Immunochemical studies on biosynthesis of rat plasma angiotensinogen and its regulation by cortisol

Glucocorticosteroid hormones increase the level of rat plasma angiotensinogen by increasing its rate of synthesis. Two forms of plasma angiotensinogen have been purified differing with respect to molecular weight and affinity to concanavalin A. Immunochemical studies using antibodies raised against the separated forms of angiotensinogen revealed cross-reactivity with both antigens. Both antibodies were able to quantitatively precipitate the angiotensinogen activity present in rat serum samples. Cortisol increased the total amount of plasma renin substrate without changing the relative amounts of both angiotensinogen forms. mRNA coding for plasma angiotensinogen was determined by in vitro translation of poly(A)-containing RNA and immunochemical analysis of translation products. Angiotensinogen mRNA could be detected in total poly(A)-containing RNA isolated from rat liver, but not in mRNA isolated from brain, although angiotensinogen has been reported to be present in the latter organ. The level of hepatic mRNA coding for plasma angiotensinogen was high in rats treated with cortisol, but not detectable in animals depleted from endogenous glucocorticosteroids by bilateral adrenalectomy.     

Biosynthesis of Angiotensinogen and Angiotensins by Brain Cells in Primary Culture

This study focuses on the ability of primary rat brain cells in culture to synthesize angiotensinogen, angiotensin I, and angiotensin II. HPLC in combination with radioimmunoassay was used to characterize these compounds. Following incubation with 3H-labeled isoleucine, radioactively labeled angiotensinogen with an approximate molecular weight of 25,000 was identified in both glial and neuronal cells. Other molecular weight forms of angiotensinogen with molecular weights of about 300 and 160,000 were present in both cell types. In addition to angiotensinogen, radioactively labeled angiotensin I and angiotensin II were also synthesized by neuronal and glial cells. These results suggest that glial and neuronal cells can synthesize angiotensinogen, angiotensin I, and angiotensin II in a similar manner shown for the peripheral renin angiotensin system.     

Characterization of human angiotensinogen

In this study of the physical and chemical properties of human angiotensinogen were determined. Human angiotensinogen is a glycoprotein containing 14% carbohydrate. The molecular weight as determined by sedimentation equilibrium studies was 56,800. A higher molecular weight was obtained on sodium dodecyl sulfate electrophoresis. Ferguson-type plots indicated that angiotensinogen is another glycoprotein which behaves anomalously on sodium dodecyl sulfate electrophoresis. The COOH-terminal amino acid was found to be serine while two NH2-terminal amino acids, alanine and aspartic acid (or asparagine), were detected. The specific angiotensin I content of angiotensinogen preparations can vary considerably with no effect on the apparent homogeneity of the isolated protein. A protein with negligible angiotensin I content has been obtained from a preparation of human angiotensinogen. The COOH-terminal amino acid of this protein was serine while the only NH2-terminal amino acid detected was alanine.     

Binding of renin and angiotensinogen to macromolecules in plasma studied by sedimentation equilibrium centrifugation

Plasma from normal and aggressive mice was subjected to sedimentation equilibrium centrifugation in an air-driven centrifuge. The aim was to study the apparent molecular weight of renin and angiotensinogen in undiluted plasma. Both renin and angiotensinogen appeared heterogeneous with respect to molecular size, suggesting binding to plasma macromolecules. Subsequent high pressure liquid chromatography, using a size exclusion column, demonstrated molecular homogeneity and molecular weights as found in noncentrifuged plasma, indicating that the binding is easily reversible. It is concluded that renin and angiotensinogen in undiluted and unfractionated plasma are weakly bound to plasma macromolecules. This may reduce their activity and to some extent explain the previously observed apparent inhibition of the enzymatic activity.     

Immunocytochemical localization of albumin,transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation

Summary Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochemically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes expressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strikingly in parallel with the hepatocyte differentiation. Albumin and transferrin are highly expressed comparatively to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.     

Immunocytochemical localization of albumin, transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation

Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochemically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes expressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strikingly in parallel with the hepatocyte differentiation. Albumin and transferrin are highly expressed comparatively to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.     

The effects of kallikrein, plasmin, and thrombin on hog kidney renin.

Pretreatment of hog high molecular weight renin for 30 min at 37 degrees C with 0.12 unit of either kallikrein or thrombin significantly increased (p less than 0.001) the amount of angiotensin I formed during subsequent incubations with homologous angiotensinogen. However, the thrombin-treated hog renin had 13 times more activity than the kallikrein-treated enzyme. Aprotinin did not inhibit the kallikrein-mediated activation of renin; the results indicated that aprotinin inhibited renin preferentially. Plasmin (0.25 unit) had little effect on the activity of high molecular weight renin. The molecular weight of hog renin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was not altered after exposure to either kallikrein, thrombin, or plasmin. These results do not exclude the occurrence of a limited proteolytic event or a conformational change beyond the detection of the current method. The data show that the activation of hog high molecular weight renin by thrombin and kallikrein was not associated with the conversion of renin to Mr = 43,000.     

Involvement of a pertussis toxin-sensitive G protein in the regulation of angiotensinogen production by an angiotensin II analog in HepG2 cells

The cellular mechanism by which the angiotensin II (AII) agonist, Sar1-AII, inhibits production and release of angiotensinogen in human hepatoma HepG2 cells was examined. Pretreatment of HepG2 cells with pertussis toxin attenuated the ability of Sar1-AII to block angiotensinogen production. This effect could be correlated with the in situ ADP-ribosylation of a protein(s) of apparent molecular weight 39,000-41,000 on SDS-PAGE, and attenuation of the ability of Sar1-AII to inhibit cAMP accumulation. The role of cAMP in angiotensinogen production was examined. A transient increase in cAMP accumulation above basal could be evoked by forskolin (8-fold) or by glucagon (5-fold) using insulin-deficient media. Although neither forskolin nor glucagon had a significant effect on angiotensinogen production agents producing a sustained increase in intracellular cAMP (8-bromo-cAMP, dibutyryl-cAMP, cholera toxin) were able to increase angiotensinogen production. Although these data indicate that intracellular cAMP is a regulatory factor in angiotensinogen production other evidence suggests that modulation of intracellular cAMP is not entirely responsible for the effects of Sar1-AII.     

Effects of Glycosylation of the Residue at Position 14 in Ovine Angiotensinogen on the Human Renin Reaction

A mutant angiotensinogen, S14N, in which Ser¹⁴ of ovine angiotensinogen was replaced by Asn to form a N-glycosylation site, was produced in CHO cells. The molecular weight was about 3,000 larger than that of wild-type ovine angiotensinogen, indicating that S14N angiotensinogen was glycosylated at Asn¹⁴. In the reaction with human renin, the K _m of mutant angiotensinogen was 3 times increased, but the V _max was not affected by the mutation.     

The angiotensinogen gene of Swiss mice is closely linked to a retrovirus-like element

Angiotensinogen is cleaved by renin and angiotensin-converting enzyme to liberate the potent vasocontrictor peptide angiotensin II. We have recently identified a cis-acting genetic lesion associated with high levels of angiotensinogen mRNA in the testis and salivary gland of Swiss mice. To determine the molecular basis of this mutation, the Swiss angiotensinogen gene was cloned, and its structure was compared to that from a low-expressing strain (BALB/c). I show that a retrovirus-like element belonging to the intracisternal A-particle gene family has been inserted 9 kb upstream from the cap site of the Swiss angiotensinogen gene. This intracisternal A-particle, named IAP-Agt, segregated concordantly with angiotensinogen expression phenotypes in CXB recombinant inbred mice. However, genomic Southern analysis showed that IAP-Agt was present in some, but not all, inbred laboratory mouse strains displaying high levels of angiotensinogen gene expression. On the basis of this evolutionary evidence, it is unlikely that IAP-Agt is the cause of the angiotensinogen mutation. It is intriguing that Ren-2, the duplicated mouse renin gene, is expressed to high levels in the male salivary gland and also contains a transposed intracisternal A-particle genome.     

Purification and characterization of human kidney renin

By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8,000-fold purification was attained by means of only pepstatin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37 degrees C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37,000 by SDS-PAGE and 38,000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.     

Endogenous angiotensin II regulates hepatic angiotensinogen production

We have investigated the effects of endogenous angiotensin II (ANG II) on hepatic angiotensinogen mRNA levels in rats. Changes in endogenous ANG II were induced by various sodium intakes (standard-, low-, and high-sodium) or by enalapril treatment. In a low sodium state for 2 weeks, angiotensinogen mRNA levels and plasma ANG II concentration increased 1.3-fold and 1.6-fold compared to those in standard sodium state, respectively. In a high sodium state, angiotensinogen mRNA levels and plasma ANG II concentration decreased by 42% and 56% compared to the standard sodierm state, respectively. Four hours after treatment with enalapril (3 mg/kg), angiotensinogen mRNA level and plasma ANG II concentration decreased by 25% and 12% compared to the standard sodium state, respectively. There was a close correlation between angiotensinogen mRNA level and plasma ANG II concentration (r = 0.79, P less than 0.01). These results suggest that endogenous ANG II may play an important role in the regulation of hepatic angiotensinogen synthesis.     

Purification and characterization of rat angiotensinogen

1. Angiotensinogen (renin substrate) was purified from plasma of nephrectomized rats by a four step procedure using ammonium sulfate fractionation, chromatography on Blue Sepharose CL-6B and SP-Sephadex C-50, and gel filtration on Sephadex G-150. 2. The final preparation had a specific concentration of 9.3 microgram angiotensin I/mg (mean of six separate runs). The best preparation so far obtained contains 14.6 microgram angiotensin I/mg protein, which represents a purity of 62%. 3. By sodium dodecyl sulfate disc electrophoresis an apparent molecular weight of 56,400, and by isoelectric focusing an isoelectric point of 4.85 has been determined. These properties of rat angiotensinogen are similar to those reported for human angiotensinogen.     

Purification and partial characterization of rat brain acid proteinase (isorenin).

1. Isorenin was purified 2000-fold from rat brain by a simple 3-step procedure involving affinity chromatography on pepstatinyl-Sepharose, The preparation appears as a homogenous protein in analytical polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis indicated an apparent molecular weight of 45 000. Isoelectric focusing separated isoenzymes with isoelectric points at pH 5.45, 5.87, 6.16 and 7.05. 2. The enzyme generates antiotensin I from tetradecapeptide (pH optimum 4.7) and from sheep angiotensinogen (pH optima 3.9 and 5.5). The rate of angiotensin I formation from tetradecapeptide was 30 000 times higher than that from sheep angiotensinogen. The enzyme has acid protease activity at pH 3.2 with hemoglobin as the substrate and pepstatin is a potent inhibitor of the enzyme with a Ki of less than 10(-9) M. 3. The properties of the enzyme strongly suggest that it is identical with cathepsin D.     

Effects of glucocorticoids and antiglucocorticoid on angiotensinogen production by hepatoma cells in culture

Summary 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 dosedependent 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. This work was supported in part by a grnat from Inserm (CRL 824022).     

Peptide nucleic acids specifically cause antigene effects in vivo by systemic injection

Peptide nucleic acids (PNAs) are uncharged DNA analogs that hybridize to complementary sequences with high affinity and stability. We previously showed that PNAs, after intraperitoneal injection into rats, are effective antisense compounds in vivo. The present study was designed to test whether PNAs also have antigene effects in vivo. The renin-angiotensin system is critical in the control of blood pressure. We designed and synthesized sense (antigene) PNAs to angiotensinogen, which is the precursor protein that leads to angiotensin I and II. Spontaneously hypertensive rats received intraperitoneal injections of either 20 mg/kg sense-angiotensinogen-PNA, mismatch-angiotensinogen PNA, or saline. Only the sense-angiotensinogen PNA treatment resulted in a significant decrease in plasma angiotensin I, systolic blood pressure, and liver and brain angiotensinogen mRNA levels. Thus, these results demonstrate on the molecular, protein, and physiological levels that antigene PNAs are effective in vivo upon systemic administration.     

Low molecular weight renin as a storage form in renin granules of the dog

The molecular weight of renin extracted from isolated renin granules of the dog was estimated by gel filtration, using tetradecapeptide as substrate, and was approximately 43,000 daltons. Neither big renin nor big big renin was demonstrable. On the other hand, crude extract of kidney cortex showed angiotensin I generating enzymes other than 43,000 dalton form of renin, whose molecular weight were over 100,000 and around 70,000 daltons. They seemed nonspecific proteases, since they hydrolyzed tetradecapeptide but not plasma angiotensinogen. Therefore renin is stored in the renin granules as a low molecular weight form.     

Effects of tempol on renal angiotensinogen production in Dahl salt-sensitive rats

We have recently reported that Dahl salt-sensitive rats (DS) on high salt diet (HS) have an inappropriate augmentation of intrarenal angiotensinogen. Recent studies also reported that the augmented superoxide anion formation plays important roles in this animal model of hypertension. This study was performed to address the hypothesis that an inappropriate augmentation of intrarenal angiotensinogen by HS is caused by the augmented reactive oxygen species. Male DS (200-220 g) were maintained on low salt diet LS (N = 7) or HS (N = 27) for 4 weeks. The HS group was subdivided into three subgroups to receive null (N = 12), superoxide dismutase mimetic, tempol (3 mmol/l, N = 8), or vasodilator, hydralazine (0.5 mmol/l, N = 7) in drinking water during the period. Systolic BP was significantly increased in the DS+HS group compared to the DS+LS group (184+/-7 mmHg vs. 107+/-5 at 4-week). Tempol or hydralazine treatment equivalently attenuated the hypertension (128+/-3 and 127+/-5 at 4-week, respectively). Urinary excretion of thiobarbituric acid reactive substances at 4-week was significantly increased in the DS+HS group compared to the DS+LS group (0.66+/-0.05 micromol/day vs. 0.14+/-0.01). Tempol treatment prevented this effect (0.24+/-0.04) but hydralazine treatment only partially prevented the effect (0.40+/-0.03). Kidney angiotensinogen levels, measured by Western blot analysis, were significantly increased in the DS+HS group compared to the DS+LS group (32+/-5 densitometric units vs. 21+/-1). Tempol (14+/-3) but not hydralazine (32+/-5) treatment prevented the intrarenal angiotensinogen augmentation. The evidence suggests that the enhanced intrarenal angiotensinogen in DS challenged with HS is associated with the augmented reactive oxygen species.     

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.     

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.