Rapid communication
Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy

https://doi.org/10.1016/j.regpep.2004.04.004Get rights and content

Abstract

Angiotensin II (Ang II) has a significant role in regulating cardiac homeostasis through humoral, autocrine and paracrine pathways, via binding to the plasma membrane AT1 receptor. Recent literature has provided evidence for intracrine growth effects of Ang II in some cell lines, which does not involve interaction with the plasma membrane receptor. We hypothesized that such intracrine mechanisms are operative in the heart and likely participate in the cardiac hypertrophy induced by Ang II. Adenoviral and plasmid vectors were constructed to express Ang II peptide intracellularly. Neonatal rat ventricular myocytes (NRVMs) infected with the adenoviral vector showed significant hypertrophic growth as determined by cell size, protein synthesis and enhanced cytoskeletal arrangement. Adult mice injected with the plasmid vector developed significant cardiac hypertrophy after 48 h, without an increase in blood pressure or plasma Ang II levels. This was accompanied by increased transcription of transforming growth factor-β (TGF-β) and insulin-like growth factor-1 (IGF-1) genes. Losartan did not block the growth effects, excluding the involvement of extracellular Ang II and the plasma membrane AT1 receptor. These data demonstrate a previously unknown growth mechanism of Ang II in the heart, which should be considered when designing therapeutic strategies to block Ang II actions.

Introduction

Classically, peptide growth factors and hormones act in the extracellular space by binding to specific receptors on the cell surface and initiating intracellular signaling events, which lead to the biological effect. However, there is evidence for another mechanism in which some of these peptides act intracellularly, without binding to receptors on the extracellular surface of the cell [1], [2]. The latter represents an intracrine mechanism, a term more commonly used in conjunction with steroid hormone actions [3]. The significance of an intracrine action is that the intracellular presence of a peptide hormone can either modulate traditional signaling events, originating from the plasma membrane receptor, by potentiation or antagonism or have an altogether different effect [4], [5].

The renin–angiotensin system (RAS) has a significant role in cardiac hypertrophy. Angiotensin II (Ang II), the bioactive component of the RAS, binds to two types of specific cell surface receptors, AT1 and AT2, which are seven-transmembrane G-protein coupled receptors (GPCRs). AT1, the predominant receptor in adult tissue, links to growth stimulatory signaling; while AT2 is more prominent in fetal tissue and is generally growth inhibitory [6]. In addition to plasma membrane, AT1-like Ang II receptors have been detected in the sarcolemma, T-tubules and nuclei of rat cardiac myocytes using electron microscopic and immunofluorescence–cytochemistry techniques [7]. Several studies have shown that intracellular Ang II receptors are functionally active. In rat hepatocytes, the AT1-like nuclear receptor couples to gene transcription and is blocked by the specific nonpeptide receptor antagonist, losartan [8], [9]. In vascular smooth muscle cells, intracellular application of Ang II leads to a rapid increase in intracellular calcium concentration and induces cell growth [10], [11]. In cardiomyopathic hamsters, at an advanced stage of the disease, microinjection of Ang II in cardiac myocytes abolishes cell coupling [12]. These effects of iAng II could be suppressed by intracellular, but not extracellular losartan or candesartan, suggesting a role for intracellular AT1. Ang II may also have effects independent of the AT1 receptor. Utilizing immunohistochemical techniques, Ang II was localized to nuclei in cerebellar neurons and to the transcriptionally active euchromatin in endothelial and granule cells, suggesting that Ang II directly regulates gene expression [13]. Ang II binding to solubilized rat liver chromatin fragments, the existence of a discrete Ang II-binding nucleoprotein particle and direct effects of nuclear localized Ang II on transcription have also been demonstrated [14], [15]. In hepatoma cells, intracellularly generated Ang II is mitogenic, a response that involves Ang II interaction with an intracellular AT1-like receptor [16], [17].

In cardiac tissue, Ang II has been depicted as an endocrine, as well as an autocrine and a paracrine factor. The possibility of an intracrine mode of action has not been studied with respect to cardiac hypertrophy. Since RAS components in the heart are increased in cardiac hypertrophy [18], [19], it is likely that Ang II levels are also increased inside the cell which might induce intracrine effects. In this study, we tested the hypothesis that intracellular angiotensin II (iAng II) participates in the cardiac hypertrophic process via an intracrine mechanism. We produced Ang II peptide intracellularly using recombinant adenoviral and plasmid expression vectors and studied the hypertrophic growth in vitro and in vivo.

Section snippets

Adenoviral expression vectors

The adenoviral expression vectors, for in vitro studies, were constructed using the Adeno-X Tet-Off gene expression system (Clontech, Palo Alto, CA). Two complimentary oligonucleotides containing coding sequence for the eight amino acids of Ang II, start and stop codons and flanked by BamHI and XbaI sites (sense strand: 5′-GATCCATGGACCGCGTATACATCCACCCCTTTTAAT-3′), were annealed and cloned into the pTRE-Shuttle plasmid. The expression cassette from the recombinant shuttle plasmid was ligated to

Intracellular expression of Ang II

To express Ang II intracellularly, we used vectors that coded only for the Ang II peptide, instead of the complete angiotensinogen (Ao) gene, to make certain that Ang II generation did not depend upon the availability of other RAS components. The omission of the signal sequence ensured that the recombinant Ang II was retained intracellularly. Adenoviral vectors, controlled by a tetracycline regulated minimal CMV promoter (Fig. 1), were used for in vitro experiments to overcome the inefficient

Discussion

In the present investigation, we identify a novel mechanism of Ang II induced cardiac hypertrophy that does not require Ang II-AT1 receptor interaction at the plasma membrane. Ang II was expressed intracellularly by removing the signal peptide. The possibility that iAng II ‘leaked’ into the extracellular space and had a growth effect was excluded by the fact that no significant increase in extracellular Ang II was observed in culture medium of Ad-AngII infected myocytes. Similarly, in vivo

Acknowledgements

This work was supported by NIH grant HL44883.

References (56)

  • W.C De Mello et al.

    Correlation between changes in morphology, electrical properties, and angiotensin-converting enzyme activity in the failing heart

    Eur. J. Pharmacol.

    (1999)
  • D.A Jans et al.

    Nuclear targeting by growth factors, cytokines, and their receptors: a role in signaling?

    Bioessays

    (1998)
  • N.M Fiaschi-Taesch et al.

    Minireview: parathyroid hormone-related protein as an intracrine factor—trafficking mechanisms and functional consequences

    Endocrinology

    (2003)
  • R.N Re

    Implications of intracrine hormone action for physiology and medicine

    Am. J. Physiol. Heart Circ. Physiol.

    (2003)
  • M Nakajima et al.

    The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
  • M.L Fu et al.

    Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect

    Receptors Channels

    (1998)
  • G.W Booz et al.

    Angiotensin-II-binding sites on hepatocyte nuclei

    Endocrinology

    (1992)
  • P Eggena et al.

    Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA

    Hypertension

    (1993)
  • H Haller et al.

    Effects of intracellular angiotensin II in vascular smooth muscle cells

    Circ. Res.

    (1996)
  • C.M Filipeanu et al.

    Intracellular angiotensin II and cell growth of vascular smooth muscle cells

    Br. J. Pharmacol.

    (2001)
  • W.C De Mello

    Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes

    Hypertension

    (1998)
  • B Erdmann et al.

    Subcellular localization of angiotensin II immunoreactivity in the rat cerebellar cortex

    Hypertension

    (1996)
  • R.N Re

    Cellular biology of the renin–angiotensin systems

    Arch. Intern. Med.

    (1984)
  • J.L Cook et al.

    In vitro evidence for an intracellular site of angiotensin action

    Circ. Res.

    (2001)
  • F Pieruzzi et al.

    Expression of renin–angiotensin system components in the heart, kidneys, and lungs of rats with experimental heart failure

    Circulation

    (1995)
  • Y.A Lee et al.

    Local stress, not systemic factors, regulate gene expression of the cardiac renin–angiotensin system in vivo: a comprehensive study of all its components in the dog

    Proc. Natl. Acad. Sci. U. S. A.

    (1996)
  • N Zhu et al.

    Systemic gene expression after intravenous DNA delivery into adult mice

    Science

    (1993)
  • G.W Booz et al.

    Role of type 1 and type 2 angiotensin receptors in angiotensin II-induced cardiomyocyte hypertrophy

    Hypertension

    (1996)
  • Cited by (134)

    • Aspects of the intracellular renin–angiotensin system

      2023, Angiotensin: From the Kidney to Coronavirus
    View all citing articles on Scopus
    View full text