Ajala, A.; Rangel, M.; Febba, A.; Strufaldi, Mw.; Sesso, R.; Casarini, De.; Franco, M. Author Information

Diabetes mellitus (DM) is characterised by alterations in the intrarenal renin-angiotensin system (RAS). Insulin treatment may reverse these changes by an unknown mechanism. We aimed to verify the association between somatic ACE with 136 kDa (sACE) and N-domain ACE with 69 kDa (nACE) from Wistar (W) rat tissue with DM.Three groups were studied: control (CT), insulin treated diabetic (DT) and untreated (D). ACE activity was determined using Hippuryl-His-Leu and Z-Phe-His-Leu as substrates. In D group, urine ACE activity increased for both substrates when compared with CT and DT, despite the decreased activity of renal tissues. Immunostaining of renal tissue demonstrated that ACE is more strongly expressed in the proximaltubule of D than in the same nephron portion in the other groups. Angiotensin (Ang) 1-7 and Ang II are less expressed in DT group when compared with CT and D. Ang II levels decreased in the D and DT groups showed when compared to the control. Ang 1-7 was detected in all studied groups with low levels in DT.The modulation of angiotensin peptides suggests that sACE, nACE, ACE 2 and NEP could have important functions in renal RAS regulation through a counter-regulatory mechanism to protect the kidney in diabetes mellitus.

The influence of drug concentrations on the development of persistent posttransplant hyperlipidemia was investigated in 82 patients who received cyclosporin A (CsA) and prednisone plus sirolimus (SRL) (52) or azathioprine (AZA) (30) during the first year after transplantation. Blood levels of CsA and SRL, daily doses of AZA and prednisone, and cholesterol, triglyceride, and glucose concentrations were determined during each visit (pretransplant and 30, 60, 90, 120, 180, and 360 days posttransplant). Persistent hyperlipidemia was defined as one-year average steady-state cholesterol (CavCHOL) or triglyceride (CavTG) concentrations above 240 and 200 mg/dL, respectively. Mean cholesterol and triglyceride concentrations increased after transplantation (P < 0.01) and were higher in patients receiving SRL compared to AZA (P < 0.001). Patients receiving SRL showed a significantly higher number of cholesterol (>229 or >274 mg/dL) and triglyceride (>198 or >282 mg/dL) determinations in the upper interquartile ranges. CsA and SRL interquartile ranges correlated with cholesterol concentrations (P = 0.001) whereas only SRL interquartile ranges correlated with triglyceride concentrations (P < 0.0001). Only pretransplant cholesterol concentration >205 mg/dL was independently associated with development of persistent hypercholesterolemia (CavCHOL >240 mg/dL, relative risk (RR) = 20, CI 3.8-104.6, P = 0.0004) whereas pretransplant triglyceride concentration >150 mg/dL (RR = 7.2, CI 1.6-32.4, P = 0.01) or >211 mg/dL (RR = 19.8, CI 3.6-107.9, P = 0.0006) and use of SRL (RR = 3, CI 1.0-8.8, P = 0.0049) were independently associated with development of persistent hypertriglyceridemia (CavTG >200 mg/dL). Persistent hypercholesterolemia was more frequent among patients with higher pretransplant cholesterol concentrations and was dependent on both CsA and SRL concentrations. Persistent hypertriglyceridemia was more frequent among patients with higher pretransplant triglyceride concentrations and was dependent on SRL concentrations.

Heart Failure is a clinical syndrome characterized by skeletal muscle abnormalities induced early fatigue and exercise intolerance (EI). We investigated the muscular phenotype in mice lacking both α2A/2C AR subtypes (KO), which develop cardiomyopathy (CM) induced by sympathetic hyperactivity with increased mortality rate at 5 mo of age. A cohort of KO and wild type (WT) mice were studied at 3 and 5 mo of age (early and moderate stage of CM). Exercise capacity was measured using a graded treadmill protocol. Heart rate (HR) by tail cuff. Fractional shortening (FS) by echocardiography and noradrenaline (NE) levels by HPLC. The fiber typing (FT) and cross-sectional area (CSA) by mATPase histochemistry. At 3 mo, KO mice presented higher HR (+15%) and decreased FS (-20%). At 5 mo, FS was even more deteriorated (-30%) followed by HR (+20%) and NE (+53%) increased in KO vs WT. In this fase, KO displayed EI accompanied by an increased FT IIa (+8%) and decreased FT intermediary (-82%) in soleus. Collectively, these preliminary results indicate that in early stage of CM KO mice presented ventricular dysfunction without muscle abnormalities. Furthermore, in moderate stage of CM, the ventricular dysfunction worsening is followed by morphological and functional alterations within skeletal muscle. Supported by FAPESP

Objective: To get some additional insight on the mechanisms of the effect of salt intake on body weight.Design and methods: Rats were fed a tow (LSD), normal (NSD), or high (HSD) salt diet. In a first set, body weight, tail-cuff blood pressure, fasting plasma thyroid-stimulating hormone, triiodothyronine, L-thyroxine, glucose, insulin, and angiotensin II were measured. Angiotensin II content was determined in white and brown adipose tissues. Uncoupling protein 1 expression was measured in brown adipose tissue. In a second set, body weight, food intake, energy balance, and plasma leptin were determined. In a third set of rats, motor activity and body weight were evaluated.Results: Blood pressure increased on HSD. Body weight was similar among groups at weaning, but during adulthood it was tower on HSD and higher on LSD. Food intake, L-thyroxine concentration, uncoupling protein 1 expression and energy expenditure were higher in HSD rats, white non-fasting leptin concentration was lower in these groups compared to NSD and LSD animals. Plasma thyroid-stimulating hormone decreased on both HSD and LSD white plasma glucose and insulin were elevated only on LSD. A decrease in plasma angiotensin II was observed in HSD rats. On LSD, an increase in brown adipose tissue angiotensin II content was associated to decreased uncoupling protein 1 expression and energy expenditure. In this group, a low angiotensin II content in white adipose tissue was also found. Motor activity was not influenced by the dietary salt content.Conclusions: Chronic alteration in salt intake is associated with changes in body weight, food intake, hormonal profile, and energy expenditure and tissue angiotensin II content. (C) 2005 Elsevier B.V. All rights reserved.

Mice lacking á2A/á2C adrenoceptors (KO), which develop sympathetic (SNS) hyperactivity induced cardiomyopathy (CM), were used to assess the control of systemic and cardiac SNS and renin angiotensin system (RAS) during the development of heart failure (HF). KO and wild type(WT) mice were studied at early (3mo) and late stage(7mo) of CM. Fractional shortening (FS) was evaluated by echocardiography, cardiac myocyte width (CW) and collagen content (CC) by microscopy. Ang II by immunohistochemistry, plasma renin activity (PRA) by RIA, ACE activity by fluorometric assay. Noradrenaline(NE) levels by HPLC. At 3 mo, KO displayed decreased FS(−20%) accompanied by increased CW (+23%), cardiac ACE activity (+24%), Ang II expression (+66%), and NE levels (+28%). Similarly, plasma NE (+29%) and PRA(+60%) were also increased in KO vs WT. At 7 mo, KO displayed further deteriorated FS (−33%) with increased CW (+27%), CC (+3 fold)and cardiac Ang II levels(+68%). Conversely, both cardiac NE (−38%) and cardiac ACE activity (−10%) decreased in KO vs WT. PRA was also decreased (−3 fold), whereas plasma NE (+58%) and serum ACE activity (+32%) increased in KO vs WT. Collectively, these results show that in early stage of CM,the cardiac dysfunction is associated to overt systemic and cardiac activation of SNS and RAS. In contrast, in late stage the further compromised cardiac function is restricted to increased systemic NE, and cardiac AII levels. Supported by FAPESP.

The need to adapt optimal conditions of sirolimus blood level monitoring in laboratories led us to optimize an high-performance liquid chromatography-ultraviolet method and compare the elution performances using the mobile phase A, 68% MeOH/2% acetonitrile (ACN)/30% H(2)O and mobile phase B, 30% MeOH/42% ACN/28% H(2)O. Samples were assayed with 1-chlorobutane, redissolved in MeOH/water and injected onto a C-18 column at 50 degrees C. The assay achieved sensitivity of 2.5-150 ng/ml (CV = 10.6%) and recovery of 92-103.6%. The intra- and interassay precisions ranged from 3.3% to 13% and from 5.9% to 15% for quality controls of 7.5, 60 and 120 ng/ml. The mobile phase A was unable to elute and recover sirolimus and internal standard in the expected retention time and concentration. Under our working conditions, the assay was precise, accurate and sensible, stressing the importance of establishing for the best working conditions according to the staff and demands of the laboratory.

1. The present study sought to evaluate cardiovascular adaptations, such as blood pressure (BP), heart rate (HR) and cardiac hypertrophy, to resistance training (RT) in a rat model. 2. The training protocol consisted of four sets of 10-12 repetitions of the squat exercise performed at 65-75% of one repetition maximum (1RM) over 4 weeks. Animals were randomly divided into three groups: control (n = 8, CO), electrically stimulated (n = 8, ES) and trained (n = 8, TR; also electrically stimulated). Blood pressure and HR were measured by a direct method in conscious rats after the training period. 3. All groups began with similar 1RM and 1RM/bodyweight (BW) ratio, however, at the end of the protocol only the TR group was different from the beginning (56% and 50%, respectively; both P < 0.01). The CO and ES groups had similar values for cardiac chambers weight/BW ratio, HR and diastolic, systolic and mean BP. Left ventricular hypertrophy (LVH) determined by the left ventricle (LV) weight/BW ratio was increased in the TR group (12%) when compared to CO (P < 0.01) or ES groups (P < 0.01). No changes were found in the weights of the atrium or right ventricle. Diastolic (14%) and mean BP (13%) were lower in the TR group (P < 0.05), whereas systolic BP and HR remained unchanged. 4. Collectively these results demonstrate that the rat RT model used is associated with significant development of cardiac hypertrophy and lowering of resting BP. These cardiovascular adaptations seem to a result of the training exercise and not influenced by stress since circulating catecholamine levels and adrenal gland weights remained unchanged in all groups.

Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and peaked on days 8 and 9. Measurement of [Ca2+]i in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application, but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture medium, and respond to bradykinin application with a transient increase in [Ca2+]i. However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1–M3 muscarinic acetylcholine receptors, indicating crucial functions of the B2BKR during neuronal differentiation.

Background Angiotensin I-converting enzyme (ACE) is a protein containing two active sites, called N- and C-domains, according to their position in the protein. Aim The aim of the present study was to verify whether the expression of the N-domain ACEs detected in the urine of Wistar and spontaneously hypertensive (SHR) rats was restricted to the kidney. Methods Adrenal, aorta, heart, liver, lung, kidney and testicle tissue from Wistar rats and spontaneously hypertensive rats were homogenized in assay buffer and analyzed by gel filtration, Western blotting and radio-immunoassay. Results Two peaks (at 136 and 69 kDa) with ACE activity upon ZPhe-His-Leu were separated by gel filtration from homogenate tissues of Wistar rats, in contrast with the tissue from hypertensive rats, which showed ACE forms of 96 and 69 kDa. The bands detected by Western blotting for all studied tissue from Wistar and spontaneously hypertensive rats showed a correspondence with the two peaks containing ACE activity detected in the polyacrylamide gel slices. Angiotensin II levels were increased in hypertensive rat tissue when compared with Wistar rat tissues. In addition, captopril 3 μmol/l inhibited the enzymic activity, where the Km was in the order of mmol/l and μmol/l using hippuryl-His–Leu and Abz-Ser-Asp-Lys(Dnp)Pro-OH as substrates, respectively. All tissues from Wistar rats presented ACE with 136 kDa, similar to somatic ACE, and N-domain ACE with 69 kDa. In the same tissue of spontaneously hypertensive rats, 96 and 69 kDa N-domain ACEs were detected. Conclusions Our results demonstrated that N-domain ACEs were not exclusively produced in the kidney and excreted in the urine; they were expressed in all tissue studied, suggesting that these enzymes could influence local angiotensin II production, contributing to organ-specific regulation.

Renin-angiotensin system activation is recognized to play an important role in atherosclerosis. This study aimed to verify the antiatherosclerotic effects of ACE inhibition on an experimental model of diabetes and hypercholesterolemia. Diabetes was induced in New Zealand male rabbits with a single dose of alloxan (100 mg/kg, i.v.), and, according to plasma glucose levels obtained after 1 week, the animals were divided into 2 groups (> or =250 mg/dL or <250 mg/dL). Each group was randomly assigned to receive or not quinapril (30 mg/d) added to a 0.5% cholesterol-enriched diet. Animals with high glucose levels at 1 week and that remained high after 12 weeks presented higher triglyceride levels (P < 0.02 versus basal). Those initially hyperglycemic but presenting <250 mg/dL glucose at the end of study formed an additional group. Plasma ACE activity was lower in quinapril-treated animals (P < 0.01 versus untreated groups). However, aorta intima/media ratio and intima area were lower only in the subgroups of quinapril-treated animals with low glucose levels (P < 0.05). Our results support the hypothesis that high plasma glucose may abolish the antiatherosclerotic effect of ACE inhibitors.

Studies were conducted in rats to determine the effect of maternal diabetes and the consequent hyperglycemia on cardiovascular function in the offspring. Diabetes was induced in pregnant Wistar rats through streptozotocin injection (50 mg/kg). Cardiovascular parameters were measured in 2-mo-old offspring animals of diabetic (OD, n=12) and control rats (OC, n=8). Arterial pressure (AP), heart rate (HR), baroreflex sensitivity, and vascular responsiveness to phenylephrine (PH) and sodium nitroprusside (SN) were measured. Angiotensin-converting enzyme (ACE) activity in heart, kidney, and lung was determined. OD rats exhibited increases in systolic AP (138+/-8 vs. 119+/-6 mmHg, OD vs. OC), with no change in HR (342+/-21 vs. 364+/-39 beats per minute (bpm), OD vs. OC). The reflex tachycardia elicited by SN was reduced in OD rats, as indicated by the slope of the linear regression (-2.2+/-0.4 vs. -3.6+/-0.8 bpm/mmHg, OD vs. OC). Vascular responsiveness to PH was increased 63% in OD rats compared with OC. OD rats showed increases in ACE activity in heart, kidney, and lung (1.13+/-0.24, 3.04+/-0.86, 40.8+/-8.9 vs. 0.73+/-0.19, 1.7+/-0.45, 28.1+/-6 nmol His-Leu.min-1 mg protein-1, OD vs. OC). Results suggest that diabetes during pregnancy affects cardiovascular function in offspring, seen as hypertension, baroreflex dysfunction, and activation of tissue renin-angiotensin system.

The effect of swimming training (ST) on vagal and sympathetic cardiac effects was investigated in sedentary (S, N = 12) and trained (T, N = 12) male Wistar rats (200-220 g). ST consisted of 60-min swimming sessions 5 days/week for 8 weeks, with a 5% body weight load attached to the tail. The effect of the autonomic nervous system in generating training-induced resting bradycardia (RB) was examined indirectly after cardiac muscarinic and adrenergic receptor blockade. Cardiac hypertrophy was evaluated by cardiac weight and myocyte morphometry. Plasma catecholamine concentrations and citrate synthase activity in soleus muscle were also determined in both groups. Resting heart rate was significantly reduced in T rats (355 +/- 16 vs 330 +/- 20 bpm). RB was associated with a significantly increased cardiac vagal effect in T rats (103 +/- 25 vs 158 +/- 40 bpm), since the sympathetic cardiac effect and intrinsic heart rate were similar for the two groups. Likewise, no significant difference was observed for plasma catecholamine concentrations between S and T rats. In T rats, left ventricle weight (13%) and myocyte dimension (21%) were significantly increased, suggesting cardiac hypertrophy. Skeletal muscle citrate synthase activity was significantly increased by 52% in T rats, indicating endurance conditioning. These data suggest that RB induced by ST is mainly mediated parasympathetically and differs from other training modes, like running, that seems to mainly decrease intrinsic heart rate in rats. The increased cardiac vagal activity associated with ST is of clinical relevance, since both are related to increased life expectancy and prevention of cardiac events.

P321 Aims: In a phase III trial black patients receiving CSA and prednisone required a higher dose (5 mg) of SRL to show reduced incidence of acute rejection (AR) compared to azathioprine. Due to the large inter-individual variability in SRL concentrations, the purpose of this prospective trial was to define target SRL whole blood trough concentrations in this high-risk population. Methods: Kidney transplant recipients of black ethnicity received CSA 8-10 mg/kg/day, prednisone (30 mg), and a 15 mg loading dose followed by 5 mg fixed daily doses of SRL till day 7 when they were randomized to target SRL trough blood concentration of 8-12(GI) or 15-20(GII) ng/mL (HPLC). Results: 70 patients (64 living/6 cadaveric, mean age 34.411.6 years, 67% male, mean HLA mm 3.31.1) were randomized to GI (N=34) and GII (N=36). At 1, 3, and 6 months, mean CSA concentrations were 178±85 vs. 163±89, 113±96 vs. 98±55, and 77±57 vs. 66±36 ng/mL (ns), respectively. Accordingly, mean SRL trough concentrations were 17.7±9.2 vs. 19.2±9.8 (ns), 12.1±5.4 vs. 20.7±8.2 (p< .001), 10.6±6.1 vs. 17.9±5.5 ng/mL (p<0.001). At 12 months, mean prednisone doses were comparable (7.4±5.0 vs. 8.1±5.4 mg/day, ns). The overall incidence of biopsy-proven acute rejection at month 12 was 13% [GI: 17 % (1IA, 2 IIA and 2 IB) vs. GII: 8% (1 IA, 1 IB and 2 IIB), p=0.482]. There were no deaths and 3 graft losses (GI=1; GII=2). There were no differences in mean creatinine (1.6±0.5 vs. 1.6±1.1 mg/dL, ns), cholesterol (250±60 vs. 250±64 mg/dL) or triglyceride concentrations (279±156 vs. 269±180 mg/dL), and hemoglobin values (14.0±2.5 vs. 13.4±3.0 g/dL, p=.0376). There were no differences comparing the incidence of infections (56%), thrombocytopenia (15%), leukopenia (6%) or in the number of SAEs in each group (GI=23 vs. GII=41, ns). Conclusions: High-risk transplant patients of black ethnicity showed very low incidences of acute rejection and good graft function using a SRL/CSA concentration-controlled strategy and early reduction in cyclosporine exposure. SRL target therapeutic concentration early after transplantation appears to be at least 15 ng/mL.

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides were used for the analyses of the S(3) to S(1)' subsites of the somatic angiotensin I-converting enzyme (ACE). Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to design selective substrates for the C-domain. Initially, we defined the S(1) specificity by preparing a library with the general structure Abz-GXXZXK(Dnp)-OH [Abz = o-aminobenzoic acid, K(Dnp) = N(epsilon)-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19 natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P(1) position had higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P(1) so we could define the C-domain selectivity of the S(1)', S(2), and S(3) subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domain selectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, which demonstrated the highest selectivity for the recombinant ACE C-domain (k(cat)/K(m) = 36.7 microM(-1) s(-1)) versus the N-domain (k(cat)/K(m) = 0.51 microM(-1) s(-1)). The substrate binding of Abz-LFK(Dnp)-OH with testis ACE using a combination of conformational analysis and molecular docking was examined, and the results shed new light on the binding characteristics of the enzyme.

Bersanetti, Patricia Alessandra; Andrade, M. C.C.; Juliano, M. A.; Casarini, D. E.; Sturrock, E. D.; Juliano, L.; Carmona, A. K. Author Information