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Hypertension is a global health problem, and angiotensin I (ANG I)-converting enzyme (ACE) inhibitors are largely used to control this pathology. Recently, it has been shown that ACE can also act as a transducer signal molecule when its inhibitors or substrates bind to it. This new role of ACE could contribute to understanding some of the effects not explained by its catalytic activity only. In this study, we investigated signaling pathway activation in Chinese hamster ovary (CHO) cells stably expressing ACE (CHO-ACE) under different conditions. We also investigated gene modulation after 4 h and 24 h of captopril treatment. Our results demonstrated that CHO-ACE cells when stimulated with ANG I, ramipril, or captopril led to JNK and ERK1/2 phosphorylation. To verify any physiological role at the endogenous level, we made use of primary cultures of mesangial cells from spontaneously hypertensive rats (SHR) and Wistar rats. Our results showed that ERK1/2 activation occurred mainly in primary cultures of mesangial cells from SHR rats upon captopril stimulation, suggesting that this signaling pathway could be differentially regulated during hypertension. Our results also showed that captopril treatment leads to a decrease of cyclooxygenase 2, interleukin-1β, and β-arrestin2 and a significant increase of AP2 gene expression levels. Our findings strengthen the fact that, in addition to the blockage of enzymatic activity, ACE inhibitors also trigger signaling pathway activation, and this may contribute to their beneficial effects in the treatment of hypertension and other pathologies.

Physical touch can help to decrease the effects of stress. The aim of this study was to evaluate the influence of tactile stimulation on the hormonal and behavioral responses of young adult rats submitted to chronic mild unpredictable stress (CMS), considering the role of angiotensin II (Ang II). In Experiment 1, male rats were divided into 4 groups: control, stress, tactile stimulation (TS), and stress + TS. CMS was applied for three weeks. Tactile stimulation was applied for seven weeks, five days a week. After the CMS protocol, depression-like behaviors were evaluated by forced swimming and sucrose consumption tests. Learning and memory were evaluated using the Y-maze test. Fifteen days after the CMS procedure, the animals were euthanized and the levels of stress hormones were determined. The hypothalamus was isolated for determination of the Ang II concentration. In Experiment 2, control and stressed rats, with or without treatment using losartan (angiotensin II type 1 receptor blocker), were evaluated using the same behavioral tests and the hypothalamus Ang II concentration was also determined. CMS increased plasma corticosterone, norepinephrine, and epinephrine concentrations, induced depression-like behaviors, impaired learning and memory, and increased the Ang II concentration in the hypothalamus. Tactile stimulation attenuated these stress-induced effects. Losartan treatment effectively prevented increase of the hypothalamic Ang II concentration and the development of depression-like behavior, and also reduced the impairment of learning and memory in the stressed animals. The results indicated that tactile stimulation seemed to protect adult rats against hormonal and behavioral chronic stress effects, and that Ang II could be involved in the CMS effects.

Diabetes mellitus and its complications have become a major health concern in Western countries. Increased activity of the intrarenal renin-angiotensin system (RAS) contributes to diabetic nephropathy (DN). We previously reported that in mesangial cells, the high glucose concentration (HG) leads to upregulation of angiotensin-converting enzyme (ACE) messenger RNA, suggesting that ACE was modulated by angiotensin II (Ang II) release. However, this relation in the collecting duct has not yet been studied. We, therefore, aimed to evaluate RAS modulation in inner medullary collecting duct cells (IMCD) exposed to HG. The IMCD were divided into normal glucose (5 mM D-glucose, NG), high glucose (30 mM, HG), and mannitol (30 mM, M) groups. The cells were cultured 48 hr in their respective media. The intracellular and extracellular ACE activity was measured using hippuryl-His-Leu as substrate via a fluorimetric assay and expression was analyzed using western blot analysis. ACE activity, intracellular (27%) and extracellular (22%), was significantly lower in the HG group than in NG and M. ACE2 activity and Ang 1-7 levels were higher in the intracellular compartment. Our data suggest that the HG cannot modify ACE synthesis in IMCD cells but can modulate its activity. The decrease in ACE activity may result in decreased levels of Ang II to protect the IMCD against proliferative and inflammatory deleterious effects of this peptide. Conversely, the increase of ACE2 generating high levels of Ang 1-7, a vasodilator peptide, suggesting that this peptide can induce glucose uptake and protect cells against oxidative stress, which can elicit insulin resistance.

The increased intrarenal renin-angiotensin system (RAS) activity contributes to diabetic nephropathy. A high glucose (HG) extracellular concentration (EC) induces activation of RAS in mesangial cel...

OBJETIVOS: Avaliar o valor prognóstico da fibronectina plasmática (FN), comparativamente à classificação numérica de Child-Pugh e os parâmetros bioquímicos que a compõem, no acompanhamento prospectivo de portadores de cirrose alcoólica durante 18 meses. MÉTODOS: Incluídos 50 pacientes com cirrose alcoólica, diagnosticada por biópsia ou critérios clínico-bioquímicos, excluídos aqueles com hepatocarcinoma ou hemorragia digestiva, infecção ou ingestão alcoólica continuada nos últimos 30 dias. A idade média do grupo foi 51,3±12,6 anos, 72% deles do sexo masculino e classificados 17 como Child-Pugh A, 18 como B e 15 como C. Os valores das bilirrubinas foram dosados pelo método automatizado, eletroforese de proteínas em acetato de celulose e o tempo de protrombina pelo método de Quick. A FN plasmática foi dosada por imunodifusão radial, com anticorpos contra FN humana em géis de agarose a 1%. RESULTADOS: Um paciente foi excluído por óbito de causa não natural e 12 foram a óbito por doença hepática. Os melhores preditores de óbito foram a pontuação de Child-Pugh [escore>10, risco relativo (RR) de 11,33) e os valores de bilirrubina (>2,5mg/dL, RR=9,47). A concentração de FN foi significantemente maior nos sobreviventes que naqueles que foram a óbito (185±66 mg/L x 131±38mg/L, p 165mg/L foram melhores indicadores de sobrevida que a classificação de Child-Pugh e seus parâmetros bioquímicos isolados.BACKGROUND: To assess the prognostic value of plasmatic fibronectin (FN), compared to numeric Child-Pugh classification and its biochemical parameters in patients with alcoholic cirrhosis followed prospectively during a 18 months-period. METHODS: Fifty patients with the diagnosis of cirrhosis by hepatic biopsy or clinical and biochemical criteria, were included in the study after the exclusion of hepatocarcinoma and GI bleeding, infection or continous alcohol ingestion in the last 30 days. The mean age was 51.3±12.6 years, being 72% males and 17 of them were classified as Child-Pugh A, 18 as B and 15 as C. Serum bilirrubin concentration was measured in autoanalyzer, protein electrophoresis was performed on cellulose acetate and prothrombin time by the Quick test. Plasmatic FN was assessed by radial immunodifusion with anti-human FN in 1% agarose gel slabs. RESULTS: One patient was excluded because no natural death and 12 died owing to hepatic disease. The numeric Child-Pugh [score>10, Relative Risk (RR) = 11.33] and total bilirrubins (> 2.5mg/dL, RR=9.47) were the best predictors of death. Mean plasmatic FN concentration was significantly higher among those who survived when compared with those who died (185±66 mg/L x 131±38mg/L, p165mg/L was better predictor of survival than Child-Pugh score or any one of its biochemical parameters.

OBJETIVOS: Avaliar o valor prognóstico da fibronectina plasmática (FN), comparativamente à classificação numérica de Child-Pugh e os parâmetros bioquímicos que a compõem, no acompanhamento prospectivo de portadores de cirrose alcoólica durante 18 meses. MÉTODOS: Incluídos 50 pacientes com cirrose alcoólica, diagnosticada por biópsia ou critérios clínico-bioquímicos, excluídos aqueles com hepatocarcinoma ou hemorragia digestiva, infecção ou ingestão alcoólica continuada nos últimos 30 dias. A idade média do grupo foi 51,3±12,6 anos, 72% deles do sexo masculino e classificados 17 como Child-Pugh A, 18 como B e 15 como C. Os valores das bilirrubinas foram dosados pelo método automatizado, eletroforese de proteínas em acetato de celulose e o tempo de protrombina pelo método de Quick. A FN plasmática foi dosada por imunodifusão radial, com anticorpos contra FN humana em géis de agarose a 1%. RESULTADOS: Um paciente foi excluído por óbito de causa não natural e 12 foram a óbito por doença hepática. Os melhores preditores de óbito foram a pontuação de Child-Pugh [escore>10, risco relativo (RR) de 11,33) e os valores de bilirrubina (>2,5mg/dL, RR=9,47). A concentração de FN foi significantemente maior nos sobreviventes que naqueles que foram a óbito (185±66 mg/L x 131±38mg/L, p 165mg/L foram melhores indicadores de sobrevida que a classificação de Child-Pugh e seus parâmetros bioquímicos isolados.

Local activation of the renin–angiotensin system (RAS) has been implicated in the pathogenesis of several renal disorders. In this study we investigated how chronic kidney disease (CKD) modulates RAS components in an experimental model. Male Wistar rats were divided into three groups: sham, nephrectomized, and nephrectomized receiving losartan. Chronic kidney disease animals presented decreased renal N-domain angiotensin-converting enzyme (ACE) activity but overexpression of N-domain ACE in urine. Remnant kidneys presented high angiotensin II levels. Losartan treatment increased urine and tissue ACE activity and tissue levels of angiotensins, mainly angiotensin (1–7), and improved renal and histopathologic parameters. Taken together, the authors’ results indicate that pathophysiological changes due to CKD could lead to an increased expression of somatic and N-domain ACE, mainly the 65 kDa isoform, suggesting that this enzyme could be used as a biological urinary marker in CKD.

The objective of this study is to evaluate pregnancy’s action on glycosaminoglycans (GAGs) profile in bladder and urethra of female adult rats. Twenty adult female rats were divided into four groups: control, day 10 pregnancy, day 20 pregnancy, and day 5 after delivery. Sulfated GAGs content were determined by densitometry. Hyaluronic acid was assessed by fluorimetric enzyme-linked immunosorbent serologic-like assay. Radioactive GAGs were quantified by measuring radioactivity in electrophoresis gel. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney tests. In all groups, dermatan sulfate was predominant followed by heparan sulfate and hyaluronic acid. Significant decrease in total GAGs in bladder was observed at the beginning of pregnancy, with progressive increase. Biosynthesis of such molecules showed a similar behavior. In urethra, changes in GAGs content were restricted to the end of pregnancy. Hyaluronic acid content showed a significant increase in bladder during puerperium. We observed different composition in vesical and urethral tissues during pregnancy and in puerperium.

To assess the prognostic value of plasmatic fibronectin (FN), compared to numeric Child-Pugh classification and its biochemical parameters in patients with alcoholic cirrhosis followed prospectively during a 18 months-period.Fifty patients with the diagnosis of cirrhosis by hepatic biopsy or clinical and biochemical criteria, were included in the study after the exclusion of hepatocarcinoma and GI bleeding, infection or continous alcohol ingestion in the last 30 days. The mean age was 51.3+/-12.6 years, being 72% males and 17 of them were classified as Child-Pugh A, 18 as B and 15 as C. Serum bilirubin concentration was measured in autoanalyzer, protein electrophoresis was performed on cellulose acetate and prothrombin time by the Quick test. Plasmatic FN was assessed by radial immunodiffusion with anti-human FN in 1% agarose gel slabs.One patient was excluded because no natural death and 12 died owing to hepatic disease. The numeric Child-Pugh [score10, Relative Risk (RR)=11.33] and total bilirubins (2.5 mg/dL, RR=9.47) were the best predictors of death. Mean plasmatic FN concentration was significantly higher among those who survived when compared with those who died (185+/-66 mg/L x 131+/-38 mg/L, p0.01), with a RR=6.59, for FN165 mg/L. Higher levels of FN, on the other hand, were the best variable to predict survival, since 96% of these 29 patients were alive at the end of follow-up.Although having less accuracy in predicting the risk of death of these patients, plasmatic FN165 mg/L was better predictor of survival than Child-Pugh score or any one of its biochemical parameters.

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.