Search PubMed⌕ Search

Biomedical subjects

E Clauser

Publications and source records attributed to E Clauser.

At least 109 records · Page 6Linked to original sources
PubMed1985

Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA.

A human term (third trimester) placental alkaline phosphatase (PLAP; EC 3.1.3.1) cDNA was isolated from a human placental lambda gt11 cDNA library. The expression library was screened by using rabbit antibodies against PLAP and oligonucleotide probes. DNA sequence analysis of a positive clone with an insert of 2.7 kilobase pairs allowed us to predict the complete amino acid sequence of PLAP (530 residues), which coincided with the reported 42 N-terminal amino acid sequence of PLAP except at position 3. Contrary to the previous supposition that there was no amino acid sequence homology between PLAP and Escherichia coli alkaline phosphatase (471 residues), we found 30% overall homology, with regions of strong homology including the putative active site and the metal-binding sites. The 44-residue C-terminal extension of PLAP has a stretch of 17 hydrophobic amino acids, which presumably anchors the protein to the plasma membrane, a change perhaps necessary for the transition from a bacterial periplasmic enzyme to a mammalian membrane-associated enzyme. We have also localized PLAP-related DNA sequences mainly on chromosome 2 and to a lesser degree on chromosome 17. It seems likely therefore that the PLAP gene resides on chromosome 2 and other member(s) of the alkaline phosphatase family may exist (on this chromosome and) on chromosome 17.

Alkaline Phosphatase↗
PubMed1985

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↗
PubMed1984

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↗
PubMed1984

Influence of converting-enzyme inhibition on rat des-angiotensin I-angiotensinogen.

The effects of high plasma renin levels on plasma levels of both total immunoreactive angiotensinogen (direct radioimmunoassay) and intact angiotensinogen measured by angiotensin I released by renin (indirect assay) were studied in sodium-depleted rats both with and without captopril treatment and in adrenalectomized rats. The direct assay measures both intact angiotensinogen and des-angiotensin I-angiotensinogen, its residue cleaved by renin. The indirect assay measures only intact angiotensinogen. Neither sodium depletion, captopril treatment, nor adrenalectomy modified the circulating levels of total angiotensinogen. However these treatments produced a decrease in intact angiotensinogen that was in proportion to the elevation of renin levels. The difference between the two assays for angiotensin represents the level of des-angiotensin I-angiotensinogen and correlated satisfactorily with the plasma levels of renin. Identical correlations were observed in adrenalectomized rats and captopril-treated rats. We conclude that des-angiotensin I-angiotensinogen levels are an index of activation of the renin-angiotensin system dependent on the circulating level of renin.

Adrenal Glands↗
PubMed1984

Characterization of precursor and secreted forms of rat angiotensinogen.

Angiotensinogen precursors synthesized by rabbit reticulocyte lysate primed with rat liver RNA were compared with angiotensinogen secreted by rat hepatoma cells and rat hepatocytes using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of glycosylation with tunicamycin permitted identification of the nonglycosylated form of secreted angiotensinogen. Whereas angiotensinogen secreted by hepatoma cells and hepatocytes showed electrophoretic heterogeneity (mol wt, 52-62 X 10(3], tunicamycin-treated cells secreted only a single angiotensinogen species [mol wt, 48.3 +/- 0.7 X 10(3) (mean +/- SD)], which could be cleaved by renin. Two putative angiotensinogen precursors were synthesized in the reticulocyte lysate: a major protein of 52.5 +/- 1.0 X 10(3) mol wt and a minor protein of 55.7 +/- 1.3 X 10(3) mol wt. Evidence that these proteins represent separate angiotensinogen precursors includes the following. 1) Both proteins were recognized by five different polyclonal antibodies and two monoclonal antibodies. 2) Both proteins increased in parallel in reticulocyte lysates primed with liver RNA from rats nephrectomized and given hormones that increase liver angiotensinogen production. 3) Both proteins were cleaved by renin to produce a single protein of 47.6 +/- 0.8 X 10(3) mol wt. 4) The des-angiotensin I-angiotensinogen generated by renin treatment of the lysate had an electrophoretic mobility identical to that of des-AI-angiotensinogen produced by renin treatment of nonglycosylated angiotensinogen secreted by tunicamycin-treated hepatoma cells and hepatocytes. These studies suggest that rat liver synthesizes two separate angiotensinogen precursors which may differ only in the size of their prepro sequence. The heterogeneity of secreted angiotensinogen can be fully accounted for by differences in N-glycosylation of asparagine residues of the molecule. Glycosylation of angiotensinogen is not essential for its synthesis, processing, and secretion or its hydrolysis by renin.

Angiotensinogen↗
PubMed1983

Immunocytochemical localization of angiotensinogen in rat liver and kidney.

The renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat angiotensinogen. This substrate was also examined in rats after bilateral nephrectomy, which is known to increase plasma angiotensinogen, and in rats treated with colchicine, which inhibits serum protein secretion. In normal rat liver, light microscopy showed the presence of immunoreactive material in a very few cells. The number of stained hepatocytes rose in rats treated with colchicine or after bilateral nephrectomy. Immuno-staining increased further when rats were both nephrectomized and colchicine treated. In the kidney, angiotensinogen was specifically located as granular formations in nephrocytes of the proximal tubule but never in the granular cells of the juxtaglomerular apparatus. The localization of these granular formations under the brush border suggests that angiotensinogen is reabsorbed from the glomerular ultrafiltrate rather than synthesized in the kidney.

Angiotensinogen↗
PubMed1983

Regulation of angiotensinogen in cerebrospinal fluid and plasma of rats.

The origin and regulation of angiotensinogen in cerebrospinal fluid (CSF) was investigated in rats by measuring renin substrate in plasma and CSF under different experimental conditions. Nephrectomy (NX) increased the circulating and the central angiotensinogen levels. There was no correlation between the individual values of plasma and CSF. Adrenalectomy (ADX) diminished and hydrocortisone treatment augmented the angiotensinogen levels in plasma and CSF. The combination of ADX and NX caused a dissociation between peripheral and central angiotensinogen, since the values were elevated in plasma but unchanged in CSF. After the application of the converting-enzyme inhibitor captopril a significant decrease of angiotensinogen was observed in plasma only. A specific radioimmunoassay for renin substrate of rat plasma also recognized CSF angiotensinogen. There was a linear correlation between the CSF substrate levels obtained by direct and indirect measurement. In conclusion, CSF angiotensinogen appears to be immunologically similar to the plasma molecule. The angiotensinogen levels in CSF and plasma may be affected in parallel but can nevertheless be dissociated from each other.

Adrenalectomy↗
PubMed1983

Synthesis and release of immunoreactive angiotensinogen by rat liver slices.

Hepatic storage and secretion of angiotensinogen was studied using rat liver slices and a new direct angiotensinogen RIA. This assay permitted the demonstration of a significant hepatic storage of angiotensinogen, largely underestimated until now by the enzymatic method of angiotensinogen measurement. Angiotensinogen release by rat liver slices was linear with time and was associated with a significant increase in hepatic content of angiotensinogen. The measurement of both release and changes in hepatic content permitted the measurement of de novo synthesis of angiotensinogen by rat liver slices in vitro. Both hepatic content and release of angiotensinogen were decreased by thyroidectomy and increased by ethinyl estradiol, dexamethasone, thyroid hormones, and binephrectomy.

Angiotensinogen↗
PubMed1983

Biochemistry and regulation of angiotensinogen.

Angiotensin II and angiotensin III, the active peptides of the renin-angiotensin system, are produced by a cascade of enzymatic reactions, whose initial step is the reaction between renin and its substrate, angiotensinogen. In plasma, the concentration of angiotensinogen is a limiting factor: the Km of the enzymatic reaction is between 1 and 2 microM depending on the species. It is therefore of interest to measure its level in plasma and tissues and to examine the main factors which may influence its synthesis and release. The complete purification of angiotensinogen has made possible the preparation of specific antibodies which cross-react with both angiotensinogen and its residue, des-angio I-angiotensinogen, and are currently used in radioimmunoassays and immunohistochemical studies. A small amount of angiotensinogen is stored in hepatic cells, where it can be detected by immunofluorescence and measured by radioimmunoassay. It is also present in proximal tubular cells of the kidney, probably reabsorbed from glomerular filtrate, but it is absent from juxtaglomerular cells. Several hormones are able to increase liver synthesis of angiotensinogen and its release. Thyroxine, angiotensin II, dexamethasone, ethinyl-estradiol and binephrectomy increase both synthesis and release. Adrenalectomy and converting-enzyme inhibition are accompanied by an increased peripheral consumption of plasma angiotensinogen, and by accumulation of des-angio I-angiotensinogen whose metabolism and role are unknown. The major role of angiotensinogen in renal hemodynamics is demonstrated by its effects on the isolated perfused kidney, an experimental observation which parallels the clinical observation of women on estroprogestative therapy, whose renal blood flow is reduced, even in the absence of a detectable increase in their blood pressure. A better knowledge of renin substrate structure in various species is a necessary requirement for the design of inhibitory analogs of angiotensinogen which will have application for the treatment of hypertension and oedema.

Adrenalectomy↗
PubMed1983

Regulation of angiotensinogen in the central nervous system.

Several interventions known to alter plasma renin substrate in rats such as nephrectomy (NX), adrenalectomy (ADX) and glucocorticoid treatment changed the angiotensinogen content in the cerebrospinal fluid (CSF) in the same direction. However, peripheral and central angiotensinogen could be dissociated from each other by ADX and NX in combination, as well as by chronic converting enzyme blockade. The regulation of brain angiotensinogen was further investigated in stroke-prone spontaneously hypertensive rats (SHR-sp) in comparison with normotensive Wistar Kyoto (WKY) rats. The angiotensinogen levels of the anterior hypothalamus and of the septal area showed strain and age-related differences. Chronic converting enzyme blockade, which kept SHR-sp normotensive, stimulated angiotensinogen in the anterior hypothalamus of both SHR-sp and WKY rats, but suppressed plasma renin substrate. A specific radioimmunoassay (RIA) for renin substrate of rat plasma also recognized the CSF angiotensinogen, and a linear correlation existed between direct and indirect measurements. In conclusion, angiotensinogen in the central nervous system appears to be immunologically similar to plasma angiotensinogen. Its regulation is not directly related, however, to circulating renin substrate, although adrenal steroids stimulate both central and peripheral angiotensinogen. A differential regulation of angiotensinogen in the brain of SHR-sp as compared to WKY is evident and could be linked to blood pressure control.

Aging↗
PubMed1983

The effects of converting enzyme inhibitors on plasma angiotensinogen and plasma aldosterone in sodium-depleted rats.

Plasma angiotensinogen was measured by two different methods in three groups of sodium-depleted rats: control rats, captopril-treated rats and enalapril-treated rats. The enzymatic method of measurement is an exhaustion technique which measures the amount of angiotensin I liberated from plasma by an excess of renin. Direct radioimmunoassay measures both angiotensinogen and its inactive residue, des-angio I-angiotensinogen, and the difference between the results of both methods can be used as an indirect measurement of plasma des-angio I-angiotensinogen. Both converting enzyme inhibitors decreased blood pressure, stimulated renin secretion, decreased plasma angiotensinogen and increased des-angio I-angiotensinogen. Plasma des-angio I-angiotensinogen levels were positively correlated with plasma renin levels. In converting enzyme inhibitor-treated rats, plasma aldosterone was slightly, but not significantly, lower than in control rats. Although plasma aldosterone was significantly correlated with plasma renin both in control rats and converting enzyme inhibitor-treated rats, plasma renin was elevated 10-fold in treated rats.

Aldosterone↗
PubMed1982

Direct radioimmunoassay of rat angiotensinogen and its application to rats in various endocrine states.

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 enable 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 radio-immunoassay 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.

Angiotensinogen↗
PubMed1982

[Changes in angiotensinogen and des-angiotensin I-angiotensinogen in man and rat upon inhibition of converting enzyme].

The effects of converting enzyme inhibition on plasma renin substrate concentration were studied in man and rat. This study use new direct radioimmunoassays of angiotensinogen completing the classical enzymatic methods. In human investigation converting enzyme is inhibited after Captopril treatment. Our results demonstrated that resulting increase of plasma renin concentration enhanced the consumption of renin substrate as shown by the fall of angiotensinogen levels measured by indirect method. In the rat, we observed the same drop of renin substrate during MK421 administration. The fall of angiotensinogen levels, measured by indirect method, was not in agreement with results of direct radioimmunoassay. This discrepancy can be explained by the accumulation of des-angiotensin I-angiotensinogen in plasma. These modifications are potentiated by sodium depletion.

Angiotensin I↗
PubMed1981

Rat angiotensinogen and des(angiotensin I)angiotensinogen: purification, characterization, and partial sequencing.

Rat angiotensinogen was completely purified by a six-step procedure including (1) ammonium sulfate precipitation, (2) affinity chromatography on Affi-gel blue, (3) chromatography on DEAE-Sephacel, (4) chromatography on hydroxylapatite, (5) chromatography on Ultrogel AcA 54, and (6) isoelectric focusing. Two peaks of pure angiotensinogen were obtained, distinguishable by their isoelectric points (4.55 and 4.75). Both contained 23 microgram of angiotensin I/mg of protein. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak with pI = 4.55 revealed two protein bands (respectively Mr 57000 and 59000) and a single protein band (Mr 57000) for the peak with pI = 4.75. The molecular weight of the latter homogeneous form, as determined by sedimentation equilibrium, was 55000. Only one immunoprecipitin line was observed when antiserum reacted with the heterogeneous angiotensinogen in Ouchterlony gels. The first 17 amino acids of the N-terminal region of the angiotensinogen with pI = 4.75 are reported. The amino acids in positions 10 and 11 which correspond to the renin cleavage site are leucyl-leucyl. The des(angiotensin I)angiotensinogen obtained after hydrolysis of angiotensinogen with pure mouse submaxillary gland renin was found to consist of a single protein band with an Mr of 56000 as revealed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Only one N-terminal residue (leucyl) was obtained for this des(angiotensin I)angiotensinogen. These findings establish that renin only cleaves angiotensinogen at a single site.

Amino Acid Sequence↗
PubMed1981

The renin-angiotensin system in thyroidectomized rats.

The influence of thyroidectomy on the renin-angiotensin system was studied in the rat. From 1-6 weeks after thyroidectomy, PRA and plasma renin substrate (PRS) decreased, but the plasma renin concentration remained unchanged, and the renal renin content increased. T3 injection corrected the changes in the plasma renin-angiotensin system of thyroidectomized rats within 20-40 h. After ethinylestradiol treatment, the PRS in thyroidectomized rats rose in the same proportion as that in normal rats, but remained below the normal level. After binephrectomy, on the other hand, the PRS was high, and PRS levels in normal and thyroidectomized animals were similar. Isoproterenol increased PRA and plasma renin concentration in control animals but had no effect on thyroidectomized rats. From the above results it may be concluded that angiotensinogen production is dependent on thyroid hormones and that renin release depends on beta-adrenergic receptor sensitivity to catecholamines, which is reduced by thyroidectomy. (Endocrinology 108: 647, 1981)

Angiotensinogen↗
PubMedDate not supplied

Role of angiotensinogen in blood pressure homeostasis.

The role of angiotensinogen in blood pressure control was assessed in normotensive rats by observing the changes resulting from inhibition by specific rat angiotensinogen antiserum. The antiserum decreased blood pressure in rats on normal sodium as well as sodium-free diets (respectively delta BP = -30 +/- 6 mm Hg and -42 +/- 8 mm Hg). In binephrectomized sodium-replete rats, administration of antiserum did not reduce blood pressure, whereas in sodium-depleted animals it slightly decreased blood pressure by 11 +/- 3 mm Hg. These results suggest that angiotensinogen participates in the regulation of blood pressure in normotensive rats, even in the sodium-replete state.

Angiotensinogen↗
PubMedDate not supplied

Production and characterization of monoclonal antibodies to rat angiotensinogen.

Three stable monoclonal antibodies to rat angiotensinogen were obtained by fusing myeloma cells with spleen cells from Balb/c mice injected with pure rat angiotensinogen. They were screened by their binding to pure iodinated angiotensinogen and to insolubilized angiotensinogen in a solid phase assay. The titers of the three antibodies varied from 1/3500 to 1/35000, their dissociation constants from 2.5 X 10(-8) M to 3.8 X 10(-10) M, and the sensitivity of the assay ranged from 200 to 10 pmol of pure angiotensinogen. These monoclonal antibodies did not recognize either angiotensin peptides or angiotensinogen from other species, except for mouse angiotensinogen, which cross-reacted with the different antibodies from 0 to 25%. Rat cerebrospinal fluid angiotensinogen, plasma des-angiotensin I-angiotensinogen, and plasma angiotensinogen were equally recognized by these monoclonal antibodies. Contrary to what was observed for a polyclonal antiserum, the monoclonal antibodies failed to inhibit the renin-angiotensinogen reaction in vitro.

Angiotensinogen↗