Expression of the High-affinity Choline Transporter CHT1 in Rat and Human Arteries
Institutes for Anatomy and Cell Biology (KSL,UP,KR,CR,RVH,WK) and Neuropathology (KK), Department of Internal Medicine/Cardiology (RCBD), and Biotechnology Centre (RS), Justus-Liebig-University Giessen, Giessen, Germany
Correspondence to: Katrin Susanne Lips, Inst. for Anatomy and Cell Biology, Justus-Liebig-University, Aulweg 123, D-35385 Giessen, Germany. E-mail: Katrin.S.Lips{at}anatomie.med.uni-giessen.de
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Summary |
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Key Words: acetylcholine artery choline transporter choline uptake endothelial cell vascular smooth muscle
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Introduction |
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Materials and Methods |
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Western Blotting
Rat aorta, hippocampus (positive control), and cerebellum (negative control) (n=5 animals) were lysed in 2 x Laemmli buffer, followed by heating to 65C (10 min). The protein solution was centrifuged for 15 min at 14,000 rpm. An aliquot (515 µl) of supernantant was subjected to 10% SDS-PAGE under reducing conditions. The protein was transferred to PVDF membrane (Immobilon-P; Millipore, Bedford, MA) by semi-dry blotting. The membrane was incubated in 25 mM Tris-buffered saline with 0.05% Tween-20 (TTBS, pH 8.0) for 1 hr at room temperature (RT). The affinity-purified CHT1 antibody (Pfeil et al. 2003) was diluted 1:1000 in 5% non-fat dry milk in TTBS and incubated for 12 hr at 4C. Monoclonal alkaline phosphatase-conjugated anti-rabbit IgG from goat (Sigma, Deisenhofen, Germany; 1:15,000 in 2.5% non-fat dry milk in TTBS, 1 hr at RT) was used as secondary antibody and 4-nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolyl-phosphate (Kirkegaard & Perry Laboratories; Gaithersburg, MD) served as a chromogen. Specificity of immunolabeling was validated by (a) omitting the first antibody and (b) preabsorption of the primary antibody with CHT1 peptide coupled to Sepharose 4B on an affinity column.
Immunofluorescence
Rat (n=5) ascending, thoracic, and abdominal aorta, pulmonary trunk, superior mesenteric artery, common carotid artery, basilar artery, circulus arteriosus cerebri, lingual artery, and human (n=5) common carotid artery, basilar artery, medial cerebral artery, and posterior cerebral artery were shock-frozen in melting 2-methylbutane. Samples of human arteries were obtained from the same subjects as described above, and from an additional female (aged 92 years) and male (aged 70 years) subject during dissection at the students' course of macroscopic anatomy. Cryosections were fixed with acetone for 10 min at -20C, and incubated for 1 hr in 10% normal swine serum containing 0.5% Tween-20, 0.1% bovine serum albumin in 0.05 M PBS, pH 7.4. Primary antisera were diluted in PBS (crude anti-CHT1 serum from rabbit 1:16,000, affinity purified anti-CHT1 antibody from rabbit 1:1500, crude anti-CHT1 serum from guinea pig 1:2000), (Lips et al. 2002; Pfeil et al. 2003
). Secondary reagents were Cy3-coupled donkey anti-rabbit IgG (1:1000 in PBS; Dianova, Hamburg, Germany), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100; Dianova), or biotinylated goat anti-guinea pig IgG (1:50; Sigma); all were applied for 1 hr. Sections incubated with the biotinylated secondary antibody were subsequently labeled with StreptavidinTexas Red (1:400; Amersham Pharmacia Biotech, Freiburg, Germany). The specificity of immunolabeling was validated by (a) omission the first antibody, (b) incubation with the preimmune serum instead of the primary antiserum, (c) preabsorption of the primary antiserum with CHT1 peptide coupled to Sepharose 4B on an affinity column, and (d) liquid-phase preabsorption with the CHT1 peptide (10200 µg/ml) that was used for immunization. Sections were rinsed, coverslipped with carbonate-buffered glycerol (pH 8.6), and evaluated with an epifluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany) and a confocal laser scanning microscope (Leica TCSSP; Mannheim, Germany).
Cell Culture
Microvascular endothelial cells of forebrain and lung and of rat aorta (n=3) were isolated by a magnetic bead (Dynabead)-coupled endothelial cell-specific antibody (anti-RECA; Serotec, Düsseldorf, Germany). For this purpose, the RECA antibody was diluted 1:20 in 0.05 M PBS and incubated with a 5% solution of 4 x 108 Dynabeads pan mouse IgG (Dynal Biotech; Hamburg, Germany) for 16 hr at 4C. Adult Wistar rats were sacrificed by inhalation of sevofluran (Abbott; Wiesbaden, Germany). Samples were minced and incubated end over end with Dynabead-coupled anti-RECA solution for 4 hr at RT. Endothelial cells coupled with the Dynabead-conjugated RECA antibody were separated by using a magnet and suspended in Endothelial Cell Growth Medium-MV (Promocell; Heidelberg, Germany). The cells were characterized by their binding capacity for the isolectin-B4 (biotin-labeled I-B4 lectin, 1:50, Sigma, followed by StreptavidinTexasRed 1:400, Amersham, Braunschweig, Germany) (Porter et al. 1990), expression of CD31 (mouse monoclonal antibody MEC13.3, 1:100; Pharmingen, Hamburg, Germany, followed by donkey anti-mouse IgG, Cy3 conjugate, 1:1000, Dianova) and
-smooth muscle actin (FITC-conjugated monoclonal antibody 1A4, 1:500; Sigma) (Muzykantov et al. 1999
; Jones et al. 2000
), and by uptake of acetylated LDL (DiI-conjugated Ac-LDL; Paesel and Lorei, Hanau, Germany) applied to the culture at 10 µg/ml for 4 hr) (Craig et al. 1998
). More than 90% of the cells were I-B4-positive, showed immunoreactivity for CD31 but not for
-smooth muscle actin, and contained acetylated LDL.
Vascular smooth muscle cells were isolated from rat common carotid artery. The endothelium and the adventitial layer of the artery were abraded before incubation in 1 ml 0.05 M PBS with 2.5 mg collagenase CLS II (Biochrom AG; Berlin, Germany), 200 µl 0.1% trypsin/0.05% EDTA (PAA; Cölbe, Germany), and 50 µg DNase I (Boehringer, Ingelheim, Germany) for 2 hr at 37C. The pellet was washed twice with PBS/DNase I, and the cells cultured to confluence in RPMI 1640 tissue culture medium (GibcoBRL) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin/amphotericin at 37C. To remove remaining fibroblasts, cells were detached with 0.1% trypsin/EDTA and incubated with 500 µl fibroblast-specific antibody solution (1:100, F4771; Sigma) and 25 µl Dynabeads for 1 hr at 37C. Vascular smooth muscle cells were washed twice and resuspended with 1640 RPMI medium with 10% fetal bovine serum and 1% penicillin/streptomycin/amphotericin. The purity of these cultures was verified by immunolabeling for -smooth muscle actin. Roughly 1% of cells remained unlabeled with this antibody.
For isolation of fibroblasts, common carotid artery and lung were incubated for 30 min in 1 ml 0.05 M PBS with 100 µl 0.1% trypsin/EDTA and 2.5 mg collagenase CLS II, and for 90 min in 1 ml PBS with 200 µl trypsin and 10 µg DNase I at 37C. The pellet was washed twice with PBS/DNase I and the cells cultured to confluence in RPMI 1640 tissue culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin/amphotericin at 37C. For IHC, the cells were detached with 0.1% trypsin/EDTA and resuspended in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin/amphotericin. Five thousand cells per well were grown in eight-well chamber slides. After 2 days, cells were fixed in phosphate-buffered 4% paraformaldehyde solution for 20 min, followed by washing steps and incubation with primary and secondary antisera as described above for tissue sections.
In Vitro [3H]-choline Uptake
Rat thoracic aorta (n=3) was dissected into three specimens and washed in NaCl-free buffer containing 5 mM Tris, 10 mM Hepes, 2 mM KCl, 1 mM MgCl2, and 1 mM CaCl2 (pH 7.4). The first specimen was incubated in 200 nM [3H]-choline (545 mCi/mg; Amersham Pharmacia Biotech) diluted in the same buffer as used for washing but with 200 mM NaCl. The second specimen was incubated in [3H]-choline-containing buffer without NaCl, and the third in [3H]-choline-containing buffer with 50 µM hemicholinium-3 (Sigma) for 10 min. After washing, the tissue was frozen and cryosections (20 µm) were prepared, dried, and analyzed for 24 hr with a Microimager (Zinsser Analytic; Frankfurt/Main, Germany).
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Results |
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[3H]-Choline Uptake by Aortic Rings
Specimens of the rat thoracic aorta were used to investigate the [3H]-choline uptake in vitro. In the presence of 200 mM NaCl in the incubation buffer, [3H]-choline uptake was prominent in the medial layer (Figure 4A)
. The spatial resolution of the autoradiographic technique (~20 µm) did not allow us to discriminate the intimal layer unequivocally from the media. Spots of [3H]-choline uptake were inconsistently observed in the adventitial layer. Specimens incubated in [3H]-choline buffer without NaCl displayed a weak signal throughout the vascular wall (Figure 4B). When specimens were incubated with [3H]-choline in the presence of hemicholinium-3, incorporation of radioactivity was reduced to a minimum (Figure 4C).
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Discussion |
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Nevertheless, choline uptake kinetics (Km, Vmax) differing from those of neuronal CHT1 have also been reported using the in situ rat brain perfusion technique (Allen and Smith 2001) and in studies using isolated bovine brain capillaries (Galea and Estrada 1992
) and endothelial cells (Estrada et al. 1990
). Sodium dependence, one of the characteristics of neuronal CHT1 (Haga 1971
), has been reported in some of the studies (Estrada et al. 1990
; Galea and Estrada 1992
) but not in others (Allen and Smith 2001
). Sensitivity of choline uptake to hemicholinium-3, a key feature of neuronal CHT1 (Chang and Lee 1970
), has been observed in all types of studies, including endothelial cell lines and extracerebral, i.e., aortic endothelial cells, but with slightly different inhibition constants (Allen and Smith 2001
; Friedrich et al. 2001
; Lipton et al. 1988
). The reasons for the discrepancies between the expected functional characteristics due to the presence of CHT1 mRNA and protein (this study) and those observed in previous functional studies remain to be determined. Because even a single nucleotide polymorphism in human CHT1 can have a profound impact on choline uptake (Okuda et al. 2002
), one, but not necessarily the only, possible explanation is offered by cell type-specific modifications of CHT1, as suggested by the Western blotting data, or by association with different modifying proteins.
The strongest immunolabeling for CHT1 in the arterial wall was observed in smooth muscle cells of the tunica media. Cultured vascular smooth muscle cells expressed CHT1 mRNA as determined by RT-PCR and also exhibited CHT1 immunoreactivity. At first sight these findings might appear surprising because smooth muscle cells are not classically considered as being cholinergic cells. However, there are several reports documenting the ability of muscle cells to synthesize ACh, either via ChAT or via the enzyme carnitine acetyltransferase. ACh-synthesizing activity and/or ACh release have been demonstrated in myoblasts (Krause et al. 1995; Fu et al. 1998
), skeletal muscle fiber (Tucek 1982
; Miledi et al. 1982
), and smooth muscle fibers of the human skin (Wessler and Kirkpatrick 2001
). Moreover, airway smooth muscle cells have been shown to display ChAT immunoreactivity (Wessler and Kirkpatrick 2001
). In support of CHT1 expression in vascular smooth muscle cells, [3H]-choline uptake was prominent in the aortic tunica media, as demonstrated by autoradiography in the present study, and this uptake shared crucial characteristics with neuronal CHT1, i.e., sensitivity to hemicholinium-3 and dependence on sodium. Hence, there is ample evidence for CHT1 expression in vascular smooth muscle cells, and further detailed kinetic studies will be required to elucidate the extent to which their high-affinity choline uptake matches that of the nervous system.
In culture, fibroblasts prepared from the common carotid artery and from the lung also exhibited distinct CHT1 immunoreactivity and expressed CHT1 mRNA. Fibroblasts and myofibroblasts are well-established targets of ACh, carrying both muscarinic and nicotinic ACh receptors (Andre et al. 1988; Sekhon et al. 2002
; Jacobi et al. 2002
; Oben et al. 2003
). Evidence for ACh synthesis by fibroblasts, however, is sparse and as yet has been presented only in abstract form for lung fibroblasts (Proskocil et al. 2000
) while several other investigators measured ACh release from fibroblasts only after transfection with ChAT (Fisher et al. 1993
; FalkVairant et al. 1996
). These findings fit best with the assumption that fibroblast ACh synthesis may be of low level in general and depends in its extent on culture conditions. Consistent with this notion, CHT1 immunolabeling of adventitial fibroblasts in the intact arterial wall and [3H]-choline uptake in the adventitial layer were inconsistent and of low labeling intensity. Moreover, adventitial [3H]-choline uptake cannot be attributed to fibroblasts with certainty because a clear discrimination between vasa vasorum, small nerve fiber bundles, and fibroblasts was not possible with by the present autoradiographic technique.
In conclusion, the present data show that expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.
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Acknowledgments |
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We thank Ms S. Tasch, Ms K. Michael, Mr M. Bodenbenner, and Mr G. Weigand for skillful technical assistance, and Ms Ch. Becker (Institute for Pathology, Justus-Liebig-University Giessen) for her help in collecting the human samples.
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Footnotes |
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