Restoration of urine concentrating ability and accumulation of medullary osmolytes after chronic diuresis.

Restoration of urine osmolality (Uosm) and medullary osmolyte contents after chronic diuresis was studied in rats infused for 6 days with furosemide and subsequently given the vasopressin analogue, 1-desamino-8-D-arginine vasopressin (DDAVP). Papillary tip intra- and extracellular electrolyte concentrations were measured by electron microprobe analysis, tissue contents of methylamines (glycerophosphorylcholine, betaine), polyols (myo-inositol, sorbitol), and several amino acids in different kidney zones by high-performance liquid chromatography. Administering DDAVP continuously after diuresis increased Uosm from (means +/- SE) 348 +/- 8 to 1,265 +/- 127 after 1 day and 2,485 +/- 186 mosmol/kgH2O after 3 days. The sum of all osmolytes at the papillary tip rose from 309.2 +/- 28.9 to 690.9 +/- 105.8 and 1,282.8 +/- 21.0 mmol/kg protein after days 1 and 3, respectively. Although interstitial tonicity (sum of Na, Cl, and K concentrations) was increased by 116 and 223% after 1 and 3 days DDAVP, intracellular tonicity was similar in chronic diuresis and following 1 or 3 days DDAVP. Coadministration of DDAVP with betaine, myo-inositol, and choline ("osmolyte treatment") did not accelerate the restoration of Uosm but caused significantly higher contents of osmolytes (except myo-inositol) in inner medulla and/or papilla after 3 days. In a minority of animals, restoration of Uosm and reaccumulation of medullary osmolytes were impeded in both DDAVP- and DDAVP/osmolyte-treated rats. These data indicate that, after chronic diuresis, accumulation of organic osmolytes and restoration of Uosm proceed in parallel. Capacity for transport and/or synthesis of organic osmolytes, rather than their availability, appear to limit reaccumulation on the first day of recovery. By the third day, delivery of some osmolytes or their precursors may limit the restoration of medullary osmolyte content. The failure of some rats to attain sufficient concentrating ability within this time period may be related to deficient reaccumulation of medullary osmolytes.

Isolation and characterization of trypsin-like and chymotrypsin-like proteinases from human cholesteatoma.

The mast-cell-specific proteolytic enzymes tryptase and chymase were identified in and isolated from cholesteatoma in a ratio similar to that found in human skin. We assume that this ratio reflects a similar distribution of tryptase-containing and tryptase/chymase-containing mast cells in both these tissues. It seems conceivable that mechanisms able to trigger excessive and/or continuous mast cell degranulation in the middle ear might be causative for the formation of cholesteatoma either directly or via primed chronic inflammatory reactions. By their ability to amplify degranulation of mast cells, mast cell proteinases, in particular chymase, may contribute to the chain of events leading to the formation of cholesteatoma.

Amino acid sequences of mammalian kazal-type proteinase inhibitors from salivary glands.

1. The amino acid sequences of bikazins (the double-headed Kazal-type proteinase inhibitors from submandibular glands) isolated from the snow leopard (Unica unica), the European mink (Mustela lutreola), and the European pine marten (Martes martes) were determined. 2. N-terminal domains of bikazins are characterized by a cysteine residue spacing that differs from that of C-terminal domains of bikazins and other Kazal-type proteinase inhibitor domains. 3. N-terminal sequences of bikazins seem to be specific for, and highly conserved within, each Carnivora family.

Osmotic adaptation of renal medullary cells during transition from chronic diuresis to antidiuresis.

The cells of the renal medulla adapt osmotically to high extracellular tonicities by high concentrations of organic osmolytes. Intracellular accumulation of these substances is, however, relatively slow. The aim of the present study was to assess the effect of an abrupt rise in extracellular tonicity on intracellular osmotically active substances after prior reduction of medullary contents of organic osmolytes by chronic diuresis. Intra- and extracellular electrolyte concentrations at the papillary tip and the tissue contents of methylamines (glycerophosphorylcholine, betaine), polyols (myo-inositol, sorbitol), and several amino acids were determined in the different kidney zones by electron microprobe analysis and high-performance liquid chromatography in control animals, in rats infused for 6 days with furosemide via osmotic minipumps, and in rats given the vasopressin analogue [deamino-Cys1,D-Arg8]vasopressin (DDAVP) after the chronic furosemide treatment. Chronic diuresis greatly reduced interstitial tonicity and inner medullary contents of methylamines and polyols and moderately reduced inner medullary amino acid contents but did not significantly affect intracellular electrolyte concentrations. When the diuretic rats were infused with DDAVP for 2 h, interstitial tonicity more than doubled and intracellular K and Cl concentrations rose by approximately 60 and 160%, while inner medullary contents of methylamines, polyols, and amino acids were not changed significantly. These data demonstrate that after effective depletion of medullary organic osmolytes by long-term diuresis, the cells of the renal papilla adapt osmotically to an abrupt increase in extracellular tonicities by elevated cell electrolyte concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

[Protease inhibitors as tumor-associated growth factors]. / Proteaseninhibitoren als tumorassoziierte Wachstumsfaktoren.

Two apparent endothelial cell growth factors were isolated and characterized from serum free cell culture medium of hepatoma cells by McKeehan et al. The factors were identified as proteinase inhibitors with known primary structure. They are the pancreatic secretory trypsin inhibitor (HPSTI) and the double headed inhibitor HI-30, the inhibitory active part of the inter-alpha-trypsin inhibitor complex. We were able to isolate acid resistant inhibitory active material from serum free culture medium of 4 out of 11 tumor cell lines, which we have analyzed. The cell lines were not derived from liver cells. The inhibitory active material was identified as the inhibitor HI-30 by N-terminal amino acid sequence analysis. The results indicate that HI-30 is a real growth factor, since it is expressed as well in tumor cells which are not derived from liver cells.

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, X. The amino-acid sequences of the trypsin-released inhibitors from horse and pig inter-alpha-trypsin inhibitors.

The amino-acid sequences of the acid-resistant inhibitors released from horse and pig inter-alpha-trypsin inhibitor (ITI) by tryptic proteolysis were determined. They are composed of two covalently linked Kunitz-type domains. In both cases the reactive site of their C-terminal antitryptic domains is occupied by arginine as in the homologous human and bovine inhibitors. The reactive site of their N-terminal domain exhibits only a weak interaction with polymorphonuclear granulocytic elastase and is occupied by leucine as in the strong elastase inhibitor released from bovine ITI. The differences between inhibitory activities of the ITI-derived inhibitors from horse, pig, and cattle are discussed on the basis of sequence differences in position P'2.

Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase.

The N-terminal amino-acid sequence of human ITI has been found to be identical with that of the acid-stable human 30-kDa inhibitors (HI-30) from urine, serum, and those released from inter-alpha-trypsin inhibitor by trypsin or chymotrypsin. Serum HI-30 and HI-30 released by trypsin differ from the urinary inhibitor by an additional C-terminal arginine residue. Compared to these two inhibitors the inhibitor released by chymotryptic proteolysis is elongated C-terminally by an additional phenylalanine residue. These results strongly favour HI-30 as the N-terminus of the inter-alpha-trypsin inhibitor and its release from this inhibitor in vivo by cleavage of the Arg123-Phe124 peptide bond by trypsin-like proteinases.

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination.

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IX. Isolation and characterization of the inhibitory parts of inter-alpha-trypsin inhibitors from several mammalian sera.

The inhibitory parts of inter-alpha-trypsin inhibitor-like proteins from several mammalian sera (sheep, goat, horse, donkey, pig, rabbit, rat and dog) were released by limited proteolysis with trypsin and were isolated by reversible binding to immobilized trypsin. The inhibitors are very similar with respect to their stability in acids, molecular masses and amino-acid compositions. They are different, however, in their inhibitory properties. In view of the known covalent structures of the inhibitory parts of the human and bovine inhibitors, homologous covalent structures consisting of two tandem Kunitz-type domains are suggested also for the isolated inhibitors. Bovine trypsin, bovine chymotrypsin and porcine plasmin are inhibited by all investigated inhibitors, most likely via their C-terminal domain. The inhibitors from horse, donkey, rabbit, rat and dog serum interact also with elastase from human polymorphonuclear granulocytes, those from sheep, goat and pig serum inhibit in addition porcine pancreatic elastase and bovine chymotrypsin via their N-terminal Kunitz-type domain. It is supposed that the amino-acid residue in position P1 of the N-terminal Kunitz-type domain is responsible for the characteristic inhibitory properties of each inhibitor.

Elastase inhibition by the inter-alpha-trypsin inhibitor and derived inhibitors of man and cattle.

The inhibitory properties of HI-14 and BI-14, the active 14-kDa parts released from the corresponding human and bovine inter-alpha-trypsin inhibitors, are compared. The structurally homologous inhibitors composed of two tandem Kunitz-type domains differ in their inhibitory specificity, although the reactive site residue in position P1 is occupied by identical (arginine in the C-terminal domain II) or similar (methionine and leucine in the N-terminal domain I of HI-14 and BI-14, respectively) amino-acid residues. The N-terminal domain I of HI-14 is completely inactive against chymotrypsin and pancreatic elastase, whereas BI-14 is a strong inhibitor of these enzymes. Elastase from polymorphonuclear granulocytes interacts with both inhibitors but with different affinities. Compared with the bovine inhibitor, the human inhibitor shows a much lower affinity from this enzyme. Human ITI and its physiological 30-kDa derivative (HI-30) show the same inhibitory properties as HI-14. The differences between human and bovine inhibitors might be explained by a preceding oxidation of Met in vivo of the reactive site residue in position P1 and/or by the influence of the environmental parts connected with this antielastase reactive site region in human ITI or in the active domains thereof.

An elastase-specific inhibitor from human bronchial mucus. Pathogenesis in lung emphysema.

Under physiological conditions human bronchial mucus contains an elastase-specific inhibitor which is quite different from hitherto known inhibitors: alpha 1-antitrypsin, HI-30, and BSI-TE. In bronchial mucus of 100 patients suffering from obstructive airway disease the amount of this inhibitor specific against elastase from both pancreas and polymorphonuclear neutrophilic leucocytes was measured. A group of 18 patients showed no inhibitor in their native mucus rather than free elastolytic activity. Another group of 82 patients had no elastolytic activity in their native mucus but free anti-elastolytic activity together with varying amounts of elastase-specific inhibitor bound to proteases. In a total of 20 cases from both groups the inhibitor was not detectable, neither in a free nor in a complexed form. Presuming that there is no hereditary deficiency involved, a strong importance of inactivating reactions such as oxidation is concluded.

An elastase-specific inhibitor from human bronchial mucus. Isolation and characterization.

It was shown previously that human bronchial mucus contains an acid-stable proteinase inhibitor directed against trypsin and chymotrypsin, polymorphonuclear granulocyte elastase and cathepsin G. In addition to this well-characterized inhibitor, designated here as BSI-ATE (identical with the inhibitor HUSI-I from human seminal plasma or antileucoprotease), another acid-stable inhibitor BSI-E is present in the mucus which exerts inhibitory activity towards porcine pancreatic and human granulocytic elastase, but not against trypsin, chymotrypsin, or granulocytic cathepsin G. This elastase-specific inhibitor was isolated by affinity chromatography. Its molecular mass and its amino acid composition are very similar to those of BSI-TE. An immunological cross-reactivity between both inhibitor species was not observed. In the mucus of patients suffering from obstructive airway disease the elastase-specific inhibitor is not present in the free form but can be liberated by acidification.

Effect of various compounds on the reaction of tris-(2-chloroethyl)amine with ribonucleic acid in vitro and on its toxicity in mice.

The effect of tris-(2-chloroethyl)-amine (HN-3) on RNA and DNA was investigated spectrophotometrically. The shift in the absorbance spectrum caused by the addition of HN-3 was used to test a variety of compounds for their ability to inhibit RNA alkylation. The effect of HN-3 on the activity of several enzymes was also investigated. The activities of ribonuclease A, desoxyribonuclease I, acetylcholinesterase, diaphorase, glutathione reductase, adenosine desaminase, glyoxalase I, 3-hydroxyacyl-CoA-dehydrogenase, xanthine oxidase, glucose-6-phosphate dehydrogenase, hexokinase and the microsomal N-oxygenation of aniline were not changed by HN-3, whereas the activity of cytochrome-c-reductase exhibited a dose dependent diminution in the presence HN-3. Of 105 compounds tested only 14, namely, sodium thiosulfate, dithioxanthine, thiosalicylic acid, 1,2,4-triazole-5-thiol, 2-thiocytosine, 2-thiohistadine, 2,3-dithiosuccinic acid, thioglycolic acid, 3-mercapto-D-valine,6-amino-2-thiouracil, thionicotine amide, dithiothreitol, sodium sulfite, and ergothioneine prevented the alkylation of RNA. All of them also reacted with HN-3 in absence of RNA. No correlation was found between the reaction constant of the reaction compound:HN-3 in the absence of RNA and the concentration of the compound which inhibited RNA alkylation by 50%. The compounds which were effective in vitro were also tested in mice for their ability to reduce HN-3 toxicity in vivo. Only sodium thiosulfate, d-penicillamine, and dithiosuccinic acid were effective. A 3.9fold increase in the LD50 of HN-3 was achieved in mice treated with sodium thiosulfate 3330 mg/kg i.p., a 1.7fold with 2125 mg dithiosuccinic acid/kg, and a 2fold increase with 2500 mg/kg d-penicillamine. The compound tested was injected i.p. 0.5 to 1 min after the s.c. injection of HN-3.