The discovery that the renin-angiotensin system (RAS) contains counter-regulatory components has revolutionized thinking about its functionality. Angiotensins II and III (Ang II &Ang III) act at the AT1 Ang II receptor subtype to cause pressor, fibrotic, mitogenic, and inflammatory responses that damage the cardiovascular system as well as susceptible target organs, e.g. kidney, heart and brain. However, when Ang II and Ang III act at the AT2 receptor subtype they have depressor and antifibrotic effects. Recently, another, more powerful counter-regulatory arm of the RAS has been shown to exist: the ACE-2/Ang 1- 7/mas axis. The monocarboxypeptidase angiotensin-converting enzyme-2 (ACE-2), discovered in 2000, acts upon Ang II to form Ang 1-7, which is an agonist ligand for the deorphanized G protein coupled receptor encoded by the mas oncogene. Acting upon the mas receptor, Ang 1-7 has depressor, antifibrotic and anti-inflammatory actions that counteract the pathophysiological effects of Ang II and Ang III at the AT1 angiotensin receptor. Since ACE- 2 has a dual function in this counter-regulatory arm of the RAS: it inactivates Ang II and forms a physiological antagonist of the actions of Ang II and Ang III at the AT1 receptor, it is of therapeutic interest in treating diseases associated with overactivity of the Ang II/Ang III/AT1 receptor axis. This proposal will develop and apply a radioligand that can be used to measure functional ACE-2 activity to complement current assay methodologies and compensate for the shortcomings of these methodologies. The concept for this proposal comes from the use of 351A, an analog of lisinopril (the widely used antihypertensive ACE inhibitor) which can be radioiodinated (125I-351A) to measure functional ACE protein and determine its distribution in tissues in the body. MLN-4760 is a specific, high affinity (IC50 ~ 440 pM) antagonist of ACE-2, but it is not readily amenable to radioiodination. The hypothesis of this proposal is that an analog of MLN-4760 in which a leucinyl side chain is replaced with a tyrosinyl side chain at the carboxy terminal side of this pseudodipeptide will retain the high affinity and specificity of MLN-4760 for ACE-2, with and without an iodine-125 molecule attached to the tyrosinyl side chain. The proposed studies involve 1) synthesis of MLN-4760 analogs (JFS101/102) that meet the specifications of being a selective, high affinity ACE-2 inhibitor, 2) radioiodination and improved HPLC purification of the mono125I (and 127I) analog, and 3) application of this radioligand for measurement of functional ACE-2 protein and its modulation in a variety of animal models of cardiovascular disease and stroke. Initial studies will focus on measurement of ACE-2 in the brain under conditions thought to affect ACE-2 expression, e.g., overexpression or deletion of ACE-2 in transgenic mice or rats virally transfected with ACE-2 mRNA or with a small interfering RNA that inhibits ACE-2 synthesis. Studies will also include determination of the distribution of ACE-2 in the rodent brain using autoradiography and quantitative densitometric analysis of 125I-MLN-4760 analog binding. A future planned outgrowth of these studies will include assay of ACE-2 in other tissues, e.g. heart, kidney, lung, pancreas, reproductive organs in animal models of disease. It is anticipated that our understanding of the expression and functional significance of ACE-2 as a counter-regulator of the traditional RAS will be considerably enhanced by this research. And, that novel therapeutic agents targeting ACE-2 might be developed to promote the beneficial aspects of its antagonism of the pathophysiological actions of the RAS.
High blood pressure continues to be one of the major health problems afflicting Americans. This research project will develop a tool to study an enzyme that can lower blood pressure and improve cardiovascular health.
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|Giese, Michael J; Speth, Robert C (2014) The ocular renin-angiotensin system: a therapeutic target for the treatment of ocular disease. Pharmacol Ther 142:11-32|
|Speth, Robert C; Vento, Peter J; Carrera, Eduardo J et al. (2014) Acute repeated intracerebroventricular injections of angiotensin II reduce agonist and antagonist radioligand binding in the paraventricular nucleus of the hypothalamus and median preoptic nucleus in the rat brain. Brain Res 1583:132-40|