Is prorenin of physiological and clinical significance?
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Animals↗
Biomedical subjects
K Poulsen
Publications and source records attributed to K Poulsen.
Alternative routes for angiotensin-I conversion: kinetics of the renin-angiotensin system in mouse plasma.
Angiotensin-I in plasma may be converted to angiotensin-II or alternatively metabolized through other routes. The aim of the present work has been to quantitate the role of the alternative routes in mouse plasma. A kinetic model for metabolism of the angiotensins through the renin-angiotensin system is proposed, the model allows for alternative routes and for simultaneous reactions to occur. Exogenously added angiotensin-I and angiotensin-II, or endogenously formed angiotensin-I and angiotensin-II from the action of exogenously added renin, were measured in plasma with time. The concentrations of angiotensin-I and angiotensin-II were measured by radioimmunoassay after separation by Dowex cation exchange and HPLC reverse-phase chromatography. The rate constants for the enzymatic reactions were experimentally determined and fitted to the kinetic model. The data were incompatible with a quantitative conversion of angiotensin-I to angiotensin-II, and indicate that about half of the angiotensin-I formed in plasma is metabolized in plasma through alternative routes.
Angiotensin I↗
Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenzae serotype b strains.
Immunoglobulin A1 (IgA1) proteases are thought to be important virulence factors in certain bacterial infections, including meningitis, and may have potential usage in vaccines. In this study, we compared the locations of EcoRI, BamHI, and PstI restriction endonuclease sites in the IgA1 protease gene (iga) region of whole-cell DNA from 76 Haemophilus influenzae strains. The analysis was performed by using isolated fragments of the cloned iga gene, which encodes the IgA1 protease originating from a H. influenzae serotype d strain, as probes in Southern blot experiments. All strains, including three without detectable IgA1 protease activity, had DNA sequences with a high degree of homology to the iga probes. The numbers and sizes of the DNA fragments hybridizing with the probes indicated that only three strains, none of which was of serotype b, had more than one iga gene. The iga restriction fragment length patterns of 60 clinical isolates of serotype b were of only four distinct types, which correlated with previously observed clusters of multilocus genotypes (electrophoretic types). This correlation supports the concept of the clonal population structure of H. influenzae. Three of the iga gene restriction types, which appear to represent 98% of the H. influenzae serotype b population, encode IgA1 proteases that were inhibited by antisera to any one of these types and therefore could form the basis for the development of a vaccine against H. influenzae meningitis.
Antibodies, Bacterial↗
Identification of renin in resident alveolar macrophages and monocytes: HPLC and immunohistochemical study.
Normal, unstimulated alveolar macrophages (AM) and monocytes (Mo) from both mice and rats have been shown to exhibit an angiotensin-I-forming enzyme. This protease has a similar or identical molecular weight to plasma renin as judged by HPLC analysis. In addition, the enzyme activity could be almost completely inhibited by a specific antibody to pure mouse submaxillary gland renin. This indicates that the angiotensin-I-generating protease in AM and Mo is identical to true renin. These results are in accordance with those of immunocytochemical studies, since the cells sharing immunoreactive renin are macrophages and monocytes. The low percentage of renin positive cells may indicate a functionally different and probably distinct subpopulation of AM and Mo. The presence of renin and angiotensins and angiotensin-I-converting enzyme in normal macrophages and monocytes seems to be common. Hence, the data presented raise the possibility that an intracellular renin-angiotensin system may infact exist in a certain subpopulation(s) of normal macrophages and monocytes.
Animals↗
Evidence for a predominant renal secretion and clearance of inactive plasma renin, studied by in vivo inhibition of protein synthesis.
The aim was to investigate the contribution of the kidneys and the extrarenal tissues to the concentration of inactive renin in the circulation. Renin was determined in plasma from normal, conscious, male mice in strains with low (BALB/c) and high (Theiller) renin content in the submandibular glands. In untreated, conscious males, inactive and active renin concentration in plasma was 24.9 GU/l (range 13.6-40.1 GU/l) and 4.2 GU/l (0.6-8.8 GU/l), respectively in the BALB/c strain (n = 28). These values were not significantly different from the concentrations of inactive (29.2 GU/l, range 15.6-88.3 GU/l) and active (5.1 GU/l, range 1.9-10.4 GU/l) renin in Theiller's strain (n = 10). Thus, inactive renin comprised 80-90% of the total renin in plasma from normal, conscious, male mice of both strains. The percentage of inactive renin was similar to that found in humans. Following sialo-adenectomy and bilateral nephrectomy, the plasma concentration of inactive renin was unchanged. The effect of in vivo inhibition of the protein synthesis by intraperitoneal injection of emetine was also investigated. After 6 h of inhibition, inactive renin in plasma declined from 21.0 to 9.9 GU/l in normal mice. In contrast, inactive renin was unchanged in sialo-adenectomized and nephrectomized mice (29.1 versus 28.6 GU/l). Our findings suggest that the kidneys are the main source of inactive renin in the circulation. The data also indicate that the kidneys simultaneously secrete and remove inactive renin from the blood at approximately the same rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Aggression↗
Quantitative activation and determination of inactive renin by high performance liquid chromatography.
Assay of inactive renin in unfractionated mouse plasma is difficult and often impossible because of high concentrations of active renin and plasma protease inhibitors. Therefore, 0.025 to 0.05 ml of plasma or amniotic fluid from mice was separated by high performance liquid chromatography (HPLC) using a silica-based size exclusion column. The eluates were examined for enzymatically active renin before and after limited proteolysis with trypsin. Since inactive renin eluted as a single peak corresponding to a molecular weight of 38,000 daltons and the elution of active renin was markedly retarded, inactive and active renin were partially separated. compared with plasma, inactive renin in amniotic fluid eluted as a broader and sometimes diphasic peak, suggesting heterogenicity. The rapid and reliable separation by HPLC provided a more than 300-fold purification of inactive renin. Despite low concentrations of plasma protease inhibitors, a 1 000 000-fold molar excess of trypsin (1 mg/ml) was needed for optimal activation. The necessity for high trypsin concentrations for activation may partly be explained by enzyme kinetic considerations. By combining HPLC with trypsin activation, inactive renin was readily measured and found to be 9.4 (5.1-13.2) GU (Goldblatt units)/l in normal and 8.0 (5.1-12.2) GU/l in sialoadenectomized and nephrectomized mouse plasma, which is higher than previously determined in our laboratory. The concentration was 12.4 (8.8-16.1) GU/l in amniotic fluid. Thus, the concentration of inactive renin in plasma is almost as high as active renin in normal mice (17.6 GU/l) and is uninfluenced by the decrease of active renin to 0.3 GU/l after sialoadenectomy and nephrectomy.
Amniotic Fluid↗
Absence of processing enzymes for inactive renin in mouse blood but polymorphonuclear neutrophils contain active renin.
The possible role of enzymes in plasma and blood cells for the conversion of inactive renin was investigated in mice of the Balb C strain. The concentrations of both inactive and active renin were unchanged in unfractionated plasma and whole blood during incubation at 37 degrees C for 5 h, indicating, that inactive renin had not been activated. Purified platelets, mononuclear cells (monocytes and lymphocytes), and polymorphonuclear leukocytes were incubated separately with partially purified inactive renin at 37 degrees C for 1 h. Inactive renin was neither activated nor degraded by whole blood cells. Polymorphonuclear neutrophils were also obtained in mice after i.p. injection of fluid thioglycollate medium. Extracts of the polymorphonuclear neutrophils were incubated with partially purified, inactive renin. During incubation at pH 7.5 for 1 h at 37 degrees C, inactive renin was stable and no activation was registered, whereas degradation, without a concomitant increase in active renin, occurred at pH 4.5. Extracts of polymorphonuclear neutrophils were found to contain active, but no inactive renin. The mean amount of renin per cell was 0.5 X 10(-11) Goldblatt units (GU) (range 0.3 X 10(-11) GU - 0.9 X 10(-11) GU; n = 6). These findings argue against a role for the blood cells and plasma in a physiological activation of inactive renin, and do not support the concept that activation of inactive renin in the circulation may participate in homeostasis.
Amniotic Fluid↗
Coordination of murine parotid secretory protein and salivary amylase expression.
PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.
Amylases↗
Is angiotensinogen a renin inhibitor and not the substrate for renin?
The cleavage of the angiotensinogen molecule by renin is slow. The rate constant for cleavage of the enzyme-substrate complex, Kcat (turnover number) is lower than usual for enzymes (0.6/s for the homologous human renin reactions and 0.15/s for the homologous mouse renin reaction). Thus each round of catalysis occurs in 1.7 s in the human and 6.7 s in the mouse reaction. The Kcat/Km values (10(4)-10(6) l/mol per s; Km, Michaelis constant) are high and in the range for protein inhibitors. The association constants (Ka) are (10(6)-10(7) l/mol) close to that of inhibitors. The slow rate seems to be favourable since homologous reactions are slower than heterologous. Angiotensinogen is structurally related to alpha1-antitrypsin. We conclude that kinetic as well as structural data indicate that angiotensinogen might be considered a protease inhibitor for renin.
Angiotensinogen↗
Inhibition of the enzymatic reaction of renin in aggressive mice.
Aggressive behaviour provokes a very high increase in plasma renin in aggressive male mice. We demonstrate that this occurs in mice with high as well as low concentrations of renin in the submaxillary glands, although the highest values were seen in the former. Plasma renin and angiotensinogen concentrations, as well as plasma renin activity, were measured before and after aggression. The theoretical plasma renin activity (generation rate of angiotensin l) was calculated from kinetic parameters and the renin and angiotensinogen concentrations. The Km (Michaelis constant) was 3 mumol/l and the kcat (turnover number) was 0.15/s for the reaction between pure submaxillary mouse renin and mouse angiotensinogen. The measured plasma renin activity was much lower than that calculated from both the kinetics and the fall in angiotensinogen concentration. The decrease in angiotensinogen in vivo was independent of the renin concentration. Intraperitoneally injected renin mimicked the aggression-provoked renin increase and resulted in better agreement between measured and calculated parameters. This indicates, that during aggression, the mouse can inhibit and control the renin-angiotensinogen reaction.
Aggression↗
Characterization of angiotensin I, II, and III from mouse as position-5 isoleucine [Ile5] angiotensins. An HPLC Study.
Angiotensin I was generated in vitro in mouse plasma by addition of mouse submaxillary renin. After deproteinization on Dowex 50W-X2 ion exchange resin, the samples were separated by reverse phase liquid chromatography and the fractions analysed by radio-immunoassay for angiotensin I. Angiotensin I from mouse was injected into the jugular vein of a nephrectomized rat in order to generate angiotensin II and III of mouse origin. Carotid arterial blood was collected immediately, centrifuged and the plasma deproteinized and separated on high pressure liquid chromatography (HPLC). The fractions were analysed for angiotensin I, II and III by radioimmunoassays. Retention times for mouse angiotensins in the liquid chromatography system were compared to synthetic [Val5] and [Ile5] angiotensin I, II and III. All three mouse angiotensins coincided with the corresponding synthetic [Ile5] angiotensins during elution, suggesting the same type of angiotensin in mouse and man. An additional study on rat angiotensins confirmed previous findings by other investigators, indicating that the angiotensins of this animal were also of the [Ile5] type.
Angiotensin I↗
Mouse salivary glands secrete a glucagon-degrading enzyme, not glucagon.
Salivary glands have been reported to synthetize glucagon in various animal species. We therefore studied the glucagon-like immunoreactivity (GLI) in mouse saliva. Stimulation with phenylephrine evoked a 15-fold increase of salivary GLI output. On Sephadex G-50 gel filtration, the salivary GLI was significantly larger than glicentin, the hitherto largest known glucagon-related peptide; furthermore, the immunoreactivity was not absorbable on glucagon immunoadsorbent. 125I-Labeled glucagon incubated with high GLI containing saliva, and subjected to gel filtration and immunoadsorption was degraded to low molecular weight, nonimmunoreactive moieties. Among EDTA, phenylmethylsulfonylfluoride, Pepstatin-A, and N-ethylmaleimide, only N-ethylmaleimide inhibited the degradation. Renin, also found in mouse saliva, degraded the tracer but did not cochromatograph with salivary GLI. In conclusion, GLI of mouse saliva is not a peptide containing a glucagon-immunoreactive sequence but represents tracer-degrading activity, probably composed of sulfhydryl enzymes.
Animals↗
Demonstration of kallikrein in a rat pancreatic acinar cell carcinoma.
Kallikrein was identified immunohistochemically and biochemically in a transplantable pancreatic acinar cell carcinoma of the rat. The concentration of immunoreactive kallikrein in tumor homogenates was the same as in the pancreas. Kallikrein in tumor cells exists as a proenzyme and is released into blood in high concentrations. The impact of the presence of a kallikrein-producing tumor on other kallikrein-containing organs and other possibly interrelated systems was investigated. The concentration of kallikrein in the submandibular gland and pancreas of host rats was not significantly different from that of control rats. Urinary kallikrein secretion was significantly increased, although this may be a result of the high plasma glandular kallikrein concentration combined with kidney damage. The plasma concentration of kininogen, kininase, and renin was not significantly different from control rats. Rats with tumor had significantly lower blood pressure than did control animals, and blood pressure was inversely related to the concentration of glandular kallikrein in plasma. However, it was not proven that the low blood pressure was due to the high concentration of kallikrein. Nephrectomized tumor rats gave a smaller hypotensive response to kininase inhibition than was expected from their high concentration of circulating kallikrein. This may be explained by the absence of the "free kallikrein" fraction in plasma of host rats.
Amylases↗
Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands.
Renin and epidermal growth factor (EGF) are synthesized in large amounts by the male mouse submandibular glands. We report the peptides to be secreted mainly in an exocrine manner. The highest values in saliva are obtained upon stimulation with the alpha-adrenergic agonist phenylephrine. The median value for renin is 54 700 nmol/l and the median value for EGF is 211 800 nmol/l. Aggressive behaviour and beta-adrenergic stimulation also increase salivary output of both peptides, while vasoactive intestinal polypeptide (VIP) plus pilocarpine selectively stimulate the secretion of renin. The pattern of increase in plasma is comparable to that in saliva though the substance concentration is lower by a factor of 2 to 70 for renin and a factor of 280 to 12 000 for EGF.
Animals↗
Characterization of inactive renin in mouse amniotic fluid.
We have demonstrated high concentrations of inactive renin in mouse amniotic fluid compared with mouse plasma and other organs. Its molecular weight was similar to that of prorenin from the mouse submaxillary gland (42 000 daltons). The activated renin had a Km value of 1.48 greater than M which is identical to that of active amniotic fluid mouse renin. The time course and concentration dependence of trypsin activation was studied, and compared with activation by direct acidification and acidification by dialysis. The pH optimum for acid activation varied with temperature between pH 3 and 4, probably because of simultaneous activation and degradation. Acid activation was almost completely reversible.
Amniotic Fluid↗
The enzymatic renin reaction in vivo is inhibited in mice during inter-male aggressiveness.
In aggressive male mice with very high plasma renin concentrations the plasma angiotensin II (ANG II) and III (ANG III) concentrations were measured in single mice in order to study whether this renin was active in vivo. The angiotensins were purified and identified by ion-exchange chromatography and High Pressure Liquid Chromatography (HPLC) and quantitated by a specific and sensitive radioimmunoassay. Plasma ANG II and ANG III values were not elevated in aggressive mice compared to normals, in spite of the fact that the plasma renin concentration was increased up to 100 fold and almost equal to the plasma renin-substrate concentration. This indicates an inhibition of the enzymatic renin reaction in vivo during aggression, which is in accordance with the lack of blood pressure increase, and an unchanged pressure sensitivity to ANG II.
Aggression↗
Synthesis and conversion of the renin precursor in vitro.
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Animals↗