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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 473-477, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0190OC

Expression of the High-Affinity Choline Transporter, CHT1, in the Rat Trachea

Uwe Pfeil, Katrin Susanne Lips, Lars Eberling, Veronika Grau, Rainer Viktor Haberberger and Wolfgang Kummer

Institutes for Anatomy and Cell Biology, Justus-Liebig-University, Giessen; and Philipps-University, Marburg, Germany

Address correspondence to: Dr. Uwe Pfeil, Institute for Anatomy and Cell Biology, Aulweg 123, 35385 Giessen, Germany. E-mail: uwe.pfeil{at}anatomie.med.uni-giessen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rate limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline by the high-affinity choline transporter (CHT1). Here, we investigated the distribution of CHT1 in the rat trachea. CHT1-mRNA was detected by reverse transcriptase–polymerase chain reaction in trachea without epithelium, abraded tracheal mucosa, and in epithelial cells obtained by laser-assisted cell-picking. Accordingly, CHT1-mRNA could also be detected in tracheal epithelial cells by in situ hybridization. Recently obtained polyclonal rabbit and guinea-pig antisera against a synthetic peptide corresponding to amino acid residues 29–40 of the rat CHT1 sequence localized CHT1 protein in combination with antisera against the vesicular acetylcholine transporter in cholinergic fibers innervating tracheal glands and the tracheal muscle. In case of the tracheal epithelium, CHT1 was restricted to the apical membrane of the ciliated cells, as demonstrated by confocal laser scanning and electron microscopy using an affinity-purified CHT1 antiserum. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favor of ACh synthesis being fuelled by choline uptake via CHT1 after release and breakdown of ACh at the luminal surface. Accordingly, cholinergic regulation of tracheal epithelial function is governed by local release and recycling of ACh by ciliated cells.

Abbreviations: acetylcholine, ACh • high-affinity choline transporter, CHT • choline acetyltransferase, ChAT • digoxygenin, DIG • polymerase chain reaction, PCR • reverse transcriptase, RT • Tris-buffered saline with 0.05% Tween-20, TTBS • vesicular acetylcholine transporter, VAChT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The airways contain both a neuronal and a non-neuronal cholinergic system. The parasympathetic nerve fibers innervating predominantly the airway smooth muscle and mucosal glands use acetylcholine (ACh) as their major transmitter to initiate muscle constriction and secretion, respectively (1). At these cholinergic terminals, ACh undergoes a well-characterized cycle of synthesis, storage, release, cleavage, re-uptake of its precursor choline, and re-synthesis. ACh is synthesized in the axoplasm from acetyl-CoA and choline by choline acetyltransferase (ChAT) and imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Upon release, ACh is cleaved by choline esterases into acetate and choline, the latter being taken up into the synaptic terminal via a high-affinity choline transporter. This re-uptake of choline is the rate-limiting step in neuronal ACh synthesis (2). The high-affinity choline transporter expressed by central cholinergic neurons has recently been cloned and termed CHT1 (3–6). In accordance with the high requirement for ACh synthesis in cholinergic nerve terminals, CHT1 has a higher affinity to choline than the organic cation transporters OCT1 and OCT2 that are used for choline uptake by other cell types, e.g., in choroid plexus and kidney (7–9).

An additional source of ACh in the airways is the respiratory epithelium (10). The presence of multiple cholinergic receptors in this epithelium (11, 12), together with the stimulatory role of ACh on ciliary beat frequency (13, 14) and on release of granulocyte-macrophage colony-stimulating factor (15), are strongly indicative for a locally acting epithelial cholinergic signaling system. The molecular components of this epithelial cholinergic system are not as well characterized as those of cholinergic neurons. Airway epithelial cells contain ACh, exhibit ChAT activity (10), and a ChAT-immunoreactive protein has been localized to ciliated cells by means of immunohistochemistry (16, 10). The identity of the transporter(s) utilized for choline uptake in these cells is still unclear. Recently, we have identified the high-affinity choline transporter, CHT1, that has originally been assumed to be restricted to cholinergic neurons, in another cholinergic epithelium, i.e., skin keratinocytes (17). Thus, we hypothesized that CHT1 might be expressed in the cholinergic airway epithelial cells as well, and we tested this hypothesis in the rat trachea by reverse transcriptase (RT) polymerase chain reaction (PCR), laser-assisted cell-picking, in situ hybridization, and light and electron microscopic immunohistochemistry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RT-PCR
Total RNA from abraded tracheal cells, trachea without epithelial cells and spinal cord of adult Wistar rats (n = 5; Harlan Winkelmann, Borchem, Germany) were isolated by using the RNazol technique (WAK-Chemie, Bad-Homburg, Germany) according to the manufacturer's protocol. Messenger-RNA was isolated from total RNA with oligotex (Qiagen, Hilden, Germany). The mRNA was reverse transcribed using Superscript RNase H- Reverse transcriptase (200 U/onset; Gibco-BRL, Karlsruhe, Germany) for 50 min at 42°C. For subsequent PCR, 2.5 µl buffer II (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 1.5–2 µl MgCl2 (15 mM), 0.6 µl dNTP (10 mM each), 0.6 µl of each primer (10 µM; MWG Biotech, Ebersbach, Germany) (Table 1), 0.25 µl AmpliTaq Gold polymerase (5 U/µl; all reagents from Perkin Elmer, Langen, Germany) were supplemented with H2O to a final volume of 25 µl. Cycling conditions were 12 min at 95°C, 40 cycles with 30 s at 95°C, 30 s at 60–70°C, 30–120 s at 72°C, and a final extension at 72°C for 7 min. Control reactions included the absence of DNA template and the absence of RT. The PCR products were separated by electrophoresis on a 1.2% TRIS-acetate-EDTA gel. Sequencing of the PCR products was done by MWG Biotech.


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  		TABLE 1 Oligonucleotide primers for CHT1 in RT-PCR analysis

 
Cell Picking
Tracheas (n = 5) were quick frozen in melting isopentane. Serial cryosections (8 µm) were collected on coverslips, stained for 1.5 min with haemalaun, and transferred to 100% ethanol. Within 2 h after sectioning, the supranuclear regions of {bsim} 25 epithelial cells were collected by laser-assisted microdissection. This was achieved by an ultraviolet laser microbeam (337 nm wavelength; PALM, Wolfratshausen, Germany) coupled to an inverted microscope. The epithelial sections were transferred into first strand buffer and RNA was reverse transcribed using MuLV Reverse transcriptase (50 U/onset; all reagents from Perkin Elmer) for 60 min at 42°C. For subsequent PCR, 4 µl buffer II, 4 µl MgCl2 (15 mM), 1 µl dNTP (10 mM each), 1 µl of each primer (10 µM, primer set 1; Table 1), and 0.4 µl AmpliTaq Gold polymerase (5 U/µl; all regents from Perkin Elmer) were supplemented with H2O to a final volume of 50 µl. Cycling conditions were 5 min at 94°C, 50 cycles with 45 s at 94°C, 45 s at 60°C, 45 s at 72°C, and a final extension at 72°C for 7 min. Control reactions included the absence of DNA template and the absence of RT. The PCR products were separated by electrophoresis on a 1.2% TRIS-acetate-EDTA gel.

In Situ Hybridization
In situ hybridization experiments were done as described in detail elsewhere (18). In brief, PCR products of primer set 1 were used for preparing digoxigenin (DIG)-labeled antisense and sense RNA probes with T7-polymerase and DIG-labeling mix (Boehringer, Mannheim, Germany). Cryostat sections (10 µm) of shock frozen tracheas (n = 5) were fixed in 4% phosphate-buffered paraformaldehyde solution, permeabilized, acetylated, prehybridized, and hybridized with 10 µg/ml probe. Detection of the DIG-labeled probe was performed as recommended by the manufacturer, with alkaline phosphatase–conjugated DIG-antibody.

Preparation of Antisera
Antisera were produced by immunization with a peptide of 12 amino acids (TKNSGNAEERSE) corresponding to amino acids 29–40 of the rat CHT1 protein (18). CHT1-specific Ig was purified from antisera by affinity chromatography. The affinity matrix consisted of the peptide used for immunization, coupled to Sepharose 4B (Pineda Antikörper-Service, Berlin, Germany). The antisera were diluted 1:5 in binding buffer (sodium phosphate buffer, 20 mM, pH 7.0) before application. The column was washed with binding buffer until no more protein emerged. Bound Ig was eluted with 100 mM glycine, 500 mM NaCl, pH 3.0.

Western Blot
For Western blot analysis, abraded tracheal epithelial cells (n = 5 tracheas) were lysed in 2x Laemmli buffer. Following heating to 65°C (10 min) the protein solution was centrifuged for 15 min at 14,000 rpm. Fifteen microliters of supernatant was subjected to 10% SDS-PAGE under reducing conditions. The protein was transferred to PVDF membrane (Immobilon-P; Millipore, Bedford, MA) by semidry blotting. The membrane was incubated in 25 mM Tris-buffered saline with 0.05% Tween-20 (TTBS) for 1 h at room temperature. The first antibody was diluted 1:1,000 in 5% nonfat dry milk in TTBS. Incubation was for 12 h at 4°C. Monoclonal alkaline phosphatase–conjugated anti-rabbit IgG from goat (1:15,000 in 2.5% nonfat dry milk in TTBS, 1.5 h at room temperature; Sigma, Deisenhofen, Germany) was used as secondary antibody, and 4-nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolyl-phosphate (NBT-BCIP; Kirkegaard and Perry Laboratories, Gaithersburg, MD) served as a chromogen. Negative controls were done by (i) omitting the first antibody, and (ii) preabsorption of the primary antiserum on an affinity column of CHT 1 peptide coupled to Sepharose 4B.

Immunofluorescence
Tracheas of Wistar rats (n = 5) were shock frozen in isopentane cooled in liquid nitrogen. Cryosections (10 µm) were cut, fixed with acetone for 10 min at -20°C, and incubated for 1 h in 10% normal swine serum containing 0.5% Tween-20, 0.1% bovine serum albumin in 0.05 M phosphate-buffered saline (PBS). Primary antisera were diluted in PBS (anti-CHT1 from rabbit: 1:16,000; anti-VAChT from goat: 1:500; Biotrend, Cologne, Germany) and applied at room temperature. These antisera were applied either singly or in combination for double-labeling immunofluorescence. Secondary antisera used in this study were: Cy3-conjugated donkey–anti-rabbit-Ig (1:1,000; Dianova, Hamburg, Germany), and FITC-conjugated mouse–anti-goat-Ig (1:800; Sigma), all were applied for 1 h. Negative controls were done by (i) omitting the first antibody, (ii) incubation with the preimmune serum instead of the primary antiserum, and (iii) preabsorption of the primary antiserum on an affinity column of CHT 1 peptide coupled to Sepharose 4B. Sections were rinsed, coverslipped with carbonate-buffered glycerol (pH 8.6), and evaluated with a confocal laser scanning microscope (TCSSP; Leica, Mannheim, Germany).

Ultrastuctural Immunohistochemistry
Wistar rats (n = 4) were killed by inhalation of halothane and transcardiacly perfused with rinsing solution (19) followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Segments of the trachea were dissected, cryoprotected in 18% sucrose in 0.1 M phosphate buffer, frozen, and cut at a thickness of 40 µm. These cryosections were subjected to a routine pre-embedding immunohistochemistry protocol as described in detail elsewhere (20), using the anti-CHT1 from rabbit at a dilution of 1:20,000–80,000, a peroxidase-conjugated F(ab)2-fragment of a donkey–anti-rabbit IgG (1:100, 1 h at room temperature; Amersham Pharmacia Biotech, Freiburg, Germany) as secondary reagent, and a diaminobenzidine developing solution (125 µg/ml) containing 75 mM nickel ammoniumsulphate. Ultrathin sections were counterstained with lead citrate and viewed with a EM 902 transmission electron microscope (Zeiss, Jena, Germany). Negative controls were done by incubation with the pre-immune serum instead of the primary antiserum.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High-affinity choline transporter (CHT1) mRNA was detected with RT-PCR in trachea without epithelium, abraded tracheal epithelium, tracheal epithelial cells from laser-assisted cell picking, and spinal cord (Figure 1A) . CHT1 mRNA was detected with primer set 1, spanning 150 bp within the coding region. PCR products showed no difference in size and sequence. For laser-assisted cell picking, only the supranuclear sections of tracheal epithelial cells were collected for RT-PCR to minimize the load of genomic DNA to the samples (Figure 1C). Primer set 2 was used to ascertain the entire coding sequence of the CHT1 mRNA from abraded tracheal epithelium. As shown in Figure 1B, products with expected size of 1,761 bp and sequence homogeneity of 99% as identified by sequencing were detected in abraded tracheal epithelium and in spinal cord.



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  		Figure 1. RT-PCR. (A) A single band of 150 bp was amplified with CHT1-specific primers (primer set 1) from cDNA of trachea without epithelium, abraded tracheal epithelium, tracheal epithelial cells obtained by laser-assisted cell picking and spinal cord. {phi} RT: control for cells obtained by laser-assisted cell picking run without reverse transcriptase. (B) Long PCR analysis using cDNA from abraded tracheal epithelial cells and spinal cord to amplify the entire coding region of CHT1 using 5'-terminal and 3'-terminal primers (primer set 2). A single band with expected size of 1,761 bp was detected in both abraded tracheal epithelial cells and spinal cord. H2O: control run without template. (C) Laser-assisted microdissection. The supranuclear regions of {bsim} 25 epithelial cells were separated from the underlying row of epithelial nuclei by an ultraviolet laser microbeam and then collected by a micromanipulator for subsequent RT-PCR (for technical details, see Ref. 21). The full height of the epithelium is indicated by E.

 
In accordance with the RT-PCR results, CHT1 mRNA could also be detected in tracheal epithelial cells by nonradioactive in situ hybridization (Figure 2) .



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  		Figure 2. In situ hybridization. (A) The mRNA of CHT1 was detected in tracheal epithelial cells with a digoxigenin-labeled antisense riboprobe. (B) Negative control with sense riboprobe. Bar: 20 µm.

 
CHT1 antisera were raised in rabbit and guinea-pig against a synthetic peptide corresponding to amino acid residues 29–40 of the rat CHT1. Western blotting experiments demonstrated a single protein of 51 kD in a tracheal epithelium homogenate (Figure 3A) . CHT1 immunoreactivity was localized in axons innervating the tracheal glands (Figure 4) and trachealis muscle. Cholinergic axons innervating tracheal glands were visualized by incubation of the tracheal sections with an antibody against the vesicular acetylcholine transporter (Figure 4). Overlay of the staining profiles showed that CHT1 and VAChT immunoreactivity were colocalized in the same axons (Figure 4). In the tracheal epithelium, CHT1 protein was found in the apical section of the ciliated cells (Figure 3B). Exact localization of the CHT1 protein to the microvilli and apical membrane of the ciliated cells was possible by electron microscopy (Figure 3C). Sera obtained before immunization of the animals showed no reaction (Figure 3D).



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  		Figure 3. Immunohistochemistry and western blotting, epithelium. (A) With western blotting, a single 51-kD protein was detected in tracheal epithelium homogenate with the affinity-purified CHT1 antiserum. (B) The CHT1 protein was localized at the apical region of the ciliated cells by immunohistochemistry and confocal laser scanning microscopy. Bar: 20 µm. (C) By electron microscopy, an exact localization of the CHT1 protein to the apical membrane and microvilli (arrows) was possible. Bar: 0.5 µm. (D) Staining was absent with serum taken before immunization. Bar: 0.5 µm.

 


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  		Figure 4. Immunohistochemistry, autonomic nerve fibers. The CHT1 antiserum labeled axons innervating tracheal glands. Cholinergic axons were visualized by labeling with an antibody against VAChT. Overlay of the CHT1 and VAChT staining profile showed colocalization of both proteins in axons innervating tracheal glands. Bar: 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates the expression of the high-affinity choline transporter CHT1 by ciliated cells of the rat tracheal epithelium and the targeting of the CHT1 protein to the apical cell membrane.

In principle, each cell requires an uptake system for the organic cation choline, because it is essential for biosynthesis of the ubiquitous membrane phospholipid phosphatidylcholine. Accordingly, the known organic cation transporters OCT1, OCT2, and OCT3, which all interact with choline with varying affinities, are expressed in a broad spectrum of tissues, with OCT1 being the most widely distributed (22).

A highly restricted expression pattern, however, has been ascribed to the recently cloned high-affinity choline transporter CHT1 that subserves to mediate the rate-limiting step of ACh synthesis in cholinergic neurons (23). It has been detected by a Northern blot screen exclusively in spinal cord and brain areas containing cholinergic neurons (5, 6), and by RT-PCR in keratinocytes (17) that are also known to synthesize and release ACh (24). In support of these findings, CHT1 protein has been detected by immunohistochemistry in rat cholinergic neurons of the brain (25), rat cholinergic spinal motoneurons and their motor endplates (26, 27), cholinergic parasympathetic neurons (rat tongue [27], rat trachea [this study]), and in human and rat keratinocytes (17).

Here, we demonstrate the expression of mRNA spanning the entire coding region of CHT1 in the rat tracheal mucosa. There are several lines of evidence that this CHT1 mRNA is expressed by the ciliated epithelial cells. First, cholinergic parasympathetic axons that are found in low numbers in the basal layer of the respiratory epithelium (28) can be eliminated as a source of CHT1 mRNA in the mucosa, because neurons are metabolically compartimentalized in that all mRNA and translation is restricted to the neuronal perikaryon and stem dendrites, whereas axons receive their protein from these sites via axonal transport. The cell bodies of the tracheal cholinergic neurons, however, are located within ganglia at the outer surface of the trachealis muscle or even more peripherally in the adventitia, and, consequently, cannot be harvested by abrading the epithelium from the luminal side for RT-PCR analysis. Second, CHT1 mRNA was also detected when only the supranuclear regions of epithelial cells were collected from frozen tissue sections by laser-assisted cell picking for subsequent RT-PCR. By this approach, lymphocytes that migrate into the epithelium (29) and also express components of a cholinergic system (30, 31) were also excluded from the sample. Third, in situ hybridization clearly localized CHT1 mRNA to the epithelial cells.

Subcellularly, CHT1 protein is targetted to the apical membrane of the ciliated epithelial cells, as demonstrated by immunohistochemistry using an affinity-purified CHT1 antiserum and confocal laser scanning and electron microscopy. This localization corresponds to the enrichment of CHT1 at cholinergic terminals (27), because the sorting mechanisms targeting proteins to the apical membrane of polarized epithelia are largely identical to those targeting proteins to axons in neurons (32). Interestingly, ChAT immunoreactivity is concentrated also at the apical part of the ciliated epithelial cells, although its localization is considered to be cytoplasmic (33, 34). Thus, CHT1 located in the apical membrane is situated in an appropriate position to fuel ACh synthesis by the underlying ChAT via re-uptake of choline after release and breakdown of acetylcholine at the luminal surface.


   Acknowledgments
 
The authors thank Mr. M. Bodenbenner, Ms. K. Michael, and Ms. S. Tasch for skilful technical assistance. Supported by the Deutsche Forschungsgemeinschaft (SFB 547, project C2, to R.V.H. and W.K.), and a HMWK-fellowship (to K.S.L.).

Received in original form September 18, 2002

Received in final form November 6, 2002


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2003 American Thoracic Society.