RESUMO
In the course of cloning abundant cDNAs from the South American coral snake Micrurus corallinus venom gland, we characterized a cDNA coding for a putative natriuretic peptide. All the natural natriuretic peptides described so far, possess a ring structure composed of 17 amino acids formed through an S-S bridge which is extended at the N-terminus by few to several amino acids and may be extended at the C-terminus, usually 4-7 amino acids. In contrast, the M. corallinus natriuretic peptide presents several distinct features: (a) the preform of the deduced natriuretic peptide displays an unusual C-terminus extension. This implies that the mature peptide has a long C-terminal tail or it is further extensively processed to result in the mature natriuretic peptide with the expected 4-7 amino-acid extension. (b) the deduced natriuretic peptide presents an unusual internal Cys within the ring structure. This raises the possibility of natriuretic peptides with a smaller ring structure. (c) the putative natriuretic peptide is flanked by two homologous peptides of unknown function. In addition, an analogous peptide was synthesized and assayed on perfused rat kidney, showing a dose-dependent response in urinary volume and sodium excretion. Moreover, northern-blot studies showed that M. corallinus natriuretic peptide transcripts were highly expressed in venom glands, but they were not detectable in other tissues like heart and brain, suggesting a main role for this M. corallinus natriuretic peptide in the venom gland or in the envenomation by this coral snake's bite
Assuntos
Masculino , Animais , Ratos , Elaps corallinus/intoxicação , Peptídeos Natriuréticos/farmacologia , Peptídeos Natriuréticos/genética , Rim , América do Sul , Análise de Sequência de DNA , Clonagem Molecular , Proteínas do Tecido NervosoRESUMO
In the course of cloning abundant cDNAs from the South American coral snake Micrurus corallinus venom gland, we characterized a cDNA coding for a putative natriuretic peptide. All the natural natriuretic peptides described so far, possess a ring structure composed of 17 amino acids formed through an S-S bridge which is extended at the N-terminus by few to several amino acids and may be extended at the C-terminus, usually 4-7 amino acids. In contrast, the M. corallinus natriuretic peptide presents several distinct features: (a) the proform of the deduced natriuretic peptide displays an unusual C-terminus extension. This implies that the mature peptide has a long C-terminal tail or it is further extensively processed to result in the mature natriuretic peptide with the expected 4-7 amino-acid extension. (b) the deduced natriuretic peptide presents an unusual internal Cys within the ring structure. This raises the possibility of natriuretic peptides with a smaller ring structure. (c) the putative natriuretic peptide is flanked by two homologous peptides of unknown function. In addition, an analogous peptide was synthesized and assayed on perfused rat kidney, showing a dose-dependent response in urinary volume and sodium excretion. Moreover, northern-blot studies showed that M. corallinus natriuretic peptide transcripts were highly expressed in venom glands, but they were not detectable in other tissues like heart and brain, suggesting a main role for this M. corallinus natriuretic peptide in the venom gland or in the envenomation by this coral snake's bite.
Assuntos
Venenos Elapídicos/química , Elapidae , Peptídeos/genética , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/química , Sequência de Bases , Northern Blotting , Clonagem Molecular , Rim/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Natriurese , Peptídeo Natriurético Encefálico , Peptídeo Natriurético Tipo C , Proteínas do Tecido Nervoso/química , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Sinais Direcionadores de Proteínas/química , Proteínas/química , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Análise de Sequência de DNA , América do SulRESUMO
Isolated rat kidneys were perfused with T-kinin (TK, Ile-Ser-BK) and bradykinin (BK). HPLC analysis of perfusate samples taken at 2-10 min during the TK perfusion (0.5 nmol/mL initial concentration) showed two peptide peaks, the first one eluting at 14.42 min, the same retention time for standard BK, and the second at 16.20 min, corresponding to that of TK. When BK (0.5 nmol/mL) was perfused, only its corresponding peak was obtained although total BK recovery was reduced quickly, as expected. Using both HPLC analysis and a kinin bioassay on the isolated guinea pig ileum, it was found that 12% of the added TK was converted to BK during the first perfusion cycle (2 min). While the BK recovered (12-14% from the initial TK concentration) was maintained at a similar proportion between the 2nd and the 10th min of perfusion, the rate of TK disappearance, as well as its full recovery from the perfusate, indicated further fragmentation of peptides during kinin perfusion. In the presence of 5 microM DL-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (Mergetpa), an inhibitor of plasma carboxypeptidase N (EC 3.4.17.3), the rate of conversion of TK to BK was not affected. On the other hand, the kinase II inhibitor bradykinin potentiating peptide 9a (BPP9a) increased both the proportion of TK converted to BK and the disappearance rate of TK from the perfusate. In the presence of BPP9a, the rate of BK production increased from 1.5 +/- 0.2 to 7.6 +/- 0.9 nmol/min. Furthermore, the recovery of BK was reduced during the first 2 min of perfusion to 7.6% and the conversion rate to 0.9 nmol/min when TK was perfused into the kidney in the presence of 10 microM bestatin, a known inhibitor of aminopeptidases. These data indicate that in the kidney TK is converted to BK, probably by aminopeptidase M, thus suggesting that BK is, in fact, an additional and functional kinin, inducing physiological and/or pathophysiological effects in the rat kidney in which TK is the main kinin released.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/metabolismo , Rim/metabolismo , Ácido 3-Mercaptopropiônico/análogos & derivados , Ácido 3-Mercaptopropiônico/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Carboxipeptidases/antagonistas & inibidores , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Perfusão , Ratos , Ratos WistarRESUMO
1. The objective of the present study was to determine whether chronic salt load or depletion leads to adaptive changes in kinetics of atrial natriuretic factor (ANF) binding and/or responsiveness to ANF. We measured the equilibrium binding to and the steady-state dose-response effects of ANF1-28 on isolated kidneys from rats kept on a high (H) or low (L) salt diet for 15 days. 2. Twenty-four hour sodium excretion was 5.90 +/- 0.46 mEq for H vs 0.06 +/- 0.01 mEq for L (P less than 0.01). Plasma levels of immunoreactive ANF of H (42.2 +/- 3.9 pg/ml) were not significantly different from those for L (35.2 +/- 5.3 pg/ml). 3. There was no significant differences in distribution, apparent density or affinity of ANF specific binding sites determined in non-filtering isolated kidneys from rats kept on the H or L salt diet. 4. Dose-response curves for the hemodynamic and excretory effects of ANF1-28 in filtering isolated kidneys from rats kept on the H salt diet were not different from those of rats kept on the L salt diet. In contrast, the vasorelaxant response to ANF1-28 in isolated kidneys preconstricted by adding serum from 24-h nephrectomized rats to the perfusate (generation of angiotensin II) was significantly more pronounced in kidneys front rats chronically adapted to the high-salt diet. 5. This effect of ANF may contribute to the increased renal plasma flow and glomerular filtration rate occurring under conditions of chronic salt loading in intact animals.
Assuntos
Fator Natriurético Atrial/fisiologia , Rim/fisiologia , Sódio na Dieta/farmacologia , Animais , Fator Natriurético Atrial/sangue , Ligação Competitiva , Taxa de Filtração Glomerular , Masculino , Ratos , Ratos Endogâmicos , Sódio/metabolismo , Sódio na Dieta/administração & dosagem , Resistência Vascular , VasodilataçãoRESUMO
1. The objetve of the present study was to determine whether chronic salt load or depletion leads to adaptive changes in Kinetics of atrial natriuretic factor (ANF) binding and/or responsiveness to ANF. We measured the equilibrium binding and the stead-state dose response effects of ANF1-28 on isolated kidneys from rats kept on a high (H) or low (L) salt diet for 15 days. 2. Twenty-four sodium excretion was 5.90 ñ 0.46 mEq for H vs 0.06 ñ 0.01 mEq for L(P<0.01). Plasma levels of immunoreactive ANF for H (42.2 ñ 3.9pg/ml) were not significantly different from those for L (35.2 ñ 5.3 pg/ml). s. There were no significant differences in distribution, apparent density or affinity of ANF specific binding sites determined in non-filtering isolated kidneys from rats kept on the H or L salt diet. 4. Dose-response curvas for the hemodynamic and excretory effects of ANF1-28 in filterin isolated kidneys from rats kept on the H salt diet were not different from those of rats kept on the L salt diet. In contrast, the vasorelaxant response to ANF 1-28 in isolated kidneys preconstricted by adding serum from 24-h nephrectomized rats to the prefusate (generation of angiotensin II) was significantly more pronounced in kidneys from ratas chronically adapted to the high-salt diet. 5. This effect of ANF may contribute to the increased renal plasma flow and glomerular filtration rate occuring under conditions of chronic salt loading in intact animals