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Correspondence to: Sönke Behrends, University Clinic HamburgEppendorf, Pharmacology, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: behrends@uke.uni-hamburg.de
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Summary |
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Nitric oxide (NO)-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Nevertheless, a detailed analysis of the expression of the NO target enzyme soluble guanylyl cyclase (sGC) in the heart is missing. The aim of the current study was to elucidate the expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Using a novel antibody raised against a C-terminal peptide of the rat ß1-subunit of sGC, the enzyme was demonstrated in early postnatal and adult hearts by Western blotting analyses, showing maximal expression in 10-day-old animals. Measurements of basal, NO-, and NO/YC-1-stimulated sGC activity revealed an increase of sGC activity in hearts from neonatal to 10-day-old rats, followed by a subsequent decrease in adult animals. As shown by immunohistochemical analysis, sGC expression was present in vascular endothelium and smooth muscle cells in neonatal heart but expression shifted to endothelial cells in adult animals. In isolated cardiomyocytes, sGC activity was not detectable under basal conditions but significant sGC activity could be detected in the presence of NO. An increase in expression during the perinatal period and changes in the cell types expressing sGC at different phases of development suggest dynamic regulation rather than constitutive expression of the NO receptor in the heart. (J Histochem Cytochem 50:13251331, 2002)
Key Words: nitric oxide, soluble guanylyl cyclase, cardiomyocyte, endothelium, smooth muscle cell
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Introduction |
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Nitric oxide (NO) or endothelium-derived relaxing factor relaxes blood vessels by activation of soluble guanylyl cyclase (sGC) in smooth muscle cells (
sGC represents the primary target of NO-releasing drugs, such as glyceryl trinitrate, that have been used in the treatment of ischemic heart disease for more than a century. Although the role of sGC in the control of vascular tone is widely appreciated, the importance of the enzyme in endothelial cells of blood vessels or myocytes of the heart is less well documented. In cardiomyocytes, sGC is believed to mediate the positive inotropic effects of NO (
Recently, the development of YC-1 and its derivative BAY41-2272, both NO-independent modulators of sGC, rekindled strong scientific interest in the NO target enzyme (1- and a ß1-subunit (
2/ß1 heterodimeric enzyme that is equally responsive to activation by NO and YC-1 has also been shown to occur in vivo (
A number of studies focus on cardiac expression of the different isoforms of NO synthases under physiological and pathological conditions, but very little is known about expression of sGC in the heart. The aim of the current study was to elucidate expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Although dynamic regulation of sGC in other tissues than the heart has previously been demonstrated (see
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Materials and Methods |
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Tissue Preparation and Determination of Protein Concentrations
Heart tissue was minced in liquid nitrogen and further homogenized in a teflon vessel containing wolfram balls in a microdismembrator (Braun; Melsungen, Germany). A 0.25 ml homogenization buffer (50 mM TEA, pH 7.4, 2 mM DTT, 2 mM EDTA) was used for approximately 50 mg of tissue. Cytosol was obtained by centrifugation at 45,000 x g for 1 hr at 4C. Protein concentrations were determined by the method of Bradford with bovine plasma gamma globulin as standard (Biorad; Hercules, CA).
Animals
All procedures and care of the rats were in accordance with institutional guidelines for the use of animals in research. Male Wistar rats (20 1-day-old, 12 5-day old, eight 10-day old, eight 20-day-old, eight 30-day-old, and eight adult or 90 day-old) were sacrificed by cervical dislocation. Animals were obtained from the animal facilities of the university.
Western Blotting Analyses
SDS-PAGE was performed in 10% slab gels and transferred electrophoretically onto nitrocellulose membranes (Biorad Minigel System). Sixty µg protein was loaded per lane. The membrane was reversibly stained with Ponceau S and unspecific binding sites were saturated by immersing the membrane for 1 hr in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) containing 5% non-fat dry milk. The membranes were then incubated for 1.5 hr in TBST buffer containing sGCß1-87 antibody (
Determination of sGC Activity
sGC activity of heart cytosol (50 µg protein per assay tube) was determined by incubation for 10 min at 37C in the presence of 1 mM cGMP, 0.5 mM [32P]-GTP (about 0.2 µCi), 3 mM MgCl2, 50 mM TEA-HCl, pH 7.4, 0.25 g/liter creatine kinase, 5 mM creatine phosphate, and 1 mM 3-isobutyl-1-methylxanthine in a total volume of 0.1 ml as described by
Statistical Analysis
All results were controlled for their statistical significance by one-way ANOVA followed a NewmanKeuls post test. A value of p<0.05 was considered statistically significant.
Immunohistochemistry
Hearts of Wistar rats were fixed by immersion in 3.7% formaldehyde solution for 24 hr at 20C and subsequently embedded in paraffin. Paraffin sections (6 µm) mounted on Superfrost slides were deparaffinized and rehydrated. The slides were then treated with 1.2% H2O2 in methanol to inhibit endogenous peroxidase activity. Antigen retrieval was performed using microwave cooking for 30 min in 0.1 M citrate buffer, pH 6.0. To block nonspecific binding sites, slides were treated with 2% normal swine serum in PBS. Then sections were incubated in a humid chamber overnight at 4C with an antiserum against the ß1-subunit of sGC (see above) diluted 1:600 in PBS, containing 0.2% (w/v) bovine serum albumin and 0.1% (w/v) NaN3. Slides were then treated for 60 min at 20C with biotinylated anti-rabbit IgG (1:250 in PBS; DAKO, Hamburg, Germany) followed by incubations at 20C for 30 min each with rabbit peroxidase anti-peroxidase (PAP, 1:200 in PBS; DAKO) and ABC (1:250 in PBS; Vector, Burlingame, CA). For visualization of the peroxidase activity, the nickelglucose oxidase approach was used (
Specificity of the anti-sGC ß1 antibody in immunohistochemistry had already been tested by analyses of sections from rat lungs of different age (
Cardiomyocyte Preparation
Preparation of cardiomyocytes was performed essentially as described earlier (
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Results |
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Occurrence and Activity of sGC in Rat Heart and Its Developmental Regulation
To investigate cardiac sGC expression and to screen for a potential difference in expression during development, hearts were harvested from early postnatal (1-, 5-, 10-, 20-, 30-day-old) and adult rats. In the respective cardiac cytosols, sGC activity determinations and Western blotting analyses were performed in parallel. Fig 1 shows the result of four enzyme assay determinations performed independently with separate cytosol preparations under basal, NO-, and NO/YC-1-stimulated conditions. Guanylyl cyclase activity was significantly higher in heart cytosol from 10-day-old vs neonatal or vs adult rats.
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By use of the novel sGC antibody directed against the ß1-subunit (
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Localization of sGC in Heart. In hearts of adult rats and during each period of postnatal development, the most prominent sGC-specific staining was found in the arteries of the myocardium (Fig 3). At postnatal day 1, cardiac arteries displayed strong sGC immunoreactivity in their smooth muscle cell layer, whereas endothelial cells of these vessels showed only weak staining (Fig 3a). At day 10, staining of comparable intensity was present both in smooth muscle cells and in endothelial cells of cardiac arteries (Fig 3b). Compared to neonatal and 10-day-old rats, a remarkable decrease of sGC expression in arterial smooth muscle cells was apparent in adult heart (Fig 3c). This was accompanied by a parallel increase of sGC-specific staining in endothelial cells of the same cardiac vessels (Fig 3c). During each period of development, capillaries of rat heart also displayed sGC immunoreactivity (Fig 3), whereas sGC expression in cardiomyocytes was barely detectable by immunohistochemical analyses.
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Occurrence and Activity of sGC in Cardiomyocytes
In the described immunohistochemical experiments, a subtle staining pattern could be observed in cardiomyocytes throughout the developmental stages. To determine whether cardiomyocytes express sGC, we performed experiments using isolated cardiomyocytes of adult rats. Comparison of sGC activity in cytosol from isolated cardiomyocytes and cytosol from whole heart revealed that sGC activity in cytosol from cardiomyocytes was below the detection limit under basal conditions (Fig 4). In contrast, activity was clearly detectable after activation with NO or YC-1/NO, showing the integrity of the enzyme in isolated cardiomyocytes. The finding of low but detectable expression of sGC in cardiomyocytes compared to whole heart is consistent with the immunohistochemical data showing only faint staining of cardiomyocytes. The combination of YC-1/NO led to a significant increase vs NO alone in whole heart but not in cardiomyocytes.
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Discussion |
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NO-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Symptoms of angina pectoris are primarily relieved by a decrease in venous tone and a reduction in cardiac preload (
Enzyme activity measurements of isolated cardiomyocytes showed undetectable basal but significant NO-stimulated sGC activity. This finding supports data that postulate a role for cGMP-dependent effects of NO in cardiomyocytes. These include modulation of cardiac contractility (
In rat lung, a significant increase of sGC expression has been shown during the perinatal period, with a peak at the early postnatal period (days 816) (
In conclusion, changes in expression levels, activity and the cell types expressing sGC at different times of development suggest dynamic regulation rather than static expression of the NO target enzyme in the heart. The shift of expression from smooth muscle cells to endothelial cells during postnatal development and its expression in cardiomyocytes support the concept that the cardiac functions of the NO-sensitive enzyme go beyond mere control of vascular tone.
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft.
Received for publication November 14, 2001; accepted May 1, 2002.
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