ARTICLE |
Correspondence to: Katarina Persson, Dept. of Clinical Pharmacology, Lund University Hospital, S-221 85 Lund, Sweden.
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Summary |
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We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells. (J Histochem Cytochem 47:739749, 1999)
Key Words: NADPH diaphorase histochemistry, nitric oxide synthase, bladder, urothelium, LPS, NADPH cytochrome P450 reductase
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Introduction |
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Nitric oxide (NO), originally identified as an endothelium-derived relaxing factor, is now considered to be a messenger molecule involved in several biological functions (
There is now considerable evidence that NADPH-d activity stains cells that lack NOS and that NADPH-d histochemistry cannot be considered an accurate marker for the presence of NOS (
The aim of the present study was to investigate which enzyme(s) is involved in the NADPH-d activity in the rat and pig urothelium. The subcellular localization and distribution of NADPH-d staining were investigated by electron microscopy to gain further insight into the characteristics of the NADPH-d staining in urothelial cells. In addition, a biochemical measurement of NOS activity was performed by using the conversion of radiolabeled L-arginine to L-citrulline (
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Materials and Methods |
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Tissue Handling
Female SpragueDawley rats (body weight 200250 g) were sacrificed by CO2 asphyxia and the bladder was removed. Bladders from female pigs were obtained at a local slaughterhouse.
To induce NOS in vivo, some rats were given IP LPS from E. coli serotype 0127:B8 (Sigma Chemical; St Louis, MO) at a dose of 15 mg/kg in saline. The rats were sacrificed 3, 6, 9, or 12 hr after injection and the bladder fixed at each time for immunohistochemistry. Rats given IP saline served as controls. The experimental protocol was approved by the Animal Ethics Committee of Lund University.
To stimulate synthesis of cGMP, bladder pieces were placed in PBS at 37C and bubbled with a mixture of 95% O2/5% CO2 as previously described (
NADPH-diaphorase Histochemistry
Tissue sections were preincubated in 50 mM Tris-HCl buffer for 10 min at room temperature (RT) and then incubated with 1 mM ß-NADPH and 0.5 mM nitroblue tetrazolium (both from Sigma) dissolved in 50 mM Tris-HCl buffer containing 0.2% Triton X-100 for 90 min at 37C. Control experiments were performed without addition of ß-NADPH. After rinsing in PBS, the sections were mounted in Kaiser's glycerol gelatin (Merck; Darmstadt, Germany). The effects of the flavoprotein inhibitor DPI (1050 µM; Sigma) and the alkaline phosphatase inhibitor levamisole (510 mM; Sigma) on the NADPH-d staining were investigated after preincubation of the sections with the inhibitors for 30 min.
Immunohistochemistry
The tissues were immersion-fixed for 4 hr in cold 4% formaldehyde in 0.1 M PBS and then rinsed in PBS containing 15% sucrose for 23 days. Both fixation and rinsing were performed at 4C, after which the specimens were frozen in isopenthane at -40C and stored at -70C before sectioning. Tissue sections were cut at a thickness of 10 µm and preincubated with PBS containing 0.25% Triton X-100 for 2 hr at RT. Incubation with primary antisera was performed overnight in the presence of one of the following antisera: rabbit antisera raised against a C-terminal fragment of nNOS (1:1500; Eurodiagnostica, Malmö, Sweden), an N-terminal fragment of nNOS (1:1000; Eurodiagnostica), iNOS (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), eNOS (1:1000; Santa Cruz Biotechnology), NADPH-cytochrome P450 reductase (1:1000; Stress Gen Biotechnologies, Victoria, BC, Canada), or mouse antisera raised against eNOS (1:500; Transduction Laboratories, Lexington, KY), cytokeratin 8.13 (1:200; Sigma), ED-1 (1:500; Serotec, Oxford, UK), or sheep antisera raised against cGMP (1:1000; a generous gift from Dr J. de Vente, Limburg University, Maastricht, Netherlands). The cGMP antiserum has been described in detail previously (
For double label immunofluorescence of iNOS and ED-1, sections were first incubated with iNOS antiserum overnight, rinsed in PBS, and then incubated with ED-1 antiserum. After rinsing, the sections were incubated for 90 min with FITC-conjugated donkey-anti rabbit IgG, rinsed, and then incubated for 90 min with Texas Red-conjugated donkey anti-mouse IgG. After rinsing, the sections were mounted as previously described. In control experiments, no immunoreactivity could be detected in sections incubated without primary antibodies. Because crossreactions with antigens sharing similar sequences cannot be excluded, the structures demonstrated are referred to as, e.g., NOS-immunoreactive.
Electron Microscopy
For visualization of NADPH staining at the electron microscopic level, the specimens were fixed with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M Sørensen's phosphate buffer (SPB, pH 7.2) for 4 hr at 4C. Several washes in SPB followed, after which the specimens were embedded in 3% agarose and sectioned at 150 µm on a vibratome. Vibratome sections were incubated with 1 mM ß-NADPH and 0.5 mM 2-[2'-benzothiazolyl]-5-styryl-3[4'-phthalhydrazidyl]-tetrazolium chloride (BSPT; Sigma) in 0.05 M Tris-HCl buffer (pH 8.0). Incubation was performed in darkness for 34 hr at RT. After several washes in Tris-HCl buffer and 0.15 M sodium cacodylate buffer, the sections were postfixed for 1 hr at 4C in 1% osmium tetroxide in 0.15 M sodium cacodylate buffer. A dehydration through a graded series of ethanol followed, and the specimens were embedded, sectioned, and contrasted according to conventional electron microscopic procedures. All grids were viewed and examined with a JEOL 1200 EX electron microscope. Control experiments were performed in the presence of BSPT but without addition of ß-NADPH.
Measurement of Nitric Oxide Synthase Activity
NOS activity was determined by measuring the formation of L-[14C]-citrulline from L-[14C]-arginine according to the procedure described by
Tissues were homogenized in ice-cold buffer using a glass pestle homogenizer. Homogenization buffer (pH 7.2) contained 20 mM HEPES, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 µM pepstatin A, and 2 µM leupeptin. Homogenates were centrifuged at 20,000 x g for 30 min at 4C and the resultant cytosolic fractions were passed over a column of AG50W-X8 (Na+ form, prepared from the H+ form) to remove endogenous arginine. The pellet was resuspended in homogenization buffer containing 1 M KCl for 5 min to remove loosely bound cytosolic proteins and after centrifugation (20,000 x g for 20 min at 4C) the particulate fraction was resuspended in homogenization buffer. NOS activity was determined in the cytosolic and the KCl-washed particulate fractions. Incubation mixtures contained 75 µl of cytosolic or particulate fractions and 25 µl of homogenization buffer containing, 2 mM NADPH, 1 mM CaCl2, 30 U/ml calmodulin, 3 µM tetrahydro-L-biopterin (BH4), 40 µM L-arginine, and 0.8 µM L-[14C]-arginine. Samples were incubated for 45 min at 37C in a shaking water bath. Preliminary experiments showed that the reaction was linear during this time. Incubation was terminated by the addition of 1.5 ml ice-cold stop buffer (5 mM HEPES, 2 mM EDTA, pH 5.5). Samples were passed through a 1-ml column of AG50W-X8 and eluted with 2 ml of stop buffer. Radioactivity of the effluent was measured by liquid scintillation spectrometry. A parallel set of incubations was performed on ice and was considered as nonspecific activity. To examine Ca2+ dependence, some experiments were performed in the absence of CaCl2 and calmodulin but in the presence of 2 mM EGTA. The activity in the presence of the NOS inhibitor L-NOARG (0.1 mM) also was studied. Ca2+ independent activity was determined as the difference between samples containing EGTA and samples containing L-NOARG. Ca2+-dependent activity was calculated by subtracting the Ca2+-independent activity from total NOS activity. Protein concentrations were determined according to Bradford using bovine serum albumin as standard. All measurements were made in duplicate and the specific activity was expressed as pmol citrulline/mg protein/min.
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Results |
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Histochemical Demonstration of NADPH-diaphorase
Histochemical demonstration of NADPH-d showed a solid blue reaction product. The urothelial cell layer in rats and pigs displayed an intense and uniform NADPH-d activity (Figure 1A and Figure 1B). Nerve fibers and endothelial cells were also stained. The urothelial NADPH-d reaction was further characterized in the rat bladder. The presence of the flavoprotein inhibitor DPI (1050 µM) and the alkaline phosphatase inhibitor levamisole (510 mM) concentration-dependently decreased the intensity of the urothelial NADPH-d labeling (Figure 2). Preincubation with levamisole decreased the intensity of the urothelial NADPH-d reaction, whereas levamisole seemed to have no effect on the neuronal and endothelial NADPH-d staining (Figure 2B). At the highest concentration of DPI, weak NADPH-d staining persisted in the urothelium, whereas all neuronal and endothelial staining seemed to have disappeared (Figure 2C).
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Immunohistochemical Investigation of NOS and Related Enzymes
Cytokeratin 8.13.
The anti-cytokeratin 8.13 antiserum, which reacts with a wide variety of epithelial cells (
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nNOS and eNOS. The C- and N-terminal antisera against nNOS labeled nerve fibers in the rat and pig bladders. However, no nNOS labeling was seen in either rat or pig urothelial cells (Figure 4A and Figure 4B). A polyclonal antibody against eNOS was used in the rat bladder after confirmation of endothelial cell staining, while a monoclonal antibody was found to give a more reliable staining of endothelial cells in the pig bladder. However, no immunoreactivity to eNOS was found in the pig or rat urothelium (Figure 4C and Figure 4D).
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NADPH Cytochrome P450 Reductase. In rats, the urothelium was uniformly immunoreactive for cytochrome P450 reductase (Figure 3C). In addition, some endothelial cells occasionally showed immunoreactivity to cytochrome P450 reductase. Although the majority of the pig bladders investigated showed no urothelial cytochrome P450 reductase immunoreactivity, a low-intensity staining of urothelial cells was detected in some specimens.
cGMP. No immunoreactivity to cGMP was detected in the urothelium of SNP-stimulated rat bladders. However, in SNP-stimulated bladders the endothelium and the smooth muscle of intramural vessels showed intense cGMP immunoreactivity (not shown). Unstimulated control preparations did not show any cGMP immunoreactivity in vascular structures.
iNOS.
Urothelial cells in control rats (given saline injection) were devoid of iNOS immunoreactivity (Figure 5A). In LPS-treated rats, the bladder urothelium displayed positive iNOS immunoreactivity (Figure 5B). The iNOS labeling showed a cytoplasmatic distribution and was found predominantly in cells located in the basal layer of the urothelium. Initial time course studies revealed that the immunoreactive response to iNOS was most prominent 69 hr after injection of LPS. There was no induction of eNOS or nNOS in urothelial cells in response to LPS. The monocyte/macrophage marker ED-1 (
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Ultrastructural Demonstration of NADPH-d in Rat Urothelial Cells
Ultrastructurally, the urothelial cells showed good tissue preservation of subcellular organelles, including the plasma membrane, nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus (Figure 6A). After incubation, the NADPH-d reaction product was found predominantly on membranes of the nuclear envelope and endoplasmic reticulum (Figure 6B). The reaction product also was found to be associated with the membranes of mitochondria. Omission of ß-NADPH in control experiments resulted in unstained sections (Figure 6A).
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Biochemical Detection of NOS in Pig Bladder
NOS activity was measured in specimens from the mucosa and the smooth muscle layer. Histological sectioning of the mucosal tissue used in the enzyme assay verified that it consisted of NADPH-d-positive urothelial cells. However, it was not possible to isolate the urothelial cell layer by dissection, and therefore the mucosal specimens also contained fractions of the suburothelial layer, i.e., nerves and vessels. The Ca2+-dependent NOS activity in the cytosolic fraction of mucosal tissue averaged 5.6 ± 2.0 pmol/mg protein/min (n = 3), whereas the activity in the particulate fraction amounted to 1.6 ± 0.18 pmol/mg protein/min (Figure 7). The NOS activity was markedly reduced in the absence of Ca2+ (cytosolic 0.35 ± 0.05; particulate 0.57 ± 0.32 pmol/mg protein/min).
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In samples representing the bladder smooth muscle, the cytosolic and the particulate Ca2+-dependent NOS-activity amounted to 7.5 ± 1.5 and 2.4 ± 0.7 pmol/mg protein/min (n = 3), respectively (Figure 7). Ca2+-independent NOS activity was negligible in both fractions.
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Discussion |
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NADPH-d and NOS
The histochemical technique for NADPH-d activity has been widely used as a marker for the presence of NOS (-NADPH (
NADPH-d and Alkaline Phosphatase
Because the presence of NOS is unlikely to explain the urothelial NADPH-d staining, the involvement of other plausible enzymes that may account for the NADPH-d staining was further investigated. Alkaline phosphatase, which is expressed by urothelial cells (
NADPH-d and Cytochrome P450 Reductase
NOS has a significant amino acid sequence homology to NADPH cytochrome P450 reductase (
DPI is expected to inhibit flavoprotein-dependent enzymes (
NADPH-d at the Ultrastructural Level
Nitroblue tetrazolium salt is considered to be unsuitable for ultrastructural localization of NADPH-d because it shows a tendency to dislocate. For ultrastructural NADPH-d studies, we used the BSPT salt, which yields an osmiophilic formazan on reduction and therefore reveals a more precise localization of the enzymatic reaction (
iNOS in the Urothelium
The inducible NOS isoform, iNOS, is Ca2+-independent and is induced by bacterial endotoxins and cytokines in a number of cell types, including epithelial cells (
In conclusion, despite the strong NADPH-d staining of the rat and pig urothelium, no immunoreactivity to constitutively expressed NOS was detected in the urothelium. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. In LPS-treated rats, the urothelial cells express iNOS. The significance of NO production by urothelial cells in urinary tract infections remains to be established.
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Acknowledgments |
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Supported by the Swedish Medical Research Council (grants 12601 and 11205), the Royal Physiographic Society, the Swedish Medical Society, the National Board of Health and Welfare, the Foundations of Crafoord, Magnus Bergwall, Tage Blücher and Memorial Lars Hierta, and the Medical Faculty, University of Lund, Sweden.
We thank Dr J. de Vente (Section of Neuropsychology, Limburg University, Maastricht, The Netherlands) for supplying the cyclic GMP antibody.
Received for publication October 6, 1998; accepted January 26, 1999.
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