© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Modulation of cardiac natriuretic peptide gene expression following endothelin type A receptor blockade in renovascular hypertension
Cardiovascular Endocrinology Laboratory, University of Ottawa Heart Institute, Ottawa, Ont., Canada
* Corresponding author. Present address: Cardiac Cell and Molecular Biology Laboratory, University of Ottawa Heart Institute, 40 Ruskin St., H-247, Ottawa, Ont., Canada, K1Y 4W7. Tel.: +1-613-761-4265; fax: +1-613-761-1597
Received 21 June 2000; accepted 30 October 2000
 |
Abstract |
|---|
 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
Objective: Increased expression of the cardiac natriuretic peptides (NP), atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) is observed during chronic hemodynamic overload. The mechanisms underlying this process are not fully understood. In vitro, endothelin 1 (ET-1) is a powerful stimulator of cardiac NP and, therefore, has been assumed to be one possible mediator of increased NP gene expression following chronic pressure or volume overload. In the present work we investigated the possible role of ET-1 in mediating the observed upregulation of cardiac NP in two kidney-one clip (2K-1C) Goldblatt hypertensive rats treated for 6 weeks with the ET-1 type A (ETA) receptor subtype receptor antagonist ABT-627. Methods: 2K-1C hypertension was induced in male Sprague–Dawley rats weighing 100–125 g by placing a silver clip (internal diameter 0.25 mm) around the left renal artery through a flank incision. The right kidney was left undisturbed. Sham operated rats underwent the same experimental procedures but no clip was placed on the left renal artery. ABT-627 was administered (10 mg/kg per day) in the drinking water for 6 weeks. Results: In hypertensive rats, ABT-627 prevented a further rise in blood pressure beginning at 3 weeks after clipping and reduced the ventricular hypertrophy observed at the end of the experiment. ETA blockade prevented enhanced NP gene expression in the right ventricle and partially prevented it in the left ventricle. No modifications in atrial NP gene expression were observed in either control or 2K-1C animals. ETA blockade decreased BNP circulating levels but did not affect ANF plasma levels in clipped rats. ABT-627 increased
-myosin heavy chain gene expression and decreased the abundance of the β isoform transcript. Conclusion: The results obtained in the present investigation show the participation of ET-1 in the increased expression of ventricular NP in 2K-1C renovascular hypertension and an apparent lack of effect of ETA blockade on atrial NP gene expression in both control and hypertensive animals thus showing that in vivo, atrial and ventricular NP gene expression are differentially regulated.
KEYWORDS Gene expression; Natriuretic peptide; Hypertension; Hypertrophy; Blood pressure
|
1 Introduction |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
Cardiac gene expression and secretion of the natriuretic peptides (NP), atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are stimulated in response to chronic volume and pressure hemodynamic overloads [1]. This response plays important modulating functions on systems that tend to increase extracellular fluid volume and systemic blood pressure, such as the renin–angiotensin–aldosterone and the sympathetic systems. Indeed, blockade of NP receptors results in cardiorenal function impairment in heart failure [2] and during mineralocorticoid escape [3]. The mechanisms underlying the stimulation of the cardiac NP system in a chronic setting are not fully understood. We have previously reported, using a model of renovascular hypertension, that there are two components that determine increased ventricular NP gene expression observed following coarctation of the abdominal aorta. One component occurs together with the hypertrophic process and is independent of load while the other is dependent upon the hemodynamic load [4]. The expression of ANF and BNP in the atria and ventricles, however, is regulated differently. In the atria, expression appears to be determined by volume-induced stretch of the atrial muscle whereas in the ventricles the level of expression seems to be largely determined by the neuroendocrine environment [4,5].
Both in vivo and in vitro data support a prominent regulatory role for endothelin-1 (ET-1) in the determination of NP gene expression through actions mediated by its ETA receptor subtype [6]. We have recently observed [5] that in DOCA-salt hypertensive rats — a model that is characterized by low plasma renin activity and high circulating endothelin — chronic blockade of ETA receptors induces significant reduction in ventricular ANF and BNP gene expression as well as normalization of blood pressure and reduction of ventricular hypertrophy. In the atria however, ETA receptor blockade lasting 5 weeks had no apparent effect on either NP stores or NP steady state mRNA levels in hypertensive or in control animals.
In hypertension models other than the high endothelin, low renin DOCA-salt model, such as in renovascular hypertension, ET-1 may be expected also to play a role in determining NP gene expression and in the development of high blood pressure and cardiac hypertrophy given several earlier observations. ETA receptor blockade largely attenuates the hypertrophic effects induced by ANG II, mechanical load or ET-1 [7,8]. The activation of a Raf-1- and MAP kinase-dependent pathway by ANG II, leading to the upregulation of genes like ANF and β-MHC, is strongly potentiated by ET-1 [8,9].
In the present work we studied the effect of the blockade of the ETA receptor with ABT-627 (a selective ETA antagonist) over 6 weeks on NP gene expression and production in order to assess the contribution of ET-1 in mediating stimulated cardiac NP production in vivo in the two kidney-one clip (2K-1C) Goldblatt renovascular hypertension model. As we have previously found that increased expression of NP in the ventricles may occur independently of hypertrophy [10], we also determined the levels of the genetic expression of and β myosin heavy chains (MHC) and collagen III as independent molecular markers of cardiac remodelling.
|
2 Methods |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
Male Sprague–Dawley rats weighing 100–125 g were housed under conditions of constant temperature and humidity with a 12-h light–dark cycle and fed ad libitum. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996) as well as the recommendations by the Canadian Council of Animal Care. 2K-1C hypertension was induced by placing a silver clip (internal diameter 0.25 mm) around the left renal artery through a flank incision. The right kidney was left undisturbed. Sham operated rats underwent the same experimental procedures but no clip was placed on the left renal artery. Animals were individually caged and allowed to recover from surgery. ABT-627, the orally active enantiomer of A-12722 ETA antagonist, was administered (10 mg/kg per day) in the drinking water for 6 weeks. The pharmacological characterization of this selective ETA antagonist has been previously reported [11]. It was found that 10 mg/kg per day was an effective oral dose to block the effects elicited by ET-1 administration. Blood pressure was monitored every week by tail-plethysmography. After 6 weeks animals were killed by decapitation and trunk blood samples were harvested into ice-cold tubes containing 1.5 mg/ml K3-EDTA. Blood samples were centrifuged at 2000xg for 30 min at 4°C and plasmas were kept at –80°C until assayed for immunoreactive ANF and BNP. The hearts were rapidly excised and the four chambers dissected, weighed and rapidly frozen in liquid nitrogen and kept at –80°C. The interatrial or interventricular septum was included with the respective left chamber.
2.1 Total RNA extraction and Northern blot analysis
Total RNA extraction and Northern blot analysis were performed as described previously [5]. Briefly, total RNA was extracted and electrophoretically separated in agarose-formaldehyde gel followed by blotting to nylon membranes (Hybond N+, Amersham). Membranes were hybridized with cDNA and oligonucleotide probes as previously detailed. The cDNA probes used were: (a) a 900-bp EcoR1/HindIII fragment containing the full-length rat ANF cDNA; (b) a 595-bp SalI fragment containing the full-length BNP cDNA; (c) a 5-kb EcoRI/SalI fragment of the mouse 28S rRNA probe; (d) a 2-kb BamHI/Bg1II fragment of the mouse PGK gene cDNA; and (e) rat 1-collagen III cDNA containing 1300 bp of the 3' non-coding and coding regions. The two oligonucleotides used were 39 and 24 base fragments specific for unique regions in the 3' untranslated regions of the rat -MHC and β-MHC genes.
The cDNA were labeled with 5' [-32P]dCTP (3000 Ci/mmol, Amersham) using the Megaprime DNA labeling system (Amersham). The oligonucleotides were labeled with [32P]ATP (3000 Ci/mmol, Amersham) using a 5' end labeling kit (Amersham). Before additional probing, bound radioactivity was stripped off from the membranes by washing with 10 mM sodium citrate (pH 6.8) and 0.25% SDS at 100°C for 10 min. Autoradiographs were scanned with an Ultrascan XL laser densitometer (LKB Produkter) and LKB 2400 Gelscan software package. The scanning values for ANF, BNP, collagen III and MHC isoforms mRNA were normalized to 28S and PGK mRNA as internal signals to correct for differences in the amount of RNA applied and transfer efficiency.
2.2 Extraction and radioimmunoassay (RIA) for ANF and BNP in plasma and tissue samples
NP were extracted from plasma and tissue samples and assayed as previously described [10,12]. Anti-rat ANF99–126 and anti-rat BNP64–95 serum were purchased from Peninsula Laboratories (Belmont, CA) and showed less than 0.01% cross-reactivity with BNP and ANF peptides, respectively.
2.3 Analysis of results
Data are expressed as mean±S.E.M. Statistical analysis was performed by one-way ANOVA followed by a post-hoc Bonferroni test using Systat®. A P-value of 0.05 or less was considered statistically significant
|
3 Results |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
Blood pressure increased significantly above controls after 1 week of clipping and remained elevated until the end of the experiment (6 weeks). ETA receptor blockade did not modify systolic blood pressure during the first 3 weeks but it partially prevented a further rise in blood pressure in the last 4 weeks (Fig. 1).
|
, Sham-operated; 
, clipped; 
, ABT-627; 
, clipped+ABT-627. *P<0.05, **P<0.01 and ***P<0.001 versus sham; 
P<0.05, Relative left and right ventricular weights were increased after 6 weeks in 2K-1C hypertensive rats and were reduced by treatment with the ETA antagonist. There was no modification in body weight in any of the groups (Table 1).
|
Plasma ANF and BNP were significantly increased in 2K-1C hypertensive rats (
56% for both peptides). Treatment with the ETA antagonist decreased BNP circulating levels but did not affect plasma ANF (Fig. 2).
|
Clipped rats showed enhanced ANF and BNP ventricular gene expression in both left and right ventricles without any modification of NP transcripts in the atria (Figs. 3–5
). The stimulation of ANF and BNP gene expression in the left ventricle evoked by clipping was around twofold for both transcripts. However the stimulation of ANF in the right ventricle was about fourfold that of BNP in the same chamber. ETA receptor blockade totally prevented the increase in ANF and BNP gene expression in the right ventricle and partially reduced it in the left ventricle.
|
|
|
In 2K-1C hypertensive rats, immunoreactive (ir) ANF and BNP was increased in the right and left ventricles (Figs. 6 and 7
). Chronic ETA receptor blockade significantly reduced irANF and irBNP content in both ventricles. No modification in irNP content was observed in any atrium of any studied group.
|
|
Collagen III gene expression was significantly enhanced in the right and left ventricle of clipped rats and was not affected by ABT-627 (Fig. 8).
|
Clipped rats showed the characteristic switch in MHC isoforms compatible with the hypertrophic process. Chronic blockade with ABT-627 increased
-MHC gene expression and decreased the β isoform (Fig. 9).
|
|
4 Discussion |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
Chronic hemodynamic overload such as that induced in various hypertensive animal models as well as that observed in human essential hypertension leads to enhanced cardiac ANF and BNP gene expression resulting in increased circulating NP levels. The factors that contribute to increased NP gene expression and elevated NP plasma levels in a chronic setting are not fully understood but the fact that this increase occurs in hypertensions of different etiologies suggests that pressure overload is the main stimulus for such an increase. Aortic-banded hypertensive rats, however, treated with a low dosage schedule of the ACE inhibitor ramipril, a procedure known to inhibit tissue but not the circulating RAS, remain hypertensive during the treatment but show a decrease in ventricular NP gene expression in concert with regression of the anatomical hypertrophy [4]. This suggests that in addition to mechanical factors, neurohumoral factors participate in the regulation of NP gene expression during chronic pressure overload.
Renovascular hypertension is a humoral-factor mediated pressure overload model in which cardiac remodelling occurs due to an increased afterload imposed on the left ventricle by systemic hypertension and neurohormonal stimulation, including RAS activation. Activation of RAS has been shown to result in secondary activation of the endothelin system [13]. Therefore, pharmacological intervention of the endothelin system should be expected to exert significant effects in renovascular hypertension. Humoral and tissue ANG II play a significant role in the triggering and maintenance of high blood pressure during an early phase of 2K-1C hypertension (1–4 weeks). In a second phase (5–8 weeks), there is a decline of ANG II circulating levels and fluid retention.
In the present work we found that blood pressure in 2K-1C rats increased after the 1st week of clipping and remained elevated up to the end of the experiment (6 weeks) in untreated animals. Treatment with the ETA antagonist partially prevented a further rise in blood pressure occurring in the last 2 weeks of the experiment observed in the untreated 2K-1C animals. Several authors have reported no change in blood pressure using different selective and non-selective ET-1 antagonists in 2K-1C hypertension [14–16] but most of these studies were carried out during the early phase in the development of hypertension. Others found attenuation of the rise in blood pressure following treatment with selective ETA antagonists, even at 2 days after clipping [17].
In contrast to findings in the present work and others mentioned above, selective ETA blockade has been found to completely prevent the increase in blood pressure induced by the administration of exogenous ANG II suggesting that renal artery stenosis activates additional hypertensive mechanisms that do not respond to ETA blockade. In the present work ETA blockade did not lower blood pressure during the early phase of hypertension but it attenuated the course of hypertension in weeks 5 and 6, suggesting that ET-1 may have a role in the maintenance of hypertension when circulating ANG II declines.
ANF and BNP gene expression was increased in the right and left ventricle but not in the atria of 2K-1C hypertensive rats. ETA receptor blockade totally prevented NP gene expression increase in the right ventricle and partially reduced it in the left ventricle. These results are similar to previous findings reported by us following ETA blockade in the DOCA-salt hypertensive rat model [5] and following ACE inhibition in the hypertensive aortic-banded rat. Thus, atrial and ventricular NP gene expression is consistently differentially regulated in the different experimental models of hypertension that that we have investigated in this and previous work [4,5].
Changes in cardiac ANF and BNP transcript abundance in 2K-1C rats were mirrored by modifications in content but not by ANF or BNP plasma levels. The enhanced ventricular ANF and BNP contents observed in clipped rats were totally prevented by ETA blockade in both right and left ventricles. No modifications were observed in atrial ANF or BNP content in any of the groups studied. Circulating ANF and BNP levels were markedly elevated in 2K-1C rats, the increase being similar for both cardiac hormones. ABT-627 treatment partially prevented the increase in circulating BNP but did not affect the increased plasma ANF levels. The reason for this dissociation in the circulating levels of the cardiac hormones after ETA blockade is not apparent but it is possible that the decrease in ventricular BNP gene expression following administration of the ETA blocker exposes an atrial response due to an underlying volume expansion. Volume expansion results in increased atrial stretch, which brings about a mainly ANF secretory response [10] due to a predominant ANF content of atrial granules over that of BNP.
ETA receptor blockade reduced left and right ventricular weight/body weight ratio in 2K-1C animals suggesting the participation of ET-1 in the triggering and maintenance of the hypertrophic process. Similar findings have been reported in other models of hypertension using ETA antagonists [5,14,17].
Untreated hypertensive rats in the present investigation showed increased collagen III gene expression in both right and left ventricles but no modifications in collagen expression were observed after selective ETA blockade. This is in line with previous findings showing that although cardiac fibroblasts exhibit both ETA and ETB receptor subtypes [18] and ET-1 stimulates collagen synthesis and turnover in cultured cardiac fibroblasts [19], cardiac fibrosis is mediated by ETB receptor-coupled mechanisms [15].
Clipped rats showed an increase in β-MHC mRNA levels and a decrease in the isoform transcript. The administration of the ETA antagonist increased the expression of the isoform and reduced the expression of the β isoform. We previously reported [5] an increase in -MHC mRNA levels after ETA blockade without significant changes in the β isoform in DOCA-salt hypertensive rats suggesting that ET-1 through the ETA receptor modulates only the genetic expression of the isoform. However, in the present work both MHC isoforms were modified by ABT-627. This difference may arise from the differences in the hypertensive models used.
In summary, our results indicate that ET-1 participates in the enhanced ventricular NP gene expression in 2K-1C renovascular hypertension. Furthermore, ET-1 regulates ventricular ANF and BNP gene expression independently of the type of hypertension since we observed similar findings previously after ETA blockade in DOCA-salt hypertension [5]. On the whole, these results support the hypothesis that atrial and ventricular ANF and BNP production and gene expression are differentially regulated in atria versus ventricles and, furthermore, both hemodynamic and endocrine stimuli contribute to such regulation. The lack of effect of 6 weeks of ETA receptor blockade as used in this work on atrial NP gene expression in either normal or hypertensive animals suggest that, in vivo, the contribution of endothelin to NP atrial expression is minimal or non-existent. This is in contrast with the strong upregulation of NP gene expression and release observed in vitro in whole tissue or cardiocyte culture following exposure to ET-1 [1,20], and warrants further inquiry.
Time for primary review 33 days.
|
Acknowledgments |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
We thank Dr T. Oppegnorth from Abbott Laboratories for the supply of ABT-627. We also thank Michelle Stevenson, Carole Frost and Amalia Ponce for their technical support.
|
References |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
|---|
- de Bold A.J., Bruneau B.G., de Bold M.L. Mechanical and neuroendocrine regulation of the endocrine heart. Cardiovasc Res (1996) 31:7–18.
[Abstract/Free Full Text] - Fukai D., Wada A., Tsutamoto T., Kinoshita M. Short-term and long-term inhibition of endogenous atrial natriuretic peptide in dogs with early-stage heart failure. Jpn Circ J (1998) 62:604–610.[CrossRef][Medline]
- Yokota N., Bruneau B.G., Kuroski de Bold M.L., de Bold A.J. Atrial natriuretic factor significantly contributes to the mineralocorticoid escape phenomenon. Evidence for a guanylate cyclase-mediated pathway. J Clin Invest (1994) 94:1938–1946.[Web of Science][Medline]
- Ogawa T., Linz W., Stevenson M., et al. Evidence for load-dependant and load-independant determinants of cardiac natriuretic peptide production. Circulation (1996) 93:2059–2067.
[Abstract/Free Full Text] - Bianciotti L., de Bold A.J. Effect of selective ETA receptor blockade on natriuretic peptide gene expression in DOCA-salt hypertension. Am J Physiol (2000) 279:H93–H101.[Web of Science]
- Suzuki E., Hirata Y., Kohmoto O. Cellular mechanisms for synthesis and secretion of atrial natriuretic peptide and brain natriuretic peptide in cultured rat atrial cells. Circ Res (1992) 71:1039–1048.
[Abstract/Free Full Text] - Ito H., Hirata Y., Adachi S., et al. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes. J Clin Invest (1993) 92:398–403.[Web of Science][Medline]
- Yamazaki T., Komuro I., Kudoh S., et al. Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem (1996) 271:3221–3228.
[Abstract/Free Full Text] - Thorburn J., McMahon M., Thorburn A. Raf-1 kinase activity is necessary and sufficient for gene expression changes but not sufficient for cellular morphology changes associated with cardiac myocyte hypertrophy. J Biol Chem (1994) 269:30580–30586.
[Abstract/Free Full Text] - Yokota N., Bruneau B.G., Fernandez B.E., et al. Dissociation of cardiac hypertrophy, myosin heavy chain isoform expression, and natriuretic peptide production in DOCA-salt rats. Am J Hypertens (1995) 8:301–310.[CrossRef][Web of Science][Medline]
- Opgenorth T.J., Adler A., Calzadilla S.V., et al. Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist. J Pharmacol Exp Ther (1996) 276:473–481.
[Abstract/Free Full Text] - Sarda I.R., de Bold M.L., de Bold A.J. Optimization of atrial natriuretic factor radioimmunoassay. Clin Biochem (1989) 22:11–15.[CrossRef][Web of Science][Medline]
- Moreau P., d'Uscio L.V., Shaw C., Takase H., Barton M., Luscher T.F. Angiotensin II increases tissue endothelin and induces vascular hypertrophy. Reversal by ETA receptor antagonist. Circulation (1997) 96:1593–1597.
[Abstract/Free Full Text] - Ehmke H., Faulhaber J., Munter K., Kirchengast M., Wiesner R.J. Chronic ETA receptor blockade attenuates cardiac hypertrophy independently of blood pressure effects in renovascular hypertensive rats. Hypertension (1999) 33:954–960.
[Abstract/Free Full Text] - Hocher B., George I., Rebstock J., et al. Endothelin system-dependent cardiac remodeling in renovascular hypertension. Hypertension (1999) 33:816–822.
[Abstract/Free Full Text] - Li J.S., Sventek P., Schiffrin E.L. Effect of antihypertensive treatment and N-omega-nitro-L-arginine methyl ester on cardiovascular structure in deoxycorticosterone acetate-salt hypertensive rats. J Hypertens (1996) 14:1331–1339.[CrossRef][Web of Science][Medline]
- Schricker K., Scholz H., Hamann M., Clozel M., Kramer B.K., Kurtz A. Role of endogenous endothelins in the renin system of normal and two-kidney, one clip rats. Hypertension (1995) 25:1025–1029.
[Abstract/Free Full Text] - Guarda E., Katwa L.C., Myers P.R., Tyagi S.C., Weber K.T. Effects of endothelins on collagen turnover in cardiac fibroblasts. Cardiovasc Res (1993) 27:2130–2134.
[Abstract/Free Full Text] - Katwa L.C., Guarda E., Weber K.T. Endothelin receptors in cultured rat cardiac fibroblasts. Cardiovasc Res (1993) 27:2125–2129.
[Abstract/Free Full Text] - Ogawa T., Vatta M., Bruneau B.G., de Bold A.J. Characterization of natriuretic peptide production by adult heart atria. Am J Physiol Heart Circ Physiol (1999) 276:H1977–H1986.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||