Elsevier

Peptides

Volume 22, Issue 11, November 2001, Pages 1859-1866
Peptides

Effects of adrenomedullin on hypertrophic responses induced by angiotensin II, endothelin-1 and phenylephrine

https://doi.org/10.1016/S0196-9781(01)00505-8Get rights and content

Abstract

We examined whether adrenomedullin (AM), a vasoactive peptide with significant expression and binding sites in the heart, modulates the hypertrophic response in cultured neonatal rat ventricular myocytes. Myocyte hypertrophy was induced by treating the cells with angiotensin II (Ang II), endothelin-1 (ET-1) or α-adrenergic agonist, L-phenylephrine (PHE). All treatments resulted in a hypertrophic response as reflected by increased protein synthesis and expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) genes. AM treatment resulted in a complete inhibition of the Ang II-induced increase in ANP and BNP gene expression and secretion. In contrast, no inhibitory effect was seen in either ET-1-induced natriuretic peptide gene expression or PHE-induced ANP and BNP gene expression and secretion. AM had only a modest effect on basal levels of natriuretic peptide secretion and gene expression. When AM mRNA levels in isolated neonatal rat myocytes treated for 48 h with Ang II, ET-1 or PHE were measured, only Ang II induced a consistent increase in AM gene expression. These results indicate that AM is not invariably associated with attenuation of the hypertrophic response but its effect is dependent on the stimulus activating myocyte hypertrophy. AM may form an important autocrine/paracrine growth-inhibitory loop in Ang II-induced myocyte hypertrophy.

Introduction

Myocardial hypertrophy is one of the basic mechanisms by which the heart compensates for hemodynamic overload. The hypertrophic response is characterized by an increase in cell size and protein synthesis and altered expressions of several cardiac-specific genes [3], [24]. The signaling mechanisms involved in the hemodynamic overload-induced myocyte hypertrophy are not known; however, several neurohormonal and autocrine/paracrine factors such as angiotensin II (Ang II), endothelin-1 (ET-1) and catecholamines appear to be critical in the development of myocyte hypertrophy and heart failure [1], [3], [30], [34], [41].

Although several neurohormonal factors produce myocyte hypertrophy, less is known about inhibitory factors acting against the development of pathologic growth and hypertrophy in the heart. Candidate molecules include the members of the natriuretic peptide family, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which have direct antigrowth effects in vascular smooth muscle cells (VSMC), endothelial cells and cardiac fibroblasts [20], [21], [2]. The kallikrein-kinin system has been suggested to mediate beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the prevention of the development of cardiac hypertrophy via increased plasma and tissue bradykinin levels [13]. Recently, several reports have suggested a role for adrenomedullin (AM) in paracrine and/or autocrine regulation of cardiac function, since high mRNA expression [35], a considerable amount of AM-like immunoreactivity [36] and a high 125I-AM binding [31] have been identified in the heart. Both circulating immunoreactive (ir)-AM and cardiac concentration of AM mRNA increase in heart failure [23] suggesting a role for AM in modulating the function and structure of failing myocardium. Consistent with this hypothesis, AM exerts a direct inotropic effect in vitro [43] and attenuates Ang II- and serum-stimulated protein synthesis in cardiac myocytes [46]. In contrast, the role of AM in the signaling of cardiac growth in response to other hypertrophic stimuli has yet to be elucidated.

The aim of the present study was to investigate the effects of AM on myocyte hypertrophy in cell culture and to test whether the inhibitory effect of AM on hypertrophy depends on the stimulus for hypertrophic activation. Myocyte hypertrophy was induced in cultured neonatal rat ventricular myocytes by administration of Ang II, ET-1 or the α1-adrenergic receptor agonist, phenylephrine (PHE). Increased ANP and BNP gene expression was used as a marker of the hypertrophic response, since up-regulation of these genes represents the reprogramming of cardiac gene expression in response to hemodynamic overload [10], [26], [33]. Our results show that AM inhibits natriuretic peptide gene expression in cultured neonatal rat cardiac myocytes differentially depending on the hypertrophic stimulus, thereby showing that endogenous autocrine/paracrine factors expressed in the heart can act as stimulus-dependent modulators of myocyte cell hypertrophy.

Section snippets

Materials

Rat AM (1–50) was purchased from Phoenix Pharmaceuticals Inc. (Mountain View, CA, USA); PHE and ET-1 were purchased from Peninsula Laboratories (St. Helens, WA, USA); and Ang II and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma Chemicals (St. Louis, Missouri, MO, USA). Other molecular biology and cell culture reagents were of the highest commercial grade and purchased from standard suppliers.

Cell preparation

Cells obtained from neonatal rat ventricular myocardia were prepared as described

Effect of AM on ANP secretion from neonatal rat ventricular myocytes

To study whether AM affects myocyte hypertrophy in cell culture, we used the induction of ANP and BNP gene expression and secretion as markers of the hypertrophic response [10], [24], [26], [33]. Treatment with AM for 48 h at concentrations of 100 nM and 300 nM decreased basal ir-ANP secretion by 24% (P < 0.05, n = 39) and 25% (P < 0.05, n = 19), respectively (Fig. 1A). Lower concentrations of AM (1 nM to 30 nM), or treatment for 24 h, had no statistically significant effect on ir-ANP

Discussion

The mechanisms by which hemodynamic overload is transduced by the cardiac muscle cell and translated into myocyte hypertrophy are not completely understood, but candidates include neurohormonal factors such as Ang II, ET-1, and α-adrenergic agonists. Our present results showed that AM is a potent inhibitor of natriuretic peptide gene expression and secretion, as well as protein synthesis stimulated by Ang II. However, no inhibitory effect was seen in either ET-1-induced natriuretic peptide gene

Acknowledgments

We thank Marja Arbelius, Tuula Lumijärvi and Sirpa Rutanen for their expert technical assistance. This work was supported by Ida Montin Foundation, Academy of Finland, Sigrid Juselius Foundation and the Finnish Foundation for Cardiovascular Research.

References (47)

  • H.E. Shubeita et al.

    Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy

    J Biol Chem

    (1990)
  • T. Takahashi et al.

    Increasing cAMP antagonizes hypertrophic response to angiotensin II without affecting Ras and MAP kinase activation in vascular smooth muscle cells

    FEBS Letters

    (1996)
  • H. Tokola et al.

    Basal and acidic fibroblast growth factor-induced atrial natriuretic peptide gene expression and secretion is inhibited by staurosporine

    Eur J Pharm

    (1994)
  • O. Vuolteenaho et al.

    Atrial natriuretic polypeptides (ANP)rat atria store high molecular weight precursor but secrete processed peptides of 25–35 amino acids

    Biochem Biophys Res Commun

    (1985)
  • N.H. Bishopric et al.

    Induction of the skeletal α-actin gene in alpha1 adrenoceptor- mediated hypertrophy of rat cardiac myocytes

    J Clin Invest

    (1987)
  • L. Cao et al.

    Natriuretic peptides inhibit DNA synthesis in cardiac fibroblasts

    Hypertension

    (1995)
  • K.R. Chien et al.

    Regulation of cardiac gene expression during myocardial growth and hypertrophymolecular studies of an adaptive physiologic response

    FASEB J

    (1991)
  • J.M. Chirgwin et al.

    Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease

    Biochemistry

    (1979)
  • A. Clerk et al.

    Regulation of phospholipases C and D in rat ventricular myocytesstimulation by endothelin-1, bradykinin and phenylephrine

    J Mol Cell Cardiol

    (1997)
  • G.I.V. Cooper

    Basic determinants of myocardial hypertrophya review of molecular mechanisms

    Annu Rev Med

    (1997)
  • H.A. Coppock et al.

    Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP, and inhibit mitogen-activated protein kinase activity

    Biochem J

    (1999)
  • A.J. de Bold et al.

    Mechanical and neuroendocrine regulation of the endocrine heart

    Cardiovasc Res

    (1996)
  • T.G. Flynn et al.

    Alignment of rat cardionatrin sequences with the preprocardionatrin sequence from complementary DNA

    Science

    (1985)
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