EVALUATION OF MEMBRANE INTERACTION AND CARDIOVASCULAR CHANGES OF NATIVE, AMIDATED AND ACETYLATED ANGIOTENSINS I AND II

The Renin-Angiotensin-Aldosterone System (RAAS) regulates blood pressure, sodium and water levels. Dysregulation can cause hypertension, renal and cardiovascular disorders. Although natural amidation and acetylation of angiotensin I and its analogs are not reported, peptides in the nervous and endocrine systems require C-terminal amidation for full activity. This study analyzed the influence of N terminal amidation and C-terminal acetylation of angiotensins I and II on peptide membrane interaction and blood pressure elevation. Native, amidated, and acetylated angiotensins I and II were synthesized via solid-phase peptide synthesis (Fmoc), purified by HPLC, and characterized by mass spectrometry (ESI). The interaction with phospholipid membranes was investigated using zwitterionic vesicles (POPC or DMPC) and ITC and DSC techniques. In ITC experiments, 25 µM peptide solutions were titrated with 20 mM POPC LUVs. The dilution heat was approximately 0.03 µcal/s per titration point. Native and acetylated peptides released heat similar to the LUVs dilution heat into the buffer. On the other hand, amidated forms showed higher heat flow, which decreased with the addition of LUVs with an association constant between 103 and 104 M-1. Both amidated forms showed negative enthalpy values of membrane interaction, with greater entropic contribution for both angio I-NH2 (19.5 times) and angio II-NH2 (84.3 times) compared to enthalpy contribution. DSC experiments were carried out to evaluate the influence of peptide binding on lipid phase transition of DMPC vesicles in the presence of angiotensins I and II, and their derivative forms. The Tm of DMPC vesicles virtually does not change with angio I and II or acetylated forms, whereas the Tm of complexes with amidated analogs increased around 2 °C. Administering peptides to Wistar rats produced an immediate increase in blood pressure (BP) and a brief reduction in heart rate for about 1 minute. Amidated angiotensin I shows a less intense pressor response (10 mmHg) compared to the native peptide (38 mmHg at 100 ng/kg). For angiotensin II, a proportional relationship between peptide concentration and vasoconstrictor effect is observed, with saturation around 75 ng/kg and an increase of approximately 40 mmHg. Consequently, amidation reduce BP four times compared to the native peptide. For acetylation, angiotensin I showed a smaller BP increase than the native peptide, although close values is noted in increased concentration. For angiotensin II, acetylation significantly increased BP, reaching its maximum effect at 100 ng/kg. These results suggest that amidation drive to a higher membrane interaction and may influence the conversion of angiotensin I to II by ACE, highlighting the importance of the C-terminal region in interaction with ACE and BP increase.