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Biomedical subjects

T Inagami

Publications and source records attributed to T Inagami.

At least 397 records · Page 22Linked to original sources
PubMed1983

Renin: structural features of active enzyme and inactive precursor.

To determine the structural basis for the unique catalytic mechanism of renin and the mechanism of activation of inactive renin, renin and inactive renin were isolated in pure form. The active site of renin consists of two aspartyl residues, two tyrosyl residues, and one arginyl residue, analogous to pepsin and other acid proteases. The complete amino acid sequence of mouse submaxillary gland renin was determined. Of the amino acids, 43% were identical to those in porcine pepsin. Combination of various chromatographic techniques permitted the separation of inactive renin from active renin in human plasma and kidney. Inactive renin of hog kidney was completely purified. Inactive renin consists of a single polypeptide chain and is activated by proteolysis but not by dissociative reagents such as 4 M NaCl or detergent. Thus it was concluded that the inactive renin in these tissues is renin zymogen rather than a renin-inhibitor complex.

Amino Acid Sequence↗
PubMed1982

Endogenous renin inhibitor in neuroblastoma cells.

Cloned neuroblastoma cells (Neuro-2a) in culture were found to contain a renin inhibitory substance. The inhibitor in the extract of cloned neuroblastoma cells was separated from renin activity by anti-renin IgG-Sepharose and selectively concentrated by adsorption to renin-agarose gel. The present study demonstrated the coexistence of renin and its inhibitor in the same cell and suggested a possible regulatory mechanism of intracellular renin activity by an endogenous renin inhibitor in neuronal cells.

Animals↗
PubMed1982

Structure of mouse submaxillary gland renin. Identification of two disulfide-linked polypeptide chains and the complete amino acid sequence of the light chain.

Reduction and carboxymethylation of mouse submaxillary gland renin produced two polypeptide chains which were readily separated by gel filtration or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of the two chains, termed heavy chain and light chain, were determined to be 30,000 and 5,458, respectively. Carboxymethylation of renin with radiolabeled iodoacetic acid followed by chain separation after reductive cleavage of disulfide bridges revealed the presence of two free cysteine residues in the heavy chain. Based on the finding of one half-cystine in the light chain and three half-cystine residues in the heavy chain. It was concluded that the heavy and light chains are linked by one disulfide bridge and that the heavy chain contains an intrachain disulfide bridge. The complete 48-amino acid residue sequence of the light chain was determined using peptide fragments obtained by cyanogen bromide cleavage and digestion with Staphylococcus aureus protease. The sequence showed 46% homology with the carboxyl-terminal region of the porcine pepsin sequence.

Amino Acid Sequence↗
PubMed1982

Brain renin from bovine anterior pituitary. Isolation and properties.

Brain renin has been purified 1,670,000-fold to homogeneity from bovine anterior pituitary in seven steps, including affinity chromatography on pepstatin-aminohexyl-agarose. The molecular weight of this enzyme is 36,000 as determined by gel filtration on Ultrogel AcA 44 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an isoelectric point of 5.25 and works best at physiological pH. In contrast to renal renin, bovine brain renin fails to bind to concanavalin A and four other lectins. The angiotensin I-forming activity of the purified brain renin was completely neutralized by anti-hog renal renin antibody. Rabbit antisera against pure brain renin showed a low degree of species and tissue specificity, reacting readily with hog brain renin and bovine and hog kidney renins. These results provide definite evidence for the presence of a functional brain renin-angiotensin system.

Amino Acids↗
PubMed1982

Vascular and tubular renin in the kidneys of mice.

Monospecific antisera and the immunocytochemical PAP-method have been used to localize renin in the kidneys of mice. With this procedure, reaction product was not only observed in the epitheloid cells of kidney vessels but also in kidney tubules: in the apical portion of proximal tubule cells and in some cells of the connecting and the cortical collecting tubule. To answer the question, whether the occurrence of renin in kidney tubule cells is the consequence of tubular synthesis or that of glomerular filtration of plasma renin followed by its uptake from the tubular lumen, tracer experiments with radioiodinated renin and with horseradish peroxidase were undertaken. The results of these studies as well as other arguments suggest reabsorptive pinocytosis of the filtered hormone as the source of tubular renin.

Animals↗
PubMed1982

Markedly elevated specific renin levels in the adrenal in genetically hypertensive rats.

The specific renin (EC 3.4.99.19) activity in the adrenal of spontaneously hypertensive rats was determined by a method that is capable of distinguishing renin from nonspecific renin-like activity of proteases by using specific antibody to renin. The renin level in the adrenals of adult spontaneously hypertensive rats with established hypertension was found to be 6-8 times as high as that of the normotensive control Wistar-Kyoto strain. The large difference in the adrenal renin level was observed even in 3-wk-old rats in which hypertension has not yet developed. The adrenal renin level was increased by bilateral nephrectomy in both the hypertensive and normotensive strains. A larger quantity of renin was found in the adrenal cortex than in the medulla, and the difference between the hypertensive strain and the normotensive strain was more prominent in the cortex than in the medulla. These results suggest possible involvement of adrenal renin in the development and in the early maintenance phase of hypertension in this animal mode of human essential hypertension by affecting the adrenocortical or adrenomedullary activity, or both.

Adrenal Cortex↗
PubMed1982

Amino acid sequence of mouse submaxillary gland renin.

The complete amino acid sequences of the heavy chain and light chain of mouse submaxillary gland renin have been determined. The heavy chain consists of 288 amino acid residues having a Mr of 31,036 calculated from the sequence. The light chain contains 48 amino acid residues with a Mr of 5,458. The sequence of the heavy chain was determined by automated Edman degradations of the cyanogen bromide peptides and tryptic peptides generated after citraconylation, as well as other peptides generated therefrom. The sequence of the light chain was derived from sequence analyses of the peptides generated by cyanogen bromide cleavage or by digestion with Staphylococcus aureus protease. The sequences in the active site regions in renin containing two catalytically essential aspartyl residues 32 and 215 were found identical with those in pepsin, chymosin, and penicillopepsin. Comparison of the amino acid sequence of renin with that of porcine pepsin indicated a 42% sequence identity of the heavy chain with the amino-terminal and middle regions and a 46% identity of the light chain with the carboxyl-terminal region of the porcine pepsin sequence. Residues identical in renin and pepsin are distributed throughout the length of the molecules, suggesting a similarity in their overall structures.

Amino Acid Sequence↗
PubMed1982

Renin in the brain and neuroblastoma cells: an endogenous and intracellular system.

A major portion of renin-like activity in extracts of brain tissues is due to nonspecific action of proteases. True renin has been separated from the proteases by various affinity chromatographic methods and true renin was identified by its inhibition by specific antirenin antibodies. Brain renin has been purified to varying extents. Renin in bovine pituitary was completely purified. Certain properties of brain renin are different from renal renin. The presence of inactive prorenin was also found in many regions. Immunohistochemical studies with renin antibodies showed intracellular localization of renin in many regions of the brain. Renin was also localized in LH gonadotrophs in the adenohypophysis. Many cloned neuroblastoma cell lines contain not only renin but also all other components of the renin-angiotensin system, indicating the existence of an intracellular mechanism of angiotensin formation within neurons.

Angiotensin I↗
PubMed1982

Juxtaglomerular cells grown as monolayer cell culture contain renin, angiotensin I-converting enzyme, and angiotensin I and II/III.

A monolayer cell culture of juxtaglomerular cells (JGC) was derived from the renal cortex of neonatal rats. The JGC had the characteristics of those within the kidney, including peripheral dense bodies and myofibrils indicating a smooth muscle origin; rough ER containing fluffy material consistent with protein synthesis; a prominent Golgi apparatus for packaging granules, and granules having the characteristics of secretory granules and lysosomes. Transplants of the cultured cells into syngeneic recipients survived for 2 weeks or longer and retained the features of JGC. The JGC granules fluoresced when treated with a rabbit antibody against pure rat renin, followed by fluorescein isothyocyanate conjugated F(ab')2 fragment of goat antirabbit IgG (Fc fragment) heavy chain specific. The latter indicated the presence of renin. The JGC were lysed in the presence of DFP, captopril, leupeptin, and EDTA, and were extracted in the presence of pepstatin. The lysate contained renin activity that was inhibited by a specific renin antibody. Nonspecific proteases were excluded by the antibody and its pH optimum. Angiotensin I-converting enzyme was detected in the lysate prepared without the use of EDTA and captopril. Angiotensins I and II/III were derived from the extract by additional extractions, TLC, and RIA, using highly specific antibodies. The angiotensins were confirmed by chromatography monitored by authentic angiotensins. We concluded that the cultured JGC contained renin, angiotensin I-converting enzyme, and angiotensin I and II/III.

Angiotensin I↗
PubMed1982

Localization of kallikrein in the rat kidney and its anatomical relationship to renin.

The anatomical relationship between kallikrein and renin in the rat kidney was investigated immunohistochemically by the peroxidase-antiperoxidase method. Kallikrein was localized to the convoluted distal tubule, starting at a point, distal to the juxtaglomerular apparatus, where the thick ascending limb of loop of Henle transformed into the convoluted distal tubule. The thick ascending limb was identified by its content of uromucoid (Tamm-Horsfall glycoprotein). Kallikrein was never observed within the juxtaglomerular apparatus itself. The kallikrein-containing tubule ended where the distal tubule submerged into the collecting duct. Renin was found in epitheloid cells of the afferent arteriole. When neighboring sections were stained for kallikrein and renin, respectively, no close anatomical relationship was observed between the kallikrein-containing and the renin-containing structures.

Animals↗
PubMed1982

Immunohistochemical localization of renin in the human kidney.

By using an antiserum to purified human renal renin, renin was localized immunocytochemically in the human kidney under normal and various pathological conditions by the unlabeled antibody enzyme light microscope: (LM) and protein A-gold colloid electron microscope (EM) procedures. In the normal kidney, renin was confined to the epithelioid cells of the afferent arteriole of the juxtaglomerular apparatus (JGA). These cells were small and few, and always in the immediate neighborhood of the glomerulus. Fine structural analysis showed renin only in the secretion granules of the epitheloid cells. All granules within a given cell were stained with comparable intensity. In cases of renal artery stenosis (ischemic kidney) and of Bartter's syndrome, renin-positive epithelioid cells were larger, showed increased staining intensity, and were often found along the afferent arteriole at some distance from the glomerulus. Again, by electron microscopic observation, renin was seen only in secretion granules of epithelioid cells. In all of the above pathologic cases, plasma renin activity was very high. However, in the other nephropathies studied, renin staining in the kidney resembled that seen in normal kidneys, even when levels of plasma renin activity were quite high.

Fluorescent Antibody Technique↗
PubMed1982

Human plasma inactive renin: purification and activation by proteases.

A new affinity chromatographic procedure was devised to purify inactive renin by using a selective hydrophobic interaction of inactive renin to octyl-Sepharose. Additional extensive purification was accomplished by immunoaffinity chromatography on antihuman renin immunoglobulin G-Sepharose. A trace amount of active renin was removed by chromatography on pepstatin-Sepharose. Human plasma inactive renin purified by this method was free from protease inhibitors and permitted the investigation of protease-mediated activation without the acid treatment which was used previously to remove inhibitors. Human plasma kallikrein, human plasmin, cathepsin B1, and arginine esteropeptidases associated with mouse epidermis growth factor and nerve growth factor were effective activators. Human urinary kallikrein, hog pancreatic kallikrein, and rat urinary esterase A were inefficient activators of low potency. Thrombin, factor Xa, factor XIIa, and urokinase did not activate inactive renin. The in vitro activation of 56,000-dalton inactive renin by these proteases was not accompanied by a recognizable reduction in molecular weight. Activation required plasma albumin, presumably as a protecting substance. These results suggest that human inactive renin can be activated by a minimum change in its molecular size.

Cathepsin B↗
PubMed1982

Brain renin.

Although the brain contains cathepsins at high concentrations which exhibit a non-specific renin-like activity at acidic pH, the presence of specific renin in the brain has been demonstrated by characterizing its specific properties. Renin was separated from cathepsin by affinity chromatography on casein-Sepharose. Brain renin showed neutral pH optima for the reaction to generate angiotensin I. The presence of inactive prorenin was also found. The isoelectric points of brain renin were significantly lower differences from that of renal or plasma renin. Immunohistochemical studies demonstrated a wide-spread localization of renin in many different regions. Angiotensin II, the final product of the prohormone-to-hormone conversion reaction mediated by renin and angiotensin converting enzyme, was found to exist in the same cell as renin by immunohistochemical studies of brain sections and with cloned and cultured neuroblastoma cells. This is the first demonstration of the mechanism of peptide hormone formation in neuronal cells. Similar intracellular formation was demonstrated in gonadotrophs of adenohypophysis. Coexistence of renin and angiotensin II was demonstrated in some cells. Electrophysiological studies have shown that angiotensin II functions to disinhibit the inhibition of neuronal response to electrical stimuli in the hippocampus.

Angiotensin II↗
PubMed1982

Molecular characterization of inactive renin: complete purification of prorenin in hog kidney and isolation of inactive renin from neuroblastoma cells: evidence for 2 different types of inactive renin.

To determine the molecular properties of inactive renin and its relationship to active renin, inactive renin in hog kidney was purified by devising affinity chromatography. Electrophoretically homogeneous inactive renin was prepared by 3 million-fold purification. It consists of a single polypeptide chain and undergoes reduction in molecular weight from 50,000 to 38,000 upon activation by proteases but not by dissociative treatment. This type of inactive renin is considered as a zymogen. However, a stable complex of renin and its inhibitor with a molecular weight of 110,000 was found in cultured neuroblastoma cells indicating the presence of a second type of inactive renin.

Animals↗
PubMed1982

Cultured juxtaglomerular cells cause hypertension by secreting angiotensin.

Cultured JGC contain renin, angiotensin I, angiotensin I-converting enzyme, angiotensin II, and, by implication, the entire RAS. JGC, as transplants, appear to secrete angiotensin II/III directly into the bloodstream to cause hypertension when the renal mass is reduced. There are two main phases of the hypertensive state, an angiotensin-dependent developmental phase and a non-angiotensin-dependent maintenance phase. This model may be useful in attempts to evaluate pro-hypertensive actions of angiotensin other than those due to direct systemic vasoconstriction. Certain of these actions appear to be intrarenal and include the stimulation of sodium reabsorption, a decrease in renopapillary blood flow, the stimulation of prostaglandin synthesis, and a constraint on the antihypertensive function of the RIC.

Angiotensin II↗