zed under Specific Aims 1-4, stem from our initial investigations of enzymes that metabolize peptides such as bradykinin (BK), angiotensins (Ang) and others. Inhibitors of Ang I converting enzyme (kininase II; ACE) are currently used to treat millions of patients for hypertension, and it is widely assumed that their therapeutic action is due to blocking the activation of a hypertensive peptide (Ang II) while inhibiting the inactivation of the hypotensive one (BK). Our research indicates that ACE inhibitors have other important actions as well. They potentiate BK and its peptidase-resistant analogue agonists of the B2 receptor and induce a form of an enzyme-receptor crosstalk. We shall use cultured cells with either transfected or constitutively expressed enzymes and receptors and study, with various techniques, the heterodimer formation between human ACE and the B2 receptor and how ACE inhibitors affect this complex by enhancing BK effects (indirect activation) on the receptor. This leads to an increased release of vascular mediators such as NO and arachidonic acid (prostaglandins;
Specific Aim 1) -- ACE inhibitors also directly activate the B1 receptor of desArg BK and desArg-Lysl-BK (Specific Aims 2 and 3) by combining with a Znbinding pentameric sequence on an extracellular domain of the receptor, in the absence of ACE and peptide agonists. Activation of the B1 receptor (Specific Aims 2 and 3) leads to a prolonged release of NO and possibly other cardiovascular mediators from cells. Their prolonged action on the B1 receptor may affect collagen synthesis. We will investigate the initiation of different transduction pathways by the different modes of activation of the B2 (Specific Aim 1) and B1 receptors using calcium channel blockers and protein kinase inhibitors. We shall continue our study of peptidases and learn more about the structure of human kininase I, which releases the peptide ligands of B1 receptors by cleaving Arg from BK and Lys-Bk.
We aim to crystallize the active subunit of kininase I (carboxypeptidase N) (Specific Aim 4). To obtain mg quantities, we will use an improved fermentation techniques with Pichia pastoris cells. Results of our experiments will help to understand how widely used ACE inhibitors interact with receptors and consequently influence vascular reactivity.
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