The location of renin (EC 3.4.99.19) in rat pituitary was determined by the peroxidase-antiperoxidase immunohistochemical technique. By using antisera prepared with purified rat renal renin, an immunoreactive substance was localized within ovoid cells scattered throughout the anterior pituitary. These cells were shown to be luteinizing hormone-producing cells by staining with anti-luteinizing hormone antisera in adjacent sections. By using the double staining method, the renin-containing cells were differentiated from cells containing corticotropin, thyrotropin, growth hormone (somatotropin), and prolactin (mammotropin). These results suggest a possible local role for renin in the anterior pituitary.Renin (EC 3.4.99.19) (5), while a large amount ofprotease with nonspecific renin-like activity is found in both the anterior and the posterior lobes (unpublished data). Furthermore, in our immunohistochemical studies of the pituitary of the mouse (7) and rat (8), renin-specific staining was observed only in the anterior lobes.A similar lack of agreement is apparent among previous reports with respect to the intrahypophysial localization of angiotensin (9, 10). The consideration of the physiological function of a renin-angiotensin system in the pituitary has been largely focused on the posterior pituitary, presumably in association with the well-known effect of angiotensin II on vasopressin release (11), though the effect may not be exerted in the pituitary. The effect ofangiotensin II on the release ofadrenocorticotropic hormone (ACTH; corticotropin) from the anterior pituitary is also known (12), but the physiological role ofpituitary renin has remained unclear.With the objectives of resolving the existing controversies concerning the location of renin and obtaining clues for the physiological significance of renin in the pituitary, we have applied an immunohistochemical staining technique for the identification of the renin-containing cells in the pituitary.MATERIALS AND METHODS Antisera. Specific antibodies to rat renin were produced in Dutch Belted rabbits by using pure rat renin as antigen. The pure renin was prepared by a published method (13). The enzyme preparation satisfied multiple criteria ofpurity, which included single bands upon polyacrylamide gel electrophoresis, sodium dodecyl sulfate gel electrophoresis, isoelectric focusing, and double immunodiffusion and symmetric chromatographic elution patterns. This preparation (1.0 mg) was conjugated to 0.5 mg of tetanus toxoid with 25 ,ug of water-soluble carbodiimide and exhaustively dialyzed, and then an aliquot containing 80 ug of renin was mixed with an equal volume of complete Freund's adjuvant and injected intradermally at multiple sites in the back ofeach rabbit. After six biweekly boosters, each with the conjugate equivalent of 10 ,.g of renin, antisera were collected. Tested at dilutions greater than 1:500, these antibodies did not crossreact with human renin or rat cathepsin. Used at 1:2000 dilution for immunohistochemical staining of ra...
a-Glucosidase 1 of Bacillus thermoamyloliquefaciens KPI 071 (FERM P8477, facultative thermophile) was purified to homogeneity. The relative molecular mass was estimated to be 62000 Da. From its catalytic properties, the enzyme has been assigned to an exo-a-1 ,4-glucosidase. The enzyme shares its antigenic groups in part with Bacillus stearotherrnophilus ATCC12016 (obligate thermophilc) exoa-1 ,4-glucosidase.These exo-a-1 ,4-glucosidases strikingly resemble oligo-l,6-glucosidases from B. thermoamyloliquefuciens KP1071 and from Bacillus cereus ATCC7064 in the molecular properties tested, including rclative molecular mass, N-terminal sequence of 15 residues, amino acid composition and structural parameters calculated from amino acid composition. We have suggested that bacillary exo-a-l,4-glucosidases take the same folded conformation, i.e. an (a/fl)8-barrel super-secondary structure in its N-terminal domain, as bacillary oligo-I ,6-glucosidases.A p-nitrophenyl-a-D-glucopyranosidase from Bacillus stemothcrmophilus ATCCl2016 (obligate thermophile) had been assigned to a novel exo-a-1,4-glucosidase [l]. However, no enzyme of similar function was found in othcr bacteria. Bacillus thrrmotrmyloIiquefuciens KP1071 (FERM P8477, facultative thermophile) produces a series of thermostable enzymes responsible for the complete hydrolysis of amylopectin, pullulan and cyclodextrin [2]. Of these enzymes, a maltotriogenic a-amylase I, a maltogenic a-amylase I1 capable of hydrolyzing a-1,6-bonds in amylopectin and of cleaving cyclodextrin rings, and an oligo-2,6-glucosidase have already been characterized [3 -51. However, serologically distinct a-glucosidases I, I1 and I11 at least remained to be isolated. In the present study, we have purified a-glucosidase 1 to homogeneity and assigned it to an exo-a-1,4-glucosidase. We have found a striking similarity of bacillary exo-a-l,4-glucosidases to bacillary oligo-I ,6-glucosidases in the N-terminal sequence and in structural parameters derived from the amino acid compositi on.
The rates of gastric emptying for highly branched cyclic dextrin (HBCD) and other carbohydrate (CHO) solutions were examined using ultrasonograph techniques. Ten healthy volunteers ingested water, physiological saline, or solutions containing various CHO, such as HBCD, glucose, maltose, sucrose, and commercially available dextrin. After a subject drank one of the solutions, the relaxed cross-sectional area of the pylorus antrum was measured at rest by real-time ultrasonography. The time required for gastric emptying was correlated with the relaxed cross-sectional area of the pylorus antrum. Among all of the solutions tested, physiological saline was transferred fastest from the stomach to the small intestine. For solutions of the same CHO, 5 % solution was transferred faster than 10 % solution. For CHO solutions other than HBCD, a low osmotic pressure was associated with rapid transfer from the stomach. The gastric emptying time (GET) of HBCD solution increased with an increase in its concentration. A shorter GET was observed for the CHO solutions at 59 to 160 mOsm regardless of their concentration. A sports drink based on 10 % HBCD adjusted to 150 mOsm by the addition of various minerals, vitamins, and organic acids was evacuated significantly (p < 0.05) faster than a 10 % HBCD solution or a sports drink based on 10 % commercially available dextrin (DE16), which has a higher osmotic pressure (269 mOsm). Our results suggest that a shorter GET could be achieved with CHO solutions with osmotic pressures of 59 - 160 mOsm. Therefore, a sports drink based on 10 % HBCD adjusted to 150 mOsm by the addition of minerals, vitamins, and organic acids could supply adequate quantities of CHO, fluid, and minerals simultaneously in a short time, without increasing GET.
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.
Renin-like activity was detected in various endocrine glands and other extrarenal tissues. Both nonspecific proteolytic activity of proteases and the specific action of renin contribute to this activity. In order to estimate the true renin component in these extrarenal tissues of rats, we determined the renin activity which is sensitive to inhibition by a specific antibody produced against pure rat renal renin. A widespread distribution of true renin was revealed. The adrenal gland was found to have the highest true renin activity per unit weight of protein among the various extrarenal tissues. Its activity was several thousand times as great as the other tissues and was only one fifteenth of that in the renal cortex. The pH optimum of this adrenal renin was approximately 7.0. These findings suggest a control mechanism for adrenal function mediated by an intraadrenal renin-angiotensin system in addition to the plasma renin-angiotensin system.
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