Immunocytochemical Localization of NaK-ATPase Isoforms in the Rat and Mouse Ocular Ciliary Epithelium

Abstract

purpose. Ion gradients established by NaK-adenosine triphosphatase (ATPase) in the ocular ciliary epithelium (CE) contribute to the production of aqueous humor. Modulation of NaK-ATPase activity in the CE may alter aqueous inflow, aqueous turnover, and intraocular pressure. To understand the role of NaK-ATPase, it is necessary to examine the distribution of NaK-ATPase subunit isoforms within the epithelium.

methods. Isoform-specific antibodies and scanning laser confocal microscopy were used to localize NaK-ATPase subunit isoforms in the CE of the mouse and rat.

results. The nonpigmented epithelium (NPE) expressed α2 and β3 at very high levels on its basolateral surface, and α1 and β2 at much lower levels. The pigmented epithelium (PE) expressed α1 and β1 subunits on its basolateral surface along its entire length, whereas α3 was expressed in the pars plana only. The distribution and apparent expression levels of isoforms were similar for mouse and rat, with only minor discrepancies, most likely caused by antibody sensitivity.

conclusions. The results indicate that sodium pumps in the NPE are primarily composed of α2 and β3, whereas those in the PE are α1 and β1. This specialization in isoform expression implies that NaK-ATPase has distinct physiological functions in the two epithelia and that its activity is likely to be regulated by different mechanisms.

The ciliary epithelium (CE) is a polarized epithelium in the anterior portion of the eye that is involved in ion transport and the production of aqueous humor. This bilayered epithelium is composed of an inner nonpigmented epithelium (NPE) that is continuous with the neural retina and an outer pigmented epithelium (PE) that is continuous with the retinal PE. Because of the manner in which they develop, these epithelia are arranged with their apical surfaces adjacent to each other. Blood vessel capillaries reside on stromal side of the PE. 

Ion gradients established by NaK-ATPase pumps on the basolateral surfaces of the NPE and PE are essential components of the movement of fluid from the vascular stroma across the CE into the vitreous. ¹ Modulation of NaK-ATPase activity in the CE affects the rate of aqueous inflow, which may then affect aqueous and vitreous humor turnover or intraocular pressure. In fact, agents such as ouabain, digoxin, and 12(R)-hydroxyeicosatetraenoic acid that affect NaK-ATPase activity in the CE have been shown to affect aqueous inflow and intraocular pressure. ^{2 3 4 5}  

To fully understand the mechanism of this regulation, it is necessary to identify the specific cellular and subcellular location of NaK-ATPase subunit isoforms in the CE. Different isoform combinations are thought to function differently in various ionic conditions and to be differentially regulated (for review, see References ⁶ and ⁷ ). In one comprehensive study, the isoforms were found to differ not only in their affinities for the substrates Na⁺ and K⁺ but notably in their voltage dependence as well. ⁸ There are four α subunits (α1, α2, α3, and α4), four β subunits (β1, β2, β3, and β4), and two splice variants of a small regulatory protein known as the γ subunit (γa andγ b). ⁹ However, α4, β4, and the γ subunits have not been found in ocular tissues to date. 

The distributions of three α (α1, α2, and α3) and two β (β1 and β2) subunits have been investigated before in human and bovine CE. ^{10 11 12 13} In contrast, most NaK-ATPase isoform localization in the retina has been performed with rat and mouse (Reference ¹⁴ and references therein), and it is in the mouse that transgenic experiments will be performed. The β3 isoform was discovered relatively recently ¹⁵ and has not been examined in the CE at all. In the present study, we used isoform-specific antibodies and confocal fluorescence immunocytochemistry to examine in detail the distribution of all NaK-ATPase subunit isoforms in the CE of the mouse and rat. Our results indicate a specific distribution of the β3 isoform in the NPE. 

Materials and Methods

The isoform-specific monoclonal and polyclonal antibodies used are summarized in Table 1 . In all procedures we adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eyes from adult male BALB/c mice were bisected and fixed by immersion in 2% paraformaldehyde in a periodate-lysine buffer (PLP) ¹⁶ for 2 hours at room temperature with gentle agitation. Adult male CD rats were anesthetized and perfused with 100 ml of phosphate-buffered saline (PBS; 0.1 M sodium phosphate, 0.15 M NaCl [pH 7.2]) followed by perfusion with 300 ml of PLP. The eyes were removed, bisected, and postfixed by immersion in fresh PLP for 1 hour at room temperature with gentle agitation. After fixation, mouse and rat eyes were rinsed in several changes of PBS. The lenses and vitreous were carefully removed, and the anterior eyecups were immersed in 30% sucrose in PBS for 3 to 4 hours at room temperature, embedded in tissue-freezing medium (TBS; Triangle Biomedical Sciences, Durham, NC) in aluminum boats, frozen on liquid nitrogen, and stored at −20°C. Cryostat sections (12 μM) were picked on positively charged microscope slides (ProbeOn Plus; Fisher Scientific, Pittsburgh, PA) and stored at −20°C until use. 

Slides were brought to room temperature and a PAP pen (Kiyota, Elk Grove, IL) was used to draw a hydrophobic ring around the sections. Slides were rinsed in PBS for 10 minutes and laid flat in a dark moist box for all subsequent incubations. The sections were covered (approximately 100 μl per slide) with 5% normal goat serum (to block nonspecific binding) in PBS with 0.3% Triton X-100 (PBS-Triton) and incubated for 1 hour at room temperature. The blocking solution was removed with an aspirator, primary antisera diluted in PBS-Triton (see Table 1 for dilution) were applied to the sections, and slides were incubated overnight at 4°C. Slides were rinsed in PBS (three times, 10 minutes each) and incubated for 2 hours at room temperature in the appropriate secondary antibody diluted in PBS-Triton, Cy3-conjugated goat anti-mouse IgG (1:300; Accurate, Westbury, NY), tetrarhodamine isothiocyanate (TRITC)–conjugated goat anti-rat IgG + IgM (1:300; Sigma, St. Louis, MO), or fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit IgG (1:1000; Accurate). The slides were rinsed as before in PBS, coverslipped with fluorescence mounting medium (Vectashield; Vector, Burlingame, CA) and observed on a fluorescence microscope (DMRB; Leica, Deerfield, IL) equipped with a scanning laser confocal system (MRC 1024 Laser Sharp, ver. 2.1A; Bio-Rad, Richmond, CA). 

Results

In both mouse and rat, NaK-ATPase immunocytochemical stain was exclusively localized on the basolateral surfaces of the NPE and PE. Unique combinations of predominant isoforms were seen in the CE of the two animals. The distribution of isoform-specific stain was similar between rat and mouse, although there were slight differences. 

Figure 1 shows low-magnification images of the mouse CE stained with α1, α2,α 3, β1, β2, and β3 isoform-specific antibodies. On the right side of each image the retina can be seen. This serves for comparison with the previously described distribution of the retinal NaK-ATPase isoforms, which have a distinctive cell specificity in this tissue. ¹⁴ The basolateral surface of the NPE was prominently labeled with α2 and β3 antibodies and the basolateral surface of PE with α1 and β1 antibodies, along the entire length of the CE. Higher magnification images revealed some differences in distribution between pars plana and pars plicata (Figs. 2 and 3) . Staining for α1 was not present in the NPE in the pars plana (Fig. 2) but was present in portions of the NPE in the pars plicata (Fig. 3) . Staining for β2 was present in the NPE in the region of the pars plana (Fig. 2) , but not in the pars plicata (Fig. 3) . Bright staining for α3 was seen on the basolateral surface of the PE in the pars plana (Fig. 2) but was lighter in the pars plicata (Fig. 3) . 

Low-magnification images of the rat CE showed that, as with the mouse, the entire length of the NPE contained bright staining for α2 andβ 3, whereas the PE contained staining for α1 and β1 (Fig. 4) . Regional differences in the distribution of α1 in the NPE were similar to those seen in the mouse (Figs. 5 and 6) : In the NPE, staining for α1 was absent from the pars plana (Fig. 5) but lightly present in the pars plicata (Fig. 6) . Staining for β2, however, was absent from the pars plana (Fig. 5) but was lightly present in the pars plicata (Fig. 6) . This is the opposite of staining seen in the mouse. Also, unlike the mouse, the rat CE was not labeled at all with α3 antibodies, although the adjacent retina was clearly labeled (see Fig. 5 ). In other experiments, we used a biotinyl-tyramide amplification (which is known to increase sensitivity up to 1000-fold ¹⁷ ) with the monoclonal antibody. Although intensity of staining for α3 in the rat retina increased dramatically, we still saw no staining for α3 in the rat CE (data not shown). It should be noted that the α3 antibodies used in this study, unlike the McBX3 antibody used in prior work, ^{11 12} bind directly to protein and do not show the same inconsistencies in detection of α3 (see the Discussion section). 

The γ subunit is a modulatory protein expressed with the NaK-ATPase in the kidney in some nephron segments, ^{18 19} but thus far there are no published reports of γ localization in ocular tissue. We probed mouse and rat sections with monoclonal and polyclonal anti-γ antibodies that recognize γ in the rat kidney but saw no immunocytochemical label in either mouse or rat CE (data not shown). 

Discussion

Distribution of NaK-ATPase Isoforms in Mammalian CE

We have used isoform-specific antibodies to examine the distribution of NaK-ATPase subunits in the rat and mouse. Our results indicate that pumps in the NPE are predominantly composed of α2/β3, whereas those of the PE are composed of α1/β1 (Fig. 7) . In addition, the NPE expresses some α1 and β2, and the PE expresses α3, although the expression levels of these isoforms appeared to vary between species and also along the length of the CE within a particular species. 

The CE distribution of NaK-ATPase subunits other than β3 has been examined in other species. The results of the earlier immunocytochemistry and of the present study are summarized in Table 2 . In human and monkey CE, α1 and α2 were immunocytochemically localized in the NPE, whereas only α1 was seen in the PE. ¹⁰ The distribution of α3 in the CE has not been examined in the monkey or human immunocytochemically, but a combination of Western and Northern blot analyses on cell populations derived from CE indicates that the human NPE and PE both express α1 and α3, but not α2 (although α2 was localized immunocytochemically in the NPE in that same study). ¹⁰ Western blot analysis on cell populations confirmed immunocytochemistry in bovine tissue. The distribution of β1 has not been examined in the CE of the human or monkey, but β2 has been localized immunocytochemically in the human NPE. ¹³ In the bovine CE, α1, α2, α3, β1, and β2 have been localized immunocytochemically in the pars plicata region of the NPE. ^{11 12 13} The level of expression of all isoforms decreased in more posterior regions of the bovine NPE, and only α1,α 2, and β2 were seen in the pars plana. This expression gradient was supported by a corresponding gradient in isoform mRNAs and NaK-ATPase activity. ¹¹ The α1 and β1 isoforms were seen throughout the length of the bovine PE. In the same study, theα 1 subunit was localized immunocytochemically in the rat NPE and PE, but no other isoforms were examined. 

As seen in previous studies, the NaK-ATPase immunocytochemical label reported in this study was confined to the basolateral surfaces of the NPE and PE. However, the distribution of NaK-ATPase subunit isoforms in mouse and rat CE reported herein varies somewhat from earlier studies in other species. Previous studies in the bovine CE have shown a gradient of expression of NaK-ATPase subunits in the NPE, with the highest levels of expression in the anterior pars plicata and much lower levels in the posterior pars plana. ^{11 12} In the present study, the relatively low amount of staining for α1 in the rat and mouse NPE and of staining for β2 in the rat NPE were brightest in the pars plicata, and decreased to undetectable levels in the pars plana, which supports the idea of a regional gradient of isoform expression. However, no gradient was seen in the major components: staining for α2/β3 in the NPE or for α1/β1 in the PE. Moreover, stainings for β2 in the mouse NPE and for α3 in the mouse PE were brightest in the pars plana and much lighter in the pars plicata. Different (species-specific) β2 antibodies were used on mouse and rat, but it seems unlikely that the apparent species-specific regional difference in β2 expression (rat, highest in pars plicata; mouse, highest in pars plana) reflects a difference in antibody specificity, because staining for β2 in the retinas of the two species was the same. In the human NPE, the intensity of β2 immunocytochemical label in the pars plicata and pars plana were similar. ¹³ It is nonetheless conceivable that β2 is posttranslationally modified in one region of the NPE in a way that favors the binding of one antibody over another. 

Additionally, staining for β2 seen on the basolateral surface of the NPE in this study was very light. Previous studies have indicated strong β2 immunocytochemical label in the bovine and human NPE. ^{12 13} However, Western blot analysis of human ocular tissues have revealed that the level of β2 expression is 10 to 20 times higher in the retina than in the CE. ¹³ One possible reason for strong β2 immunocytochemical signal in the CE in the bovine and human studies is a problem with antibody specificity. Theβ 2 antibody used to label bovine CE in the 1991 study was a polyclonal anti-adhesion molecule on glia (AMOG) antibody known to also contain antibodies that recognize α2 and α3. ²⁰ We have shown in this study that α2 expression is very high in the NPE, and it is therefore possible that some of the apparent staining for β2 seen in the bovine study was actually α2. 

The species difference in the expression of α3 seen in this study was unexpected. We used both monoclonal (XVIF9G10) and polyclonal (polyα 3) antibodies to examine the distribution of α3 in the mouse CE, and the basolateral surface of the PE was clearly labeled with both antibodies. For the rat (where only the monoclonal antibody worked), the adjacent retina was clearly labeled, but the CE was completely devoid of staining for α3. In principle the level of α3 expression in the rat CE could be below our detection threshold, but nothing was detected even with biotinyl-tyramide amplification of the monoclonal antibody. α3 mRNA has been seen in human ciliary processes on Northern blot analysis ¹⁰ but was localized immunocytochemically in the bovine NPE ¹¹ with an antibody of our own, McBX3, that was later shown to be against a posttranslational modification rather than α3-specific sequence. ²¹ McBX3 recognizes α3 in mammalian brain but does not recognize it in mammalian heart, despite an identical cDNA sequence. It also recognizes α1 in kidney NaK-ATPase from some species other than rodents. We also tested the McBX3 monoclonal antibody that had been used in the bovine study but found no specific label in either the retina or CE of mouse or rat. Therefore, the presence and possible role of α3 in the CE appears to be species specific. 

In summary, our results indicate that pumps in the NPE are composed predominantly of α2/β3, whereas those in the PE are α1/β1. Although other isoforms may be present, their expression level appears to be lower. 

Physiological Implications of NaK-ATPase Isoform Distribution in CE

The CE is an unusual structure, in that two epithelia with different properties are fused face-to-face, joined by gap junctions between their respective apical membranes. Ocular fluid is secreted across both layers. Only the NPE layer has the tight junctions typical of ion- and water-transporting epithelia, serving as the barrier for the whole structure. NaK-ATPase lines the basolateral surfaces of both PE and NPE cells, pointing, as it were, in opposite directions. Ghosh et al. ¹² proposed that the CE may in fact be capable of transport in both directions. Transport by the α1β1 isoform of the PE would reabsorb Na⁺ (and therefore ocular fluid), whereas transport by the α2β2 (their work in bovine subjects) or α2β3 (the present study) isoform of the NPE would secrete Na⁺ and ocular fluid. Current models for the mechanism of ocular fluid secretion take into consideration the distribution of other important membrane transporters—notably, Cl⁻ channels and aquaporin and bumetanide-sensitive Na⁺K⁺Cl⁻ transporter—as major players in net outward transport. ^{22 23 24} The apparently symmetrical distribution of some components ²³ raises intriguing questions, however. 

The reproducible difference in NaK-ATPase α subunit isoforms between NPE and PE suggests a difference in properties or functional role. Something has been learned about the intrinsic properties of the isoforms from expression studies. First, when expressed in Xenopus oocytes, the human α2 isoform had approximately half the turnover rate of the α1 isoform, and the α3 isoform had even less. ⁸ In addition to different intrinsic maximal rates, the isoforms differed in their affinity for Na⁺ and K⁺. The α1 isoform had a significantly higher affinity for both Na⁺ and K⁺ than did α2, whereas α3 had a K⁺ affinity similar to α1 but a Na⁺ affinity much lower than α1 orα 2. ⁸ Somewhat different results were obtained for rat isoforms expressed in insect cells, ⁷ in which α2 had a higher affinity for Na⁺ and a lower affinity for K⁺ than did α1. The α3 isoform, in contrast, had a significantly lower affinity for both ions. In mammalian cells,α 3 again had the lowest Na⁺ affinity. ²⁵ However, other investigators showed that these specific properties were found to vary, depending on the cellular context ^{26 27} and on whether the modulatory γ subunit was expressed. ¹⁹  

One isoform-specific feature that could play a role in the relative rates of transport in the NPE and PE is the voltage-dependence of the pump. The voltage dependence of NaK-ATPase is controlled, not by a voltage sensor segment analogous to voltage-dependent ion channel, but by passage of departing Na⁺ ions through an ion well wide enough to be influenced by the transmembrane field. ²⁸ Crambert et al. ⁸ reported that α2 has a steeper voltage dependence than α1, whereas α3 has a voltage dependence that is almost flat. In their experiments in Xenopus oocytes, the voltage dependencies were normalized at− 50 mV, and relative to that voltage, hyperpolarization would favor transport by α1, whereas depolarization would favor transport byα 2. Transport by α3 would be strongly favored at hyperpolarizing potentials compared with the other two isoforms, whereas at depolarizing potentials it would remain fixed, and the others would increase. Previous studies in the rabbit have shown that the conductance of the gap junctions between the cells of the NPE and PE can be modulated by adrenergic stimulation, hindering the passage of ions through these junctions. ^{29 30} Therefore, it is possible for the two layers to have different potentials. We could speculate that changes in voltage mediated by the regulation of gap junctions and K⁺ channels would affect the relative activities of the two types of NaK-ATPase in the two layers of the CE. 

The other likely reason for expressing two different kinds of NaK-ATPase in two cells joined by gap junctions is to permit their separate regulation by second messengers. ^{12 31} Regulation of the NaK-ATPase, particularly in NPE cells, is an active field, but the role of specific isoforms is yet to be investigated. Only a few studies have investigated the underlying mechanisms of regulation ofα 2 and α3 in any tissue. ^{7 32 33} This mechanism should be the focus of future work on the CE. In 1991 Ghosh et al. ¹² proposed that the α1β1 isoform of PE may be unregulated because of the perception that this subunit combination is the housekeeping form of the enzyme, whereas the other isoforms of the NPE may be specialized to respond to environmental factors. Since then, however, active regulation of α1β1 has been described through a variety of mechanisms in other tissues,34 and modulation of ocular fluid transport could as easily include inhibition ofα 1β1 as activation of α2β3. 

 Supported by National Institutes of Health Grant 5RO1 NS27653 (KJS).

 Submitted for publication August 4, 2000; revised November 3, 2000; accepted December 4, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Properties of the Isoform-Specific Antibodies

Antibody	Isoform	Host	Type	Mouse*	Rat*	Dilution	Source
6F	α1	Mouse	Monoclonal	✓	✓	1:4	DSHB 22 , †
McB2	α2	Mouse	Monoclonal	✓	✓	1:4	K. J. Sweadner 33
XVIF9G10	α3	Mouse	Monoclonal	✓	✓	1:100	Affinity Bioreagents 22 , ‡
Poly-α3	α3	Rabbit	Polyclonal	✓		1:250	R. Levenson34
BSP3	β1	Rat	Monoclonal	✓		1:4	C. Goridis35
IEC 1/48	β1	Mouse	Monoclonal		✓	1:4	A. Quaroni36
426	β2	Rat	Monoclonal	✓		1:4	M. Schachner 19
GP50	β2	Mouse	Monoclonal		✓	1:4	P. W. Beesley37
RNTβ3	β3	Rabbit	Polyclonal	✓	✓	1:2000	K. J. Sweadner38

*  Some antibodies were species specific.

†  Developmental Studies Hybridoma Bank, University of Iowa, Iowa City.

‡  Deerfield, IL.

Figure 1.

 

Low-magnification images of NaK-ATPase subunit isoform localization in the mouse CE. Staining for α1, β1, and α3 was localized primarily in the PE, whereas staining for α2, β2, and β3 was localized in the NPE. The retina is visible on the right in each picture. The brightest stain was the photoreceptor inner segments stained for α3 and β2. Bright spots in the retina with staining for α2 were due to artifactual stain of blood vessels. Stain of the retinal PE by antibodies to α1 and β1 was also visible, continuous with the PE. Scale bar, 100 μm.

Figure 2.

 

High-magnification images of NaK-ATPase subunit isoform localization in the pars plana of the mouse CE. Staining for α1, β1, and α3 was localized on the basolateral surface of the PE, whereas staining forα 2, β2, and β3 was localized on the basolateral surface of the NPE. Scale bar, 50 μm.

Figure 3.

 

High-magnification images of NaK-ATPase subunit isoform localization in the pars plicata region of the mouse CE. Staining for α1 was localized on the basolateral surface of the PE and on the NPE in limited areas. Staining for β1 and α3 was localized predominantly on the basolateral surface of the PE. Staining for α2 and β3 was restricted to the basolateral surface of the NPE. No staining for β2 was seen in the pars plicata region of the CE. Scale bar, 50 μm.

Figure 4.

 

Low-magnification images of NaK-ATPase subunit isoform localization in the rat CE. Retina is visible at the right, and staining of the RPE for α1 and β1 can be seen. In the CE, staining for α1 and β1 was primarily in the PE, whereas staining for α2 and β3 was in the NPE. Very light staining for β2 was seen in the pars plicata region of the NPE (more visible in Fig. 6 ). No staining forα 3 was seen in the CE, although the adjacent retina was clearly labeled. Scale bar, 100 μm.

Figure 5.

 

High-magnification images of NaK-ATPase subunit isoform localization in the pars plana region of the rat CE. Staining for α1 and β1 was localized on the basolateral surface of the PE, whereas α2 and β3 were localized on the basolateral surface of the NPE. Although the adjacent retina was clearly labeled, the pars plana region of the CE was devoid of staining for α3 and β2. Scale bar, 50 μm.

Figure 6.

 

High-magnification images of NaK-ATPase subunit isoform localization in the pars plicata region of the rat CE. Staining for α1 and β1 was clearly localized on the basolateral surface of the PE. Expression ofα 1 in restricted areas of the NPE was less pronounced than in the mouse (Fig. 3) . Staining for α2 and β3 was localized on the basolateral surface of the NPE. Very light staining for β2 was seen on the basolateral surface of the NPE in the pars plicata region. No staining for α3 was seen in the CE in the rat. Scale bar, 50 μm.

Figure 7.

 

NaK-ATPase subunit isoform composition of the rodent CE. The cells of the PE expressed primarily α1 and β1 on their basolateral surface, whereas those of the NPE expressed primarily α2 and β3 on their basolateral surface. In parentheses are the isoforms that make a minor contribution to each epithelial layer.

Table 2.

 

Summary of NaK-ATPase Subunit Isoform Localization by Immunocytochemistry in the Ocular CE

Species	Location	α1	α2	α3	β1	β2	β3	Study
Human	NPE	+	+					10
	PE	+	−					10
	NPEpct					++		13
	NPEpln					+		13
	PEpct					−		13
	PEpln					−		13
Monkey	NPE	+	+					10
	PE	+	−					10
Bovine	NPEpct	++	++	++	+	++		11 12 13
	NPEpln	+	+	−	−	+		11 12 13
	PEpct	+	−	−	+	−		11 12 13
	PEpln	+	−	−	+	−		11 12 13
Rat	NPEpct	+						11
	PEpct	+						11
	NPEpct	+	++	−	−	+/−	++	Present
	NPEpln	−	++	−	−	−	+	Present
	PEpct	+	−	−	+	−	−	Present
	PEpln	+	−	−	+	−	−	Present
Mouse	NPEpct	+	++	−	−	−	+	Present
	NPEpln	−	++	−	−	+/−	+	Present
	PEpct	+	−	+	+	−	−	Present
	PEpln	+	−	+	+	−	−	Present

 Pct, pars plicata; pln, pars plana. Blanks indicate that experiments were not performed; −, negative result.

The authors thank Bradley T. Hyman for use of the confocal microscope, and Robert Levenson, Christo Goridis, Andrea Quaroni, Melitta Schachner, and Phillip W. Beesley for the gift of specific antibodies.