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