Angiotensinogen production by rat hepatoma cells in culture and analysis of its regulation by techniques of somatic cell genetics.

Angiotensinogen was synthesized by cells derived from the Reuber H35 rat hepatoma. Independent clones produced similar amounts of angiotensinogen, which corresponded to about four times more than expected for normal hepatocytes. The protein was secreted rapidly but could be visualized within cells using immunofluorescence. For one clone, it is shown that maximal angiotensinogen synthesis occurred during mid-exponential growth. Somatic cell genetics techniques have been used to investigate the regulation of angiotensinogen expression. Eleven clones of dedifferentiated variant hepatoma cells that failed to produce most or all of the liver specific proteins analyzed including albumin fell into two groups: Seven clones produced only 1-3% as much angiotensinogen as the differentiated clones, and four showed a reduction to 10-30%. Clones of the latter class were the only ones among the eleven analyzed that retained the potential to give rise to revertants, showing restoration of the differentiated state. All revertants fully restored angiotensinogen production, but only some of them re-expressed albumin. Somatic hybrids between differentiated hepatoma cells and one of the variants showed a substantial reduction in angiotensinogen production, whereas for some clones, albumin synthesis was fully maintained. These results show that regulation of the expression of angiotensinogen and of a second serum protein, albumin, was independent and that angiotensinogen synthesis was a faithful indicator of the general differentiation profile of all classes of clones.

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.

Biochemical and physiological studies on two T-kininogen species using monoclonal antibodies.

Rat T-kininogens were characterized in plasma, urine and liver slice incubation medium in different physiological conditions using monoclonal antibodies that allow to distinguish between the two forms of T-kininogen. T-kininogen purified from the plasma of both normal and inflamed Wistar and Sprague-Dawley rats was found to contain the two forms of T-kininogen, TI and TII, separated by non-denaturing polyacrylamide gel electrophoresis. The two forms were also found in the plasma of several strains of normal and inflamed rats, except in the plasma of the Buffalo rat which contained only TII-kininogen. The two forms of T-kininogen were also found in the media in which liver slices from inflamed and non-inflamed wistar rats had been incubated. The urine T-kininogen of normal rats was chiefly TI-kininogen, but both forms were found in the urine of inflamed rats. T-kininogen in the plasma of normal and inflamed rats was further characterized by chromatography on Con A-Ultrogel. In normal plasma, we observed a ratio of non-retained to retained T-kininogen of 0.41. The retained T-kininogen was eluted as two peaks, one eluted with 45 mM and the other with 120 mM alpha-methyl-D-glucoside. The ratio of non-adsorbed to adsorbed T-kininogen in inflamed Wistar rat plasma was 1.40 and the retained material was almost exclusively in a single peak, which eluted with 50 mM alpha-methyl-D-glucoside. The non-adsorbed and adsorbed fractions contained both forms of T-kininogen, but the protein bands in the non-retained fraction had greater mobilities on non-denaturing PAGE. SDS-PAGE analysis of T-kininogen deglycosylated by N-glycosidase F showed a major band with a molecular mass of 50 kDa, whereas the molecular mass of the native form was 66 kDa. We concluded that both forms of T-kininogen exist in the non-inflamed and the inflamed rat plasma, except for the Buffalo rat, and that T-kininogen displays a different chromatographic pattern on Con A-Ultrogel after inflammation suggesting altered glycosylation.

Differential effects of nephrectomy and surgery on plasma kininogens and angiotensinogen in the rat.

The effects of bilateral nephrectomy or a sham operation on plasma angiotensinogen and on the different kininogens were studied in the rat. Total plasma kininogen was measured by RIA of kinins after trypsin hydrolysis. In addition, the high molecular weight (HMW) kininogen and the low molecular weight (T)-kininogen were specifically quantified by using direct RIAs. Angiotensinogen was measured by RIA of angiotensin I after exhaustion by renin. Three groups of control, nonoperated, bilaterally nephrectomized and sham-operated rats were studied in each experiment. Twenty-four hours after either a bilateral nephrectomy or a sham operation total plasma kininogen was elevated approximately 5 times when compared to control rats. Time course measurements from 0 to 48 h in 3 other groups of control, bilaterally nephrectomized and sham-operated rats demonstrated that kininogen gradually increased at 12, 24, and 48 h after the surgery and that the elevation observed in plasma kininogen appeared to be entirely due to an increase in T-kininogen levels. There was no difference in T-kininogen levels between bilaterally nephrectomized and sham-operated animals. By contrast HMW kininogen was neither influenced by surgery nor by nephrectomy. Angiotensinogen increased more than 8 times in bilaterally nephrectomized rats but displayed only little changes in sham-operated animals. During the course of this experiment it was observed that also in control animals submitted to repeated skin incision and venipuncture for blood sampling at the jugular vein, T-kininogen increased dramatically in plasma, but reached values lower than in sham-operated or bilaterally nephrectomized rats. In a third experiment performed in normal rats it was found that T-kininogen levels were more than 3 times elevated over initial values 24 h after a single blood sampling at the jugular vein. These results indicate that T-kininogen but not HMW kininogen is very sensitive to surgery, perhaps as a result of increased T-kininogen synthesis due to an inflammatory reaction. The T-kininogen might participate in the inflammatory reaction that occurs at the site of tissue injury and in the healing process. As there was no difference in T-kininogen, and in HMW kininogen levels between bilaterally nephrectomized and sham-operated rats, the kidneys do not seem to play an important role in the regulation of plasma kininogens. Angiotensinogen, HMW kininogen, and T-kininogen are therefore regulated separately after nephrectomy or surgery.

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.

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)

Liver angiotensinogen synthesis and release during captopril treatment in sodium-depleted rats.

In vivo generation of angiotensins depends upon both plasma renin and angiotensinogen concentrations. Those factors which may influence hepatic angiotensinogen synthesis and release were examined. We have evaluated in vivo the effects of converting enzyme inhibition on several plasma renin-angiotensin system components, and, using an in vitro preparation of liver slices, we also investigated the effects of converting enzyme inhibition on the synthesis and release of hepatic angiotensinogen. Angiotensinogen concentrations were determined by two different methods. The first was an indirect enzymatic assay which measures the amount of angiotensin I liberated from plasma by an excess of renin. The second was a direct RIA that measures both angiotensinogen and its inactive residue the des-angiotensin I-angiotensinogen. The difference between the methods represents the circulating levels of des-angiotensin I-angiotensinogen. Captopril administration in sodium-depleted rats increased plasma concentrations of renin, des-angiotensin I-angiotensinogen, and angiotensin I and decreased plasma angiotensinogen concentration measured by both methods. Plasma des-angiotensin I-angiotensinogen was significantly correlated to plasma renin concentration, which suggests an increase in the consumption of angiotensinogen when the renin secretion is extremely increased. The angiotensinogen liver content and in vitro angiotensinogen release were decreased in sodium-depleted rats treated with a converting enzyme inhibitor, and these parameters were negatively correlated to in vivo plasma levels of renin, angiotensin I, and des-angiotensin I-angiotensinogen. They were positively correlated to plasma angiotensinogen concentration measured by the indirect assay. These data suggest that captopril administration during sodium depletion has two simultaneous effects: it increases angiotensinogen consumption and second, decreases angiotensinogen production and release.

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 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.

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.

Characterization of plasma and cerebrospinal fluid human angiotensinogen and des-angiotensin I-angiotensinogen by direct radioimmunoassay.

Angiotensinogen and the product of its hydrolysis by renin, des-angiotensin I-angiotensinogen, were quantitated in human plasma and in cerebrospinal fluid (CSF) by a direct RIA. This assay was developed using polyclonal antibodies raised against pure human angiotensinogen. The antibodies recognized only primate angiotensinogen and des-angiotensin I-angiotensinogen. Results obtained with the direct RIA were compared with those of the indirect assay which measures angiotensinogen through angiotensin I liberated by an excess of renin. Both assays gave almost identical results in normal subjects whereas in three different conditions characterized by a high renin level (severe hypertension plus low sodium diet, converting enzyme inhibition, and adrenal insufficiency) higher results were obtained by the direct assay. This difference between the results of both methods was attributed to des-angiotensin I-angiotensinogen accumulation which is detected only in the direct assay. CSF angiotensinogen had similar immunochemical properties to plasma angiotensinogen and could also be measured by the direct RIA. Isoelectric focusing of plasma angiotensinogen and des-angiotensin I-angiotensinogen revealed a similar microheterogeneity. Microheterogeneity was also a characteristic of CSF angiotensinogen, but its isoelectric point was more basic than plasma angiotensinogen.

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.

Study of the antigenic determinants of human renin.

The primary structure of human renin, recently established from the complementary DNA sequence of its messenger RNA, shows a strong homology to other aspartyl proteases. This homology has permitted the construction of a model of the three-dimensional structure of renin based on the crystallographically determined structures of three aspartyl proteases: penicillopepsin, endothiapepsin, and rhizopuspepsin. Using an algorithm in which a spherical probe approximating the size of the antibody-binding domain (1-nm radius) was allowed to contact the surface of the renin model, we predicted 12 to 15 peptides to be immunogenic epitopes. We synthesized peptides corresponding to three different regions of the model: Cys-Gly-Ser-Asp-Pro-Gln-His-Tyr-Glu-Gly-amide (C-180-188), Tyr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-215-224; disulfide bond between cysteines) and Tyr-Gly-Ser-Ser-Thr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-211-224; disulfide bond between cysteines), and Cys-Tyr-Ser-Ser-Lys-Lys-Leu-Cys-Gly (C-290-296-G; disulfide bond between cysteines). All four peptides were tested for their binding to 11 polyclonal and 7 monoclonal antibodies raised against pure human renin, in both a solution assay and an enzyme-linked immunosorbent assay. Peptides Y-215-224 and Y-211-224 bound to all 11 polyclonal antibodies in the solution assay, and peptide Y211-224 bound to eight of them in the enzyme-linked immunosorbent assay. Therefore, region 211-224 can be identified as a major epitope of the human renin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effects of thyroidectomy of the rat on the structure and functions of skeletal muscle mitochondria]. / Effets de la thyroïdectomie du rat sur la structure et les fonctions des mitochondries des mescles squelettiques

Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some difference appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver or thyroidectomized rats.

Angiotensinogen in the developing rat fetal hindbrain and spinal cord from 18th to 20th day of gestation: an immunocytochemical study.

We have detected angiotensinogen immunoreactivity in the hindbrain and in the spinal cord of rat fetuses during the 18th to 20th day of gestation. In the 18th-day fetus, a few immunoreactive angiotensinogen cells are localized in precise brain areas. Their number sharply increase during the 19th and 20th day gestation period when there is an active cell differentiation and cell growth. These observations suggest a role of the renin-angiotensinogen system during cell growth and cell differentiation.

Differential effects of inflammation models on rat T-kininogen and rat angiotensinogen.

The effect of different experimental models of inflammation on plasma concentrations of T-kininogen and angiotensinogen was examined in the rat. T-kininogen, a major phase protein which inhibits cysteine proteinase is increased in all cases of induced inflammation: administration of lipopolysaccharide and turpentine, bilateral nephrectomy or sham-operation and intraperitoneal injection of peanut oil. Angiotensinogen, the renin-substrate, is increased by lipopolysaccharide but is decreased by turpentine. Sham-operation or peanut oil injection have no effect on angiotensinogen whereas, bilateral nephrectomy and dexamethasone increase its concentration. Therefore, angiotensinogen is regulated differently than T-kininogen during inflammation.

Early effects of thyroidectomy and triiodothyronine administration on rat-liver mitochondria.

This work was undertaken to study the action exerted by thyroid hormones on mitochondria. By day 6 after thyroidectomy, the respective activities of two inner-membrane enzymes--succinate and beta-hydroxybutyrate cytochrome c reductases--had already dropped by 32 and 50%, whereas, in the outer membrane, the activity of rotenone-insensitive NADH-cytochrome c reductase did not change significantly. The decrease in the activity of the inner-membrane enzymes closely followed the disappearance of T3 and T4 from serum. 10 h after administration of 25 micrograms/100 g T3 to thyroidectomized rats, the activity of succinate and beta-hydroxybutyrate cytochrome c reductases and the oxygen consumption rate with succinate or beta-hydroxybutyrate were significantly increased, while, in the outer membrane, the activity of monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase remained unchanged. In the thyroidectomized rat, L-[3H]leucine incorporation in vivo is diminished in all the liver mitochondrial proteins, and especially in two constituents of MW 19 000 and 28 000. The radioactivity of these two components is also decreased in the normal rat treated with chloramphenicol, a specific inhibitor of mitochondrial protein synthesis. L-[14C]leucine incorporation in isolated liver mitochondria was significantly increased in the thyroidectomized rat, 10 h after T3 treatment. Thus, thyroid hormones have an early and preferential action on the mitochondrial protein synthesizing system and on the inner-membrane enzyme activities.

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.

Colocalization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain.

Angiotensinogen is produced in the brain, but its precise localization and the cells in the central nervous system producing it are unknown. We have performed a double staining test for angiotensinogen and glial fibrillary acidic protein in rat brain and report here that these proteins colocalize in astrocytes.

Effect of sex hormones on plasma T-kininogen in the rat.

The influence of sex hormones on rat plasma T-kininogen concentration was examined. The level of T-kininogen in the post-pubertal female rat is about 3-times that of the male animal. Female rats castrated as adults or 15 days after birth, had low T-kininogen concentrations, near those of male rats. In contrast, castration of mature or immature male animals induced no change in T-kininogen. Treatment of castrated female or male rats with 17 alpha-ethinylestradiol significantly increased the T-kininogen level, whereas administration of testosterone or progesterone had no effect. The influence of estrogen was specific for T-kininogen, since plasma HMW kininogen concentration was the same in male and female rats and was not affected by castration or sex hormone treatment. T-kininogen concentration was not significantly changed in pregnant rat between the 12th and the 20th day of pregnancy, but increased after parturition. It was high in the newborn rat at birth and then decreased similarly over the next 3 weeks in males and females. It continued to decrease in the males, reaching the level of the adult rat, but it increased in the female from 3-4 weeks of age and reached the adult level at about 6-8 weeks. These data indicate that natural estrogens have a physiological influence on the plasma level of T-kininogen in female rats whereas testosterone had no effect on either male or castrated female rats. HMW kininogen is not physiologically dependent on sex hormones.