Estudo do mecanismo de ação do peptídeo hipotensor TsHpt-I isolado da peçonha do escorpião Tityus serrulatus e de seus análogos estruturalmente minimizados

Abstract

Bradykinin Potentiating Peptides (BPPs) can be found in different animal sources and, from the structural point of view, share a common signature with a Pyroglutamic acid residue in the N-terminus and a doublet Pro-Pro residues at the C-terminal portion. TsHpt-I, is a peptide isolated from the yellow scorpion Tityus serrulatus venom that, although sharing the Pro-Pro motif, do not exhibit the angiotensin converting enzyme (ACE) inhibition, as described to other known BPPs, although it is able both to potentiate bradykinin and to induce a fast and transient in vivo hypotension in normotensive rats. To better evaluate the observed hypotensive effect of TsHpt-I, CHO cells transfected with the bradykinin receptors were used to observe, by confocal microscopy, a fluorescence induced by nitric oxide production in stimulated cells by BK and TsHpt-I. Western Blot analyses in CHO B2 cells showed the phosphorilation of Ser 1177 residue, the excitatory site of eNOS, in presence of the BK and TsHpt-I, corroborating the confocal results. In a similar assay used for CHOB2 cells, the nitric oxide was measured in cardiomyocytes that express constitutively the BK receptors. In these cells, TsHpt-I, likewise BK, was able to induce the release of nitric oxide. This effect was abolished by HOE 140, an antagonist of the B2R. Synthetic minimized analogue from C-terminal portion of TsHpt-I, with only three aminoacids residues (KPP), was also able to keep the activity observed for the whole peptide. When KPP was acetylated at the lysine side-chain, the nitric oxide production was abolished, suggesting that the positive charge is essential for the bradykinin receptors agonism. The use of KPP in cardiomyocytes in presence of HOE 140, has abolished the fluorescence, as for TsHpt-I. In cardiomyocytes from double knockout mice for B1/B2, BK, TsHpt-I and KPP were not able to induce nitric oxide release. In this assay only the positive control group (Ang(1-7)) showed fluorescence.These results suggest the direct activation of BKR as a primary mode of action for TsHpt-I and the analog peptide bearing only three aminoacids (KPP). Also it was evidenced that the positive charge of the lysine is crucial for this activity.