amastatin and Hypertension

Hypertension is regulated through both the central and systemic renin-angiotensin systems. In the central renin-angiotensin system, zinc-dependent aminopeptidase A (APA) up-regulates blood pressure by specifically cleaving the N-terminal aspartate, but not the adjacent arginine, from angiotensin II, a process facilitated by calcium. Here, we determined the crystal structures of human APA and its complexes with different ligands and identified a calcium-binding site in the S1 pocket of APA. Without calcium, the S1 pocket can bind both acidic and basic residues through formation of salt bridges with the charged side chains. In the presence of calcium, the binding of acidic residues is enhanced as they ligate the cation, whereas the binding of basic residues is no longer favorable due to charge repulsion. Of the peptidomimetic inhibitors of APA, amastatin has higher potency than bestatin by fitting better in the S1 pocket and interacting additionally with the S3' subsite. These results explain the calcium-modulated substrate specificity of APA in central hypertension regulation and can guide the design and development of brain-targeting antihypertensive APA inhibitors.

Topics: Antihypertensive Agents; Binding Sites; Calcium; Catalytic Domain; Crystallography, X-Ray; Glutamyl Aminopeptidase; Humans; Hypertension; Peptides; Protease Inhibitors; Substrate Specificity

Two aminopeptidase inhibitors, amastatin (AM) and bestatin (BE), were employed in 3 strains of rats, spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD), to investigate the central angiotensinergic system. The results indicate that intracerebroventricular (i.c.v.) injections of AM and BE induced pressor elevations in all 3 strains of rats. In order to test for the possibility of spillage into peripheral vasculature, members from all 3 strains were peripherally infused with AM, BE, or 0.15 NaCl via jugular vein catheters. The SHRs were significantly more responsive to the aminopeptidases than the normotensive strains, however their overall pressor responses were only 33% of those to i.c.v. infusion. Next, in order to test the notion that these aminopeptidase inhibitors are having their effect via the central angiotensinergic system, and not some other peptidergic system, the specific angiotensin receptor antagonist, Sar1, Thr8-AII (sarthran) was employed. Intracerebroventricular pretreatment with sarthran prevented subsequent pressor responses to i.c.v. AM and BE in members of all 3 strains, thereby suggesting that these aminopeptidase inhibitors are having their effect via the central angiotensinergic system.

Topics: Aminopeptidases; Angiotensin II; Animals; Anti-Bacterial Agents; Blood Pressure; Brain; Enzyme Inhibitors; Hypertension; Leucine; Male; Oligopeptides; Peptides; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Receptors, Angiotensin