ABSTRACT
Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/chemistry , Antihypertensive Agents/chemistry , Hypertension/drug therapy , Protein Precursors/chemistry , Superoxide Dismutase/chemistry , Teprotide/chemistry , Amino Acid Sequence , Angiotensin-Converting Enzyme Inhibitors/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Antibodies/chemistry , Antihypertensive Agents/metabolism , Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Bothrops , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Guinea Pigs , Heart Rate/drug effects , Hypertension/genetics , Hypertension/metabolism , Hypertension/pathology , Male , Mice , Models, Molecular , Molecular Sequence Data , Natriuretic Peptide, C-Type/chemistry , Natriuretic Peptide, C-Type/metabolism , Natriuretic Peptide, C-Type/pharmacology , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Precursors/pharmacology , Rats , Rats, Inbred SHR , Rats, Wistar , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase/pharmacology , Teprotide/metabolism , Teprotide/pharmacologyABSTRACT
Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityus serrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64 Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1 µg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60 min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1 µg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C.