Central-to-axial chirality induction in biphenyl chiroptical probes for the stereochemical characterization of chiral primary amines.

Flexible biphenyls have been applied as efficient and practical chiroptical probes for absolute configuration assignment to chiral primary amines. The mechanism of the central-to-axial chirality transfer from the amine moiety to the conformationally flexible biphenyl system has been determined by NMR and computational studies. This allowed proposing a general non-empirical rule in order to establish, simply by looking at the sign of the 250 nm A band in the ECD spectrum of the biphenyl derivative, the torsion of the biphenyl and thus the absolute configuration of the amine. The method proved to be very reliable and sensitive, allowing treatment of samples on the µmol scale and permitting the simultaneous determination of the amine sample's absolute configuration and enantiopurity.

Resistance exercise training ameliorates chronic kidney disease outcomes in a 5/6 nephrectomy model.

Chronic kidney disease (CKD) is defined by decreased glomerular filtration rate (GFR) or increased albumin excretion leading to renal injury. However, exercise training is an important non-pharmacological intervention that ameliorates and protects against Diabetes Mellitus, cardiovascular disease, and CKD. AIM: Our aim was to evaluate the capability of resistance exercise training (RET) to improve CKD outcomes and the contribution of the renal and muscular Akt/mTOR signaling pathway for RET beneficial effects on a CKD model. MAIN METHODS: Male Wistar rats were subjected to RET, followed for 10 weeks, and randomly divided into 5 groups: Sham: Sham-operated; sedentary and nephrectomy (5/6Nx) (SNS); exercising post-5/6Nx (SNE); exercising pre-5/6Nx (ENS); exercising pre- and post-5/6Nx (ENE). The systolic blood pressure (BP) was measured. Creatinine, proteinuria, and blood urea nitrogen (BUN) were evaluated. After euthanasia Renal and muscular Akt/mTOR signaling pathways were analyzed. KEY FINDING: Our study showed that the SNS presented renal injury, hypertension, weight and muscular mass loss and a higher mortality rate. SNS group also decreased renal IL-10 and increased TNF-alfa and TGF-Beta. Renal AKT, mTOR, and rpS6 pathway were increased, PTEN was decreased on SNS. And muscular Akt and mTOR were decreased on SNS. SIGNIFICANCE: The RET before and after the 5/6Nx ameliorates all these parameters mentioned above, suggesting that RET is a good non-pharmacological approach to diminish complications frequently found in CKD. We also suggest that the AKT-m-TOR pathway can play an important role in these beneficial outcomes of RET on the CKD animal model.

Inhibition of the P2X7 receptor improves renal function via renin-angiotensin system and nitric oxide on diabetic nephropathy in rats.

AIM: To evaluate the effects of P2X7 receptor blockade on renin-angiotensin system (RAS) in rats with diabetic nephropathy (DN). MAIN METHODS: Wistar rats were unilaterally nephrectomized and received streptozotocin for diabetes mellitus (DM) induction; control animals (CTL) received the drug vehicle. The animals were submitted to P2X7 receptor silencing, forming the group (DM + siRNA). The animals were placed in metabolic cages for data collection and evaluation of renal function; at the end of the protocol, the kidney was removed for analysis of P2X7, renin, angiotensin-converting enzyme (ACE), ACE2, angiotensin, thiobarbituric acid reactive substance levels (TBARS), nitric oxide (NO) and qualitative histological. KEY FINDINGS: The metabolic profile was attenuated in DM + siRNA vs. DM and there was a significant improvement in creatinine, urea and proteinuria levels in the same group. Renin expression was significantly decreased in DM + siRNA vs. DM. ACE and ACE2 were significantly reduced in DM + siRNA vs. DM. TBARS levels were decreased and NO showed an increase in DM + siRNA vs. DM, both significant. All histological alterations were improved in DM + siRNA vs. DM. SIGNIFICANCE: Data have shown that although silencing of the P2X7 receptor did not decrease fasting glucose, it promoted an improvement in the metabolic profile and a significant recovery of renal function, revealing a protective action by the inhibition of this receptor. This effect must have occurred due to the inhibition of RAS and the increase of NO, suggesting that the use of P2X7 receptors inhibitors could be used as adjuvant therapy against DN progression.

Up-regulation of renal renin-angiotensin system and inflammatory mechanisms in the prenatal programming by low-protein diet: beneficial effect of the post-weaning losartan treatment.

Previous studies have shown that the renin-angiotensin system (RAS) is affected by adverse maternal nutrition during pregnancy. The aim of this study was to investigate the effects of a maternal low-protein diet on proinflammatory cytokines, reactive oxygen species and RAS components in kidney samples isolated from adult male offspring. We hypothesized that post-weaning losartan treatment would have beneficial effects on RAS activity and inflammatory and oxidative stress markers in these animals. Pregnant Sprague-Dawley rats were fed with a control (20% casein) or low-protein diet (LP) (6% casein) throughout gestation. After weaning, the LP pups were randomly assigned to LP and LP-losartan groups (AT1 receptor blockade: 10 mg/kg/day until 20 weeks of age). At 20 weeks of age, blood pressure levels were higher and renal RAS was activated in the LP group. We also observed several adverse effects in the kidneys of the LP group, including a higher number of CD3, CD68 and proliferating cell nuclear antigen-positive cells and higher levels of collagen and reactive oxygen species in the kidney. Further, our results revealed that post-weaning losartan treatment completely abolished immune cell infiltration and intrarenal RAS activation in the kidneys of LP rats. The prevention of augmentation of angiotensin (Ang II) concentration abolished inflammatory and fibrotic events, indicating that Ang II via the AT1 receptor is essential for pathological initiation. Our results suggest that the prenatal programming of hypertension is dependent on the up-regulation of local RAS and presence of immune cells in the kidney.

Differential sympathetic and angiotensinergic responses in rats submitted to low- or high-salt diet.

The present study was designed to evaluate, in Wistar rats, the effect of high- or low-salt diet on the hemodynamic parameters and on the renal and lumbar sympathetic nerve activity. The renal gene expression of the renin angiotensin system components was also evaluated, aiming to find some correlation between salt intake, sodium homeostasis and blood pressure increase. Male Wistar rats received low (0.06% Na, TD 92141-Harlan Teklad), a normal (0.5% Na, TD 92140), or a high-salt diet (3.12% Na, TD 92142) from weaning to adulthood. Hemodynamic parameters such as cardiac output and total peripheral resistance, and the renal and lumbar sympathetic nerve activity were determined (n=45). Plasma renin activity, plasma and renal content of angiotensin (ANG) I and II, and the renal mRNA expression of angiotensinogen, renin, AT1 and AT2 receptors were also measured (n=24). Compared to normal- and low-salt diet-, high-salt-treated rats were hypertensive and developed an increase (P<0.05) in total peripheral resistance and lumbar sympathetic nerve activity. A decrease in renal renin and angiotensinogen-mRNAs and in plasma ANG II and plasma renin activity was also found in salt overloaded animals. The renal sympathetic nerve activity was higher (P<0.05) in low- compared to high-salt-treated rats, and was associated with an increase (P<0.05) in renal ANG I and II and with a decrease (P<0.05) in AT2 renal mRNA. Plasma ANG I and II and plasma renin activity were higher in low- than in normal-salt rats. Our results show that increased blood pressure is associated with increases in lumbar sympathetic nerve activity and total peripheral resistance in high-salt-treated rats. However, in low-salt-treated rats an increase in the renal sympathetic nerve was correlated with an increase in the renal content of ANG I and II and with a decrease in AT2 renal mRNA. These changes are probably in favor of the antinatriuretic response and the sodium homeostasis in the low-salt group.

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.

High expression of human carboxypeptidase M in Pichia pastoris: purification and partial characterization.

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.

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 (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM 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 microM 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 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates.

A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2, 4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K(m) for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was approximately 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.

Angiotensin converting enzymes from human urine of mild hypertensive untreated patients resemble the N-terminal fragment of human angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE) activity was analyzed in human urine collected from mild hypertensive untreated patients. DEAE-cellulose chromatography using linear gradient elution revealed two forms of angiotensin I-converting enzyme, eluted in the conductivity of 0.75 and 1.25 mS. The fractions of each conductivity were pooled and submitted to direct gel filtration in an AcA-34 column, and the apparent molecular weights of urinary ACEs were estimated as 90 kDa (for ACE eluted in 0.75 mS) and 65 kDa (for ACE eluted in 1.25 mS). Both enzymes have a K(i) of the order of 10(-7) M for the specific inhibitors studied, and are able to hydrolyze luteinizing hormone-releasing hormone and N-acetyl-Ser-Asp-Lys-Pro as described for N-domain ACE. By Western blot analysis, both peaks were recognized by ACE-specific antibody Y4, confirming the molecular weight already described. A plate precipitation assay using monoclonal antibodies to the N-domain of ACE showed that both forms of ACE binds with all monoclonal antibodies to the active N-domain ACE, suggesting that these forms of human urine ACEs resemble the N-fragment of ACE. The HP2 ACE (65 kDa) is similar to low molecular weight (LMW) ACE from normal subjects, and the HP2 ACE (90 kDa) is different from high molecular weight (190 kDa) and LMW (65 kDa) normal ACEs. The 90 kDa ACE could have an important role in development of hypertension. It will be fundamental to elucidate the molecular mechanism responsible for the genesis of this isoform.

Specificity comparison of a serine endopeptidase (SH1) and a serine thiol endopeptidase (STH2) purified from human urine.

In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].

Angiotensin I-converting enzyme isoforms (high and low molecular weight) in urine of premature and full-term infants.

Angiotensin I-converting enzyme (ACE) isoforms in urine from healthy and mildly hypertensive untreated patients have been described in the literature. Healthy subjects have high- and low-molecular-weight ACEs (170 and 65 kDa), whereas mildly hypertensive untreated patients have only low-molecular-weight ACEs (90 and 65 kDa), both of which resemble ACE from the N-terminal domain. Previous studies have shown that ACE is regulated during development, and renal tubules of premature human infants are not completely mature, given that nephrogenesis is not complete until the 36th week of gestation. The aim of the present study was to purify and characterize ACE isoforms from urine of premature and full-term infants and to detect the presence of the N-domain form of ACE during prenatal development. Urine from premature and full-term infants was concentrated in an Amicon concentrator, dialyzed in the same equipment against 50 mmol/L Tris-HCl buffer (pH 8.0) that contained 150 mmol/L NaCl, and submitted to gel filtration on an AcA-34 column equilibrated with the buffer described above. Two peaks (P1 and P2 for premature infants; TP1 and TP2 for full-term infants) with ACE activity on hippuryl-His-Leu (K(m), 3 mmol/L) were detected. All enzymes were Cl(-) dependent and inhibited by captopril and EDTA. The peptides angiotensin-(1-7) and N-acetyl-Ser-Asp-Lys-Pro, described as specific for N-domain ACE, were hydrolyzed by P2 and TP2, which suggests that both enzymes are N-domain ACE. In premature infants, P1 activity with hippuryl-His-Leu was 12-fold lower than P2 activity, but in full-term infants, the difference between TP1 and TP2 was 1.6-fold. Chromatography profiles of urine from premature infants were analyzed on days 1, 3, 7, 14, 21, and 30 after birth. The P1 of ACE was detected around the 21st and 30th days, whereas P2 was detected from day 1. These results suggest that ACE activity is related to renal development and that N-domain ACE as well as full-length ACE is present in urine from premature infants. This may indicate that healthy subjects produce and secrete the N-domain form of ACE even before term development.

Calcium channel blockers as inhibitors of angiotensin I-converting enzyme.

Using ion-exchange chromatography of dialyzed human urine from healthy and hypertensive patients, we detected two peaks of angiotensin I-converting enzyme (ACE) activity on hippuryl-His-Leu eluted at ionic strengths of 0.7 (F1 peak) and 1.25 (F2 peak) mS. These hydrolytic activities decreased gradually in the urine of patients submitted to isradipine treatment, F2 and F1 disappearing after 12 and 24 hours, respectively. By Western blot analysis, the urine fractions corresponding to both peaks from healthy and untreated patients presenting ACE activity and from treated patients (24 hours) without this activity were recognized by an ACE-specific antibody. These results indicated that ACE was present but inhibited in the urine of isradipine-treated patients. In vitro assays with ACE isolated from human urine and guinea pig plasma demonstrated that the enzyme is inhibited by isradipine and other commercially available calcium channel blockers, such as felodipine, nifedipine, and verapamil. A noncompetitive inhibition was observed with all calcium channel blockers studied. In conclusion, these results suggest that besides the primary effect on calcium channels, the more commonly used calcium channel blockers are also ACE inhibitors. The development of efficient calcium channel blockers with higher ACE inhibitory activity could result in interesting bifunctional antihypertensive drugs.

Purification and characterization of a neutral endopeptidase-like enzyme from human urine.

OBJECTIVE: The aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP-like) in human urine and propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine. METHODS: Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column. NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FDQ-EDDnp. The Km was determined using Abz-FDQ-EDDnp as a substrate. The hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC). The mol. wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast. RESULTS: The NEP-like was purified from human urine until homogeneity and presented a mol. wt of 94000. The substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP. For this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined Km was 14 microM. The enzymatic activity was inhibited by thiorphan and phosphoramidon. In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP. CONCLUSION: A NEP-like enzyme was purified from human urine. Based on the mol. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.

Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture.

OBJECTIVE: Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture. METHODS AND RESULTS: Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130000 and ACEInt2, 68000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5. CONCLUSION: The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.

Internally quenched fluorogenic substrates for angiotensin I-converting enzyme.

OBJECTIVE: Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE). DESIGN: We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl]-ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids). METHODS: Hydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (lambda(em) = 420 nm and lambda(ex) = 320 nm). The peptides were obtained by solid-phase synthesis or by classical solution methods. RESULTS: Despite of the blocked C-terminal sequences, the internally quenched bradykinin-related peptides were hydrolysed by ACE. The best substrates for plasma guinea pig ACE were Abz-GFSPFRA-EDDnp and Abz-GFSPFFQ-EDDnp, in which the fluorescence appeared after the first cleavage that occurred at R-A and F-Q bond, respectively. This ACE activity was sensitive to NaCl concentration and the optimum pH is greater than 8.0. Measurements of ACE activity with Hip-His-Leu and Abz-GFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closely (r = 0.959). Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril. CONCLUSIONS: We describe internally quenched fluorogenic substrates for ACE devoid of free C-terminal carboxyl group. They are convenient tools for ACE studies as they permit continuous fluorimetric measurements of the enzymatic activity, even in human serum.

Conformational Studies by Dynamic NMR. 71.(1) Stereodynamics of Triisopropyl(aryl)silanes in Solution and in the Solid State.

A study has been carried out of the conformations of triisopropyl(aryl)silanes (i-Pr)(3)SiAr, (Ar = phenyl, 1-naphthyl, and 2-naphthyl) both as to the orientation of the three isopropyl groups and the conformation about the silicon-aromatic bonds. The report comprises dynamic NMR studies of conformational interconversions in solution and in the solid state as well as molecular mechanics calculations. The barriers for the stereomutation processes measured in the crystalline state were found significantly higher than those in solution.

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.

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% 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.

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% 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% and 67%, 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.