Journal of Pharmacological and Toxicological Methods
Original articleThe suitability of iodinated Angiotensin-(1–7) peptides as pharmacological tools
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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