Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals

We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 ± 5.0 vs 16.1 ± 6.0 percent in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes.

Relationship of cyclosporin and sirolimus blood concentrations regarding the incidence and severity of hyperlipidemia after kidney transplantation

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.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37°C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 æM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 æM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 æM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Swimming training increases cardiac vagal activity and induces cardiac hypertrophy in rats

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 percent 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 percent) and myocyte dimension (21 percent) were significantly increased, suggesting cardiac hypertrophy. Skeletal muscle citrate synthase activity was significantly increased by 52 percent 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.

Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 +/- 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 +/- 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 +/- 3.1 and 3.9 +/- 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II

Purification and characterization of endopeptidase H2, a kinin inactivating serine proteinase (kininase) from human urine

1. A kinin-inactivating chymotrypsin-like serine-endopeptidase was purified 202-fold from human urine by DEAE-cellulose chromatography, gel filtration, DEAE/HPLC chromatography and affinity chromatography. It hydrolyzed bradykinin at the Phe5-Ser6 peptide bond at a rate of 1.090 mumol min-1 mg protein-1 at pH 8.0 and 37 degrees C. The molecular weight of this endopeptidase H2, estimated by SDS-polyacrylamide gel electrophoresis and by gel filtration, was 60 kDa, and its optimum pH for bradykinin hydrolysis was near 8.5. 2. Bradykinin inactivating activity was inhibited 100 per cent by the serine-proteinase inhibitor PMFS (1 mM) and the chymotrypsin inhibitor TPCK (5 mM). Reagents such as 2-mercaptoethanol (3 mM) and pOH-mercuribenzoate (3 mM) inhibited the enzyme by 100 per cent and 67 per cent , respectively. 3. Endopeptidase H2 hydrolyzes the Phe-Ser bond of peptides related to bradykinin and its activity appears to be limited to peptide chains of < or = 18 amino acid residues since it does not hydrolyze BAM 22, peptide E or kininogen. 4. The molecular size and inhibition profile suggested that endopeptidase H2 differs from the serine-proteinases previously described in rat liver, rat hepatic endothelium, rat and rabbit brain. 5. The physiological role of endopeptidase H2 may be a link between the kinin and neuropeptide systems in the control of water-electrolyte balance

Serum catecholamine levels determined by high performance liquid chromatography coupled with electrochemical detection

Este trabalho apresenta um método rápido para a quantificaçäo de catecolaminas utilizando a técnica de cromatografia líquida de fase reversa acoplada à detecçäo eletroquímica. Separaçäo isocrática rápida foi obtida empregando como fase móvel a soluçäo: 0,02M de fosfato de sódio dibásico, 0,02M de ácido nítrico, pH 2,64, metanol a 10 por cento, 0,12mM de EDTA sódico e 556 mg/L de ácido heptanosulfônico. Delineou-se o procedimento de preparaçäo das amostras com extraçäo das monoaminas em alumina, para melhorar a recuperaçäo e diminuir fatores de diluiçäo. O tempo total de análise é de 15 minutos, com boa separaçäo dos picos de monoaminas. O limite de detecçäo obtido para as monoaminas séricas é de 40 a 50 pg/mL, com uma taxa de recuperaçäo de 70-75 por cento.

Endopeptidase and carboxypetidase activities in human urine which hydrolyze bradykinin

We have fractionated the bradykinin inactivating activity of human urine by stepwise elution chromatography on DEAE-cellulose and recovered 95% of the inactivating activity and 29% ofd the protein (absorbance at A280 nm). Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin convertingg activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradyykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides. Cleavage sites demonstrated for Fractioons A through G were: Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pho7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G). The relative molecular weight of the bradykininase activity present in each fraction, determined by gel filtration, was: 16 kDa (A), 70 kDa (B), 60 kDa (B) (C), 88 kDa (D), 230 kDa (E) and 49 kDa (G). Bradykinin inactiivating activity was inhibited 50--100% by 3 mM EDTA (A,B,D,E adn G), 1 mMM 2-mercaptoethanol (A,B,C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Xn2+ (C), 60 uM BPP5a and 40 uM BPP9a (D), 0.1 uM phosphoramidon (E) and 3 mM sodium p-hydroxyymercuribenzoate (G). The properties of some of these bradykinin inactivating activities correspondend to enzymes previously described in urine and tissues: carboxypeptidases (Fractions A and B, angiotensin I converting enzyme (Fraction d), neutral endopeptidase (Fraction E). However, the chymotrypsin-like activity of fractions C and F and the prolylendopeptidase activity of fraction G have not been described before in urine and they being purified in order to obtain a more accurate characterization