[Use of monoclonal antibodies for determining plasma renin].
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Antibodies, Monoclonal↗
Biomedical subjects
J Menard
Publications and source records attributed to J Menard.
Rat high-molecular-weight kininogen: purification, production of antibodies and demonstration of lack of immunoreactive kininogen in a strain of brown Norway rats.
High-molecular-weight kininogen was purified to apparent homogeneity from Wistar rat plasma by a two-steps chromatographic procedure. 3 mg of kininogen were obtained from 205 ml of plasma. The purified high-Mr kininogen had a bradykinin content of 10.2 micrograms bradykinin equivalents/mg protein. Under denatured and reduced conditions it gave a single band on polyacrylamide gel electrophoresis corresponding to an apparent molecular mass of 110 kDa. Antibodies obtained against rat high-Mr kininogen gave a single precipitation line when tested against rat plasma in double immunodiffusion and in crossed immunoelectrophoresis. Although rat high-Mr kininogen possesses physicochemical properties (molecular mass, kinin content per molecule and amino acid composition) similar to human high-Mr kininogen, its antibodies do not cross-react with human, monkey or rabbit plasma, indicating major interspecies differences in the structure of the molecule. Immunoreactive kininogen of Wistar rats was identical to that of Brown Norway rats from a strain bred in Orleans, France (BN/Orl). However, plasma from a strain of Brown Norway rats bred in Leuven, Belgium (BN/Kat), reported to be deficient in a kinin precursor (Damas, J. and Adam, A. (1980) Experientia 36, 586-587), did not contain immunoreactive material discernible by double immunodiffusion or crossed immunoelectrophoresis.
Amino Acids↗
Inhibition of human renin by synthetic peptides derived from its prosegment.
The primary structure of human preprorenin has recently been determined from its cDNA sequence. It includes a 46-amino acid NH2-terminal prosegment. Six peptides corresponding to the entire prosegment (9-40), except for the NH2-terminal (1-8) and COOH-terminal (41-46) ends have been synthesized. These peptides were tested for their inhibitory effect on human plasma renin activity. Boc-Tyr-Thr-Thr-Phe-Lys-Arg-Ile-Phe-Leu-Lys-Arg-Met-Pro-OMe (where Boc represents t-butoxycarbonyl and OMe represents methoxy) (h Y(9-20) and its fragment Boc-Leu-Lys-Arg-Met-Pro-OMe h (16-20) were the most potent inhibitors with IC50 values of 2 X 10(-4) and 3 X 10(-4)M, respectively. Peptides located near the COOH-terminus were less inhibitory. The inhibitory capacity of h (16-20) was studied further on highly purified human renin acting on either pure human angiotensinogen or a synthetic human tetradecapeptide substrate. In both of these assays its inhibitory potency was about 10-fold greater than that found on plasma renin activity. Peptide h (16-20) was 3-6 times less potent in inhibiting human renin than its mouse counterpart m (15-19) was in inhibiting mouse renin. Kinetic studies carried out with h (16-20) showed a mixed type of inhibition. When human angiotensinogen was used as substrate, Ki and K'i values were 17.7 +/- 3.9 and 2.9 +/- 0.9 microM, respectively. These studies showed that human renin, like mouse renin and pepsin, can be inhibited by peptides derived from its prosegment. In addition, as in the case of pepsin, they suggest that the NH2-terminal part of the prosegment interacts more strongly with the active enzyme.
Amino Acid Sequence↗
Assessment of the antimineralocorticoid effect of RU 28318 in healthy men with induced exogenous and endogenous hypermineralocorticism.
The antimineralocorticoid effect of a single dose of RU 28318, has been assessed in healthy men with exogenous or endogenous hypermineralocorticism. For exogenous hypermineralocorticism induced by ingestion of 9 alpha-fluorohydrocortisone (9 alpha-FHC) and aldosterone infusion, RU 28318 100 mg (9 alpha-FHC ingestion) or 200 mg (aldosterone infusion) was administered, and its effect compared with identical doses of spironolactone or a placebo. For endogenous hypermineralocorticism induced by ingestion of furosemide, RU 28318 100 and 300 mg was tested in comparison with 100 mg spironolactone or placebo. In all 3 studies, both RU 28318 and spironolactone significantly raised the urinary Na/K ratio when compared to placebo administration. No significant difference was apparent between RU 28318 and spironolactone. Thus, a single dose of RU 28318 in man has an antimineralocorticoid effect identical to those produced by the identical molar dose of spironolactone. In addition, the results show that furosemide-induced hyperaldosteronism constitutes a simple and reproducible test for assessing the antimineralocorticoid effect of a drug.
Adult↗
Processing of rat and human angiotensinogen precursors by microsomal membranes.
We have studied the processing of rat and human angiotensinogen precursors by microsomal membranes as a means of determining the number of asparagine-linked oligosaccharide units per angiotensinogen molecule, and thus the utilization of potential sites of N-glycosylation. Glycosylated, processed forms of angiotensinogen were isolated by chromatography on lentil lectin-Sepharose 4B. 35S-Methionine-labeled precursor and processed forms of angiotensinogen were compared with glycosylated and nonglycosylated 35S-methionine-labeled mature forms of angiotensinogen secreted by hepatoma cells, using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. N-Glycosylation of secreted angiotensinogen was inhibited using tunicamycin. For rat angiotensinogen, only 2 of 3 potential sites of N-glycosylation were utilized; in contrast, all 4 potential sites of N-glycosylation of human angiotensinogen were utilized. For neither rat or human angiotensinogen precursor was there any evidence for a prosequence.
Acetylglucosaminidase↗
Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture.
Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0 degree C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.
Cells, Cultured↗
Isolation of renin-producing human cells by transfection with three simian virus 40 mutants.
A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.
Antigens, Viral, Tumor↗
Can inhibition of the renin-angiotensin system have a cardioprotective effect?
The inhibition of the renin-angiotensin system (RAS) has important effects on different parameters of left ventricular function. Chronic inhibition of the RAS avoids hypokalemia and potassium losses by increasing aldosterone release. This potassium-sparing effect is likely to prevent cardiac arrhythmia. Inhibition of the RAS reverses cardiac hypertrophy in renovascular and in spontaneously hypertensive rats (SHR), but not in DOCA salt hypertensive rats. Inhibition of the RAS also reverses the decrease in myocardial contractility, as demonstrated by the reversion of isoenzyme profile of cardiac myosin in renovascular hypertensive rats. In DOCA salt hypertensive rats, RAS inhibition has no effect on blood pressure or on cardiac contractility. Despite its peripheral vasodilatory property, inhibition of the RAS does not increase heart rate in relation to a direct negative chronotropic effect of angiotensin II inhibition and to the absence of activation of the baroreflex system. When RAS is activated, its inhibition has a coronary vasodilatory effect, but this coronary vasodilation is associated with a decrease in perfusion pressure and with an increase in intrinsic cardiac contractility. These concomitant effects lead us to conclude that inhibition of RAS probably has no important beneficial effect on the oxygen demand/oxygen supply ratio in the myocardium distal to the coronary artery stenosis.
Angiotensin-Converting Enzyme Inhibitors↗
Conjugative mapping of pyruvate, 2-ketoglutarate, and branched-chain keto acid dehydrogenase genes in Pseudomonas putida mutants.
Branched-chain keto acid dehydrogenase, an enzyme in the common pathway of branched-chain amino acid catabolism of Pseudomonas putida, is a multienzyme complex which catalyzes the oxidative decarboxylation of branched-chain keto acids. The objective of the present study was to isolate strains with mutations of this and other keto acid dehydrogenases and to map the location of the mutations on the chromosome of P. putida. Several strains with mutations of branched-chain keto acid dehydrogenase, two pyruvate and two 2-ketoglutarate dehydrogenase, were isolated, and the defective subunits were identified by biochemical analysis. By using a recombinant XYL-K plasmid to mediate conjugation, these mutations were mapped in relation to a series of auxotrophic and other catabolic mutations. The last time of entry recorded was at approximately 35 min, and the data were consistent with a single point of entry. Branched-chain keto acid dehydrogenase mutations affecting E1, E1 plus E2, and E3 subunits mapped at approximately 35 min. One other strain affected in the common pathway was deficient in branched-chain amino acid transaminase, and the mutation was mapped at 16 min. The mutations in the two pyruvate dehydrogenase mutants, one deficient in E1 and the other deficient in E1 plus E2, mapped at 22 minutes. The 2-ketoglutarate dehydrogenase mutation affecting the E1 subunit mapped at 12 minutes. A 2-ketoglutarate dehydrogenase mutant deficient in E3 was isolated, but the mutation proved too leaky to map.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Characterization of precursor and secreted forms of human angiotensinogen.
To define the basis of the heterogeneity of angiotensinogen, we have characterized the immunoreactivity of high molecular weight (HMW) and low molecular weight (LMW) plasma angiotensinogen, the angiotensinogen precursor synthesized by cell-free translation, and angiotensinogen secreted by human hepatoma (Hep G2) cells. Angiotensinogen precursor synthesized by rabbit reticulocyte lysate primed with RNA prepared from liver or Hep G2 cells was compared with angiotensinogen secreted by Hep G2 cells by using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). So as to assess the contribution of N-glycosylation of angiotensinogen, Hep G2 cells were incubated in the presence of tunicamycin. Glycosylation of secreted angiotensinogen was further characterized by using chromatography on concanavalin A-Sepharose, digestion with neuraminidase, and treatment with trifluoromethane sulfonic acid. In Sephadex G-200 column chromatography, HMW plasma angiotensinogen eluted just after the column void volume and was clearly separated from LMW angiotensinogen which eluted just before bovine serum albumin. Both HMW and LMW plasma angiotensinogen were shown to bind to monoclonal and polyclonal antibodies raised against pure LMW angiotensinogen. Only one angiotensinogen precursor (mol wt 50,000) was identified by cell-free translation which, after cleavage by renin, was reduced to mol wt 45,600. Angiotensinogen secreted by Hep G2 cells showed electrophoretic heterogeneity (mol wt 53,100-65,400). Tunicamycin-treated Hep G2 cells secreted five discrete forms of angiotensinogen, a predominant form of mol wt 46,200, with other forms (mol wt 46,800, 48,100, 49,200, and 49,600) representing 10% of secreted angiotensinogen. All five forms showed a similar reduction in molecular weight after cleavage by renin. The predominant 46,200-mol wt protein represented nonglycosylated angiotensinogen in that, after cleavage by renin, it had an electrophoretic mobility (mol wt 45,600) identical to the desangiotensin I-angiotensinogen resulting from renin cleavage of the angiotensinogen precursor. The other higher molecular weight forms of angiotensinogen secreted by tunicamycin-treated Hep G2 cells were shown to represent O-glycosylated angiotensinogen in that they were reduced to 46,200 mol wt by treatment with trifluoromethane sulfonic acid. Dexamethasone (10(-7) and 10(-6)M) stimulated angiotensinogen secretion by Hep G2 cells two- to fourfold, both in the absence and presence of tunicamycin. However, a small stimulatory effect of mestranol (10(-7) M) was evident only in the presence of tunicamycin. Neither dexamethasone nor mestranol influenced the electrophoretic pattern (SDS-PAGE) of angiotensinogen secreted by Hep G2 cells. However, when incubation media were chromatographed on Sephadex G-200 with subsequent immunoprecipitation of the column fractions, both dexamethasone and mestranol were shown to stimulate the secretion of HMW angiotensinogen (eluting just after the column void volume) which, on SDS-PAGE, migrated in a position identical to LMW angiotensinogen. From these studies, we conclude that all forms of human angiotensinogen are derived from a single precursor. The heterogeneity of secreted angiotensinogen represents differences in posttranslational processing of angiotensinogen. This processing includes both N- and O-glycosylation, and also the formation of HMW complexes (HMW angiotensinogen) through association either with other angiotensinogen molecules or with some other protein(s) whose secretion by hepatocytes is stimulated by glucocorticoids and estrogens.
Angiotensinogen↗
Angiotensinogen production and consumption in the adrenalectomized rat.
The aim of this study was to investigate the mechanisms by which angiotensinogen decreases after adrenalectomy. Plasma angiotensinogen was measured by two different methods: an indirect assay, which measures angiotensin I liberated from the plasma by an excess of renin, and a direct RIA, which measures both angiotensinogen and des-angiotensin I-angiotensinogen. In the normal rat angiotensinogen concentrations were found to be slightly, but not significantly, higher using the direct assay. After adrenalectomy a large discrepancy was observed between the indirect assay, which showed a considerable drop in plasma angiotensinogen levels, and the direct assay, which revealed a small but significant decrease. This discrepancy arose from the presence of a molecule that cross-reacts with angiotensinogen antibodies, and has a more acidic pI in isoelectric focusing than angiotensinogen: des-angiotensin I-angiotensinogen. This molecule accumulates in adrenalectomized rat plasma. The decrease in plasma angiotensinogen levels, measured by the indirect assay, could not be explained by a decrease in angiotensinogen production, as this was unchanged in the in vitro liver slice system, but was caused by an increase in angiotensinogen consumption, due to a rise in the plasma concentration of renin. Renin concentration shows a negative correlation with angiotensinogen (as measured by the indirect assay), and a positive correlation with des-angiotensin I-angiotensinogen level. Moreover, mineralocorticoids were shown to correct both renin and angiotensinogen concentrations, whereas a replacement dose of glucocorticoids (dexamethasone) had no effect on the level of renin or angiotensinogen, as measured by the indirect assay. We conclude that after adrenalectomy, plasma angiotensinogen decreases, due to an increase in renin production. A parallel accumulation of des-angiotensin I-angiotensinogen is observed.
Adrenalectomy↗
Immunologic identification of both plasma and human renal inactive renin as prorenin.
Antibodies were raised against a synthetic tetradecapeptide which is a component of the non-renin portion (prosegment) of human renin precursor. Inactive renin from human kidney and plasma strongly adsorbed to a gel coupled to immunoglobulins purified from such an antiserum. These results suggest that renal and circulating inactive human renins contain in their structure the prosegment of prorenin.
Antibody Specificity↗
Acute inhibition of the renin-angiotensin system: interest and limits to detect surgically curable hypertension.
The acute blockade of the renin-angiotensin system has been made it possible to investigate its role in the maintenance of blood pressure and aldosterone secretion in normotensive and hypertensive subjects. The administration of saralasin or captopril and, in the near future, of renin inhibitors induces a fall in blood pressure that is variable from one subject to the other according to the sodium balance and the level of activation of the system. These blockers also decrease the angiotensin II-dependent aldosterone production and increase renin secretion according to the circulating level of angiotensin II and the functional state of adrenal and juxtaglomerular receptors. In practice the definition of an abnormal response to renin-angiotensin blockade is difficult to define precisely, but the hypotensive effect has been tentatively used for the diagnosis of renin-dependent hypertension, especially renovascular hypertension and primary hyperaldosteronism. In renal artery stenosis the most convincing results mainly concern the lateralization of an abnormal unilateral renin secretion, which is potentiated by an acute blockade of the renin-angiotensin system. The acute administration of converting enzyme inhibitor is also useful to detect the absence of decrease in plasma aldosterone, which is characteristic of a solitary tumor or of other anatomical and functional disorders of the adrenal glands.
Aldosterone↗
Radiologic features of renin-producing tumors. A report of two cases.
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Adult↗
[Management of severe hypertension in a patient with arteriopathy].
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Antihypertensive Agents↗
Antihypertensive effect of enalapril as first-step treatment of mild and moderate uncomplicated essential hypertension. Evaluation by two methods of blood pressure measurement.
The new angiotensin converting enzyme inhibitor enalapril (MK-421), was given in a single daily dose of 20 mg to 53 patients with uncomplicated essential hypertension. Its effects were compared with those of a placebo given to 47 patients on a double-blind randomized basis. The blood pressure was measured in all patients by a physician, using a mercury sphygmomanometer, and by an automatic device in the absence of the physician. After 15 days of treatment, enalapril induced a significant reduction in systolic blood pressure (161.4 +/- 13 versus 145.1 +/- 15, p less than 0.001) and in diastolic blood pressure (103.3 +/- 6 versus 92.9 +/- 8, p less than 0.001) measured by the physician. The magnitude of the fall in blood pressure was identical after 30 days of active treatment. The reduction in blood pressure induced by enalapril was similarly detected by both methods of measurement, despite the fact that blood pressure values were higher when measured by the physician. A placebo effect was observed with the physician's values that was not present with the automatically recorded values. A very significant correlation between blood pressure values obtained by these two methods was observed. However, among nine of 53 patients treated with enalapril, a difference in the decrease of blood pressure of 10 mm Hg or more was noted between the two methods of measurement. The decrease in blood pressure occurred with no change noted in the pulse rate or orthostatic hypotension. Plasma renin activity increased after treatment. No changes were observed in creatinine clearance and plasma electrolyte levels.
Adult↗
Kallikrein along the rabbit microdissected nephron: a micromethod for its measurement. Effect of adrenalectomy and DOCA treatment.
Active and inactive kallikrein were measured along the rabbit microdissected nephron. A sensitive and specific micromethod for the measurement of kininogenase activity was developed in order to quantify kallikrein in pieces of tubule as small as 0.3-0.5 mm. Our study confirms that active and inactive kallikrein are located to the connecting tubule (CNT). The effects on renal kallikrein of a chronic DOCA treatment and of adrenalectomy were studied. Urinary excretion of kallikrein was also monitored. After DOCA treatment, active kallikrein increased in the tubule and in urine but inactive kallikrein did not significantly change. Adrenalectomy decreased by 50% active and inactive contents of CNT, as well as reduced the excretion of total kallikrein. Kallikrein content in CNT was also measured in adrenalectomized rabbits 3 h after a single injection of either aldosterone (10 micrograms) or dexamethasone (100 micrograms). After either aldosterone or dexamethasone injections, kallikrein activities were not restored, whereas in the same animals Na-K-ATPase activity which was depressed on cortical and medullary collecting tubules after adrenalectomy returned toward normal values. These data indicate that kallikrein synthesis and activation are influenced by adrenal hormones. Renal kallikrein is, however, regulated at a much slower rate than Na+-K+-ATPase. This may suggest an indirect rather than direct action of corticosteroid hormones on kallikrein.
Adrenal Cortex Hormones↗
Effects of glucocorticoids and antiglucocorticoid on angiotensinogen production by hepatoma cells in culture.
Angiotensinogen is synthesized in large amounts by Fao cells derived from the Reuber H35 rat hepatoma in a medium enriched with 5% fetal bovine serum (FBS). Treatment of FBS with dextran-coated charcoal removed endogenous steroids without modifying angiotensinogen production. This treatment allowed the study of the effects of steroids on angiotensinogen production. Hydrocortisone increased the angiotensinogen synthesis in a dose-dependent manner. The antiglucocorticoid RU 38486 did not change the basal rate of angiotensinogen production but inhibited the stimulation by hydrocortisone. Similar results were obtained with dexamethasone. Angiotensinogen biosynthesis seems to be regulated by two distinct mechanisms: (a) glucocorticoid independent, controlling the basal rate of angiotensinogen production and (b) glucocorticoid dependent, mediating the increased rate of angiotensinogen production upon glucocorticoid treatment.
Angiotensinogen↗