(Hypertension. 1996;28:995-1004.)
© 1996 American Heart Association, Inc.
Articles |
Synthesis and Secretion of Natriuretic Peptides in the Hypertensive TGR(mREN-2)27 Transgenic Rat
the Departments of Pharmacology and Toxicology (M.M., H.R.) and Physiology (O.V.), Biocenter Oulu, University of Oulu (Finland); Max-Delbruck Center for Molecular Medicine, Berlin-Buch, Germany (D.G.); and the 2nd Department of Internal Medicine, Kyoto (Japan) University School of Medicine (K.N.).
Correspondence to Heikki Ruskoaho, MD, Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland. E-mail heikki.ruskoaho@oulu.fi.
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Abstract |
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To examine the pathophysiological mechanisms in transgenic rats carrying the murine Ren-2d renin gene, we studied atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) gene expression and secretion in 12-week-old hypertensive TGR(mREN-2)27 and normotensive Sprague-Dawley rats. Hypertension and marked left ventricular hypertrophy in TGR(mREN-2)27 rats were associated with high baseline plasma levels of immunoreactive ANP (148±18 versus 34±3 pmol/L, hypertensive versus normotensive rats; P<.001), whereas plasma immunoreactive BNP levels did not differ significantly between the strains (19±4 versus 12±3 pmol/L, P=.06). ANP mRNA and immunoreactive ANP levels in the left ventricular endocardial and epicardial layers in TGR(mREN-2)27 rats were about 20 to 40 times higher (P<.001) than those in normotensive rats. There were no statistically significant differences between atrial and ventricular BNP mRNA levels, but left ventricular immunoreactive BNP concentrations were twofold higher in hypertensive TGR(mREN-2)27 than in normotensive rats. Infusion of [Arg8]-vasopressin (0.05 µg/kg per minute IV, for 2 hours) in normotensive rats produced rapid increases (twofold, P<.05 to .01) in left ventricular BNP mRNA and immunoreactive BNP levels, whereas ventricular BNP mRNA and peptide levels did not change significantly in hypertensive rats. The increase in left atrial BNP mRNA levels in response to acute pressure overload was also significantly smaller in the hypertensive than normotensive rats (3.5-fold versus 5.2-fold, P<.01). Furthermore, the proportional but not absolute (in picomoles per liter) increase in plasma immunoreactive ANP was smaller in transgenic rats in response to acute saline and [Arg8]-vasopressin infusions (0.9% NaCl: 1.9-fold increase versus 4.4-fold increase in normotensive rats, P<.001; [Arg8]-vasopressin: 2.2-fold versus 4.8-fold increase, P<.001). These results show that baseline and cardiac overload–induced increases in BNP synthesis are markedly attenuated in transgenic rats carrying the murine Ren-2d renin gene. In addition, acute volume and pressure overload produced a smaller proportional increase in ANP secretion in hypertensive rats than normotensive rats. These alterations in the natriuretic peptide system may contribute to the pathogenesis of hypertension and cardiovascular complications in the TGR(mREN-2)27 rat.
Key Words: renin-angiotensin system • natriuretic peptides • renin • rats, transgenic • hormones • gene expression
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Introduction |
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The renin-angiotensin system plays a major role in the regulation of blood pressure, electrolyte balance, and pathomorphological alterations of cardiovascular organs via its effector peptide Ang II. The transgenic rat line TGR(mREN-2)27 is a genetic model of hypertension developed by introducing the mouse Ren-2 renin gene into the genome of the rat with the use of microinjection techniques.1 2 3 The overexpression of this gene leads to the development of severe hypertension and early morphological signs of pathological alterations within the cardiovascular system, including cardiac hypertrophy and glomerulosclerosis.4 5 It has been reported that although renin activity in the kidney is suppressed,1 6 the transgene is highly expressed in extrarenal tissues such as adrenal gland, brain, resistance blood vessels, and the gastrointestinal tract.6 7 8 Therefore, it is suggested that hypertension results from enhanced local Ang II generation in extrarenal tissues. This interpretation is supported by the findings that hypertension in TGR rats is very sensitive to low doses of converting enzyme inhibitors or Ang II receptor antagonists.8 9 10 11 However, the role of other mechanisms underlying the development and maintenance of hypertension and associated cardiovascular complications remains to be elucidated.
The three members of the natriuretic peptide hormone family, ANP, BNP, and C-type natriuretic peptide (CNP) are involved in the regulation of blood pressure and fluid homeostasis. CNP is principally found in the central nervous system and vascular endothelial cells, and ANP and BNP are mainly localized in the heart.12 13 14 15 In the normal adult heart, ANP is mainly produced by the atria, and BNP is synthesized in both atria and ventricles.16 17 ANP and BNP decrease blood pressure, increase salt and water excretion, and inhibit the release and action of several vasoactive hormones, including renin, Ang II, and aldosterone.15 18 Chronic pressure and volume overload in patients with hypertension and congestive heart failure as well as in animal models producing ventricular hypertrophy are characterized by the induction of both ANP and BNP gene expression.13 14 Most recently, studies with natriuretic peptide receptor antagonists as well as ANP inactivation by gene targeting have supported the idea that endogenous natriuretic peptides play a major role in the maintenance of hemodynamic, hormonal, and renal function in normal conditions19 20 and heart failure.21
In this study, we therefore examined the pathophysiological role of natriuretic peptide gene expression and secretion in TGR rats. We studied the release of immunoreactive ANP (ir-ANP) and ir-BNP in conscious rats under baseline conditions and in response to acute volume overload (infusion of 0.9% saline within 1 minute) and pressure overload (infusion of AVP for 2 hours). In addition, we examined the influence of acute pressure overload (infusion of AVP for 2 hours) on ANP and BNP gene expression in hypertensive TGR rats and normotensive SD rats.
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Methods |
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Materials
The materials used were AVP (Peninsula Laboratories Europe), guanidine isothiocyanate (Fluka Chemie), CsCl (Serva Feinchemica Co), formamide (JT Baker Chemicals), agarose NA (Pharmacia), heparin (Leiras), BAS 85 nitrocellulose membrane (Schleicher & Schuell), [32P]dCTP and radioiodine (Amersham), and x-ray films (Eastman Kodak). All other chemicals were obtained from Sigma Chemical Co.
Animals
TGR rats were generated and bred by Mullins and colleagues.1 Homozygous male transgenic rats were studied at 12 weeks of age. As a control group, we used age-matched, normotensive SD rats. Both strains were obtained from Møllegaard Breeding Centre Deutschland GmbH (Schonwalde, Germany). The rats were housed in plastic cages in a room with a controlled humidity of 40% and temperature of 22°C. A 12-hour light/dark cycle was maintained. The experimental design was approved by the Animal Experimentation Committee of the University of Oulu.
Chronic Instrumentation of Rats
With rats under chloral hydrate anesthesia (300 mg/kg IP), a PE-60 catheter was placed into the abdominal aorta through the left femoral artery for measurement of blood pressure and HR and collection of blood samples, as previously described.22 PE-50 catheters were inserted into the right atrium through the jugular vein for measurement of RAP and into the femoral vein for administration of drugs. All catheters were exteriorized behind the neck, filled with a heparinized (500 IU/mL) saline solution, and plugged with a stainless steel pin. After operation, the rats were housed individually in the experimental cages and had free access to food and water.
The day after the operation, the arterial and right atrial catheters were attached to pressure transducers (model MP-15, Micron Instruments) and a polygraph (model 7E, Grass Instruments) for recording of MAP, HR, and RAP. The venous catheter was connected to a syringe or infusion pump (B Braun Perfusor ED, Braun Melsungen AG) for administration of vehicle or drugs. The rats were left undisturbed for 30 minutes to become acclimatized to the laboratory before hemodynamic variables were recorded in conscious, freely moving rats.
Experimental Design in Conscious Rats
The experiments were started by measurement of MAP, HR, and RAP for 25 minutes before 1.0 mL blood was withdrawn from the arterial catheter for measurement of plasma ir-ANP and ir-BNP levels. All blood samples were immediately replaced by an equal volume of blood from a donor rat. Donor blood was obtained from conscious rats to which this volume was replaced by 0.9% NaCl. Baseline hemodynamic measurements were made 5 minutes later, when MAP, HR, and RAP were stabilized close to control values. Then, RAP was acutely increased by at least 3 mm Hg by infusion of 1.1 mL/kg physiological saline (mean, 5.1±0.1 mL saline per rat, n=17) over 1 minute. Blood samples were obtained 1 and 5 minutes after volume expansion. Next, at 30 minutes, AVP (0.05 µg/kg per minute) or vehicle was administered intravenously via an infusion pump for 2 hours at a rate of 37.5 µL/min. Blood samples were obtained before (ie, at 30 minutes) and 1 and 2 hours after the start of vehicle or drug administration. Each rat received randomly either volume expansion or AVP infusion only, and one group of cannulated TGR and SD rats received vehicle infusions only. Blood samples were taken into precooled tubes containing 1.5 mg EDTA per 1 mL blood on ice and immediately centrifuged (2000g, 10 minutes, 4°C). Plasma was stored at -20°C until radioimmunoassay.
Tissue Preparation
At the end of AVP and vehicle infusions, the rats were immediately decapitated, the abdominal cavity was opened, and the heart was removed. The aorta and pulmonary artery were carefully excised close to the ventricular surface, and the right and left atria and other tissue were removed. All ventricles were divided into a right ventricular free wall portion and a left ventricular septal portion (combined right and left septa). To avoid possible contamination of the ventricular sample by atrial tissue, we cut ventricles into the superior (about 10% to 15% of the total weight) and inferior parts; the latter were used for ventricular peptide and mRNA determinations. The left ventricle was cut into 40-µm slices on a cryostat at -20°C, and the slices were combined to represent two equal layers (endocardium and epicardium) of the left ventricular wall, as described previously.22 All cardiac tissue samples were blotted dry, weighed, immersed in liquid nitrogen, and stored at -70°C until assayed.
Assay of ir-ANP and ir-BNP in Plasma
ir-ANP and ir-BNP levels were measured by radioimmunoassay from the extracted plasma, as previously described.22 23 24 Briefly, plasma (0.45 mL) samples were extracted by Sep-Pak C18 cartridges, and eluates were redissolved in 450 µL radioimmunoassay buffer. The extracted samples were incubated in duplicates of 100 µL with 100 µL of a specific rabbit ANP antiserum (final dilution, 1:200 000)23 or a rabbit BNP antiserum (final dilution, 1:50 000)16 at 4°C. Synthetic rat ANP99-126 and rat BNP51-95 were used as standards. After incubation for 48 hours, 125I-labeled rat ANP99-126 (100 µL, 6000 cpm) or Tyr0-BNP51-95 (100 µL, 10 000 cpm) with normal rabbit serum (1 µL per tube) was added. After incubation for another 24 hours at 4°C, the immunocomplexes were precipitated with anti-rabbit 
-globulin in the presence of polyethylene glycol. The sensitivities of the ANP and BNP assays were 1.0 fmol per tube, and the within- and between-assay coefficients of variation were less than 10% and less than 15%, respectively. The 50% displacements of the standard curves were at 11 and 12 fmol per tube in the ANP and BNP assays, respectively.
ANP and BNP mRNA Determination
RNA was isolated from atrial and left ventricular tissue by the guanidine isothiocyanate/CsCl method.24 For RNA Northern and dot blot analyses, 3.0-µg samples of RNA from the atria and 20-µg samples of RNA from the ventricles were transferred to the nitrocellulose membrane. The full-length rat ANP cDNA probe (a generous gift from Dr Peter L. Davis, Queen's University, Kingston, Canada),25 a 390-bp fragment of rat BNP cDNA,16 and an oligonucleotide probe complementary to rat 18S ribosomal RNA22 were labeled with [32P]dCTP with the Quick Prime Kit (Pharmacia LKB Biotechnology). The membranes were hybridized overnight at 42°C in 5x SSC (1x SSC=0.15 mol/L NaCl and 0.015 mol/L trisodium citrate, pH 7), 0.5% sodium dodecyl sulfate, 5x Denhardt's solution, 50% formamide, and 100 µg/mL sheared herring sperm DNA. After hybridization, the membrane was washed in 0.1x SSC and 0.1% sodium dodecyl sulfate three times for 20 minutes at 55°C and was exposed to x-ray film at -70°C with intensifying screens (Cronex Lightning Plus, DuPont). Autoradiograms generated by Northern and dot blots were scanned with a densitometer (Millipore Corp Imaging Systems). The hybridization signals of ANP and BNP mRNAs were normalized to that of 18S for each sample to correct for potential differences in loading, transfer, or both.
Statistical Analysis
Results are expressed as mean±SE. Data were analyzed with one- or two-way ANOVA combined with Bonferroni's t test or Wilks' interval test. For comparison of statistical significance between two groups, Student's t test for unpaired data was used. Differences at the 95% level were considered statistically significant.
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Effects of Acute Volume Expansion on Hemodynamics and Plasma ir-ANP and ir-BNP Levels in Conscious SD and TGR Rats
Basic data are summarized in Table 1
. MAP, measured directly in chronically cannulated conscious rats, was markedly higher in 12-week-old male homozygous TGR rats than in normotensive SD rats. HR was also higher in TGR than SD rats, whereas RAP values did not differ significantly. The average body weight of control and transgenic rats was similar. Left ventricular weight was significantly greater in TGR than SD rats (Table 1). Consequently, ratios of left ventricular weight to body weight were also significantly greater in TGR rats than in age-matched normotensive SD rats. The resting value of plasma ir-ANP was 4.4-fold higher (P<.001) in conscious TGR rats than in normotensive rats. There was a tendency for higher plasma levels of ir-BNP in TGR rats, but this difference (58%) was not significant (Table 1).
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Acute volume expansion with 0.9% saline increased RAP by 3.8±0.2 mm Hg (P<.001) and HR by 52±12 beats per minute (bpm) (P=.01) in normotensive SD rats, whereas their MAP values did not change significantly (Table 2). In hypertensive TGR rats, changes in all hemodynamic variables in response to acute volume expansion were similar to those seen in controls; RAP increased by 3.3±0.2 mm Hg (P<.001) and HR by 56±14 bpm (P<.01), whereas MAP remained unchanged. In conscious normotensive rats with indwelling catheters, acute volume overload resulted in a 4.4-fold increase in plasma ir-ANP concentrations (from 30.5±0.3 to 135.0±22.9 pmol/L, P<.001), but plasma ir-BNP concentrations remained unchanged (Fig 1). Acute saline infusion in TGR rats resulted in a significantly smaller proportional increase in plasma ir-ANP levels than in control rats (1.9-fold increase; F=6.5, P<.001, TGR rats versus controls). As observed in normotensive SD rats, acute volume expansion in TGR rats did not significantly affect plasma ir-BNP concentrations (Fig 1). Baseline plasma levels of natriuretic peptides did not differ significantly between control and volume-expanded rats (Fig 1), and during blood sampling at rest in unloaded rats, plasma concentrations of ir-ANP and ir-BNP (Fig 1) as well as hemodynamic variables (Table 2) remained constant.
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To further analyze the difference of natriuretic peptide release between normotensive SD and hypertensive TGR rats, we correlated the relative increase in plasma ir-ANP and ir-BNP concentrations in response to volume overload with changes in RAP, eg, the degree of atrial stretch. The relative increase in plasma ir-ANP corresponding to the increase of 2.5 mm Hg in RAP in normotensive rats was 3.11-fold (Fig 2). In TGR rats, the relation in the changes in plasma ir-ANP concentration and RAP shifted to the right; ie, the relative increase corresponding to the increase of 2.5 mm Hg in RAP was 1.75-fold (P<.001, TGR rats versus controls). As shown in Fig 1
, despite a marked increase in RAP, plasma ir-BNP levels remained unchanged in both normotensive and hypertensive rats.
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Effects of AVP on Hemodynamics and Plasma ir-ANP and ir-BNP Levels in Conscious SD and TGR Rats
To study the effects of acute pressure overload on the release of natriuretic peptides, we infused AVP intravenously (0.05 µg/kg per minute) into conscious rats. Each rat received only one infusion (vehicle or AVP) for 2 hours. MAP, HR, and RAP were measured continuously throughout the experiments, and plasma concentrations of ir-ANP and ir-BNP were measured before and 1 and 2 hours after the infusions were started. In normotensive SD rats, AVP infusion increased MAP maximally by 30% (from 138±2 to 179±3 mm Hg, P<.05) (Fig 3). During AVP infusion, MAP reached maximal values within 30 minutes and remained elevated throughout the experimental period. In TGR rats, in response to AVP infusion, changes in MAP were similar to those in controls; MAP increased during the first 30 minutes from 176±39 to 227±24 mm Hg (28%, P<.05) and remained elevated during the entire experimental period (Fig 3). HR decreased in both normotensive SD and hypertensive TGR rats during AVP, but the change was statistically significant only in the normotensive rats (35%, from 388±4 to 254±6 bpm, P<.05). Although AVP infusion at a volume rate of 37.5 µL/min results in a volume of 4.5 mL infused over 2 hours, RAP did not change significantly in response to AVP infusion in either SD or TGR rats. Baseline hemodynamic variables did not differ significantly between vehicle- and AVP-infused rats, and during vehicle infusion in both strains, all hemodynamic variables remained constant (Fig 3).
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Acute pressure overload induced by AVP infusion in TGR rats resulted in a smaller proportional increase in plasma ir-ANP levels than in control rats (Fig 4). In normotensive SD rats, AVP infusion caused a 4.8-fold increase in plasma ir-ANP concentrations (from 38.4±7.5 to 161.8±16.0 pmol/L, P<.001) within 2 hours, whereas a 2.2-fold increase in plasma ir-ANP levels (from 174.1±55.6 to 377.7±100.3 pmol/L, P<.01) was observed in TGR rats. When the relative increase in plasma ir-ANP levels in response to AVP infusion was correlated with changes in MAP, a 4.36-fold increase in plasma ir-ANP levels corresponding to the increase of 25 mm Hg in MAP in normotensive rats was observed (Fig 2). In TGR rats, the relative increase in plasma ir-ANP levels corresponding to the increase of 25 mm Hg in MAP was 1.75-fold (P<.001, TGR rats versus controls).
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AVP infusions also produced significant increases in plasma ir-BNP concentrations in normotensive SD rats (from 17.6±2.3 to 44.3±8.5 pmol/L, P<.05), whereas plasma ir-BNP levels did not change significantly (33.0±9.3 versus 71.9±14.3 pmol/L, P=NS) during AVP infusion in TGR rats (Fig 4). The relative change in plasma ir-BNP levels corresponding to the increase of 25 mm Hg in MAP was 2.38-fold in SD rats and 1.82-fold in TGR rats (Fig 2).
Effect of AVP on Cardiac ANP and BNP mRNA and Immunoreactive Natriuretic Peptide Concentrations in Conscious SD and TGR Rats
To examine whether the increased secretion of natriuretic peptides in response to acute cardiac overload causes changes in ANP and BNP gene expression and storage, we determined ANP and BNP mRNA levels and immunoreactive natriuretic peptide concentrations from tissue samples. RNA Northern blot analysis with rat ANP and BNP cDNA probes identified a single 0.9-kb mRNA species in the atria and ventricles of both strains (Fig 5). To quantify the changes in mRNA levels, all densitometric values for ANP and BNP mRNA were normalized to their corresponding 18S densitometric values. Basic data are summarized in Table 3. ANP mRNA levels in the left ventricular endocardial and epicardial layers in TGR rats were 32 to 40 times higher than those in normotensive SD rats. Correspondingly, mean left ventricular endocardial and epicardial ir-ANP concentrations were 24- and 42-fold higher, respectively, in TGR rats than in SD rats (Table 3). In contrast, baseline right and left atrial ANP mRNA and ir-ANP levels did not differ significantly between TGR and SD rats. Atrial and ventricular BNP mRNA levels did not differ significantly, whereas left ventricular and atrial ir-BNP concentrations were higher in hypertensive TGR rats than in normotensive SD rats (Table 3).
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As shown in Figs 6 and 7, AVP infusion resulted in an early activation of BNP synthesis that was attenuated in TGR rats compared with SD rats. In the normotensive rats, AVP administration produced 1.7-fold (P<.01) and 2.0-fold (P<.01) increases in left ventricular endocardial and epicardial BNP mRNA levels, respectively, whereas left ventricular BNP mRNA levels did not change significantly in hypertensive TGR rats in response to AVP infusion (Fig 6). Furthermore, AVP infusion in normotensive rats produced 5.2-fold (P<.001) and 2.2-fold (P<.05) increases in left and right atrial BNP mRNA levels, respectively. In the AVP-infused TGR rats, left atrial mRNA levels were 3.5-fold higher (P<.001) and right atrial BNP mRNA levels 39% lower (P<.05) than those of vehicle-infused TGR rats (Fig 7). In normotensive SD rats, AVP-induced rapid activation of the BNP gene was followed by an increase in ir-BNP concentration and content in the left ventricle but not in left or right atria. In hypertensive TGR rats, ir-BNP levels did not change significantly in either atria or ventricle in response to AVP infusion (Figs 6 and 7).
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In contrast to the marked changes of BNP mRNA levels observed in normotensive rats, ANP mRNA levels during AVP infusion remained unchanged in both strains, except that ANP mRNA levels in the right atria in normotensive rats were slightly (6%, P<.05) lower than in TGR rats (Fig 7). AVP administration had no effect on ir-ANP levels in atria and left ventricle in either transgenic or control rats (Figs 6 and 7).
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Discussion |
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The present results show that transgenic rats carrying the extra mouse submandibular salivary gland renin gene (Ren-2) are characterized by altered natriuretic peptide synthesis and secretion. The insertion of the mouse renin gene in the TGR rats resulted in normal baseline cardiac BNP mRNA levels and impaired induction of BNP synthesis in response to an acute increase in blood pressure produced by AVP infusion. In addition, acute volume and pressure overload produced a smaller proportional increase in ANP secretion in hypertensive TGR rats than in normotensive SD rats. Because ANP and BNP have potent hemodynamic, renal, and hormonal actions,12 13 14 15 these alterations in natriuretic peptide synthesis and release may be involved in the pathophysiology of hypertension seen in this rat strain. In support of this hypothesis, genetic decreases in ANP gene expression in mice have been shown to result in salt-sensitive hypertension.20 Furthermore, in a canine model of left ventricular dysfunction, the elevation of circulating natriuretic peptides was shown to be inadequate for the degree of heart failure, since acute antagonism of natriuretic peptide receptors resulted in significant deleterious effects on hemodynamic, hormonal, and renal function.21
The TGR rat represents a monogenic model of hypertension with low renal renin and high renin expression in extrarenal tissues and in which the tissue renin-angiotensin system may be involved in the hypertensinogenic process (for reviews, see References 2 and 3). Consistent with other reports, we found that rats harboring an extra renin gene had significant hypertension. Previous studies have shown that untreated rats develop hypertension-related cardiovascular alterations in a variety of tissues, including the kidney (sclerosis), vasculature (hypertrophy of the media), and heart (cardiac hypertrophy) early in life.4 5 26 Our present results indicate that hypertensive TGR rats at the age of 12 weeks demonstrate a significant degree of left ventricular hypertrophy. The extent of ventricular hypertrophy corresponds to an increase in left ventricular mass of about 59%. The magnitude of hypertrophy in TGR rats is clearly greater than that in 1-year-old SHR (35% to 40% compared with WKY), with average ratios of left ventricular weight to body weight of about 3.22 27 The myocardial remodeling in transgenic rats has also been shown to be accompanied by a stiffening of the left ventricle.26
In addition to left ventricular hypertrophy, cardiac adaptation to hemodynamic stress involves changes in contractile proteins and cardiac gene expression.28 29 An analysis of changes in myocardial natriuretic peptide gene expression in TGR rats indicated increased levels of ANP mRNA in the left ventricle. Transgenic rats also had markedly higher left ventricular levels of ir-ANP than their normotensive counterparts. Furthermore, baseline plasma ir-ANP levels were higher in conscious, freely moving TGR rats compared with SD rats, whereas baseline atrial ANP mRNA and ir-ANP levels did not differ between the hypertensive and normotensive rats. These results on cardiac and plasma ANP are in line with results obtained in several other models of experimental hypertension14 and may reflect a compensatory induction of ventricular ANP gene expression that counterregulates the increased tone of vascular resistance observed in the hypertensive rats. The magnitude of induction of ANP synthesis in the left ventricle of transgenic Ren-2 rats was, however, markedly higher (20- to 40-fold) compared with that in other experimental rat models of volume and pressure overload.14 The most striking induction of ventricular ANP gene expression has been seen in the aortocaval fistula model, with an 11-fold increase in ANP mRNA,30 whereas old, markedly hypertensive SHR and Dahl salt-sensitive rats generally show a 5- to 10-fold increase in both left ventricular ir-ANP and ANP mRNA levels.14 Because Ang II is a potent stimulator of cardiomyocyte cell growth31 32 and ANP synthesis,14 overproduction of Ang II in the hearts of transgenic rats6 8 33 may, in addition to high blood pressure, explain both the marked hypertrophic process and the induction of ventricular ANP gene expression. On the other hand, aldosterone and other corticosteroids are elevated in young TGR rats during the development of hypertension,9 34 35 suggesting that corticoids also may contribute to the pathophysiological alterations in the heart.
We also examined changes in cardiac BNP synthesis and found that baseline atrial and ventricular BNP mRNA levels did not differ significantly between SD and TGR rats. Atrial ir-BNP levels were also similar in the strains, except that the left atrial ir-BNP concentration was slightly higher in TGR rats than in controls. Ventricular hypertrophy in TGR rats was associated with a 1.8-fold increase in the left ventricular concentrations of ir-BNP. Thus, during the established phase of hypertension in TGR rats, BNP synthesis is only modestly increased in the left ventricle, whereas induction of ANP gene expression is marked. The moderate induction of ventricular BNP synthesis in hypertensive rats is in agreement with the finding that TGR rats had almost normal plasma ir-BNP levels, since the major source of circulating BNP is the ventricles.16 17 Plasma and cardiac BNP are therefore insensitive markers of cardiac changes during the established phase of ventricular hypertrophy and hypertension in TGR rats. Ventricular BNP synthesis and secretion are more clearly augmented in other experimental rat models of hypertension, including SHR23 36 37 38 and stroke-prone SHR,16 particularly at the onset of the hypertensive stage.37 However, also in these hypertensive models, the plasma BNP concentration remains lower than the elevated plasma ANP concentration determined simultaneously, which is quite different from previous observations in humans17 39 and hamsters.40 Markedly increased BNP synthesis and secretion are observed in hypertrophic and dilated cardiomyopathic hamster strains40 and in patients with congestive heart failure (as much as a 300-fold increase in plasma BNP compared with controls).17 Thus, when compared with humans and hamsters, several experimental rat models of hypertension show inappropriately low baseline cardiac BNP synthesis. Furthermore, the TGR rat with severe cardiac hypertrophy and hypertension appears to be different from other hypertensive models, such as SHR and stroke-prone SHR.
To examine further the pathophysiological significance of natriuretic peptides in the maintenance of high blood pressure in TGR rats, we studied the capacity of the heart to secrete ANP and BNP in response to acute volume expansion and AVP infusion. In these experiments in conscious, freely moving rats, increases in RAP and MAP indicated that acute volume expansion and AVP infusion, respectively, increased the work load of the heart similarly in hypertensive and normotensive rats. The increased work load produced increases in plasma ANP concentration in both strains, but for a given increase of RAP or MAP (Fig 2), proportionally smaller amounts of ir-ANP were released in the hearts of TGR than in SD rats. These results differ from those in SHR, which showed that both in vitro and in vivo, the relative increase in ANP release at identical degrees of stretch was similar in SHR and WKY.22 38 It is important to note, however, that although the percent increases in plasma ir-ANP concentration were attenuated during the established phase of hypertension in TGR rats, the absolute amount of ir-ANP released (in picomoles per liter) was either similar (acute volume expansion: TGR, +77 versus SD, +105) in both strains or larger (AVP infusion: TGR, +204 versus +123) in transgenic rats. This observation agrees with a previous report22 which showed that during acute swimming exercise, the absolute increase in plasma ir-ANP levels was greater in SHR than WKY. Taken together, our results on basal and stimulated ANP release in conscious rats show that hypertension mediated by overexpression of the renin gene results in high resting levels of plasma ir-ANP and normal or enhanced absolute but attenuated proportional increases in ir-ANP release in response to increased acute cardiac workload.
We also measured plasma ir-BNP levels in conscious rats subjected to volume expansion–induced and AVP-induced acute hemodynamic stress. The increase in cardiac work load in response to AVP infusion resulted in about a twofold increase in circulating BNP levels in normotensive rats. This agrees with our recent finding that pressor doses of AVP and phenylephrine produced a delayed (within 30 minutes to 2 hours) rise in plasma ir-BNP levels in SHR and WKY.24 On the other hand, acute volume expansion, which caused an increase in RAP and marked activation of ANP secretion, did not stimulate BNP release from the heart. Thus, the regulation of BNP secretion in response to increased cardiac workload is different from that of ANP (see below). Interestingly, direct atrial stretch in the isolated perfused rat heart preparation by increases in RAP (3.6 mm Hg) similar to those used in the present study resulted in a 70% increase in BNP secretion into the perfusate, and the maximal increase in BNP release was seen after 20 minutes of distension.41 This difference between in vitro and in vivo studies may be due to the fact that plasma ir-BNP concentrations result from cumulative changes in the secretion and elimination rates.12 13 14 Furthermore, the isolated perfused rat heart model allows for the study of natriuretic peptide release in the absence of hemodynamic changes and neurohumoral factors, which have been reported to influence stretch-induced natriuretic peptide release in vivo.14
Finally, we determined the effect of acute pressure overload on cardiac ANP and BNP synthesis. These studies showed that the increased workload of the heart produced by AVP infusion caused rapid increases in atrial and ventricular BNP mRNA levels and that the ability of cardiac cells to increase BNP mRNA levels was attenuated in TGR rats. Consistent with the result that pressure overload had a smaller effect on left ventricular and atrial BNP mRNA levels in hypertensive than in control rats, the activation of ventricular BNP mRNA levels in response to AVP was accompanied by an increase in ir-BNP levels in normotensive SD rats but in not hypertensive TGR rats. These findings differ from those obtained in SHR, which showed a rapid increase in ventricular BNP mRNA levels in response to acute pressure overload similar to that in normotensive WKY.24 The normal baseline plasma BNP and cardiac BNP mRNA levels and impaired acute stimulation of BNP synthesis may be interpreted as a deficient adaptational response to reduce both the acute and chronic hemodynamic loads imposed on myocytes. Of note, the transgenic rats have a nearly 40% increase in left ventricular wall thickness, in addition to a decrease in ventricular compliance.25 It is possible that similar changes have occurred in the cardiac atria. Therefore, although acute volume overload and AVP infusion increased the workload of the heart similarly in hypertensive and normotensive rats, a smaller amount of myocyte stretch may also contribute to the attenuated synthesis and secretion of natriuretic peptides in response to volume and pressure overload in TGR rats.
We previously reported that although in vitro BNP expression is a rapid and sensitive marker of increased cardiac overload, ANP mRNA levels remained unchanged during 2 hours of constant right atrial stretch in an isolated perfused rat heart preparation.41 Also in the present study, atrial ANP mRNA levels did not differ from controls after 2 hours of pressure overload produced by AVP in both strains. Furthermore, AVP administration had no effect on left ventricular ANP mRNA levels in either transgenic or control rats. Thus, acute regulation of cardiac ANP occurs at the level of hormone release from the storage granules, whereas regulation of BNP synthesis seems to occur at the level of rapid transcription of the BNP gene. In agreement with earlier studies,16 17 the ventricle appears to be the major source of elevated levels of circulating BNP, as shown by interesting parallelisms among BNP mRNA, tissue ir-BNP, and plasma ir-BNP levels. However, since ANP and BNP are stored together in atrial granules42 and pressure overload did not cause any depletion of left atrial BNP stores (the present study), BNP of atrial origin may also contribute to an increase in plasma BNP levels during AVP infusion.
It remains to be determined whether alterations in natriuretic peptide gene expression and secretion in TGR rats derive solely from pressure overload or from its combination with the altered Ang II production. In its classic definition, the renin-angiotensin system acts through Ang II generation within the circulation. It has been suggested that hypertension in TGR rats cannot be attributed to a stimulation of the endocrine renin-angiotensin system or to an overexpression of renin in the kidney because renin gene and protein expressions in the kidney as well as plasma renin and Ang II concentrations were suppressed or unchanged in these rats compared with control rats.1 6 9 10 43 Since Ang II augments ANP secretion in vivo, enhances pressure overload–induced ANP release, and stimulates natriuretic peptide synthesis (for review, see Reference 14), attenuated proportional ANP release could be related to suppressed circulating Ang II in this hypertension model. However, other researchers have found elevated circulating renin and Ang II levels in association with increased tissue levels of angiotensin peptides.33 44 Furthermore, Ren-2 is also found in cardiac tissue,6 and homozygosity is associated with markedly increased angiotensinogen expression.8 Thus, since local formation of Ang II appears to be enhanced in transgenic rats, our results suggest that altered synthesis and secretion of natriuretic peptides may not be due to an activated intracardiac renin-angiotensin system but may be related partially to both the high blood pressure and suppressed circulating renin-angiotensin system in TGR rats. Nevertheless, the alterations in the natriuretic peptide system may explain the findings that TGR rats exhibit a tendency toward sodium and water retention45 and that despite low renal and circulating renin levels, the renin-angiotensin system actively participates in the maintenance of high blood pressure in adult TGR rats.
In summary, our results show that Ren-2 transgenic rats develop hypertension that is associated with normal baseline cardiac BNP mRNA levels and impaired induction of BNP synthesis in response to an acute increase in blood pressure produced by AVP infusion. Also, the proportional but not absolute increases in ANP secretion in response to acute volume and pressure overload are attenuated in the TGR rat strain. Because natriuretic peptides have potent hemodynamic, renal, and hormonal actions, these alterations in the natriuretic peptide system may contribute to the pathophysiology of hypertension in this rat strain. Furthermore, because of the prominent alterations in the natriuretic peptide system, the hypertensive TGR rat strain appears to be a particularly suitable model for the study of the molecular and cellular mechanisms involved in the transduction of the hemodynamic stress signal into changes in cardiac function and gene expression. Whether hypertension in the TGR rat strain is also associated with other impairments of the natriuretic peptide system, such as abnormal elimination from the circulation and altered cellular responsiveness to endogenous natriuretic peptides, remains to be studied.
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Acknowledgments |
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This study was supported by the Academy of Finland (Health Research Council) and Sigfrid Juselius Foundation. We thank Tuula Lumijarvi, Tuula Raisanen, and Marja-Leena Vainikka for their excellent technical assistance.
Received May 21, 1996; first decision July 3, 1996; accepted July 3, 1996.
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