Volume 51, Issue 4, Pages 1042-1053 (April 2007)
1. Introduction
Synthesis and release of acteylcholine (ACh) is not restricted to specialized subsets of neurons but also occurs in a broad variety of non-neuronal cells, in particular in surface epithelia [1], [2].
Recent studies suggest that this may apply for the bladder urothelium as well. Urothelial ACh has been proposed to be released into the bladder lumen to address nicotinic receptors on the luminal membrane of umbrella cells or to be released basally to act upon the detrusor and nerve fibres [3], [4], [5], [6]. So far, however, there is only indirect experimental evidence for urothelial ACh. Yoshida et al. [6], [7] measured ACh release from isolated human bladder strips and demonstrated a reduced release in preparations in which the epithelium had been removed. These findings are compatible with a urothelial release of ACh, but also with urothelial release of another factor that might trigger ACh release from deeper structures. In addition, immunolabelling of the urothelium with an antiserum directed against the ACh-synthesizing enzyme choline acetyltransferase (ChAT) has been reported [6], [7], although these data still await validation by preabsorption experiments or other independent methods, such as Western blotting, determination of ChAT activity, or reverse transcriptase–polymerase chain reaction (RT-PCR).
On this background, we set out to determine the molecular components of a putative cholinergic system in the urothelium in more detail. First, ACh was detected in the urothelial cell layer by a high-performance liquid chromatography-electrochemical (HPLC-EC) method. Next, we employed RT-PCR and immunohistochemistry to investigate urothelial expression of (1) ChAT, the classical Ach-synthesizing enzyme in the nervous system and several non-neuronal cells; (2) carnitine acetyltransferase (CarAT), which is responsible for ACh synthesis in muscle cells [8]; (3) the vesicular ACh transporter (VAChT), which shuffles ACh from the cytoplasm into synaptic vesicle in cholinergic nerve terminals [9]; and (4) polyspecific organic cation transporters (OCTs; isoforms 1–3), which are able to translocate ACh directly across the plasma membrane [10]. In view of their polyspecific properties [11], we tested whether a commonly used anticholinergic drug, trospium chloride, interferes with ACh transport by OCTs. The study was performed on human and murine urothelium in parallel to evaluate whether the mouse may serve as a suitable model for experimental approaches addressing this system in the future.
2. Materials and methods
2.1. ACh assay
FVB mice were killed by inhalation of an overdose of isoflurane (Abbott, Wiesbaden, Germany). The bladder was carefully dissected, opened, and fixed in a Petri dish with the luminal surface facing upwards. A cotton-tipped applicator (Q-tip) was gently rubbed along the luminal surface and thereafter placed in 1ml 15% formic acid in acetone (v/v). Two samples were taken from the luminal surface (about 2cm2) of human urinary bladders obtained from surgery. After standing on ice (30min), specimens (material swiped off by the Q-tip) were frozen in liquid nitrogen. The bladder was then fixed in buffered 4% paraformaldehyde and processed as described for immunohistochemistry; then serial cryosections were stained with hematoxylin-eosin and evaluated by light microscopy. Only those specimens were included in the further analysis in which the basal lamina was not disrupted.
ACh was measured by cationic exchange HPLC combined with bioreactors and electrochemical detection with a detection limit of 10fmol ACh per injection (20μl) as described in detail elsewhere [12].
2.2. Reverse transcriptase–polymerase chain reaction
Murine samples were obtained by scraping off the urothelium as described above, but specimen holders were then placed in lysis buffer (RLT-buffer; Qiagen, Hilden, Germany) instead of formic acid/acetone. Further sample processing was as described in detail earlier [10]. Primer sequences are provided in
NS20Y cells—a cholinergic murine forebrain neuroblastoma cell line (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany)—were used as a positive control for expression of components of the murine cholinergic system.
Human bladder mucosal biopsies were obtained from six female patients aged between 57 and 76 yr (mean: 67). All patients underwent endoscopic surgery primarily to remove bladder cancer or to examine the bladder wall by biopsies for carcinoma in situ or interstitial cystitis. Four additional mucosal biopsies were taken out of the trigonum (four patients) or the bottom (two patients) of the bladder with the use of endoscopic forceps. This procedure was approved by the local ethics committee, and informed patients gave signed consent. Processing of these specimens was identical to that of murine specimens, except that human specific primers were used Table 1.
Caco cells—a human colonic epithelial cell line (German Collection of Microorganisms and Cell Culture)—served as a positive control for human epithelial ChAT expression. Additional complementary DNAs generated from airways during a previous study [10] were also used as positive controls.
2.3. Immunohistochemistry
Mice were killed by isoflurane inhalation; then the bladder was rapidly dissected, embedded in optimal-cutting-temperature compound, and shock-frozen in melting isopentane. For ChAT immunohistochemistry, bladders from six additional mice were filled via a cannula with buffered 4% paraformaldehyde and immersed in the same fixative for 2h, before being rinsed several times in 0.1mol/l phosphate buffer (PB), immersed overnight in the same buffer supplemented with 18% sucrose for cryoprotection, and shock-frozen. From each patient from whom a mucosal biopsy had been obtained for RT-PCR analysis, a second biopsy was shock-frozen as described to be processed for immunohistochemistry. Cryosections (10μm) were fixed either in acetone for 10min at −20°C or in Zamboni fixative (2% paraformaldehyde, 15% saturated picric acid in 0.1mol/l PB) for 20min and then processed for routine indirect immunofluorescence as described elsewhere [10]. Antibodies are listed in The sections were evaluated by epifluorescence microscopy (Axioplan 2 imaging; Zeiss, Jena, Germany) or with a confocal laser-scanning microscope (TCS SP2; Leica, Mannheim, Germany).
| |
|
|
 |
Antigen |
Host species |
Dilution |
Fixative |
Source/reference |
|
|
Primary antibodies for use in murine tissues |
|
|
ChAT, synthetic peptide, aa 282–295 of rat “common ChAT” sequence |
Rabbit |
1:8000 |
PFA |
[23] |
|
|
VAChT |
Goat |
1:800 |
Acetone, PFA |
Biotrend, Cologne, Germany |
|
|
OCT1, synthetic 21 aa peptide, near C-terminus |
Rabbit |
1:20 |
Acetone |
Alpha Diagnostics, San Antonio, USA |
|
|
OCT2, synthetic 21 aa peptide |
Rabbit |
1:400 |
Acetone |
Alpha Diagnostics |
|
|
OCT3, synthetic 18 aa peptide |
Rabbit |
1:400 |
Acetone |
Alpha Diagnostics |
|
|
|
|
|
Primary antibodies for use in human tissues |
|
|
ChAT; synthetic peptide, aa 282–295 of rat “common ChAT” sequence |
Rabbit |
1:8000 |
Zamboni |
[23] |
|
|
VAChT |
Goat |
1:500 |
Zamboni |
Biotrend, Cologne, Germany |
|
|
OCT1, synthetic 21 aa peptide, near C-terminus |
Rabbit |
1:20 |
Acetone |
Alpha Diagnostics |
|
|
OCT2, synthetic peptide, aa 533–547 of human sequence |
Rabbit |
1:200 |
Acetone |
[10], [30] |
|
|
OCT3, synthetic peptide, aa 297–313 of human sequence |
Rabbit |
1:500 |
Acetone |
[10] |
|
| |
|
|
|
Antigen |
Host species |
Dilution |
Conjugate |
Source |
|
|
Secondary antibodies |
|
|
Rabbit-IgG |
Donkey |
1:2000 |
Cy3 |
Chemicon, Hofheim, Germany |
|
|
Goat-IgG |
Mouse |
1:400 |
FITC |
Sigma-Aldrich, Taufkirchen, Germany |
|
| |
|
|
The specificity of the primary antibodies was validated by (1) omission of the primary antibody, (2) preabsorption with their corresponding antigen at a concentration of 40μg/ml for 1h at room temperature before use in immunofluorescence, and (3) evaluation of immunofluorescence in genetically OCT-deficient mice (OCT1/2 double-knockout mice) by using tissues collected during a previous study [10]
2.4. Expression of OCTs in epithelial cells and transport measurements
Human OCT1, OCT2, and OCT3 were stably expressed in Chinese hamster ovary (CHO) cells by methods described in detail earlier [10], [13]. For transport measurements, confluent cells were washed with phosphate-buffered saline (PBS), suspended by shaking, collected by 10-min centrifugation at 1000×g, and suspended at 37°C in PBS. The cells were incubated for 1s in PBS containing the prototypic substrate 0.2μmol/l [3H]1-methyl-4-phenylpyridinium ([3H]MPP), without inhibitor or in the presence of increasing concentrations of trospium chloride. Uptake was stopped by addition of ice-cold PBS containing 100μmol/l quinine (stop solution), and the cells were washed three times with ice-cold stop solution. To measure uptake at 0-s incubation, ice-cold stop solution was added to the cells first, and radioactive substrates were added thereafter. Uptake rates were calculated from quadruplicate measurements after 0-s incubation and 1-s incubation. Uptake rates of [3H]MPP in the presence of different concentrations of trospium chloride were calculated from measurements after 0s and 1s incubation. For each transporter, three or four independent experiments were performed in which four measurements were conducted for each concentration of trospium chloride. The Hill equation for multisite inhibition was fitted to individual (not shown) or to normalized and combined experiments (Fig. 8A–D). Mean values±SD were calculated from the mean inhibitory concentration (IC50) values of the individual experiments (Fig. 8D). The significance of differences between the mean IC50 values was estimated by one–way analysis of variance followed by Tukey comparison.
3. Results
3.1. ACh and its synthesizing enzymes
ACh concentration in the murine urothelium amounted to 0.22±0.03nmol/g wet weight (mean±SEM; n=6). Eight and 14pmol, respectively, were measured in the two available samples of human urothelium. RT-PCR failed to detect ChAT messenger RNA (mRNA) both in murine (13 different primer sets tested) (Fig. 1A) and human (2 different primer sets tested) urothelium (Fig. 2A) despite positive results in human Caco cells (Fig. 2A), murine spinal cord (Fig. 1A), and murine tracheal epithelium (not shown). CarAT mRNA, however, was readily detectable (Fig. 1A). In immunohistochemistry, the ChAT antiserum distinctly labelled all cell layers in both murine and human urothelium (Fig. 1, Fig. 2). This labelling could be prevented by preabsorption of the antiserum with its corresponding synthetic antigen (Fig. 1C). In view of the RT-PCR data and the known structural similarity of ChAT with CarAT, we also preincubated the ChAT antiserum with CarAT isolated from pigeon breast muscle, which also resulted in loss of urothelial labelling (Fig. 1, Fig. 2). Hence, this immunolabelling was not suited to discriminate between ChAT and CarAT in the urothelium.
Fig. 1. ChAT in murine urothelium. (A) Reverse transcriptase–polymerase chain reaction demonstrates the urothelial expression of CarAT, but not of ChAT (primer pair murine ChAT2, Table 1). Spinal cord served as positive control for efficiency of detecting ChAT messenger RNA. (B–D) ChAT immunolabelling of the urothelium (B) can be successfully preabsorbed both with synthetic ChAT antigen (C) and with CarAT protein (D). Bar=20μm. bp: base pair. CarAT: carnitine acetyltransferase; ChAT: choline acetyltransferase.
Fig. 2. ChAT in human urothelium. (A) Reverse transcriptase–polymerase chain reaction failed to detect ChAT in the urothelium despite positive signals in Caco cells. (B, C) ChAT immunolabelling of the urothelium (B) can be successfully preabsorbed with CarAT protein (C). Bar=20μm. bp: base pair; CarAT: carnitine acetyltransferase; ChAT: choline acetyltransferase; Uro/CaCo 1+2: primer pair human ChAT1; Uro/Caco 3: primer pair human ChAT2 (Table 1).
3.2. Molecular components of the ACh release machinery
VAChT, the vesicular transporter shuffling ACh from the cytosol into synaptic vesicles in cholinergic neurons, was detected neither by RT-PCR nor by immunohistochemistry in murine and human urothelium (Fig. 3, Fig. 4). Instead, numerous VAChT-immunoreactive nerve fibres were observed immediately underneath the urothelial basal membrane in the lamina propria (Fig. 3B). Among polyspecific OCTs, isoforms OCT1 and OCT3 were readily detected by RT-PCR both in murine and human urothelium (Fig. 5). Immunohistochemistry supported these data. OCT1 immunolabelling was observed throughout the epithelial layers with pronounced labelling of the intermediate and basal cells, and OCT3 immunoreactivity was nearly restricted to the basal cells (Fig. 6, Fig. 7). Specific OCT2 immunolabelling was not obtained in the urothelium (Fig. 6, Fig. 7).
Fig. 3. VAChT in murine bladder mucosa is undetectable by reverse transcriptase–polymerase chain reaction (A; spinal cord served as positive control). GADPH-specific primers confirmed efficacy of RNA isolation and reverse transcriptase. In immunohistochemistry, VAChT is restricted to subepithelial nerve fibres (arrows in B). (C) Preabsorption control. Bar=20μm. bp: base pair; GADPH: glyceraldehyde-3-phosphate dehydrogenase; H2O: control run without template; VAChT: vesicular acetylcholine transporter.
Fig. 4. VAChT in human urothelium. Neither reverse transcriptase–polymerase chain reaction (RT-PCR) (A) nor immunohistochemistry (B) provides specific positive results. Nonspecific labelling of suburothelial structures persists after preabsorption of the antiserum (C). Human bronchial epithelium served as positive control for VAChT messenger RNA detection by RT-PCR, and GADPH-specific primers confirmed efficacy of RNA isolation and RT. Bar=20μm. bp: base pair; GADPH: glyceraldehyde-3-phosphate dehydrogenase; VAChT: vesicular acetylcholine transporter.
Fig. 5. Expression of OCT isoforms, reverse transcriptase–polymerase chain reaction. OCT1 and 3 are express |
Female Urology 
;)
;)
;)
;)
;)