Gene Expression in Rat Lacrimal Gland Duct Cells Collected Using Laser Capture Microdissection: Evidence for K⁺ Secretion by Duct Cells

Abstract

purpose. To compare gene expression profiles of lacrimal gland duct and acinar cells after laser capture microdissection (LCM) and identify molecular networks related to K⁺ secretion, testing the hypothesis that duct cells are responsible for high K⁺ levels in tears.

methods. Frozen sections of lacrimal glands from five rats were subjected to LCM to isolate pure samples of duct and acinar cells. RNA was extracted, amplified, reverse transcribed, and hybridized to rat cDNA microarrays. Paired arrays from ducts and acini of the five animals were scanned and analyzed with in-house software. Gene expression was confirmed with fluorescent antibodies and confocal microscopy.

results. A list of 10,294 genes expressed in ducts and acini was searched using gene ontologies related to ion transport. From a list of 55 genes that were expressed in ducts, a panel of genes hypothesized to be involved in basolateral-to-apical transport of K⁺ and Cl⁻ was chosen for validation by immunofluorescence and confocal microscopy. This analysis confirmed translation of the genes of interest and showed that NKCC1, Na⁺,K⁺-ATPase and the M3 cholinergic receptor are expressed on the basolateral membrane of duct cells, whereas KCC1, IK_Ca1, CFTR, and ClC3 are apically localized.

conclusions. Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying lacrimal gland duct cells about which relatively little is known at the molecular level. As demonstrated in a proposed model, the polarized expression of transporters and channels on lacrimal gland duct membranes is consistent with the hypothesis that duct cells secrete the relatively high K⁺ in lacrimal fluid.

Fluid secreted by the lacrimal gland acini flows into intralobular and interlobular ducts that anastomose to form the excretory ducts that release lacrimal fluid onto the ocular surface. ¹ Because they are abundant, easily accessible, and can be maintained in short-term culture, the lacrimal acinar cells have been studied extensively and are well characterized. They secrete fluid that is thought to be similar in electrolyte concentration to extracellular fluid. ² Secretion of proteins, ^{3 4} growth factors, ^{5 6} and vitamins ^{7 8} by the acinar cells has been studied, and the transporters, channels, signaling pathways, and intracellular membrane trafficking involved in acinar cell function are quite well understood. ^{3 9 10 11 12 13 14} In contrast, because of their relative paucity and inaccessibility, the duct cells have not been studied extensively, nor have they been isolated and placed in culture. Lacrimal gland fluid and tears have a K⁺ concentration greater than 20 mEq/L compared with a K⁺ concentration of 5 mEq/L in extracellular fluid. ^{2 15 16} Although the mechanism of action is unknown, it has been suggested that this high K⁺ is important for the health of the ocular surface epithelium, ^{17 18 19} leading to marketing of artificial tear formulations with elevated K⁺ levels. ²⁰ It has been proposed that the relatively high potassium ion concentration in lacrimal gland fluid and tears is due to potassium secretion by the duct epithelium. This hypothesis is based on an early observation suggesting that the acini secrete isotonic fluid with an electrolyte composition similar to that of the extracellular fluid, ² implying that the ducts must add potassium to the secreted fluid. A model for ion transport by the duct cells has been proposed by Mircheff, ¹² but has not been experimentally confirmed. 

The work presented in this article is part of a larger study designed to compare gene expression between duct and acinar cells. Laser capture microdissection (LCM) ^{21 22} was used to collect near homogeneous populations of lacrimal duct and acinar cells. Gene expression profiles were subsequently compared between duct and acinar cells by using cDNA microarrays. To test the hypothesis that duct cells secrete K⁺ into the lacrimal fluid, the cDNA microarray data from duct and acinar cells were mined using gene ontologies relevant to ion transport. A set of genes likely to be involved in basolateral-to-apical K⁺ secretion was identified, and expression of the channel and transport proteins was confirmed using immunofluorescence and confocal microscopy. A proposed model for K⁺ and Cl⁻ secretion by the duct epithelium of the lacrimal ducts is presented. 

Materials and Methods

Animals

Male Sprague-Dawley rats, 6 weeks old (Charles River, Portage, MI), were euthanatized by CO₂ asphyxia. The exorbital lacrimal glands were removed, embedded in OCT medium, frozen in liquid N₂, and stored at −80°C until use. Pregnant female Sprague-Dawley rats were euthanatized, and the 13-day embryos were snap frozen in liquid N₂. All work with animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Laser Capture Microdissection

Frozen sections, 6 μm thick, were cut in a cryostat (model CM 1850; Leica, Nussloch, Germany). Serial sections were cut and examined unstained on a phase-contrast microscope to locate regions of the gland with numerous ducts. Sections from these regions to be used for LCM were mounted on aminoalylsilane-coated slides (HistoGene; Arcturus Engineering, Inc., Mountain View, CA; or Silane-Prep Slides; Sigma, St. Louis, MO), fixed in 70% ethanol, stained with hematoxylin and eosin, and dehydrated through graded alcohols and xylene. They were then air dried and stored in a desiccator. 

Acinar and duct cells were captured using a Pix Cell IIe LCM System and CapSure Macro LCM caps (Arcturus Engineering, Inc.). Total RNA was purified from the captured cells using a PicoPure RNA Isolation Kit (Arcturus Engineering, Inc.). 

cDNA Microarray Analysis

Lacrimal cell RNA was subjected to two rounds of amplification (Message Amp aRNA Kit; Ambion, Austin, TX). After the first round of cDNA synthesis, before in vitro transcription of aRNA, the integrity of the cDNA was checked by GAPDH PCR of a 500-bp sequence. All RNA samples used in the study yielded a prominent GAPDH PCR product and had a 260/280 ratio >2:1. 

Rat cDNA microarrays (a 20,000 gene set; Lion Bioscience, Inc., Cambridge, MA), were printed in the Microarray Core Facility at the Van Andel Research Institute (Grand Rapids, MI). The aRNA (10 μg) from duct and acinar cells was reverse transcribed (Superscript II RT; Invitrogen, Carlsbad, CA), in the presence of Cy3-dCTP (PerkinElmer, Boston, MA) and random primers (Invitrogen). Reference RNA from 13-day rat embryos that had undergone one round of amplification was reverse transcribed in the presence of Cy5-dCTP in a parallel fashion, to provide a common reference across all arrays. 

The Cy-3- and Cy-5-labeled cDNA products from duct or acinar cells and reference samples were cohybridized to microarrays, as previously described. ^{23 24} Because we used rat RNA, the blocking solution used in the hybridization protocol contained rat DNA (Hybloc; Applied Genetics Laboratories, Melbourne, FL). The slides were scanned at 532 and 635 nm (ScanArray Lyte scanner; PerkinElmer). The image files were analyzed using Gene Pix Pro 3.0 software (Molecular Devices Corp., Union City, CA) and the complete rat microarray clone list (Lion Bioscience, Inc.) to create result files (Gene Pix; Molecular Devices Corp.). The files were uploaded into XenoBase, a fully integrated genomic/proteomic/medical informatics database with associated analysis and annotation tools designed at the Van Andel Institute. (http://www.vai.org/vari/xenobase/summary.asp). ^{23 24} Data were analyzed as described in the Results section. 

Immunohistochemistry and Confocal Microscopy

Antibodies to specific pump, cotransporter, and channel proteins related to K⁺ transport, along with the appropriate FITC and rhodamine labeled secondary antibodies are described in Tables 1 and 2 . Frozen sections (6 μm) were mounted on slides (Superfrost; Fisher Scientific, Pittsburgh, PA). The sections were fixed in 70% ethanol, washed, and blocked with 1% BSA (Sigma) and 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in PBS. Primary antibodies, diluted 1:100 in buffer (Primary Antibody Dilution Buffer; Biomeda, Foster City, CA) were applied to the slides and incubated overnight in a moist chamber at 4°C. The sections were rinsed in 0.05% Tween-20 (Sigma) and secondary antibodies, diluted 1:100 in dilution buffer, were applied and incubated for 1 hour in the dark. The slides were again washed with Tween-20 and 4′,6′-diamino-2-phenylindole (DAPI; Invitrogen, Eugene, OR) was then applied for 10 minutes to stain the nuclei. The sections were then washed in deionized water, coverslipped using aqueous mounting medium (Gel/Mount; Biomeda) and allowed to dry overnight in the dark. 

Images in Figures 2to 4and 7to 9were captured with a confocal microscope (model 510 NLO confocal microscope with a META detector; Carl Zeiss, Inc., Thornwood, NY). FITC was excited at 488 nm using an argon laser and HFT UV/488/543/633 dichroic, while rhodamine was excited with a helium-neon laser at 543 nm and the same dichroic. DAPI was excited with a Ti-sapphire pulsed laser at 800 nm. Nomarski/differential interference contrast (DIC) images were acquired with the visible detector and 543 nm excitation. Images were captured and processed using the image programs inherent in the microscope system (Carl Zeiss Meditec, Inc.). Images in Figures 5 and 6were captured with a separate system (ApoTome Imaging system with Axiovision software; Carl Zeiss Meditec, Inc.). 

Results

Laser Capture Microdissection and RNA Amplification

Duct cells were cleanly separated from surrounding cells because the plastic film on the caps (Cap Sure; Arcturus Engineering, Inc.) did not adhere well to connective tissue that delineates ductal and acinar compartments (Fig. 1) . Duct cells were captured from the glands of each rat (n = 5) used in the study by using 200 to 400 laser pulses, yielding 81.9 to 241.3 μg of aRNA after two rounds of amplification. To accumulate adequate cells, it was necessary to move the cap to various regions on the same section and to capture cells from multiple sections. This manipulation of the cap did not negatively affect the efficiency of cell capture. Acinar cells captured with 500 pulses yielded 64.1 to 210.4 μg of aRNA after two rounds of amplification. 

Gene Expression in Lacrimal Gland Duct and Acinar Cells

To analyze microarray data, filters were set to include only genes with a sample intensity ≥150 in 5 of 10 arrays. (The 10 arrays consisted of arrays from duct cells of five rats and five paired arrays from acinar cells.) Data were log₂ transformed and mean centered across the arrays. A total of 10,294 cDNA probes passed these filters. The complete list of genes is provided online in Supplementary Table S1. The data were then mined by using gene ontologies for biological process activity, molecular function, and cellular components, as related to ion transport (Table 3) . This analysis, which was conducted using Affymetrix NetAff annotations (Affymetrix, Santa Clara, CA) linked to Entrez Gene ID, yielded a list of 55 genes with known functions (Table 4) . 

The probabilities were corrected to false-discovery rates (FDRs) of 0.2. The FDR is a considerably more stringent test than Student’s t-test, setting a lower threshold for making an appropriate conclusion that a gene is differentially expressed. The FDR test indicated that seven of the genes listed in Table 4were differentially expressed between ducts and acini. Notable genes in this group were those encoding the α (Αpt1a1), β (Atp1b1, Atp1b3), and γ (Fxyd2) subunits of Na⁺,K⁺-ATPase which were expressed at significantly higher levels in duct cells than in acinar cells. 

The list of transport-related genes was inspected for genes likely to be involved in transport of K⁺ into the ducts. Genes of particular interest identified in this group based on previously published studies (Mircheff, ¹² as discussed later) were a K⁺/2Cl⁻ cotransporter (SLC12a4), a Na⁺,K⁺,2Cl⁻ cotransporter (Slc12a1), an intermediate conductance Ca-activated potassium channel (Kcnn4), anion exchanger 1 (Slc4a3), chloride channel 3 (Clcn3), the muscarinic cholinergic receptor 3 (Chrm3), and aquaporin 5 (Aqp5). Three genes in the ATP-binding cassette family, of which the cystic fibrosis transmembrane regulator (CFTR) is a member, were also identified on the microarrays (Table 4) . Unexpectedly, CFTR was not present on the list, however, preliminary analysis of the data revealed an expressed sequence tag (EST) with homology to CFTR, based on its initial identification in the Unigene database (Unigene Rn.13195, internal ID 197628 in the supplementary data/ http://www.ncbi.nlm.nih.gov/UniGene; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), that was strongly expressed in both ducts and acini. 

We chose to confirm the expression of these genes and the Na⁺,K⁺-ATPase α and β subunits (Table 1)by immunofluorescence studies with the goal of confirming the translation of these genes and demonstrating that the cellular location of the gene products is consistent with vectorial transport of K⁺ into the lacrimal gland ducts. 

Immunofluorescence Confocal Microscopy

A Nomarski/DIC image of a lacrimal gland section stained with DAPI is shown in Figure 2 . Cross sections of two ducts are prominently illustrated. This section was costained with a rhodamine-conjugated donkey anti-rabbit IgG secondary antibody without a primary antibody. No rhodamine fluorescence was visible, as was true of all sections stained only with secondary antibody. 

Consistent with its detectable gene expression on cDNA microarrays (Table 4) , Na⁺,K⁺-ATPase was expressed in both lacrimal ducts and acinar cells (Fig. 3) . This pump protein was not expressed on the apical membranes of the ducts, as clearly shown with the antibody to the Na⁺,K⁺-ATPase β subunit (Fig. 4B) . 

The CFTR was present in both duct and acinar cells. In duct cells it appeared to be expressed more strongly on the apical membranes (Fig 4A) . This can be seen most clearly on sections dually labeled for both CFTR and Na⁺,K⁺-ATPase β subunit (Fig. 4B) . 

The ClC-3 chloride channel antibody stained both duct and acinar cells. Although fluorescence was detected on the entire visible area of the acinar cells, the fluorescence in the ducts was localized to the apical membranes of the cells (Fig. 5) . 

The gene for the band-3 anion exchanger (AE1) was expressed in both acinar cells and duct cells. Immunofluorescence showed that this transporter is localized to the basolateral membranes of the acini. In contrast, AE1 while clearly present in the ducts, was not preferentially localized to any particular duct cell membrane (Fig. 6) . 

The intermediate conductance calcium-activated potassium channel (IK_Ca1) gene was expressed in both duct and acinar cells of the lacrimal gland (Table 4) . The channel protein was faintly detectable in acinar cells, but was prominently localized to the apical membrane of the duct cells (Figs. 7A 7B) . The M3 cholinergic receptor gene was also expressed in ducts as detected on microarrays. Because acetylcholine can mediate release of internal Ca²⁺ stores in lacrimal gland, ^{3 4} sections were dually labeled for the M3 receptor and IK_Ca1. M3 showed basolateral localization in ducts, whereas IK_Ca1 was confined to the duct cell apical membranes (Fig. 7C) . 

Of major interest with regard to secretion of K⁺ into the ducts were the NKCC1 and KCC1 cotransporters. By immunofluorescence, little evidence of expression of these transporters was detectable in the acinar cells (Fig. 8A) . In contrast, the KCC1 cotransporter was strongly expressed in duct cells and was limited to the apical membranes of the ducts (Fig. 8) . The NKCC1 cotransporter was strongly expressed on the basolateral membrane, as seen in the image without the Nomarski overlay (Fig. 8B) . No colocalization of these transporters was evident on the confocal micrographs. 

The ducts and acini also stained prominently for aquaporin 5, which was also highly expressed at the transcriptional level (Table 4)and has been shown to be present in lacrimal gland by immunostaining. ³⁵ Aquaporin 5 was limited to the apical membranes of duct cells (Fig. 9) . 

Discussion

Laser Capture Microdissection

The development of the fields of genomics and proteomics and use of microarrays allows global investigation of cellular characteristics, but, as pointed out by Emmert-Buck et al., ²¹ and Bert and Emmert-Buck ²² optimization of these techniques requires pure populations of cells. In their original paper on LCM, the use of LCM to isolate glomeruli from kidney, amyloid plaques from brain, and precursor cells of breast neoplasm from mammary tissue was demonstrated. ²¹ Subsequently, this technique has been used to study numerous tissues, permitting the isolation of specific cell types such as primary spermatocytes from testes, ³⁶ specific regions of the retina and brain ^{37 38 39} and lung cancer cells. ⁴⁰  

In the present study we have demonstrated the utility of LCM for separation of duct cells from the surrounding acinar cells and connective tissue. Although there is one report of patch clamp recording from isolated duct cells, ⁴¹ the identity of the cells was not unequivocally confirmed. This is, to our knowledge, the first report on isolated duct cells with retention of histologic structure and organization that accurately identifies the cell type in question. LCM has excellent prospects for future studies that will distinguish between ducts and acinar cells in animal models of lacrimal gland disease as well as for investigations of human lacrimal gland biopsy or pathology specimens. 

cDNA Microarray Analysis

The efficient use of the vast amount of data produced by cDNA microarray analysis requires that the data be approached with specific hypotheses or problems in mind. ⁴² Although analysis of the microarray data yielded data on expression of many similarities and differences in gene expression between lacrimal ducts and acini (Supplementary Table S1), we chose to use the microarrays as an efficient method for screening duct and acinar cells for expression of genes related to K⁺ secretion. This analysis led to identification of genes expressed in the ducts that are potentially involved in vectorial basal-to-apical transport of K⁺ and Cl⁻ by duct cells including the intermediate conductance Ca-activated K⁺ channel which has not been identified in the lacrimal gland. Statistical analysis of microarray data also revealed relatively high levels of Na⁺,K⁺ ATPase gene expression in ducts, supporting the hypothesis that the ducts secrete K⁺. These data on channels, transporters, and receptors, which were confirmed by immunofluorescence, led to the construction of a model of K⁺ secretion by the ducts, as is discussed in the remainder of this report. 

Expression of Transporters, Channels, and Receptors in Duct Cells

It is well known that secretion of fluid by the lacrimal gland is dependent on the activity of Na⁺,K⁺-ATPase. Early studies of fluid secretion by the lacrimal gland showed that secretion is inhibited by ouabain. ⁹ Based on findings in other secretory epithelia it was expected that the Na⁺,K⁺-ATPase would be localized to the basolateral membrane of lacrimal epithelial cells. Although there was controversy for a time as to whether the pump is also present on the apical membranes, ^{43 44} there is now general agreement that Na⁺,K⁺-ATPase is basolaterally located. ^{9 45} This is confirmed in the present study, in which we demonstrate by immunofluorescence the expression of the α and β subunits of Na⁺,K⁺-ATPase in duct and acinar cells and the absence of these proteins from the apical membranes of ducts (Figs. 3 4B) . In the first report on Na⁺,K⁺-ATPase in lacrimal gland Dartt et al., ⁹ using ³H-ouabain, suggested that the number of pump sites was greater in duct cells than in acinar cells. This was confirmed quantitatively by Okami et al., ⁴⁵ who labeled the α subunit using immunogold and demonstrated that expression of Na⁺,K⁺-ATPase is three to five times higher on duct cell basolateral membranes than on acinar cell basolateral membranes. Our novel observation that the expression of the mRNA for both the α and β Na⁺,K⁺-ATPase subunits, as well as the γ subunit (FXYD domain-containing ion transport regulator) ⁴⁶ is higher in the duct cells is in agreement with the reports by Dartt et al. ⁹ and Okami et al. ⁴⁵ As originally proposed by Dartt and Okami, increased gene and protein expression for all the subunits of the Na⁺,K⁺ pump in the duct cells (Table 4)is consistent with a high level of K⁺ transport by the lacrimal gland ducts. 

Na⁺,K⁺,2Cl⁻ Cotransport.

Coupled cotransport of sodium, potassium, and chloride occurs across the membranes of most animal cells and, as reviewed by Russell, ⁴⁷ is mediated by Na⁺,K⁺,Cl⁻; K⁺,Cl⁻; and Na⁺,Cl⁻ cotransporters. These types of transporters are inhibited by furosemide and based on inhibition of lacrimal fluid secretion in vivo by furosemide, Dartt et al. ⁹ and Micheff ¹² suggested that lacrimal acinar and duct cells may express a basolateral Na⁺,Cl⁻ cotransporter. Subsequently, Singh ⁴⁸ presented in vitro evidence for Na⁺,Cl⁻ cotransport in the lacrimal gland, but an electrophysiological study by Ozawa et al. ⁴⁹ demonstrated stimulation of Cl⁻ uptake into mouse lacrimal acinar cells with both Na⁺ and K⁺, suggesting a basolateral Na⁺,K⁺,Cl⁻ cotransporter. 

The Na⁺,K⁺,2Cl⁻ cotransporter exists as two isoforms, both mediating ion influx: NKCC1, expressed in many cell types, and NKCC2, found only in the kidney. ⁴⁷ Walcott et al. ⁵⁰ recently demonstrated the presence of NKCC1 in duct and acinar cells of mice with localization to the basolateral membranes. In agreement with Walcott et al. the present study presents evidence for expression of NKCC1 on the basolateral membranes of duct cells of rat lacrimal glands (Fig. 8) . This transport protein was barely detectable in rat acinar cells. This finding suggests that the Na⁺,Cl⁻ cotransporter proposed by Dartt et al. ⁹ and Mircheff ¹² may be more active in acinar cells, whereas expression of NKCC1 is consistent with K⁺ secretion by the duct cells. 

K⁺,Cl⁻ Cotransport.

The K⁺,Cl⁻ cotransporters of the CCC family are represented by at least four members, KCC1 to -4, ^{26 27 51 52 53} which are widely distributed. ^{54 55} The KCC transporters mediate efflux of K⁺ and Cl⁻ from cells and are furosemide sensitive. The characteristics of KCC1 ⁵³ are consistent with the properties of a K⁺,Cl⁻ transporter identified in the distal convoluted tubule (DCT) and cortical collecting duct (CCD) using microperfusion techniques. ^{56 57} Both of these nephron segments secrete K⁺ into the lumen of the tubule. Evidence obtained in the present study suggests a similar function of KCC1 in the lacrimal gland ducts. The expression of KCC1 mRNA in duct cells (Table 4)and localization of KCC1 to the apical membrane (Fig. 8)is of particular importance, because in kidney, where KCC1 mRNA is present in DCT and CCD cells, ⁵⁵ the protein has not yet been conclusively localized to the apical membrane, although physiologic evidence exists for the apical location of KCC1 in kidney tubules. ⁵³  

The furosemide sensitivity of KCC1 suggests that furosemide may decrease K⁺ levels in lacrimal gland fluid. Although Dartt et al. ⁹ showed a decrease in fluid secretion when the gland was exposed to furosemide, a concomitant decrease in K⁺ concentration has not been investigated. 

Cl⁻ Transport.

Apical chloride channels in lacrimal acinar and duct cells were postulated by both Dartt et al. ⁹ and Mircheff. ¹² Evans and Marty ⁵⁸ demonstrated the existence of a calcium-dependent chloride current in rat lacrimal acinar cells and more recently, Herok et al. ⁵⁹ have studied effects of osmotic stress on Cl⁻ currents in acinar cells; however, chloride channels, including the cystic fibrosis transmembrane regulator (CFTR) and the ClC family have not been systematically studied in the lacrimal gland. 

Microarray data identified at least three genes from the ATP-binding cassette family, which were multidrug resistant proteins (Table 4) . Although micorarray evidence for the expression of the CFTR gene was not compelling, confocal microscopy confirmed that the CFTR chloride channel ^{28 60} is strongly expressed in both duct and acinar cells. As expected, based on its location in other secretory epithelial such as pancreas, ⁶⁰ CFTR is strongly localized to the apical membrane of the duct cells (Fig. 4) . This is, to our knowledge, the first evidence of expression of CFTR in lacrimal gland. The only other report concerning CFTR in lacrimal gland is the observation of lacrimal gland disease in a mouse model of cystic fibrosis with a null mutation for CFTR. ⁶¹ According to the authors these mice had dilated acini, suggesting back pressure due to blocked ducts. Apparently, no further research has addressed this problem, although there is one report of dry eye in patients with cystic fibrosis. ⁶²  

The ClC family of chloride channels has at least eight widely expressed members, ClC0 to -7, ⁶³ plus two channels expressed primarily in kidney, ClC-K1 and ClC-K2. ⁶⁴ These represent channels involved in secretion, volume regulation, and control of membrane potential. On microarrays, we detected two Cl⁻ channels of the ClC family (Table 4) , a ClC-K1-like channel that is usually considered to be primarily expressed in kidney, ⁶⁵ and ClC-3, which is expressed in many transporting epithelia, including intestine, airways, kidney, ⁶³ and salivary gland. ⁶⁶ The ClC channels have not been studied in detail in the lacrimal gland, but Majid et al. ⁶⁶ have reported that in contrast to salivary gland, the ClC-3 channel mRNA and protein is apparently not expressed in lacrimal gland acinar cells. In contrast, we have demonstrated strong immunofluorescence on the apical membranes of duct cells exposed to a ClC-3 antibody. This is in agreement with apical localization of ClC-3 in ducts of the Cl⁻ transporting epididymal epithelium. ⁶⁷ Although we cannot rule out the possibility that the ClC-3 antibody used in this study cross-reacts with a related ClC channel, our observations place ClC-3 or a related channel in the apical membrane of the lacrimal duct cells (Fig. 5)where it would be well-positioned for conductance of Cl⁻ between the cytoplasm and the duct lumen. 

The band 3 anion exchanger (AE1) that mediates Cl⁻/HCO₃ ⁻ antiport was also identified on microarrays in both ducts and acini. Confirmation of its expression by immunostaining localized this transport protein to the basolateral membranes of acinar cells (Fig. 6) . Ozawa et al. ⁶⁸ and Lambert et al. ⁶⁹ provided physiologic evidence for expression of the anion exchanger in mouse and rat lacrimal acini. The latter group proposed a basolateral location, which we have confirmed. The anion exchanger is also strongly expressed in ducts, but a clear basolateral or acinar localization could not be confirmed. Therefore, we have not included this transporter in our proposed model for vectorial K⁺ and Cl⁻ transport by lacrimal ducts. 

Potassium Channels and Cholinergic Receptors.

Electrophysiological evidence for the presence of K⁺ channels on the luminal membranes of acinar cells has been reported by Tan et al. ⁷⁰ In the present study, microarrays revealed many K⁺ channels expressed in lacrimal duct cells (Table 4) . Most of these channels are probably electrogenic channels that may or may not be involved in K⁺ secretion. For this reason, we chose to focus on a channel that can clearly be associated with K⁺ secretion, the intermediate conductance calcium-activated K⁺ channel (Kcnn4, IK_Ca1), which is strongly expressed in lacrimal duct cells, as shown in the present study, and is also known to be expressed in pancreas. ⁷¹ In addition to the IK_Ca1 channel, potassium secreting cells have also been shown to express a large-conductance calcium activated channel (BK_Ca) and small conductance channel (SK_Ca). ³¹ The BK_Ca channel has been identified in the apical membrane of lacrimal acinar cells and may also be present in ducts. ⁷² Although it is an abundant and common channel, the BK_Ca channel was not included on the rat microarray used in this study, so we have no gene expression data on the BK_Ca channel in duct cells. Attempts to identify the BK_Ca channel in ducts by immunofluorescence yielded equivocal results (data not shown). 

The IK_Ca1 channel was strongly expressed in the apical membrane of the duct cells (Fig. 7)and based on data from the proximal colon it may be very important in regulated K⁺ secretion. The proximal colon regulates plasma [K⁺] by secreting K⁺ into the intestinal lumen. Joiner et al. ³¹ have shown that IK_Ca1 is expressed by epithelial cells of the proximal colonic crypts with greater expression in the apical membrane than in the basolateral membrane. Elevated internal Ca²⁺ in response to cholinergic stimulation opens IK_Ca1 channels, as well as BK_Ca and SK_Ca channels, resulting in apical secretion of K⁺. Although BK_Ca and SK_Ca are also present in the apical membrane of the intestinal crypt cells, IK_Ca1 predominates and is the primarily regulator of K⁺ secretion. The strong localization of IK_Ca1 to the apical membranes of the lacrimal gland duct cells in the present study suggests that this channel plays a similar role in K⁺ secretion by the lacrimal ducts. 

The expression of M3 cholinergic receptors on the basolateral membranes of duct cells (Fig. 7C)is consistent with cholinergic control of the IK_Ca1 channels. The M3 receptor was the only cholinergic receptor detected on microarrays (Table 4) , which is consistent with previous reports that M3 receptors are the predominant cholinergic receptors in the lacrimal gland. ^{73 74} Cholinergic control of the acinar cells of the gland is well established and quite thoroughly understood. ^{3 4 9 15} Ding et al. ⁷⁵ have previously demonstrated close association of cholinergic nerve fibers with ducts in mouse lacrimal glands. Localization of the receptor to the duct cell basolateral membrane confirms that the ducts as well as the acini are under cholinergic control. 

A Model of K⁺ and Cl⁻ Secretion by Lacrimal Duct Cells

Mircheff ¹² proposed a model for K⁺ secretion by lacrimal ducts that included the Na⁺,K⁺ pump and a Na⁺,K⁺ cotransporter on the basolateral membrane with undefined K⁺ and Cl⁻ channels on the apical membrane. Taken together the various transporters and channels just described can be used to propose a mechanism for K⁺ and Cl⁻ secretion by the lacrimal ducts (Fig. 10)that expands on the Mircheff’s hypothesis. As in all secretory epithelia the basolateral Na⁺,K⁺ pump extrudes Na⁺ from the cell while pumping K⁺ into the cell. The relatively high level of pump expression in the duct cells may cause elevated intracellular [K⁺], promoting secretion at the apical membrane. A high level of pump activity will increase the influx of Na⁺ via the basolateral NKCC1 transporter, thereby loading the cell with K⁺ and Cl⁻ via cotransport. The apical KCC1 transporter in turn secretes K⁺ and Cl⁻ into the lumen, in agreement with the proposed mechanism in the kidney cortical collecting duct. ⁵³ Gillen and Forbush ⁷⁶ used overexpression of KCC1 in HEK-293 cells to show that the resultant decrease in intracellular Cl⁻ stimulates NKCC1 activity, and suggested apical–basolateral cross-talk between transporters. In light of this finding, the significantly higher expression of KCC1 in lacrimal gland duct cells than in acinar cells argues for a role of the duct cells in KCl secretion. Potassium also enters the lumen of the duct through apical IK_Ca1 channels under the influence of intracellular Ca²⁺ released in response to cholinergic stimulation. Regulation of these channels via the M3 receptor insures that K⁺ secretion increases in parallel with parasympathetically stimulated fluid flow from the lacrimal acini. As K⁺ is secreted via IK_Ca1, and probably BK_Ca channels, ⁶⁷ Cl⁻ follows via the apical CFTR channels as well as ClC channels. 

In conclusion, this study demonstrates how LCM and cDNA microarrays, combined with immunohistochemistry can be used to study the relatively inaccessible lacrimal duct cells. The data provide a reasonable solution to long-standing questions about the secretory function of lacrimal duct cells and the elevated K⁺ levels in tears. This demonstrates the feasibility of using these techniques for further studies of duct cells addressing problems related to lacrimal gland diseases such as Sjögren’s syndrome and tumorigenesis. 

 Supported by National Eye Institute Senior Fellowship F33 EY014783, a sabbatical leave and research fellowship from Calvin College, and financial support from the Van Andel Research institute (JLU), and a Michigan Life Sciences Corridor-Core Technology Alliance (JHR). HMH was supported by a summer research fellowship from the Howard Hughes Medical Institute (Grant 52002664) and by a Grant-in-Aid of Research from Sigma Xi. Calvin College is the recipient of National Science Foundation Major Research Instrumentation Grant 0421120.

 Submitted for publication March 22, 2005; revised June 22 and December 21, 2005; accepted March 6, 2006.

 Disclosure: J.L. Ubels, None; H.M. Hoffman, None; S. Srikanth, None; J.H. Resau, None; C.P. Webb, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primary Antibodies Used for Immunofluorescence Studies

Antigen	Name of Antibody	Species	Source	References
Na+/K+/2Cl− cotransporter	NKCC1 NKCC11-A	Rabbit anti-rat	Alpha Diagnostic International	25
Furosemide-sensitive K+/Cl− Cotransporter	KCC1 P-20	Goat anti-human	Santa Cruz Biotechnology	26 27
CFTR chloride channel	CFTR Ab3 L12B4	Mouse anti-human	NeoMarkers Lab Vision	28
Na+-K+-ATPase α subunit	Na/K-ATPase α1 N-15	Goat anti-human	Santa Cruz Biotechnology	29
Na+-K+-ATPase β Subunit	Na/K-ATPase β1 C464.8	Mouse anti-rabbit	Santa Cruz Biotechnology	29
C1C3 chloride channel	CLC-3 K-17	Goat anti-human	Santa Cruz Biotechnology	30
Intermediate conductance Ca-activated K+ channel	Anti-KCa3.1	Rabbit anti-rat	Alomone Labs, Ltd.	31
Anion exchanger 1 (AE1)	Band 3 C-17	Goat anti-human	Santa Cruz Biotechnology	32
M3 cholinergic receptor	mAChR M3 C20	Goat anti-human	Santa Cruz Biotechnology	33
Aquaporin 5	AQP5	Goat anti-human	Santa Cruz Biotechnology	34

Table 2.

 

Secondary Antibodies Used in Immunofluorescence Studies

Fluorophore	Species
Rhodamine red X	Donkey anti-mouse IgG
Rhodamine red X	Donkey anti-rabbit IgG
Fluorescein (FITC)	Donkey anti-goat IgG
Fluorescein (FITC)	Donkey anti-rabbit IgG
Fluorescein (FITC)	Donkey anti-mouse IgG

 All antibodies were from Jackson ImmunoResearch Laboratories, Inc.

Figure 2.

 

Rat lacrimal gland frozen section (6 μm) stained with DAPI and viewed with Nomarski optics. The section was also exposed to a rhodamine-conjugated donkey anti-rabbit IgG secondary antibody alone, to ensure minimal nonspecific staining and is representative of control experiments performed in all immunofluorescence studies. Large arrows: indicate ducts. Small arrow: blood vessel. Bar, 50 μm.

Figure 3.

 

Rat lacrimal gland stained for Na⁺,K⁺-ATPase α (A) and β (B) subunits. Both acinar and duct cells are positive for Na⁺,K⁺-ATPase. Localization to the basolateral membrane is clearly seen in the section stained for the β subunit (arrow). Bar, 50 μm.

Figure 4.

 

Expression of CFTR in lacrimal gland. (A) CFTR (rhodamine) was strongly localized to apical membranes of duct cells (arrow) as shown in this image with Nomarski/DIC overlay. (B) Dual labeling of CFTR (rhodamine) and Na⁺,K⁺-ATPase β subunit (FITC). Color was enhanced from the original to emphasize apical localization of CFTR (large arrow) and basal localization of Na⁺,K⁺-ATPase (small arrow) in ducts. Bar, 50 μm.

Figure 7.

 

(A, B) Intermediate conductance Ca-activated potassium channel (IK_Ca1) was expressed primarily on the apical membrane of duct cells (arrows). (C) Dual labeling of the IK_Ca1 channel (rhodamine, large arrow) and M3 cholinergic receptor shows basolateral location of the M3 receptors (FITC, small arrow). Bar, 50 μm.

Figure 8.

 

Expression of NKCC1 (rhodamine) and KCC1 (FITC) transporters by lacrimal duct cells. (A) The basolateral expression of NKCC1 (large arrow) and apical expression of KCC1 (small arrow) overlaid onto the Nomarski/DIC image. (B) Fluorescence without the Nomarski/DIC overlay. Note well-defined basolateral location of NKCC1. Bar, 50 μm.

Figure 9.

 

Aquaporin 5 was expressed in duct and acinar cells. The channel was prominently localized to the apical membranes of duct cells (arrow). Bar, 50 μm.

Figure 5.

 

The ClC-3 chloride channel was strongly localized to the apical duct membrane and was also expressed by acinar cells. Bar, 50 μm.

Figure 6.

 

The anion exchanger (AE1) was expressed in both ducts and acini and was localized to the basolateral side of the acinar cells. Bar, 50 μm.

Figure 1.

 

LCM of lacrimal gland duct cells. The laser spot size was 7.5 μm and was set at 7.5- to 8-ms pulse duration at a power of 75 to 85 mW. (A) Longitudinal section of a duct (arrow) before laser capture. (B) The same section after capture of duct cells. (C) Duct cells on the laser capture cap. Bar, 25 μm.

Table 3.

 

Gene Ontology Lists Used to Mine Microarray Data for Transport-Related Genes

Biological process activity ontologies

Ion transport

Sodium ion transport

Potassium ion transport

Chloride transport

Cation transport

Water transport

Synaptic transmission

Molecular function ontologies

Ion channel

Ion transporter

Symporter

Antiporter

ATP binding

Cellular component activity ontologies

Integral to membrane

Integral to plasma membrane

Table 4.

 

Ion Transport-Related Genes Expressed in Rat Lacrimal Gland Duct and Acinar Cells, as Detected on cDNA Microarrays

Gene	Ducts	Acini	t Statistic	P	Symbol
FXYD domain-containing ion transport regulator 2*	0.2	−2.372	−7.593	0.0001	Fxyd2
Inositol 1,4,5-triphosphate receptor 3*	1.377	1.827	4.04	0.0037	Itpr3
ATPase, Na+/K+ transporting, beta 3 polypeptide *	−1.01	−1.884	−3.819	0.0051	Atp1b3
ATP-binding cassette, sub-family C, member 5*	0.006	−1.505	−3.067	0.0154	Abcc5
ATPase, Na+/K+ transporting, alpha 1 polypeptide *	1.419	0.743	−2.565	0.0334	Atp1a1
Glycine receptor, alpha 2 subunit*	0.116	−0.227	−2.466	0.039	Glra2
ATPase, Na+/K+ transporting, beta 1 polypeptide *	1.681	−0.267	−2.382	0.0444	Atp1b1
Chloride channel K1-like	1.608	0.492	−2.301	0.0504	Clcnkb
Solute carrier family 12, member 4	−1.491	−2.054	−2.226	0.0566	Slc12a4
Lectin, galactose binding, soluble 9	−1.126	−1.709	−2.122	0.0666	Lgals9
Potassium inwardly rectifying channel, subfamily J, member 1	−0.28	−0.473	−1.807	0.1083	Kcnj1
Dri 27/ZnT4 protein	0.073	1.27	1.722	0.1233	Slc30a4
Inositol 1,4,5-triphosphate receptor 1	3.012	2.572	−1.709	0.1258	Itpr1
5-Hydroxytryptamine receptor 3a	−1.813	−2.248	−1.65	0.1375	Htr3a
Solute carrier family 4, member 3	0.037	−0.526	−1.628	0.1422	Slc4a3
Potassium voltage-gated channel, subfamily H, member 2	−0.773	−0.434	1.448	0.1855	Kcnh2
Calcium channel, voltage-dependent, beta 4 subunit	−0.23	−0.781	−1.39	0.202	Cacnb4
Phospholipase C, gamma 1	−2.489	−2.966	−1.346	0.2151	Plcg1
FXYD domain-containing ion transport regulator 1	−0.785	−0.249	1.299	0.2303	Fxyd1
Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4	0.776	1.185	1.235	0.2518	Kcnn4
Potassium voltage gated channel, Shab-related subfamily, member 1	−1.234	−0.022	1.213	0.2598	Kcnb1
Mitochondrial H+-ATP synthase alpha subunit	−2.198	−1.745	1.202	0.2636	Atp5a1
Solute carrier family 12, member 1	−0.755	−0.234	1.16	0.2794	Slc12a1
ATPase, Ca++ transporting, plasma membrane 1	2.207	2.514	1.071	0.3155	Atp2b1
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2	1.636	2.01	1.067	0.317	Atp2a2
Potassium channel, subfamily K, member 1	3.944	4.298	0.918	0.3857	Kcnk1
Solute carrier family 22, member 1	−1.493	−1.095	0.916	0.3867	Slc22a1
ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide	0.05	0.235	0.868	0.4108	Atp5b
Solute carrier family 22, member 5	0.942	0.482	−0.86	0.4151	Slc22a5
Sodium channel, voltage-gated, type 1, beta polypeptide	0.618	0.11	−0.771	0.4627	Scn1b
Potassium voltage-gated channel, subfamily F, member 1	−0.756	−0.951	−0.77	0.4632	Kcnf1
Phospholamban	−2.438	−2.038	0.763	0.4672	Pln
Cholinergic receptor, muscarinic 3	1.675	1.976	0.7	0.504	Chrm3
Adrenergic receptor, alpha 2a	0.206	0.035	−0.679	0.516	Adra2a
Solute carrier family 5, member 6	−0.412	−0.337	0.631	0.5454	Slc5a6
Inositol 1,4,5-triphosphate receptor 2	0.047	0.509	0.622	0.5511	Itpr2
ATP-binding cassette, sub-family C, member 1	−0.525	−0.826	−0.601	0.5646	Abcc1
ATPase, Ca++ transporting, cardiac muscle, fast twitch 1	−1.564	−0.922	0.584	0.5752	Atp2a1
Purinergic receptor P2X, ligand-gated ion channel, 3	0.291	0.546	0.536	0.6063	P2rx3
Solute carrier family 9, member 1	0.035	−0.17	−0.525	0.6141	Slc9a1
Potassium inwardly rectifying channel, subfamily J, member 3	3.097	3.525	0.522	0.6161	Kcnj3
Chloride channel, nucleotide-sensitive, 1A	−1.244	−1.38	−0.41	0.6927	Clns1a
ATP-binding cassette, sub-family C, member 9	0.182	0.092	−0.397	0.7018	Abcc9
Plasmolipin	0.12	−0.067	−0.392	0.7055	Tm4sf11
Calcium channel, voltage-dependent, alpha2/delta subunit 1	−3.216	−3.019	0.345	0.7386	Cacna2d1
Gamma-aminobutyric acid A receptor, alpha 5	0.421	0.542	0.295	0.7755	Gabra5
Chloride channel 3	0.336	0.433	0.275	0.7901	Clcn3
Glutamate receptor, ionotropic, kainate 5	0.296	0.369	0.222	0.8298	Grik5
Solute carrier family 22, member 6	−0.509	−0.465	0.111	0.9146	Slc22a6
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c, isoform 1	0.09	0.118	0.105	0.919	Atp5g1
Glutamate receptor, ionotropic, N-methyl d-aspartate 1	0.248	0.228	−0.079	0.9393	Grin1
Aquaporin 5	6.602	6.632	0.066	0.9489	Aqp5
Solute carrier family 22, member 2	−0.003	−0.006	−0.016	0.9878	Slc22a2
Purinergic receptor P2X, ligand-gated ion channel, 1	1.292	1.298	0.012	0.9904	P2rx1

 Data are log₂ transformed sample/reference intensity ratios (n = 5) where a negative value indicates reduced expression relative to the rat embryo reference. Bold entries indicate genes chosen for confirmation by immunofluorescence.

*  Significant difference between ducts and acini. False discovery rate analysis (FDR ≤ 0.2).

Figure 10.

 

Model for secretion of K⁺ and Cl⁻ by the lacrimal duct epithelium based on the channels and transporters identified in this study. Na⁺,K⁺ATPase and the Na⁺,K⁺,2Cl⁻ cotransporter (NKCC1) on the basolateral membrane load the cell with K⁺ and Cl⁻, which are then secreted into the lumen of the duct by the IK_Ca1, ClC3, and CFTR channels and the K⁺,Cl⁻ (KCC1) cotransporter. The M3 cholinergic receptor regulates intracellular Ca²⁺ levels, thereby activating the IK_Ca1 channel. *Gene expression significantly higher in ducts than in acini on cDNA microarrays.

Supplementary Materials

Supplementary Table S1 - 2.27 MB (.xls) 

The authors thank Jeremy Miller, PhD, for assistance with microarray data analysis, Paul Norton and Pete Haak for preparing microarrays, and Eric Hudson for assistance with confocal microscopy.