Direct Radioimmunoassay of Rat Angiotensinogen and Its Application to Rats in Various Endocrine States

1982 ◽  
Vol 62 (4) ◽  
pp. 355-360 ◽  
Author(s):  
J. Bouhnik ◽  
E. Clauser ◽  
J. Gardes ◽  
P. Corvol ◽  
J. Menard
Keyword(s):  
Angiotensin Ii ◽  
Rat Plasma ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Direct Assay ◽  
Indirect Assay

1. Antibodies were raised in rabbits against pure rat angiotensinogen. The antisera obtained were highly specific for rat angiotensinogen and did not bind hog, dog, rabbit, monkey or human angiotensinogen. They did not cross-react with angiotensin I, angiotensin II or synthetic hog tetradecapeptide renin substrate. However, rat des-angiotensin I—angiotensinogen cross-reacted 100% with the angiotensinogen antibody. 2. A direct radioimmunoassay for rat angiotensinogen in plasma was developed and this enabled 5 fmol of this protein to be detected. Comparison of the amounts of angiotensinogen determined by the indirect and direct assay systems indicated a 1:1.2 ratio for normal rats and rats in various endocrine states, except for adrenalectomized animals. In the latter, the angiotensinogen level measured by direct radioimmunoassay was four times that obtained by indirect assay. 3. The presence of a large amount of des-angiotensin I—angiotensinogen in adrenalectomized rat plasma is discussed.

Intrarenal Formation of Angiotensin II in the Rat: Interference by Saralasin and SQ 20881

1974 ◽  
Vol 48 (s2) ◽  
pp. 37s-40s
Author(s):  
H. Zschiedrich ◽  
K. G. Hofbauer ◽  
E. Hackenthal ◽  
G. D. Baron ◽  
F. Gross
Keyword(s):  
Angiotensin Ii ◽  
Vascular Resistance ◽  
Plasma Renin ◽  
Rat Plasma ◽  
Renal Vascular Resistance ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Vasoconstrictor Effect ◽  
Renal Vascular ◽  
Dose Dependent

1. Isolated rat kidneys were perfused with a medium free of components of the renin-angiotensin system. 2. Angiotensin II, angiotensin I, tetradecapeptide renin substrate or rat plasma renin substrate added to the medium caused a dose-dependent increase of renal vascular resistance. 3. The vasoconstrictor effect of angiotensin II was inhibited by 1-Sar-8-Ala-angiotensin II (Saralasin). The inhibition was dose-dependent, being complete at the highest doses applied. In this dose range, Saralasin increased renal vascular resistance. Saralasin also inhibited vasoconstriction induced by tetradecapeptide renin substrate. 4. The vasoconstrictor effect of angiotensin I was suppressed by SQ 20881, up to a maximum of 87% depending on the dose. Similarly the increase in renal vascular resistance induced by a purified preparation of rat plasma renin substrate was inhibited by 55%; no effect on the action of tetradecapeptide renin substrate was observed. 5. The data suggest that, within the kidney, angiotensin I is converted into angiotensin II to the extent of about 1.25%. Since no angiotensin I is formed from synthetic renin substrate, the vasoconstrictor effect of the tetradecapeptide may be either due to a direct interaction with the angiotensin II receptor or the consequence of the intrarenal formation of angiotensin II. In contrast, the results with rat plasma renin substrate suggest that angiotensin I is formed from ‘natural’ substrate and is subsequently converted into angiotensin II.

Measurement of inactive renin in normal, nephrectomized, and adrenalectomized rats

1991 ◽  
Vol 69 (9) ◽  
pp. 1381-1384 ◽  
Author(s):  
Knud Poulsen ◽  
Arne Høj Nielsen ◽  
Arne Johannessen
Keyword(s):  
Animal Model ◽  
Exchange Resin ◽  
Cation Exchange Resin ◽  
Plasma Renin ◽  
Rat Plasma ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Suitable Animal Model ◽  
Inactive Renin ◽  
Adrenalectomized Rats

In a new method for measurement of inactive rat plasma renin, the trypsin generated angiotensin I immunoreactive material, which was HPLC characterized as similar to tetradecapeptide renin substrate, is removed by a cation exchange resin before the renin incubation step. The method also corrects for trypsin destruction of endogenous angiotensinogen by the addition of exogenous angiotensinogen. When measured with this method inactive renin in rat plasma decreased after nephrectomy and increased after adrenalectomy. This is in accordance with findings in humans. A sexual dimorphism of prorenin (inactive renin) in rat plasma, similar to that reported in humans and mice, was demonstrated. Thus, inactive renin in the rat is no exception among species, and the rat might be a suitable animal model for further studies dealing with the physiology of prorenin in plasma and tissues.Key words: angiotensinogen, inactive renin, renin.

Methods for serial study of renin-angiotensin system in the unanesthetized rat

1975 ◽  
Vol 228 (2) ◽  
pp. 369-375 ◽  
Author(s):  
JS Carvalho ◽  
R Shapiro ◽  
P Hopper ◽  
LB Page
Keyword(s):  
Renin Angiotensin System ◽  
Plasma Renin ◽  
Rat Plasma ◽  
Angiotensin I ◽  
Ether Anesthesia ◽  
Renin Substrate ◽  
Angiotensin System ◽  
Arterial Blood ◽  
Sympathetic Nervous ◽  
Improved Technique

Micromethods for measurement of plasma renin concentration (PRC) and plasma renin-substrate concentration (PSC) have been developed for rat plasma with radioimmunoassay of angiotensin I. An improved technique for aortic implantation of plastic cannulas was developed for use in experiments 1-2 wk in duration. The effects on components of renin system of anesthesia and tail cutting were studied. Arterial blood was sampled through cannulas without animal manipulation. PRC varied little in unanesthetized rats, was moderately and variably increased during pentobarbital anesthesia, and was markedly and consistently elevated during ether anesthesia. PSC was unchanged during anesthesia. PRC was increased in blood obtained by tail cutting within 1-2 min after cutting. With the use of the methods and techniques described here serial studies of the renin system in plasma of unanesthetized rats are shown to be feasible. A role for the sympathetic nervous system in the mediation of renin secretion by ether is proposed.

scholarly journals Renin substrate in granules from rat kidney cortex

1976 ◽  
Vol 154 (3) ◽  
pp. 625-637 ◽  
Author(s):  
B J. Morris ◽  
C I. Johnston
Keyword(s):  
Microsomal Fraction ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Rat Plasma ◽  
Reaction Scheme ◽  
Kidney Cortex ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Rat Kidney Cortex ◽  
Molecular Weights

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.

Activation and measurement of plasma prorenin in the rat

1991 ◽  
Vol 69 (9) ◽  
pp. 1331-1340 ◽  
Author(s):  
P. Ioannou ◽  
A. Y. Loh ◽  
D. H. Osmond
Keyword(s):  
Human Plasma ◽  
Rat Plasma ◽  
Blood Collection ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Blood Samples ◽  
Functional Component ◽  
Fresh Frozen ◽  
Normal Rat

Prorenin determination in rat plasma has been problematic from the outset. Consequently, its existence is questioned by some and its quantity by others, making it difficult for knowledge to advance as to its function relative to the renin system. The present study examines major variables in the determination of rat plasma prorenin and renin, notably different prorenin activation protocols involving blood samples obtained under various conditions from animals under different anesthetics. We found that a trypsin activation step with 5 mg/mL plasma, 60 min at 23 °C, followed by a PRA step of 10 min at 37 °C, resulted in the highest prorenin estimates, up to approximately 400 ng∙mL−1∙h−1 in terms of angiotensin I, as compared with published values of 0–190, based on other protocols. These estimates were obtained despite considerable destruction of angiotensinogen (renin substrate) by trypsin. Cryoactivation of prorenin was much less effective than in human plasma but, when followed by trypsin, it facilitated greater activation than with trypsin alone. Comparable fresh and fresh-frozen plasmas had similar prorenin–renin values, but lower values were observed in plasmas that had been repeatedly frozen and thawed. Conscious rats and those anesthetized with Inactin or ether had higher renins and prorenins than those anesthetized with methoxyflurane or halothane. Rats with kidneys in place during blood collection had higher renins (but not prorenins) than those whose kidneys were clamped off, suggesting that last-minute renin release during blood collection had occurred. We conclude that (i) trypsin generates increased renin, or renin-like, activity in plasma, suggesting activation of a precursor; (ii) on this basis, high prorenin levels exist in normal rat plasma; (iii) renin and prorenin levels are variously influenced by different anesthetics and blood handling procedures; (iv) variation in prorenin levels suggests that it is a dynamic (functional?) component of the renin system; (v) prorenin measurements are heavily influenced by methodological variations during the trypsin step or the subsequent PRA step; (vi) using standardized methodology, the rat can serve as a model for investigating the function of prorenin in normotension and hypertension.Key words: tryptic activation, angiotensinogen, adrenalectomy, anesthesia.

MEASUREMENT OF PLASMA RENIN-SUBSTRATE IN MAN

1973 ◽  
Vol 56 (2) ◽  
pp. 159A-171 ◽  
Author(s):  
MALCOLM TREE
Keyword(s):  
Angiotensin Ii ◽  
Substrate Concentration ◽  
Plasma Renin ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Potential Sources ◽  
Sources Of Variation ◽  
Method Of Measurement ◽  
Inhibitor Solution

SUMMARY Values of plasma renin-substrate concentration in man vary widely according to the method of measurement used. Potential sources of variation have been tested and, as far as possible, excluded in the method described here. Blood was diluted rapidly in an angiotensinase-inhibitor solution containing EDTA and phenanthroline; plasma was separated by centrifugation and the renin-substrate in the specimen was hydrolysed by renin to angiotensin I which was identified as such by chromatography and radioimmunoassay. Angiotensin I was used as a standard to determine the amount of angiotensin formed on incubation. Use of angiotensin II for a standard, as in other methods, led to falsely low values of plasma renin-substrate concentration. Recovery of added substrate was 94%. Changes of plasma renin-substrate concentration in some physiological and pathological states are reported briefly.

Measurement and characterization of angiotensin peptides in plasma.

1988 ◽  
Vol 34 (6) ◽  
pp. 1046-1051 ◽  
Author(s):  
K Hermann ◽  
D Ganten ◽  
T Unger ◽  
C Bayer ◽  
R E Lang
Keyword(s):  
Angiotensin Ii ◽  
Human Plasma ◽  
Enzyme Inhibitor ◽  
Standard Procedure ◽  
Rat Plasma ◽  
Small Sample ◽  
Blood Collection ◽  
Angiotensin I ◽  
Angiotensin Peptides ◽  
Plasma Angiotensin

Abstract We report a method for the extraction of angiotensin peptides from plasma with a mixture of acetone, 1 mol/L HCl, and water (40/1/5 by vol). The method is highly reproducible for the measurement of angiotensin I and angiotensin II in small sample volumes, with analytical recoveries of about 80% for both peptides. We investigated the influence of sample handling and found a standard procedure for blood collection, plasma preparation, and extraction was essential. The method was used to measure angiotensin I and II in rat and human plasma. In rat plasma, the mean (+/- SEM) concentrations of angiotensin I and angiotensin II were determined to be 67 (+/- 8) and 14 (+/- 1) pmol/L (n = 10), respectively. Neither angiotensin I nor angiotensin II was detectable 24 h after bilateral nephrectomy. Acute oral administration of the converting-enzyme inhibitor ramipril caused a significant increase of angiotensin I from 85 (+/- 6) to 257 (+/- 33) pmol/L (n = 10; P less than 0.001) and a significant decrease of angiotensin II from 12 (+/- 1) to 7 (+/- 0.4) pmol/L in rat plasma (n = 9; P less than 0.001). In human plasma, angiotensin I and angiotensin II values of 21 (+/- 1) and 6.6 (+/- 0.5) pmol/L (n = 10) were found. A single oral dose of the diuretic furosemide increased angiotensin I significantly from 21 (+/- 1) to 32 (+/- 1.7) pmol/L (n = 5); P less than 0.001), whereas angiotensin II remained unchanged, 6.6 (+/- 0.5) vs 6.4 (+/- 0.4) pmol/L (n = 5). Extracted peptides could be identified as [IIe5]-angiotensin I and [IIe5]-angiotensin II by HPLC in combination with specific radioimmunoassays for angiotensin I and angiotensin II.

scholarly journals Quantification of intact plasma AGT consisting of oxidized and reduced conformations using a modified ELISA

2016 ◽  
Vol 311 (6) ◽  
pp. F1211-F1216 ◽  
Author(s):  
Ryousuke Satou ◽  
Hiroyuki Kobori ◽  
Akemi Katsurada ◽  
Kayoko Miyata ◽  
L. Gabriel Navar
Keyword(s):  
Renin Angiotensin System ◽  
Plasma Renin ◽  
Rat Plasma ◽  
Limiting Factor ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Angiotensin System ◽  
Mouse Plasma ◽  
Rats And Mice ◽  
Pleiotropic Actions

The pleiotropic actions of the renin-angiotensin system (RAS) depend on the availability of angiotensinogen (AGT) which generates angiotensin I (ANG I) when cleaved by renin. Thus, quantification of the intact AGT (iAGT) concentrations is important to evaluate the actual renin substrate available. The iAGT conformation exists as oxidized AGT (oxi-AGT) and reduced AGT (red-AGT) in a disulfide bond, and oxi-AGT has a higher affinity for renin, which may exacerbate RAS-associated diseases. Accordingly, we determined iAGT, oxi-AGT, and red-AGT levels in plasma from rats and mice. Blood samples were obtained by cardiac puncture and then immediately mixed with an inhibitor solution containing a renin inhibitor. Total AGT (tAGT) levels were measured by tAGT ELISA which detects both cleaved and iAGT. iAGT levels were determined by iAGT ELISA which was found to only detect red-AGT. Thus, it was necessary to treat samples with dithiothreitol, a reducing agent, to quantify total iAGT concentration. tAGT levels in rat and mouse plasma were 1,839 ± 139 and 1,082 ± 77 ng/ml, respectively. iAGT levels were 53% of tAGT in rat plasma but only 22% in mouse plasma, probably reflecting the greater plasma renin activity in mice. The ratios of oxi-AGT and red-AGT were ∼4:1 (rat) and 16:1 (mouse). Plasma iAGT consists of oxi-AGT and red-AGT, suggesting that oxidative stress can influence ANG I generation by the AGT conformation switch. Furthermore, the lower availability of plasma iAGT in mice suggests that it may serve as a limiting factor in ANG I formation in this species.

Simultaneous Determination of Plasma Converting Enzyme and Angiotensinase Activity by Radioimmunoassay

1971 ◽  
Vol 40 (6) ◽  
pp. 443-449 ◽  
Author(s):  
K. Poulsen ◽  
L. L. Poulsen
Keyword(s):  
Angiotensin Ii ◽  
Human Plasma ◽  
Rat Plasma ◽  
Least Square ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Plasma Angiotensin ◽  
Least Square Fit ◽  
Simple Measurement

1. Homologous angiotensin-I was added to untreated plasma. Angiotensin-II which is formed by plasma converting enzyme and subsequently degradated by angiotensinases was determined as a function of time by using radioimmunoassay. Having determined the kinetics, the activities of converting enzyme and angiotensinases were calculated by a least-square fit of the theoretical curve to the experimentally measured values. 2. The method gives a simple measurement of converting-enzyme activity in untreated plasma; in plasma from salt-depleted and renal hypertensive rats this was found to be slightly increased but was normal in plasma from nephrectomized rats. 3. The half-lives for angiotensin-I in normal rat and human plasma were found to vary between 0·8 and 2·1 min, and the possibility that plasma converting enzyme participates in the regulation of the formation of angiotensin-II in vivo cannot be excluded. 4. The angiotensinase activity in rat plasma gave half-lives for angiotensin-II between 1·5 and 3·3 min; the half-life in normal human plasma was 10 min.

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