Low-protein diet supplemented with ketoacids ameliorates proteinuria in 3/4 nephrectomised rats by directly inhibiting the intrarenal renin-angiotensin system.

Low-protein diet plus ketoacids (LPD+KA) has been reported to decrease proteinuria in patients with chronic kidney diseases (CKD). However, the mechanisms have not been clarified. As over-activation of intrarenal renin-angiotensin system (RAS) has been shown to play a key role in the progression of CKD, the current study was performed to investigate the direct effects of LPD+KA on intrarenal RAS, independently of renal haemodynamics. In this study, 3/4 subtotal renal ablated rats were fed 18 % normal-protein diet (Nx-NPD), 6 % low-protein diet (Nx-LPD) or 5 % low-protein diet plus 1 % ketoacids (Nx-LPD+KA) for 12 weeks. Sham-operated rats fed NPD served as controls. The level of proteinuria and expression of renin, angiotensin II (AngII) and its type 1 receptors (AT1R) in the renal cortex were markedly higher in Nx-NPD group than in the sham group. LPD+KA significantly decreased the proteinuria and inhibited intrarenal RAS activation. To exclude renal haemodynamic impact on intrarenal RAS, the serum samples derived from the different groups were added to the culture medium of mesangial cells. It showed that the serum from Nx-NPD directly induced higher expression of AngII, AT1R, fibronectin and transforming growth factor-ß1 in the mesangial cells than in the control group. Nx-LPD+KA serum significantly inhibited these abnormalities. Then, proteomics and biochemical detection suggested that the mechanisms underlying these beneficial effects of LPD+KA might be amelioration of the nutritional metabolic disorders and oxidative stress. In conclusion, LPD+KA could directly inhibit the intrarenal RAS activation, independently of renal haemodynamics, thus attenuating the proteinuria in CKD rats.

Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo.

Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

A study of the anti-plasmodium activity of angiotensin II analogs.

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase method on Merrifield's resin using the t-Boc strategy, purified by RP-HPLC and characterized by liquid chromatography/ESI (+) MS (LC-ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti-plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least-square analysis, assessing the position-wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C-terminus, as well as that of hydrophobic amino acids in the N-terminus, suggests that the mechanism underlying the anti-malarial activity of these peptides is attributed to its amphiphilic character.

Effects of ethanolic extract from Radix Scrophulariae on ventricular remodeling in rats.

PURPOSE: To explore the effects of ethanolic extract of Radix Scrophulariae (EERS) on ventricular remodeling in rats. METHODS: Rats with coronary artery ligation (CAL) were randomly assigned to 5 groups: CAL model; CAL plus 40 mg/kg captopril; CAL plus 60 mg/kg, 120 mg/kg, 240 mg/kg EERS. Sham operation rats were randomly assigned to 2 groups, sham-operated control and sham-operated plus 120 mg/kg EERS. The rats were orally administered with the corresponding drugs or drinking water for 14 weeks. The left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with hematoxylin and eosin or picric acid/Sirius red for cardiomyocyte cross-section area or collagen content measurements respectively. The concentrations of hydroxyproline (Hyp), matrix metalloproteinase 2 (MMP-2), angiotensin II (Ang II), aldosterone (ALD), endothelin 1 (ET-1), atrial natriuretic peptide (ANP), tumor necrosis factor α (TNF-α) and renin activity (RA) in myocardium or serum were determined. Real-time RT-PCR was used to detect the mRNA expressions of angiotensin converting enzyme (ACE), ET-1 and ANP. RESULTS: EERS could significantly reduce the LVWI and HWI, decrease heart tissue concentrations of Hyp and collagen deposition, diminish cardiomyocyte cross-section area, reduce the tissue level of Ang II, ET-1, ANP and TNF-α. EERS could also down regulate the mRNA expression of ACE, ET-1 and ANP in myocardium. CONCLUSION: EERS attenuates ventricular remodeling. The mechanisms may be related to restraining the excessive activation of RAAS, TNF-α and modulating some gene expressions associated with cardiac hypertrophy.

Differentiating N-terminal aspartic and isoaspartic acid residues in peptides.

Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(•) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+•) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+•).

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides.

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1-7). Parasite infection decreased Ang-(1-7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1-7) decreased the level of infection in an A779 (specific antagonist of Ang-(1-7) receptor, MAS)-sensitive manner. 10(-7) M PD123319, an AT(2) receptor antagonist, partially reversed the effects of Ang-(1-7) and Ang II. However, 10(-6) M losartan, an antagonist of the AT(1) receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10(-8) M Ang II or 10(-8) M Ang-(1-7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10(-7) M A779. 10(-6) M dibutyryl-cAMP increased the level of infection and 10(-7) M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1-7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1-7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus.

Modulation of cardiac Ca2+ channel by Gq-activating neurotransmitters reconstituted in Xenopus oocytes.

L-type dihydropyridine-sensitive voltage dependent Ca(2+) channels (L-VDCCs; alpha(1C)) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, was G(q)- and PLC-independent and Ca(2+)-dependent, but not Ca(2+)-mediated, as only basal levels of Ca(2+) were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations.

Antagonist effect of a receptor-mimicking peptide encoded by human angiotensin II complementary RNA.

This article reports on the binding and the angiotensin II (Ang II) antagonistic properties of a peptide, referred to as hIIA, encoded by an RNA strand complementary to the human Ang II messenger RNA. Although Ang II and hIIA (H2N-Glu-Gly-Val-Tyr-Val-His-Pro-Val-COOH) share four amino acids, the iodinated and tritiated forms of hIIA were unreactive with seven monoclonal antibodies defining four distinct epitopes on the Ang II molecule and failed to bind to Ang II hepatic and mesangial receptors. However, hIIA did inhibit binding of 125I-Ang II to rat hepatocyte membranes (IC50, 2 x 10(-7) M) and to the various monoclonal antibodies. The lowest IC50 (5 x 10(-7) M) was measured with the monoclonal antibody specific for the Ang II sequence generally considered as implicated in receptor recognition. As predicted from the binding studies, hIIA was further shown to antagonize some biological properties of Ang II. On mesangial cells, hIIA alone had no effect on intracellular calcium concentration ([Ca2+]i) and prostaglandin E2 synthesis but did abolish the transient increase in [Ca2+]i in response to 100 nM Ang II and did induce a specific dose-dependent inhibition of the Ang II-stimulated prostaglandin E2 release. Furthermore, intravenous infusion of hIIA (200 micrograms.kg-1.min-1) inhibited by 66 +/- 3% the rat hypertensive response to 100 ng.kg-1 Ang II but had no effect on the pressor activity of agents such as alpha 1-adrenergic and HT2 serotonin agonists. Our data suggest that the "complementary" peptide hIIA interacts directly with Ang II by mimicking the Ang II complementary site on the receptor and can inhibit the physiological effects of Ang II. This type of Ang II complementary peptide may serve as a model for a new class of antihypertensive drugs.