Original article
The suitability of iodinated Angiotensin-(1–7) peptides as pharmacological tools

https://doi.org/10.1016/j.vascn.2004.07.005Get rights and content

Abstract

Introduction: The heptapeptide Angiotensin-(1–7) [(Ang-(1–7)] is a biologically active component of the Renin–Angiotensin System. Pharmacological studies often involve Ang-(1–7) radioactively labelled with 125I. Given the small size of the original peptide, we investigated whether introduction of a rather bulky iodine label interferes with the biological activity of Ang-(1–7). Methods: Ang-(1–7) was labelled with nonradioactive iodine with the chloramine-T method. The reaction products were separated on HPLC and analysed with mass spectrometry. The products were tested for biological activity in two ways: The ability of labelled Ang-(1–7) to block Ang II-induced contraction in rat aortic rings was tested in an organ bath setup. The affinity of labelled angiotensin for ACE in rat plasma was examined in vitro. Results: Iodination of Angiotensin-(1–7) resulted in two main products: monoiodinated and diiodinated Ang-(1–7) that could be easily separated on HPLC. In an organ bath experiment, monoiodinated Ang-(1–7) blocked Ang II responses identical to the native compound, whereas diiodinated Ang-(1–7) had lost its ability to block Ang II responses. Likewise, monoiodinated Ang-(1–7) had retained its affinity for ACE, while the affinity of diiodinated Ang-(1–7) was greatly reduced. Discussion: Monoiodinated Ang-(1–7) has a biological activity identical to the native compound, whereas this is lost in diiodinated Ang-(1–7). Therefore, only the monoiodinated radioactive form seems suited for pharmacological studies.

Introduction

Angiotensin-(1–7) [Ang-(1–7)] is a biologically active metabolite of Ang I and II that counteracts the detrimental effects of Ang II in diseases that are characterised by high activity of the Renin–Angiotensin System, such as hypertension and heart failure (Collister & Hendel, 2003, Iyer et al., 1998, Loot et al., 2002). The mechanisms by which Ang-(1–7) exerts its beneficial effects are not fully understood. Amongst the possibilities identified are inhibition of the Angiotensin Converting Enzyme (ACE) and actions at various receptors, including antagonism of the Angiotensin II type 1 (AT1) receptor, stimulation of AT2 and bradykinin B2 receptors, and stimulation of a putative Ang-(1–7) receptor, resulting in the release of vasodilating prostaglandins and nitric oxide (NO; Gironacci et al., 1999, Porsti et al., 1994, Roks et al., 1999).

Studies into the mechanisms of action of Ang-(1–7) and its significance in disease, such as radioligand binding studies, often involve [125I]-labelled Ang-(1–7). Given the relative small size of the heptapeptide in relation to the iodine label, it is conceivable that the label alters its properties due to, e.g., steric hindrance. In this article therefore, we compare the biological activity of monoiodinated and diiodinated Ang-(1–7) to the native compound. We studied their effect on ACE activity and on Ang-II-induced vasoconstriction.

Section snippets

Chemicals and reagents

Milli-Q grade water was used to prepare all stock solutions and reaction mixtures. All solvents (HPLC quality) were obtained from Merck (Darmstadt, Germany). All salts and reagents were of analytical grade and were purchased from Merck or Sigma-Aldrich (Zwijndrecht, Netherlands). Human Ang II and Ang-(1–7) were obtained from Bachem (Bubendorf, Switzerland).

Synthesis of iodinated Ang-(1–7)

Ang-(1–7) was iodinated using the chloramine-T method (Hussain et al., 1995, Robles et al., 2001, Yamada et al., 2000). Small variations in

Synthesis of iodinated Ang-(1–7)

We optimised the chloramine-T reaction to yield high amounts of both monoiodinated and diiodinated Ang-(1–7). After three rounds of simplex optimisation, we found the optimal reaction parameters as indicated in the Methods section. At these conditions, virtually all (98.6±0.0%) original Ang-(1–7) was iodinated resulting in 27.5±0.4% monoiodinated Ang-(1–7), 47.9±0.3% diiodinated Ang-(1–7), and 23.2±0.2% triiodinated Ang-(1–7).

Effect of iodinated Ang-(1–7) on Ang II dose–response curves

The ability of iodinated Ang-(1–7) to block Ang-II-induced

Discussion

Ang-(1–7) exerts its effects through several mechanisms, including ACE inhibition (Deddish et al., 1998, Roks et al., 1999), AT1 antagonism (Mahon et al., 1994, Roks et al., 1999, Ueda et al., 2000), AT2 agonism (Muthalif, Benter, Uddin, Harper & Malik, 1998), and agonism on a d-Ala7-Ang-(1–7) sensitive receptor likely to be the Mas receptor (Santos et al., 2003). A labelled counterpart of Ang-(1–7) preferably should retain all these actions to serve as a valuable pharmacologic tool. Therefore,

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