony D. C. Macknight,1 and Mortimer M. Civan2

1Department of Physiology, University of Otago Medical School, Dunedin, New Zealand 9001; and 2Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Submitted 13 June 2003 ; accepted in final form 31 January 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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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


THE INTRAOCULAR PRESSURE (IOP) reflects a balance between the rates of inflow and outflow of aqueous humor. Net secretion of aqueous humor is performed by the bilayered ciliary epithelium (6) (Fig. 1). Reducing inflow to lower IOP is a major strategy in the medical therapy of the glaucomas. Unidirectional secretion (Fig. 1A) proceeds in three steps: 1) Na+ and Cl can be taken up from the underlying stroma by electroneutral antiports and symports of the pigmented ciliary epithelial (PE) cells; 2) solute passes from PE cells to nonpigmented ciliary epithelial (NPE) cells through gap junctions; and 3) Na+ and Cl are released from the NPE cells through Na+,K+-activated ATPase and Cl channels, respectively. In principle, transport in the opposite direction could reduce net transepithelial transport (Fig. 1B). Transport mechanisms potentially underlying such reabsorptive movement have been identified for both NPE and PE cells (6, 10, 16, 24), but reabsorptive flow across the ciliary epithelium has not yet been documented.



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Fig. 1. Transporters thought to underlie unidirectional secretion (A) and possible reabsorption (B) across the ciliary epithelium (6, 24). PE and NPE, pigmented and nonpigmented ciliary epithelial cells, respectively.

 
Gap junctions linking the adjoining apical membranes of the PE and NPE cells are thought to provide conduits for both unidirectional secretion (Fig. 1A) and reabsorption (Fig. 1B) across the ciliary epithelium. In addition, there is much structural (27, 28), biochemical (7), and functional (2, 4, 12, 22, 26, 33) evidence for gap junctions between adjacent NPE and PE cells, suggesting that the ciliary epithelium is a functional syncytium. Thus gap junctions might regulate solute and water transfer both within and between the PE and NPE cell layers. Indeed, heptanol, a gap junction blocker, reduces transepithelial ion movement, an effect that has been ascribed to interrupting PE-NPE cell communication (27). We have addressed the putative role of gap junctions in ciliary epithelial secretion by measuring the intracellular ionic content of neighboring PE and NPE cells by electron probe X-ray microanalysis.

In our previous microprobe analyses of ciliary epithelial cells, we generally found little difference in average compositions between PE and NPE cells. Although consistent with the syncytial view of the ciliary epithelium, cell composition was altered relatively little in most of these studies. One aim of the present experiments was to examine the relationships between cells displaying the much larger changes in individual composition that follow ouabain inhibition of the Na+/K+ exchange pump. We also used heptanol in an attempt to isolate effects of ouabain on the NPE cells from those on the paired PE cells. Our prediction was that, after incubation in heptanol, effects on cell composition of drugs and other agents applied to one surface of the epithelium should be limited to the cell type on that surface. If so, this would allow examination of the transport characteristics of NPE and PE cells in the intact epithelium without undue modification of the effects by the paired cell.

As illustrated by Fig. 1, studies of ciliary epithelial transport are commonly analyzed within the framework of lumped-parameter models, but some published results have suggested possible regional differences in net secretion (11, 13, 14, 1721, 25). The microprobe technique permits measurement of cell electrolyte composition in individual cells, thus providing a unique opportunity to explore this possibility directly. Recently, we reported evidence of a higher turnover rate of Cl in the anterior ciliary epithelium (22). In addition to examining gap-junctional function, the present study addressed whether cells in the anterior and posterior regions of the ciliary epithelium were equally affected by ouabain.

Our results indicate that there are differences in gap-junctional function within and between the PE and NPE cell layers, that the functional unit of the ciliary epithelium is the PE-NPE cell couplet, and that the turnover of Na and K is substantially faster in the anterior than in the posterior ciliary epithelial cells.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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The methods used in this study for tissue freezing, cryosectioning, and analysis have been described in detail elsewhere (3, 24).

Cellular model. Dutch black-belted rabbits of either sex and older than 6 wk postweaning were obtained from the Department of Laboratory Animal Sciences, University of Otago Medical School, and were treated in accordance with the Resolution on the Use of Animals in Research of the Association for Research in Vision and Ophthalmology. Animals were anesthetised with 30 mg/kg pentobarbital sodium and killed by injecting air into the marginal ear vein. After enucleation, the iris-ciliary body (ICB) was excised and cut into quarters, and each quarter was bonded with cyanoacrylate to a Mylar support frame on its stromal border. The quadrants with attached frames were mounted in incubation chambers.

Our experimental protocols, in common with many epithelial studies, required the application of different solutions to either side of the ICB. Tissues were mounted between two half-chambers designed to separate the solutions bathing the aqueous and stromal surfaces of the ciliary epithelium. Electrodes were not installed. Each support frame with attached ICB quadrant was aligned by pins between the half-chambers and an indent in the stromal half-chamber, so that the quadrant accurately occluded the common aperture of the two half-chambers. The fluid volume of each half-chamber was 1.5 ml. Fresh solution was constantly infused through each half-chamber at 0.5 ml/min, with higher flow rates during solution changes. Aspiration of excess solution maintained equal levels of fluid on each side of the epithelium, with no pressure gradient. A gas lift in each half-chamber provided gentle aeration and stirring of the solution.

Quadrants of ICB were incubated for 1–2 h at room temperature (18–22°C) under control conditions. Pairs of quadrants (one from each eye) were then incubated for at least 30 min under either control or experimental conditions. After incubation, the tissues were blotted and then plunged into liquid propane at –80°C to freeze the preparation rapidly, thus preventing any redistribution of ions and water. Blocks were fractured from the frozen tissue under a dissecting microscope (x7). Careful attention was paid to the origin and orientation of the block. Thus, after transfer of a block to the cryoultramicrotome and subsequent trimming, we could identify and accurately select the region from which the sections were cut. Generally, sections were cut tangentially. We identified the origins of the sections as either the posterior region (toward retina) or the anterior region (toward iris), respectively, by using criteria established previously (25). Occasionally, sections were cut radially, particularly when we sought to correlate changes in ion content with position along the posterior-anterior axis.

In our initial topological study (25), we analyzed three regions of the rabbit ciliary epithelium: a posterior pars plicata (30) region adjacent to the minimal (in the rabbit) pars plana (30), an anterior region including what has been referred to as the iridial portion of the primary ciliary processes (30), and a middle region between these two. In that study, the responses of the middle region tended to track those of the anterior region but also appeared to display some properties of a region transitional between the well-defined anterior and posterior regions. These two areas are indeed very well defined structurally so that, with experience, the source of the section can be easily identified in cross section by the investigator. The posterior region is characterized by long ciliary processes reaching regularly down to the iris. In contrast, the anterior region displays irregularly shaped cross-sectional areas, whose folds do not necessarily extend to the iris in the section examined but rather reach the iris in an earlier or subsequent section. In the present work, we have compared the elemental compositions of these two structurally well-defined regions: the anterior iridial portion of the primary ciliary processes and the posterior pars plicata region.

Sections of thickness 0.2–0.4 µm were cut at –80 to –90°C, freeze-dried at 10–4 Pa (equivalent to 7.5 x 10–7 Torr), and transferred for analysis to a scanning electron microscope (JEOL JSM 840) equipped with an energy-dispersive X-ray spectrometer.

Solutions and chemicals. The solutions contained (in mM) 145 Na+, 5.9 K+, 122.1 Cl, 15.0 HEPES [4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid], 1.2 Mg2+, 2.5 Ca2+, 1.2 H2PO4, 30 HCO3, and 10 glucose at pH 7.30–7.45 and 305–315 mosmol/kgH2O. Throughout incubation, 95%O2-5%CO2 was bubbled through the solution. All chemicals were reagent grade. Both ouabain and heptanol were added directly to solutions at their final concentrations and dissolved by stirring. In addition, solutions containing heptanol were sonicated for 1 h following stirring.

Data acquisition and reduction. Electron probe X-ray microanalysis permits both quantification and localization of intracellular elements. Using an electron microscope, we target a specific visualized area within the cell. The specimen is irradiated with a beam of electrons, which ionizes a small fraction of the atoms bombarded. After an electron is knocked from an inner atomic shell, an electron from an outer shell can take its place. The relaxation of the electron from a higher to a lower energy state generates a quantum of X-ray energy. Spectroscopic measurement of the characteristic energy and number of these quanta permits identification and quantification of the elements within the sample.

The dried sections were imaged with a transmitted electron detector. X-rays were collected with a Tracor Northern 30-mm2 X-ray detector using a probe current of 140–200 pA for 100 s at an accelerating voltage of 20 kV. The intracellular data were obtained by the electron beam scanning a rectangular area within the nucleus of each selected NPE or PE cell, which varied from ~0.9 x 1.2 to ~2.4 x 3.0 µm depending on the size of the nucleus analyzed. It must be emphasized that a great strength of the electron probe is the capability of directly visualizing those cells and those subcellular targets that are analyzed, thereby ensuring that only the analyzed epithelial cells and not the extracellular compartment or other cellular components contributed data on elemental composition. The individual PE and NPE cells were analyzed in pairs to enable the cellular compositions of the PE and NPE layers to be compared on an individual cell-pair basis.

The elemental peaks were quantified by filtered least-square fitting to a library of monoelemental peaks (3). The library spectra for Na, Mg, Si, P, S, Cl, K, and Ca were derived from microcrystals sprayed onto a Formvar film. In addition to the quantal element-specific X-rays, irradiating sections with an electron beam produces nonquantal white or continuous radiation or "Bremsstrahlung," arising from electron deceleration by coulombic interaction with atomic nuclei. The white counts (w), an index of tissue mass (5), were summed over the range 4.6–6.0 keV, and corrected for the nontissue contributions arising from the Al specimen holder and Ni grid. As discussed previously (24), the Na, K, and Cl signals were routinely normalized to the P signal obtained in the same scanned area of each cell, yielding molar ratios of these elements. We have previously estimated the P content in this tissue to constitute ~500 mmol/kg dry wt (2). P was chosen for normalization because of the constancy of the intracellular signal, which almost entirely reflects the covalently linked fraction in epithelial cells. For example, inorganic phosphate (Pi) is accumulated to only 3 mmol/kg intracellular water in the epithelial cells of frog skin (5). In such cells, the total pool of ATP, ADP, phosphocreatine, and Pi corresponds to only 5% of the total P pool measured in the ciliary epithelial cells (2). The validity of normalizing to P has been experimentally demonstrated by the close linear relationship we have obtained between the two largely intracellular elements K and P (see Fig. 3 in Ref. 2). As illustrated in Table 1, we have found that there is no regional difference in P content. NPE cells had the same values of P/w anteriorly and posteriorly, as did the PE cells.



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Fig. 3. Relationship between elemental compositions of adjacent paired NPE and PE cells in anterior epithelium following exposure to ouabain alone. Data obtained from experiments of Fig. 2. Lines generated from least-square fits had the following correlation coefficients for Na/P, K/P, and Cl/P, respectively: stromal ouabain: r = 0.89, 0.92, 0.12 (A); aqueous ouabain: r = 0.95, 0.95, 0.54 (B); stromal and aqueous ouabain: r = 0.76, 0.93, 0.70 (C).

 

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Table 1. Means ± SE and median with 95% confidence interval for P/w for all tissues

 
The values we report for Na/P, Cl/P, and K/P are the measured estimates of the intracellular Na, Cl, and K contents, respectively. Although it is not possible to estimate ion concentrations in millimoles per liter from these data, the changes in intracellular contents of (Na + K) or of (Na + K + Cl) must reflect changes in intracellular water content (1). For this reason, the sums of the measured ratios (Na/P + K/P) are illustrated on occasion. Data are presented graphically in the form of box plots. It is clear from the results of these experiments that variation in ion contents from cell to cell reflects appreciable real variation in transport characteristics rather than simply experimental error. Thus it is important to appreciate the magnitude of this variation, and this is most readily represented graphically. In the box plots, the medians are indicated by the central horizontal lines, the notch around the median indicates the 95% confidence interval of the sample median, the lower and upper borders of the boxes include all data between the 25th and 75th percentiles, and the whiskers display the data range between the 10th and 90th percentiles. Circles are individual data points that lie outside this range. Thus every cellular measurement is included in this visual representation. Overlap of notches in box plots of different samples indicates that the sample medians are not significantly different. If the notches of two plots just fail to overlap, then the medians are significantly different at the 0.05 probability level.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Effects of ouabain alone. Figure 2 presents the effects produced by incubating paired preparations in 100 µM ouabain on the stromal, aqueous, or both surfaces for 30–40 min. As described in materials and methods, the elemental contents of Na, K, and Cl are normalized to the P content. Over this period of incubation, ouabain produced much larger changes in Na/P (Fig. 2A) and K/P (Fig. 2B) of the NPE and PE cells in the anterior than in the posterior ciliary epithelium and had no significant effect on the Cl/P (Fig. 2C) throughout the tissue.



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Fig. 2. Effects of ouabain alone on elemental composition of NPE and PE cells in anterior and posterior ciliary epithelium. Ouabain (100 µM) was applied to stromal, aqueous, or both surfaces for 30–40 min. Here and in subsequent figures, Na (A), K (B), and Cl (C) are normalized to phosphorus content (see MATERIALS AND METHODS). In box plots, central lines indicate medians, and notches around the medians present the 95% confidence limits. If notches of 2 plots do not overlap, the medians are significantly different at the 0.05 P level. Results were obtained from 3 animals: control (anterior epithelium: 8 sections, 75 cells; posterior: 9 sections, 75 cells); stromal ouabain (anterior: 9 sections, 100 cells; posterior: 8 sections, 89 cells); aqueous ouabain (anterior: 12 sections, 92 cells; posterior: 9 sections, 93 cells); stromal and aqueous ouabain (anterior: 11 sections, 100 cells; posterior: 10 sections, 99 cells).

 
Focusing on the anterior epithelium, the Na/P (Fig. 2A) and K/P (Fig. 2B) following stromal ouabain and especially aqueous ouabain showed considerable variation from cell to cell, as evidenced by the height of the enclosed boxes (25th to 75th percentiles). In contrast, incubation with ouabain on both sides produced a significantly greater gain of Na and loss of K, and the scatter in the data was much reduced. This suggests that much of the variation from cell-pair to cell-pair after aqueous or stromal ouabain reflected differences in the functional capacity of the Na+-K+-ATPase and of passive K+ release and Na+ uptake transporters in the cell type not exposed to ouabain. The absence of an appreciable elevation in Cl/P (Fig. 2C) also indicates that, over 30–40 min of incubation, there was little cell swelling.

Despite the very large ouabain-triggered changes in Na and K content of the anterior cells, the median values of Na/P (Fig. 2A) and K/P (Fig. 2B) were the same in both NPE and PE cells. Evidently, when communication through the gap junctions linking the two cell layers is not blocked, paired NPE and PE cells are affected to similar extents under all conditions, as documented by the linear relationship between the elemental contents of PE and NPE cells following exposure to ouabain (Fig. 3, A–C).

One striking observation was that the ouabain-induced changes in Na/P and K/P varied markedly from cell to cell within the NPE and PE cell layers. The cells that gained the most Na lost the most K. This is clear from the plots of anterior NPE cell K/P as a function of Na/P following incubation in stromal, aqueous, or bilateral ouabain (Fig. 4, A–C). The deviation from linearity of the data points with the very highest Na/P of Fig. 4C likely indicates that bilateral exposure to ouabain produces the largest inhibitions of the Na+ pumps. Under these conditions, there is more complete replacement of K by Na, leading to membrane depolarization and further uptake of Na accompanied by Cl.



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Fig. 4. Relationship between Na/P and K/P in paired anterior NPE ({circ}) and PE cells ({bullet}) following exposure to ouabain alone. Data points obtained from experiments of Fig. 2. Pure exchange of Na for K should fall on the line of identity drawn from K/P = 1.1 at Na/P = 0 to K/P = 0 at Na/P = 1.1. Uptake of Na together with Cl shifts the data points to the right of the line. A: stromal ouabain: r = –0.85 (NPE cells), r = –0.87 (PE cells). B: aqueous ouabain: r = –0.91 (NPE cells), r = –0.89 (PE cells). C: stromal and aqueous ouabain: r = –0.65 (NPE cells), r = –0.61 (PE cells).

 
The variance in Na/P and K/P among pairs of PE-NPE cells did not simply reflect variation from section to section. Figure 5 demonstrates that contiguous PE-NPE cell couplets displayed similar elemental contents in control tissues (Fig. 5A), heptanol-treated tissues (Fig. 5B), and tissues exposed to ouabain without heptanol. However, in contrast to control or heptanol-exposed tissue, the Na/P and K/P levels of PE-NPE cell couplets of ouabain-treated tissues can be very different from those of neighboring cell pairs in the same section (Fig. 5C).



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Fig. 5. Na and K contents in neighboring epithelial cells within a single section from a control tissue in the absence of heptanol or ouabain (A), from a tissue incubated with 3 mM heptanol for 70 min (B), and from a tissue exposed to aqueous 100 µM ouabain alone for 30 min (C). Note that here the elemental contents of paired NPE-PE cells are comparable but that the contents can vary greatly from cell pair to cell pair.

 
Effects of heptanol alone. Incubation of tissue in 3 mM heptanol for 70 min was without appreciable effect on the elemental composition of the great bulk of the NPE and PE cells either anteriorly or posteriorly. The only exception to this general finding was that heptanol produced appreciable gains of Na and Cl and loss of K in a relatively few NPE cells, with little effect on the paired PE cells. Further analysis of the sections indicated that the NPE cells affected in this way were from the most anterior regions of the tissue (data not shown). Removal of these sections removed the scatter without significantly altering the medians calculated from the results overall.

We conclude that application of heptanol alone affects the composition only of the most anterior NPE cells. A possible explanation for this is that these cells rely on their partner PE cells to maintain their Na and K compositions; i.e., these cells cannot maintain their ion composition unaided, because the passive ion movements exceed the capacity of their pump activity.

Time course of responses to ouabain in presence of heptanol. Tissues were incubated in 3 mM heptanol and 100 µM ouabain on both sides and analyzed after 0, 20, 40, and 80 min. The time course reflects both the time taken for ouabain to reach the cells and block the Na+-K+-ATPase, and the rate at which Na is gained by, and K lost from, the cells. As in the absence of heptanol, ouabain exerted greater changes on the Na/P and K/P of the anterior (Fig. 6A) than the posterior (Fig. 6C) ciliary epithelial cells.



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Fig. 6. Time course of effects of ouabain and heptanol on anterior and posterior epithelium. A and B: responses of anterior epithelial Na/P and K/P and (Na + K)/P and Cl/P, respectively, to 100 µM ouabain and 3 mM heptanol. C: responses of Na/P and K/P of posterior epithelial cells. Responses of the posterior epithelial (Na + K)/P and Cl/P were small and therefore omitted. Experimental tissues were preincubated with heptanol in both stromal and aqueous solutions for 40 min before exposure to ouabain in the continuing presence of heptanol for 20, 40, or 80 min. Control tissues (not shown) were exposed to heptanol alone for a further 80 min. Data obtained from eyes of 1 animal. A and B: heptanol alone: 6 sections, 46 cells; ouabain 20 min + heptanol: 5 sections, 46 cells; ouabain 40 min + heptanol: 4 sections, 35 cells; ouabain 80 min + heptanol: 4 sections, 29 cells. C: heptanol alone: 3 sections, 32 cells; ouabain 20 min + heptanol: 3 sections, 32 cells; ouabain 40 min + heptanol: 3 sections, 28 cells; ouabain 80 min + heptanol: 2 sections, 30 cells.

 
The anterior NPE cells showed the greatest changes in both Na and K at all times (Fig. 6A). Note that, as Na gain exceeded K loss, Cl began to increase with the excess cell cation (Fig. 6B). This resulted in cell swelling, apparent by ~40 min. At every time, there were differences between individual cells in the extent of the gain in Na and loss of K. The anterior PE cells lost about one-third as much K content as the NPE cells by 40 min, and their Na content was much below that of the NPE cells at that time (Fig. 6A). Note that for these cells the (Na + K) and Cl did not increase over time (Fig. 6B). Thus the PE cells did not swell over this period.

Concentration-response relationship to ouabain in presence of heptanol. The tissues of Fig. 7 were exposed to ouabain on both surfaces for 30 min. As noted with the foregoing protocols, ouabain exerted the greatest effects on anterior NPE cells, with an apparently saturating concentration of 10 µM for the effects on both Na and K over this time period. For the anterior PE cells, only 100 µM ouabain produced changes approaching those of both 10 and 100 µM concentrations on the anterior NPE cells. For the posterior NPE and PE cells, the ouabain effects were much smaller; note the different vertical scale of Na/P in Fig. 7C.



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Fig. 7. Concentration-response relationship of Na/P and K/P to ouabain in presence of 3 mM heptanol. Tissues were preincubated with heptanol in both stromal and aqueous solutions for 40 min before being exposed either to ouabain for 30 min in the continuing presence of heptanol or remaining in heptanol solution alone for a further 30 min. Data were obtained from experiments using eyes from 2 animals. Tissues from the 1st animal were incubated in heptanol alone or in 5, 10, or 100 µM ouabain; tissues from the 2nd animal were incubated in heptanol alone or in ouabain 0.2, 1.0, 10, or 100 µM. A and B: anterior epithelium. Heptanol alone: 7 sections, 61 cells; ouabain 0.2 µM: 3 sections, 31 cells; ouabain 1 µM: 3 sections, 29 cells; ouabain 5 µM: 4 sections, 38 cells; ouabain 10 µM: 6 sections, 55 cells; ouabain 100 µM: 6 sections, 48 cells. C and D: posterior epithelium. Heptanol alone: 5 sections, 54 cells; ouabain 0.2 µM: 3 sections, 27 cells; ouabain 1 µM: 5 sections, 31 cells; ouabain 5 µM: 2 sections, 29 cells; ouabain 10 µM: 5 sections, 42 cells; ouabain 100 µM: 6 sections, 45 cells.

 
Sidedness of ouabain's effects in presence of heptanol. Figure 8 presents results obtained with tissues exposed to 100 µM ouabain on either the stromal or aqueous side for ~30 min. For comparison, results with ouabain on both sides, obtained by combining the data from the 100 µM ouabain dose-response experiment and the 20- and 40-min ouabain time course experiment, are also shown at the extreme right. From the studies of time (Fig. 6) and concentration dependence (Fig. 7), we expected that stromal ouabain would exert relatively small effects on anterior PE cells. This proved to be the case (Fig. 8, A–C). In contrast, addition of ouabain solely to the aqueous side resulted in significant gain in Na and in Cl and loss of K in anterior NPE cells. As with ouabain in the absence of heptanol, the effects were quite variable from cell to cell, indicated by the large spread in the data. For the anterior cells exposed only to aqueous ouabain, there was no significant correlation among the Na, K, and Cl contents of the NPE and of the PE cells (Fig. 9, A–C). The results were quite different from those for aqueous ouabain without heptanol (Fig. 3B) and provide convincing evidence that heptanol uncoupled the connection between the two cell types to a great extent. Nevertheless, some of the PE cells did appear to be affected by aqueous ouabain to a limited extent in the presence of heptanol, as judged by the greater scatter in the PE cell K/P box plot (Fig. 8B). Note that, because of the small effect of stromal ouabain, the scatter in the relationships between NPE and PE cell Na/P, K/P, and Cl/P was similar to that seen in the absence of ouabain and heptanol. Thus the lack of correlation of Na/P, K/P, and Cl/P for NPE and PE cells here were consistent with, but did not prove, uncoupling of the two cell types, even though we believe that this must have occurred. Both NPE and PE cells with higher Na/P were associated with lower K/P (Fig. 10). For the relatively few PE cells that showed appreciable changes, the Na uptake appeared to reflect largely an equimolar replacement for K. In the case of the NPE cells, the Na uptake seemed to reflect both Na/K exchange and Na/Cl couptake.



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Fig. 8. Effects of aqueous or stromal ouabain (100 µM) and heptanol (3 mM) in both solutions on Na/P (A), K/P (B), and Cl/P (C) of anterior and posterior ciliary epithelial cells from 2 animals. For comparison, this composite figure also includes the analyses (on extreme right) obtained from 3 animals following exposure to 100 µM ouabain in both stromal and aqueous solutions. The latter results were obtained by combining data from the 20- and 40-min time points of Fig. 6 with the responses to 100 µM ouabain of Fig. 7. A: anterior epithelium. Control (no ouabain, no heptanol): 7 sections, 49 cells; heptanol (no ouabain): 19 sections, 117 cells; stromal ouabain (with heptanol): 7 sections, 52 cells; aqueous ouabain (with heptanol): 10 sections, 60 cells; stromal and aqueous ouabain: 15 sections, 126 cells. B: posterior epithelium. Control (no ouabain, no heptanol): 6 sections, 51 cells; heptanol (no ouabain): 12 sections, 113 cells; stromal ouabain (with heptanol): 5 sections, 54 cells; aqueous ouabain (with heptanol): 9 sections, 69 cells; stromal and aqueous ouabain: 6 sections, 105 cells.

 


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Fig. 9. Relationship between elemental contents in paired anterior NPE and PE cells after incubation in 100 µM ouabain + 3 mM heptanol. Data obtained from experiments of Fig. 8; r < 0.4 for all linear least square fits.

 


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Fig. 10. Relationship between Na/P and K/P in anterior NPE and PE cells after incubation with 100 µM aqueous ouabain in presence of 3 mM heptanol. Data obtained from experiments of Fig. 8. To illustrate the relationship between cells exchanging Na for K compared with gaining Na with Cl, a line of identity is drawn from K/P = 1.1 at Na/P = 0 to K/P = 0 at Na/P = 1.1.

 
Comparison of the results of Fig. 3B obtained in the absence of heptanol and of Fig. 9, A–C, obtained in its presence strongly suggested that the heptanol was effectively interrupting PE-NPE communication through the gap junctions. This possibility was tested directly in the same experiment by analyzing the results of adding 100 µM ouabain for 30 min to the aqueous solution in the presence and absence of 3 mM heptanol. Both the box plot (Fig. 11) and the correlation plots (Fig. 12) illustrate the marked uncoupling effects of heptanol. Figure 11 clearly demonstrates that the PE cells gained much more Na and lost much more K in the absence of heptanol than in its presence, suggesting that PE cells have a lower permeability to Na+ and/or K+.



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Fig. 11. Effects of aqueous ouabain on anterior cells in presence or absence of heptanol. Tissues from 1 animal were incubated 30 min with 100 µM aqueous ouabain with or without 3 mM heptanol in stromal and aqueous solutions for 40 min before ouabain exposure and throughout exposure. Ouabain + heptanol: 6 sections, 38 cells; ouabain alone: 3 sections, 28 cells.

 


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Fig. 12. Effect of 3 mM heptanol on relationship between elemental compositions of paired anterior NPE and PE cells exposed to 100 µM ouabain. A: Na/P: r = 0.82 (no heptanol), r = 0.03 (with heptanol). B: K/P: r = 0.96 (no heptanol), r = 0.10 (with heptanol). C: Cl/P: r = 0.70 (no heptanol), r = 0.16 (with heptanol).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Our basic approach was to assess differences in cell transport by measuring Na, K, and Cl contents after blocking the Na+ pumps. The changes observed necessarily reflect the time required for ouabain to diffuse to the cells, the rate at which ouabain binds to and inhibits the pumps, and the rates of Na+ influx into and K+ efflux out of the cells. The major new findings are that 1) in the absence of heptanol, ouabain altered the elemental compositions of paired PE and NPE cells similarly, but dramatic differences were noted between the compositions of neighboring PE and NPE cell pairs within any one tissue; 2) ouabain produced very different changes in paired PE and NPE cells in the presence of heptanol; and 3) the anterior and posterior regions of the ciliary epithelium displayed different time courses and concentration-response relationships to application of ouabain.

Baseline measurements. In the absence of ouabain and heptanol, there was relatively little variation in Na/P and K/P of neighboring cells within a single section (Fig. 5). Under these control conditions, NPE and PE cells displayed similar median values and frequency distributions for Na/P and K/P (Fig. 2). It seems implausible that neighboring pairs of PE and NPE cells in series have precisely the same number and activity of transporters. Therefore, we interpret the relative homogeneity of Na/P, K/P, and Cl/P to reflect the ability of Na+, K+, and Cl to equilibrate by moving from one cell to its neighbor through gap junctions.

Effects of ouabain alone. Application of ouabain to both surfaces of excised rabbit iris-ciliary bodies reduces the intracellular K and increases the intracellular Na contents of the PE and NPE cells (2). In the present study, we have confirmed and extended that observation. In the presence of ouabain without heptanol, the Na, K, and Cl contents of adjoining pairs of PE and NPE cells remained very similar (Fig. 3), as predicted if the PE-NPE gap junctions remain functional. However, the variation in Na/P and K/P among neighboring cell pairs was striking (Fig. 5C). This finding was unexpected in view of structural (27, 28), biochemical (7, 9), and functional (2, 4, 12, 22, 26, 33) evidence for gap-junctional coupling between adjoining cells within the cell layers. The present results clearly indicate that the gap junctions linking PE and NPE cells are different in number or molecular composition from those linking adjoining cells within either the PE or NPE cell layers. This observation is consonant with the original observations of Raviola and Raviola (27), who noted the far greater number of gap junctions between than within the PE and NPE cell layers. Our observation is also in agreement with recent studies of the connexin (Cx) composition of ciliary epithelial gap junctions.

The prominence of Cx43 in ciliary gap junctions was initially reported by Coca-Prados et al. (7) and subsequently confirmed by several laboratories (9, 29, 32). Although Cx50 had been thought to participate in these gap junctions (32), the specificity of the antibody to Cx50 used has been questioned (9). On the basis of a recent careful RT-PCR and immunostaining study of rat ciliary epithelium (9), it is likely that PE and NPE cell couplets are linked by homotypic, homomeric Cx40 and Cx43 gap junctions. In contrast, adjoining NPE cells appear linked by separate homomeric Cx26 and Cx31 gap junctions. Interestingly, Coffey et al. (9) did not detect connexins linking adjoining PE cells, although PE-PE gap junctions have been demonstrated by freeze fracture (27). The negative result of Coffey et al. could reflect either the small plaque size of the PE-PE gap junctions or the participation of as-yet-unidentified connexins.

The inability of PE-PE and NPE-NPE gap junctions to sustain ionic equilibration between adjoining ouabain-treated cells may have reflected simply the limited expression and conductance of these junctions. Alternatively, ouabain may have triggered uncoupling of junctions between adjacent cells in the two layers. Connexins can be selectively up- and downregulated, especially through phosphorylation and dephosphorylation (23). In the current context, ouabain may have triggered a cascade of events leading selectively to interruption of gap junctions linking cells within the PE and NPE layers. For example, the increased Na+ concentration produced by ouabain should increase Ca2+ and proton concentrations secondarily by reducing the electrochemical driving forces for Na+/Ca2+ and Na+/H+ exchange. Indeed, exposure to ouabain either in the aqueous or aqueous and stromal solutions significantly reduced (Na + K – Cl)/P, consistent with a fall in intracellular pH (data not shown). Reductions in intracellular pH and increases in intracellular Ca2+ do block gap junctions (32), but future direct measurements of pH and Ca2+ will be needed to test the plausibility of this possibility.

Effects of heptanol alone. Heptanol alone did not result in appreciable changes in ion contents in either the NPE or PE cells (with the exception noted in results in relation to some NPE cells in sections from the most anterior region of the tissue). This observation, illustrated for example in Fig. 5B, indicates either that, under the conditions of the experiments, variations in ion movements into the two cell types are able to be compensated for by movements out of the same cells across their basolateral membranes, or that heptanol is not particularly effective in blocking gap junctions between adjoining PE-PE and NPE-NPE cell pairs.

Effects of ouabain in presence of heptanol. If the observed heptanol-induced reduction in current across rabbit iris-ciliary body (31) indeed reflects interruption of PE-NPE cell gap junctions, heptanol should reveal the differential responsivity of PE and NPE cell composition to ouabain. The present results verify this prediction. Despite the substantial variance in the effects of ouabain on elemental composition of PE-NPE cell pairs (Fig. 5C), the elemental composition of the paired PE and NPE cells tracked one another very well in the absence of heptanol (Fig. 3). In contrast, heptanol abolished this tracking (Figs. 10 and 12). It is evident from the analyses of time and concentration dependence that, on average, the anterior NPE cells responded more strongly to ouabain than their PE cell counterparts did. This suggests that the rate of Na+ extrusion by the NPE cell pumps might exceed the rate of net Na+ transfer from PE to NPE cells. A significant fraction of Na+ extrusion by these pumps may represent recycling across the basolateral membrane abutting the aqueous humor. If so, this recycling through the NPE cells might provide a basis for significantly regulating NaCl reabsorption through the gap junctions and PE cells back to the stroma. This putative reabsorption could then be regulated by modifying Na+ and Cl release across the PE cell basolateral membrane. Indeed, recent electrophysiological studies suggest that cAMP directly activates PE-cell maxi-Cl channels whose open probability and conductance are enhanced in Cl-loaded cells (10, 16).

Topology of net fluid transport. A number of investigators have observed regional differences in the expression of Na+-K+-activated ATPase and additional proteins and biologically active peptides. Interestingly, NPE cells of young calves display higher expression of {alpha}1/{alpha}2/{alpha}3/{beta}1/{beta}2-isoforms of Na+-K+-activated ATPase anteriorly than posteriorly, but PE cells express a constant relative concentration of {alpha}1/{beta}1 throughout the epithelium (8). Thus net vectorial Na+ extrusion through the Na+ pumps is likely higher anteriorly. Whether the net vectorial movement is actually reversed, leading to reabsorption posteriorly (20), has never been tested. We have already obtained intriguing indications of topological differences in transport from electron microprobe analyses of rabbit ciliary epithelium (25). Those indications were that 1) the ratio of K to Na content was higher in the posterior than in the anterior epithelium, consistent with a lower turnover of Na+ and K+ in the posterior region; and 2) three separate perturbations exerted greater effects on the Cl content of the anterior than of the posterior epithelium, consistent with a higher turnover rate of Cl anteriorly. The three perturbation were omission of CO2/HCO3 from the bathing solutions, inhibition of carbonic anhydrase in the presence of CO2/HCO3, and inhibition of Na+-K+-2Cl cotransport with bumetanide.

The present results provide strong support for the possibility that net transport across the ciliary epithelium is region specific. After exposure to ouabain to block the Na+-K+-activated ATPase, the epithelium responded far more vigorously anteriorly than posteriorly. Unstirred layers can play no role in this phenomenon, since the basolateral surfaces of the NPE cells are equally accessible to the aqueous solutions anteriorly and posteriorly. In addition, because the vast majority of cell K behaves as does K in free solution and the plasma membrane is not impermeable to Na, cells must gain Na and lose K if the sodium pump is inhibited. The regional specificity of the responses to ouabain could reflect the isoform distributions of Na+-K+-activated ATPase or the distribution of regulating molecules such as phospholemman (15). The rate of inhibition of the enzyme by ouabain depends on the rate of turnover of the pump. The slower the pump rate, the longer it will take for maximal pump inhibition. However, pump inhibition is not normally the rate-limiting step in determining the consequent rate of change in cell composition; rather, it is the rates at which the cells can gain Na and lose K. As illustrated in Fig. 6, it is apparent that the posterior cells gain Na and lose K continuously throughout the 80 min of incubation but at a much slower rate than the anterior cells. Whether the increased turnover of Na+, K+, and Cl underlies a higher rate of aqueous humor secretion across the anterior region of the ciliary epithelium remains to be determined.


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This study was supported in part by a Project Grant from the Health Research Council of New Zealand and Lottery Health and by National Eye Institute Grants EY-08343 and EY-01583 (for core facilities).


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, Richards Bldg., Philadelphia, PA 19104-6085 (E-mail: civan{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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