José Antonio Silva

The Brain Renin-Angiotensin System Modulates Angiotensin II–Induced Hypertension and Cardiac Hypertrophy

The potential involvement of the brain renin-angiotensin system in the hypertension induced by subpressor doses of angiotensin II was tested by the use of newly developed transgenic rats with permanent inhibition of brain angiotensinogen synthesis [TGR(ASrAOGEN)]. Basal systolic blood pressure monitored by telemetry was significantly lower in TGR(ASrAOGEN) than in Sprague-Dawley rats (parent strain) (122.5+/-1.5 versus 128.9+/-1.9 mm Hg, respectively; P<0.05). The increase in systolic blood pressure induced by 7 days of chronic angiotensin II infusion was significantly attenuated in TGR(ASrAOGEN) in comparison with control rats (29.8+/-4.2 versus 46. 3+/-2.5 mm Hg, respectively; P<0.005). Moreover, an increase in heart/body weight ratio was evident only in Sprague-Dawley (11.1%) but not in TGR(ASrAOGEN) rats (2.8%). In contrast, mRNA levels of atrial natriuretic peptide (ANP) and collagen III in the left ventricle measured by ribonuclease protection assay were similarly increased in both TGR(ASrAOGEN) (ANP, x2.5; collagen III, x1.8) and Sprague-Dawley rats (ANP, x2.4; collagen III, x2) as a consequence of angiotensin II infusion. Thus, the expression of these genes in the left ventricle seems to be directly stimulated by angiotensin II. However, the hypertensive and hypertrophic effects of subpressor angiotensin II are at least in part mediated by the brain renin-angiotensin system.

Normal blood pressure and renal function in mice lacking the bradykinin B(2) receptor.

Telemetric blood pressure determinations, heart rate measurements, and pressure-natriuresis-diuresis experiments were used to characterize cardiovascular and renal function in bradykinin B2 receptor knockout mice fed mouse chow containing 0.25% NaCl or mouse chow containing 4% NaCl. In B2 receptor knockout mice fed usual mouse chow, the mean arterial blood pressure leveled between 108±1 and 110±3 mm Hg, and the heart rate leveled between 520±26 and 525±29 bpm, values that were not different from those measured in B1 receptor knockout mice or 129Sv/J control mice. Increasing dietary salt intake did not affect mean arterial blood pressure and heart rate. Accordingly, pressure-natriuresis curves, pressure-diuresis curves, renal blood flow, and glomerular filtration rate were not different between B2 receptor knockout and 129Sv/J mice. Increasing dietary salt intake to 4% increased renal blood flow to levels between 8.41 and 9.50 mL/min per gram kidney wet weight in 129Sv/J mice, whereas in B2 receptor-deficient mice, renal blood flow was not affected and ranged between 6.85 and 7.88 mL/min per gram kidney wet weight. Other renal function parameters were not affected. Absence of B2 receptor function was verified in B2 receptor knockout mice with bradykinin infusion. These data suggest that the absence of B2 receptor function does not necessarily make B2 receptor knockout mice hypertensive or induce salt sensitivity. Presumably, differences in the genetic background or an adaptation to the loss of B2 receptor function may account for these results, in contrast with earlier reports involving B2 receptor knockout mice. We hold the latter possibility to be more likely and to be a fruitful possibility for future research.

Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained β‐adrenergic hyperactivity

Myocardial hypertrophy and dysfunction occur in response to excessive catecholaminergic drive. Adverse cardiac remodelling is associated with activation of proinflammatory cytokines in the myocardium. To test the hypothesis that exercise training can prevent myocardial dysfunction and production of proinflammatory cytokines induced by β‐adrenergic hyperactivity, male Wistar rats were assigned to one of the following four groups: sedentary non‐treated (Con); sedentary isoprenaline treated (Iso); exercised non‐treated (Ex); and exercised plus isoprenaline (Iso+Ex). Echocardiography, haemodynamic measurements and isolated papillary muscle were used for functional evaluations. Real‐time RT‐PCR and Western blot were used to quantify tumour necrosis factor α, interleukin‐6, interleukin‐10 and transforming growth factor β1 (TGF‐β1) in the tissue. NF‐κB expression in the nucleus was evaluated by immunohistochemical staining. The Iso rats showed a concentric hypertrophy of the left ventricle (LV). These animals exhibited marked increases in LV end‐diastolic pressure and impaired myocardial performance in vitro, with a reduction in the developed tension and maximal rate of tension increase and decrease, as well as worsened recruitment of the Frank–Starling mechanism. Both gene and protein levels of tumour necrosis factor α and interleukin‐6, as well as TGF‐β1 mRNA, were increased. In addition, the NF‐κB expression in the Iso group was significantly raised. In the Iso+Ex group, the exercise training had the following effects: (1) it prevented LV hypertrophy; (ii) it improved myocardial contractility; (3) it avoided the increase of proinflammatory cytokines and improved interleukin‐10 levels; and (4) it attenuated the increase of TGF‐β1 mRNA. Thus, exercise training in a model of β‐adrenergic hyperactivity can avoid the adverse remodelling of the LV and inhibit inflammatory cytokines. Moreover, the cardioprotection is related to beneficial effects on myocardial performance.

The synthesis and distribution of the kinin B1 and B2 receptors are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

Kinins, a special class of polypeptides, are represented by bradykinin (BK), kallidin (Lys-BK), as well as their metabolites. The biological actions of these polypeptides binding on their receptors (B1 and B2) have been related to inflammation process, cytokines action, glutamate release and prostaglandins production. Usually, kinin B1 receptor is not expressed at a significant level under physiologic conditions in most tissues, but its expression is induced by injury, or upon exposure in vivo or in vitro to pro-inflammatory mediators. The kinin B2 receptor subtype is constitutively and widely expressed throughout the central and peripheral nervous system. These data raise the possibility for de novo expression of those receptors during the temporal lobe epilepsy (TLE), which has been related to cell death, gliosis and hippocampal reorganization. To correlate kinin system and TLE, adult male Wistar rats were submitted to pilocarpine model of epilepsy. The hippocampi were removed 6 h, 5 and 60 days after status epilepticus (SE) onset. The collected tissues were used to study the expression of kinin B1 and B2 mRNA receptors, using Real-Time PCR. Immunohistochemistry assay was also employed to visualize kinin B1 and B2 distribution in the hippocampus. The results show increased kinin B1 and B2 mRNA levels during acute, silent and chronic periods and changes in the kinin B1 and B2 receptors distribution. In addition, the immunoreactivity against kinin B1 receptor was increased mainly during the silent period, where neuron clusters of could be visualized. The kinin B2 receptor immunoreactivity also showed augmentation but mainly during the acute and silent periods. Our results suggest that kinin B1 and B2 receptors play an important role in the epileptic phenomena.

Role of kinin B1 and B2 receptors in the development of pilocarpine model of epilepsy.

The tissue sclerosis found in epilepsy of limbic origin is characterized by shrunken gliotic hippocampus, granule cell loss in the dentate gyrus and extensive pyramidal cell loss in Ammons horn. Evidence has indicated that sprouting of dentate granule cell axons into the inner molecular layer of the dentate gyrus is related to hyperexcitability. Trying to understand the role of kinin B1 and B2 receptors in the physiopathology of temporal lobe epilepsy (TLE), the present work was delineated to study the development of the epilepsy model induced by pilocarpine in B1 and B2 knockout mice (B1KO and B2KO, respectively). Behavior parameters, cell death and mossy fiber sprouting were analyzed. B1KO mice showed increased latency for the first seizure, associated to a decreased frequency of spontaneous seizures, when compared with wild-type mice. In addition, B1KO mice showed less cell death in all hippocampal formation associated to a reduced grade of mossy fiber sprouting. Furthermore, B2KO mice presented minor duration of the silent period and an increased frequency of spontaneous seizures, when compared with wild-type mice. B2KO and their control lineage showed similar pattern of cell death in the hippocampus, which was very intense when compared with saline-treated animals. The mossy fiber sprouting was also increased in B2KO mice, when compared to wild-type mice and saline-treated animals. Taken together, these data suggest a deleterious effect for kinin B1 receptor and a protective effect for kinin B2 receptor during the development of the temporal lobe epilepsy.

Molecular and pharmacological evidence for modulation of kinin B1 receptor expression by endogenous glucocorticoids hormones in rats

The effect of endogenous glucocorticoid hormones on the expression of rat B1 receptors was examined by means of molecular and pharmacological functional approaches. Rats were adrenalectomized (ADX), and 7 days after this procedure the intradermal injection of B1 receptor agonist des‐Arg9‐BK produced a significant increase in the paw volume, while only a weak effect was observed in sham‐operated animals. A similar increase in the contractile responses mediated by B1 agonist des‐Arg9‐BK was also observed in the rat portal vein in vitro. Chemical ADX performed with mitotane (a drug that reduces corticosteroid synthesis) produced essentially the same up‐regulation of B1 receptors as that observed in ADX rats. The modulation of B1 receptor expression was evaluated by ribonuclease protection assay, employing mRNA obtained from the lungs and paw of ADX rats. Additionally, both paw oedema and contraction of portal vein mediated by B1 agonist des‐Arg9‐BK in ADX rats, were markedly inhibited by treatment with dexamethasone, or COX‐2 inhibitor meloxican, or with the NF‐κB inhibitor PDTC. Interestingly, the same degree of inhibition was achieved when the animals were treated with a combination of submaximal doses of dexamethasone and PDTC. The involvement of NF‐κB pathway was further confirmed by mobility shift assay using nuclear extracts from lung, paw and heart of ADX rats. It was also confirmed that the treatment of ADX rats with dexamethasone, PDTC or dexamethasone plus PDTC completely inhibit NF‐κB activation caused by absence of endogenous glucucorticoid. Together, the results of the present study provide, for the first time, molecular and pharmacological evidence showing that B1 kinin receptor expression can be regulated through endogenous glucocorticoids by a mechanism dependent on NF‐κB pathway. Clinical significance of the present findings stem from evidence showing the importance of B1 kinin receptors in the mediation of inflammatory and pain related responses.

Effects of circuit-based exercise programs on the body composition of elderly obese women

Aim The aim of this study was to investigate the impact of circuit-based exercise on the body composition in obese older women by focusing on physical exercise and body weight (BW) gain control in older people. Methods Seventy older women (>60 years old) voluntarily took part in the study. Participants were randomized into six different groups according to body mass index (BMI): appropriate weight (AW) control (AWC) and trained (AWT) groups, overweight (OW) control (OWC) and trained (OWT) groups, and obesity (O) control (OC) and trained (OT) groups. The exercise program consisted of 50 minutes of exercise three times per week for 12 weeks. The exercises were alternated between upper and lower body using rest between sets for 40 seconds with intensity controlled by heart rate (70% of work). The contraction time established was 5 seconds to eccentric and concentric muscular action phase. The following anthropometric parameters were evaluated: height (m), body weight (BW, kg), body fat (BF, %), fat mass (FM, kg), lean mass (LM, kg), and BMI (kg/m2). Results The values (mean ± standard deviation [SD]) of relative changes to BW (−8.0% ± 0.8%), BF (−21.4% ± 2.1%), LM (3.0% ± 0.3%), and FM (−31.2% ± 3.0%) to the OT group were higher (P < 0.05) than in the AWT (BW: −2.0% ± 1.1%; BF: −4.6% ± 1.8%; FM: −7.0% ± 2.8%; LM: 0.2% ± 1.1%) and OWT (BW: −4.5% ± 1.0%; BF: −11.0% ± 2.2%; FM: −16.1% ± 3.2%; LM: −0.2% ± 1.0%) groups; additionally, no differences were found for C groups. While reduction (P < 0.03) in BMI according to absolute values was observed for all trained groups (AWT: 22 ± 1 versus 21 ± 1; OWT: 27 ± 1 versus 25 ± 1, OT: 34 ± 1 versus 30 ± 1) after training, no differences were found for C groups. Conclusion In summary, circuit-based exercise is an effective method for promoting reduction in anthropometrics parameters in obese older women.

Lovastatin decreases the synthesis of inflammatory mediators in the hippocampus and blocks the hyperthermia of rats submitted to long-lasting status epilepticus

Statins may act on inflammatory responses, decreasing oxidative stress and also reducing temperature after a brain ischemic insult. Previous data have indicated that statins protect neurons from death during long-lasting status epilepticus (SE) and attenuate seizure behaviors in animals treated with kainic acid. In this context, the study described here aimed to investigate the effect of lovastatin on body temperature and on mRNA expression levels of hippocampal cytokines such as interleukin-1β, interleukin-6, tumor necrosis factor α, and kinin B1 and B2 receptors of rats submitted to pilocarpine-induced SE. Quantitative real-time polymerase chain reaction showed a significant decrease in mRNA expression of interleukin-1β, interleukin-6, tumor necrosis factor α, and kinin B1 receptor in animals with SE treated with lovastatin, compared with untreated animals with SE (P<0.001). Lovastatin also reduced SE-induced hyperthermia, indicating that mechanisms related to brain protection are triggered by this drug under conditions associated with acute excitotoxicity or long-lasting SE.

Ischemia/reperfusion is an independent trigger for increasing myocardial content of mRNA B-type natriuretic peptide

This study aims to determine whether a relation exists between ischemia/reperfusion and myocardial B-type natriuretic peptide (BNP) mRNA expression independent of variations in intracavitary diastolic volume and consequently, of cardiomyocyte stretching. Twenty-three rats were subjected to the following conditions: control (C), 15 min of ischemia (I15), or ischemia plus 15 (R15), 30 (R30), or 45 (R45) min of reperfusion in the in situ hearts. Isolated hearts of sixteen additional rats (sham, n = 8; occlusion, n = 8) were perfused for studies in the absence of ventricular distension. All hearts were divided in two segments (ischemic and nonischemic). Ventricular distension was avoided by excluding the atria and mitral valves. In both experiments, BNP mRNA was quantified by real-time polymerase chain reaction in both nonischemic and ischemic regions. In the in situ hearts, myocardial BNP mRNA values at R15 (4.24 ± 0.75) in the ischemic region were higher than in other groups (C: 1.43 ± 0.81, P = 0.044; I15: 3.05 ± 0.62, P = 0.048; R30: 0.76 ± 0.84, P = 0.001; R45: 1.47 ± 0.60, P = 0.046, [analysis of variance]). In isolated hearts without ventricular distension, myocardial BNP mRNA (arbitrary units) content at R15 in ischemic regions (4.54 ± 0.26) was greater than in nonischemic regions in both occlusion (3.51 ± 0.20, P < 0.001) and sham (3.38 ± 0.25, P = 0.0001 and 3.47 ± 0.19, P = 0.0001) groups. The present data show that ischemia/reperfusion is responsible for increased BNP mRNA myocardial content independent of changes of ventricular cavity diastolic volume.

Akt pathway activation and increased neuropeptide Y mRNA expression in the rat hippocampus: Implications for seizure blockade

The aim of this study was to analyze the expression of survival-related molecules such Akt and integrin-linked kinase (ILK) to evaluate Akt pathway activation in epileptogenesis process. Furthermore, was also investigated the mRNA expression of neuropeptide Y, a considered antiepileptic neuropeptide, in the pilocarpine-induced epilepsy. Male Wistar rats were submitted to the pilocarpine model of epilepsy. Hippocampi were removed 6h (acute phase), 12h (late acute), 5d (silent) and 60d (chronic) after status epilepticus (SE) onset, and from animals that received pilocarpine but did not develop SE (partial group). Hippocampi collected were used to specify mRNA expression using Real-Time PCR. Immunohistochemistry assay was employed to place ILK distribution in the hippocampus and Western blot technique was used to determine Akt activation level. A decrease in ILK mRNA content was found during acute (0.39+/-0.03) and chronic (0.48+/-0.06) periods when compared to control group (0.87+/-0.10). Protein levels of ILK were also diminished during both periods. Partial group showed increased ILK mRNA expression (0.80+/-0.06) when compared with animals in the acute stage. Silent group had ILK mRNA and immunoreactivity similar to control group. Western blot assay showed an augmentation in Akt activation in silent period (0.52+/-0.03) in comparison with control group (0.44+/-0.01). Neuropeptide Y mRNA expression increased in the partial group (1.67+/-0.22) and in the silent phase (1.45+/-0.29) when compared to control group (0.36+/-0.12). Results suggest that neuropeptide Y (as anticonvulsant) might act in protective mechanisms occurred during epileptic phenomena. Together with ILK expression and Akt activation, these molecules could be involved in hippocampal neuroprotection in epilepsy.