ARTICLE |
Correspondence to: Kenji Omori, Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 2-50, Kawagishi-2-chome, Toda, Saitama 335-8505, Japan.
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Summary |
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We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E. coli. This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum. Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats. Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas. Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues. (J Histochem Cytochem 48:685693, 2000)
Key Words: cGMP, phosphodiesterase, cerebellum, kidney, pancreas
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Introduction |
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Cyclic nucleotides act as second messengers and regulate many functions in various tissues. Many kinds of cyclic nucleotide phosphodiesterases (PDEs) have been reported as regulators of intracellular cAMP and cGMP concentrations. On the basis of amino acid sequence analysis and biochemical properties, ten PDE families have been recognized in mammalian tissues (
The cGMP-binding cGMP-specific PDE (PDE5) is highly specific for cGMP and is involved in regulation of the intracellular concentration of cGMP in various tissues. PDE5 was purified from rat and bovine lung (
A cDNA encoding PDE5 was first cloned from bovine lung and was found to encode a protein of 865 amino acid residues (
Here we raised an antibody that bound to the PDE5 for use in immunohistochemistry. The antibody recognizes both human and rat PDE5 proteins in immunoblot analysis. Moreover, the antibody described here has been utilized to show the localization of PDE5 in the brain, kidney, and pancreas at the light microscopic level.
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Materials and Methods |
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Production of Full-length Recombinant Human PDE5 Protein
To obtain a human PDE5A cDNA fragment for expression, we performed a polymerase chain reaction (PCR) with primers designed according to the sequence of human PDE5A1 reported previously (
Antibody Production
A rabbit polyclonal antiserum was raised against the purified recombinant MBP-PDE5. The MBP-PDE5 protein was mixed with Freund's complete adjuvant (Difco; Detroit, MI) for the first immunization and with Freund's incomplete adjuvant when boosted. After immunizing two Japanese White rabbits (Kitayama Laboratories; Nagano, Japan) four times, antiserum was collected. An anti-PDE5 antibody IgG fraction was prepared from the antiserum by affinity chromatography on a protein G column (Amersham Pharmacia Biotech; Poole, UK). The IgG fraction was eluted with 0.1 M glycine-HCl (pH 2.7), and the eluate was neutralized immediately with 1 M Tris-HCl (pH 9.0), and stored at -20C until use. Protein concentrations of antibodies were determined with a protein assay kit (Bio-Rad; Melville, NY) with bovine serum albumin as the standard.
For some immunohistochemical studies, an anti-PDE5 antibody was purified by affinity chromatography on an MBP-PDE5-conjugated column. The affinity column was prepared using a HiTrap NHS-activated column (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The antibody was eluted with 0.1 M glycine-HCl (pH 2.7) and then neutralized immediately as described above.
Immunoblot Analysis
Cytosolic extracts of COS-7 cells expressing recombinant human PDE5A1 and recombinant rat PDE5A2 proteins were prepared as described previously (
Immunohistochemistry
Tissues freshly excised from 12-week-old male rats were fixed overnight with 10% formalin in 0.1 M phosphate buffer (pH 7.2). The tissues were dehydrated in graded ethanol solutions and embedded in paraffin. Four-micrometer sections were cut and mounted on Superfrost Plus slides (Fisher Scientific; Springfield, MA). The paraffin was removed with xylene and the tissues were rehydrated through graded ethanol to water. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol for 20 min at room temperature. Nonspecific staining was blocked by incubation with 1.5% normal goat serum in PBS for 1 hr at room temperature in a humidified atmosphere. The sections were incubated with the anti-PDE5 antibody IgG fraction, the anti-PDE5 antibody purified by the MBP-PDE5 affinity column, or normal IgG fraction prepared from preimmune serum at the same concentration overnight at 4C. After three washes of 10 min each in PBS-T, the sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (Vector Laboratories; Burlingame, CA) for 30 min at room temperature and washed three times, followed by incubation for 30 min at room temperature with the avidinbiotinperoxidase complex (Vector Laboratories). After three washes, visualization was carried out by incubating the sections with a solution of 50 mM Tris-HCl (pH 7.5), 0.1% 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemicals; Osaka, Japan), and 0.03% hydrogen peroxide. The sections were stained with hematoxylin before they were mounted.
Reverse Transcriptase-PCR and Southern Blot Analysis
Reverse transcriptase (RT)-PCR and Southern blot analysis were performed to detect PDE5A1 and PDE5A2 transcripts in rat tissues. To examine the efficiency of PCR amplification, we first produced reference DNAs. A cDNA fragment coding for N-terminal regions of rat PDE5A1 (amino acid residues 179 in addition to the 5'-untranslated region of 21 bp) was amplified using the 5' primer 5'-AAAACTCGAGCAGAAACCCGCGGCAAACACC-3', the 3' primer 5'-GCATGAGGACTTTGAGGCAGAGAGC-3', and rat PDE5A1 cDNA isolated previously (
Rat PDE5A1 and PDE5A2 transcripts in tissues were detected by RT-PCR and Southern blot analysis. After RT reaction was carried out by using 1 µg of total RNA prepared from rat cerebellum or 1 µg of poly A + RNA from rat kidney and pancreas, PCR amplification was performed under the same conditions as described above. The PCR products were subjected to 2% agarose gel electrophoresis and the fractions were transferred onto Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). To confirm that PCR products were derived from PDE5A transcripts, we detected both PCR products by Southern blot analysis using a 32P-labeled DNA probe prepared using a Tth111I-SpeI fragment of rat PDE5A cDNA. After hybridization, the membranes were exposed to X-ray film at RT for 15 min. The resultant films were scanned and quantitated as described above.
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Results |
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Immunoblot Analysis
A polyclonal antibody towards the purified MBP-PDE5 was produced, and anti-PDE5 antibody IgG fraction was purified from the antiserum by affinity chromatography on a protein-G column. The antigenic specificity was investigated with COS-7 cells that express recombinant human and rat PDE5 proteins. Cytosolic extracts of transfected COS-7 cells were electrophoretically separated, blotted onto membranes, and incubated with the anti-PDE5 antibody IgG fraction. The IgG fraction showed immunoreactive bands of ~110 kD and ~97 kD in the cytosolic extracts of transfected COS-7 cells expressing human and rat PDE5 proteins, respectively (Fig 1). No band was detected either with preimmune rabbit IgG fraction or with the mixture of the anti-PDE5 antibody after preincubation with the purified MBP-PDE5 protein used to raise the antibody (Fig 1). These findings showed that the anti-PDE5 antibody bound to both human and rat PDE5 proteins. Immunoblot analysis of lysates from rat platelets, in which PDE5 is abundant, also gave a specific staining of a ~97-kD band (Fig 1). The band of ~97 kD was diminished with a preadsorbed anti-PDE5 antibody IgG fraction. This antibody was therefore considered to be specific to PDE5 and not to react with any other proteins in rat platelets. To reduce nonspecific staining, we purified an anti-PDE5 antibody by an affinity column conjugated with the purified MBP-PDE5. The purified anti-PDE5 antibody detected a major band at ~97 kD in cytosolic extracts from rat lung and cerebellum ( Fig 1). Therefore, the antibody was also considered to be specific to PDE5 and not to react with any other proteins in these tissues.
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Immunohistochemical Analysis
In rat cerebellum, immunohistochemistry at the light microscopic level using the anti-PDE5 antibody IgG fraction showed intense labeling of most Purkinje cell layers. However, other Purkinje cells, whose nuclei were visualized by hematoxylin, did not appear to be stained by the antibody ( Fig 2A). No specific staining was observed when the sections were incubated with preimmune IgG fraction (Fig 2B). The PDE5 protein was expressed in the cell bodies and the dendrites of cerebellar Purkinje cells, and the expression was observed in the first, second, and third branches of the dendrites ( Fig 2C). In contrast, no signal was seen in the axons of cerebellar Purkinje cells at any region of the cerebellum. The PDE5 protein was not detected in other parts of brain using the anti-PDE5 antibody IgG fraction (not shown).
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To detect PDE5 protein clearly in tissues except for the brain, the anti-PDE5 antibody purified by the MBP-PDE5 affinity column was used for immunostaining. The purified anti-PDE5 antibody demonstrated that PDE5 was localized on the proximal renal tubules and medullary collecting ducts ( Fig 3A and Fig 3C ). No signal was detected in glomeruli. Preimmune IgG fraction gave no positive signal (Fig 3B and Fig 3D).
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In the pancreas, epithelial cells of pancreatic ducts were apparently immunostained with the anti-PDE5 antibody purified by the MBP-PDE5 affinity column (Fig 3E and Fig 3G). The antibody immunostained specifically neither pancreatic islet cells nor pancreatic acinar cells. In our experiment, the PDE5 activity was not observed in RIN-m cells derived from rat pancreatic islets (not shown). This finding is in accordance with the result from the immunohistochemistry using the anti-PDE5 antibody. No signal was detected in the pancreas by using the preimmune IgG fraction (Fig 3F).
RT-PCR and Southern Blot Analysis
The relative amounts of PDE5A1 and PDE5A2 transcripts in rat tissues were examined by RT-PCR. The efficiency of PCR amplification using specific primer sets for PDE5A1 and PDE5A2 was first estimated (Fig 4A), and an optimal linear range for PCR amplification of reference DNAs was obtained up to 3 pg of pGEM-5A1N and 10 pg pGEM-5A2N, which was then used for determination of the relative amounts of PDE5A transcripts in tissues. The amplification using the primer set for PDE5A1 was found to be approximately twice as efficient as that using the primer set for PDE5A2 in the optimal range. By contrast, each PCR amplification using 10 pg and 30 pg of pGEM-5A1N and 30 pg pGEM-5A2N appeared to reach saturation.
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RT-PCR and Southern blot analysis demonstrated the presence of both PDE5A1 and PDE5A2 transcripts in the rat cerebellum, kidney, and pancreas (Fig 4B), in which the PDE5 protein was detected by immunostaining. Semiquantitation of gene expression of two types of PDE5A in rat tissues by RT-PCR fell within the optimal range of reference DNA amplification. The PCR product derived form PDE5A2 transcripts appeared to be two to three times more abundant in the rat cerebellum, kidney, and pancreas compared with that from PDE5A1 transcripts. Considering the efficiency of reference DNA amplification, the amount of PDE5A2 transcripts in these tissues was four to six times greater than that of PDE5A1 transcripts in rat tissues.
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Discussion |
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To precisely determine the cell types producing PDE5 in rat tissues, we have developed a polyclonal antibody against recombinant MBP fusion human PDE5. In immunoblot analysis, the anti-PDE5 antibody immunoreacted with both recombinant human and rat PDE5 proteins. We also detected PDE5 immunostaining by the antibody in rat platelets, lung, and cerebellum. Immunoblot analysis using the anti-PDE5 antibody showed the presence of an immunoreactive band at approximately the expected molecular size of PDE5 (~95 kD), whereas no strong signal was detected at any other molecular size. Because the band disappeared with the preadsorbed anti-PDE5 antibody, the ~97-kD protein stained by the antibody would be expected to be derived from rat PDE5.
The precise localization of PDE5 in the cerebellum was visually revealed with the anti-PDE5 antibody. The cerebellum is well known as a memory center for coordinating and learning movements. Nitric oxide (NO) and cGMP are associated with the induction of long-term depression among some neurons of the cerebellum (
PDE isozymes in suspension of renal tubules and glomeruli have been reported. As for the population of PDE1 and PDE5, a different profile was shown between tubules and glomeruli. In glomeruli, PDE5 is more abundant than PDE1 (
Epithelial cells of pancreatic ducts secrete bicarbonate into pancreatic ducts in response to cAMP elevation by the stimulation of secretin (
Previously, we reported that many regulatory elements, including cAMP response elements, were present in the 5'-untranslated region and intron of the human and rat PDE5A genes (
Received for publication September 3, 1999; accepted December 8, 1999.
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