Ouabain-like factors in human urine: identification of a Na-K-ATPase inhibitor as vanadium-diascorbate adduct.

We investigated endogenous Na-K-ATPase inhibitors, i.e. ouabain-like factors(OLFs), in the urine of salt-loaded healthy subjects. During an intake of > 30g NaCl/day 24h-urines were collected, lyophilized, redissolved and acidified to pH 3.5. With gelchromatography the inhibitory activity eluted in a post-salt fraction FIV from Sephadex G-25. When this fraction was again passed through Sephadex G-10, one of three OLFs eluted in the early subfractions FIV/1-2 close to H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with Mr around 400 eluted in a late subfraction FIV/8 which resolved after reverse-phase HPLC into a more polar OLF- (water phase) and a more apolar OLF-2 (20% acetonitrile). Only the more apolar OLF-2 cross-reacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-K-ATPase with IC50 of around 1.5 x 10(-5) M and 1.5 x 10(-4) M, respectively. Identification of OLF-2 was first attempted because most material was available for further processing. Data from mass-spectroscopy, nuclear magnetic resonance (1H-NMR) and infrared spectroscopy characterized OLF-2 as structurally unrelated to ouabain but resembling ascorbic acid derivatives, i.e. vanadium (V) diascorbates (Mr 403) with similar elution times from RP-HPLC as OLF-2. They inhibited the enzyme in its E2-configuration with IC50 of 9 x 10(-5) M and 2 x 10(-6) M for V(IV)- and V(V)-diascorbate, respectively. OLF-1, OLF-2 and V-diascorbate raise intracellular free calcium in inner medullary collecting duct and vascular smooth muscle cells which also contract in vitro. V-diascorbate was also natriuretic in a bioassay. We suggest that V-diascorbates represent one of several OLFs excreted in human urine.

Vanadium-diascorbates are strong candidates for endogenous ouabain-like factors in human urine: effects on Na-K-ATPase enzyme kinetics.

Recently, we isolated from the urine of salt-loaded healthy subjects a more polar ouabain-like factor OLF-1 and a more apolar OLF-2, the latter cross-reacted with a digoxin anti-body. They were purified to single compounds with dose-dependent Na-K-ATPase inhibition. Mass-spectroscopy (MS) showed a Mr of around 400 and 1H-NMR- and IR-spectroscopy suggested diascorbic acid salts, i.e., vanadium (V) diascorbates (Mr 403) with similar elution times from RP-HPLC as OLFs. IC50 was 9 x 10(-5)M for VIV-diascorbate as compared to 2 x 10(-6)M for Vv-diascorbate. Enzyme inhibition was non-competitive with respect to sodium and Mg-ATP; p-NPPase assay showed strong inhibition in its E2-configuration. We suggest that V-diascorbates represent endogenous OLFs excreted in human urine.

Endogenous sodium pump inhibitors in human urine. Further identification of inhibitors of Na-K-ATPase.

We investigated the presence of endogenous Na-K-ATPase inhibitor(s), ie, ouabain-like factors (OLFs), in the urine of salt-loaded healthy subjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (> 30 g NaCl/day). The samples then were lyophilized. Redissolved urine concentrates were acidified (pH 3.5) and subjected to gelchromatography on a Sephadex G-25 column where the OLFs eluted in the post-salt fraction IV. When lyophilized fraction IV was rechromatographed on Sephadex G-10, OLFs with molecular mass (M(r) of approximately 400 eluted in a late fraction IV/8 separate from added ouabain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross-reacted with a digoxin antibody. By preparative thin-layer chromatography OLF-1 and OLF-2 were purified as single compounds with potent dose-dependent Na-K-ATPase inhibition and Ki-values approximating 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L, respectively. Mass-spectroscopy (MS) showed M(r) of 391 and 1H-NMR characterized the endogenous urinary apolar OLF-2 as a compound that is structurally totally unrelated to ouabain; infrared (IR) spectroscopy of OLF-1 and OLF-2 also revealed no similarity with ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

[Endogenous natriuretic and ouabain-like factors. Their potential role in volume and blood pressure regulation]. / Endogene Natriuretische und Ouabain-ähnliche Faktoren. Ihre potentielle Rolle bei der Volumen- und Blutdruckregulation.

The existence of an endogenous natriuretic hormone and ouabain-like factors (OLF) has been postulated for many years. This postulate was based on our original observation that a small M.W. fraction in the serum after acute expansion of the extracellular fluid volume (ECFV) not only exhibited natriuretic activity but also inhibited the Na-K-ATPase enzyme in vitro similar to ouabain. Since then, numerous studies confirmed the presence of OLFs in serum, urine, cerebrospinal fluid, and various organs including the heart and hypothalamus. Some of these OLFs are well-known endogenous compounds, such as free unsaturated fatty acids, which inhibit in vitro transmembranous sodium transport, Na-K-ATPase and 3H-ouabain binding to its membrane receptor or cross-react with digoxin antibodies. Chemically yet undefined OLFs of potentially hypothalamic origin were detected in various models of experimental and clinical hypertension and are suggested to play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs isolated from the urine of salt-loaded healthy subjects strongly enhance basal and vasopressin-stimulated release of calcium in vascular smooth muscle cells and platelets similar to the effects we had observed with endothelin. This urine fraction also exhibits natriuretic activity which increases in parallel with sodium intake. Further chromatographic separation and amino acid analysis confirmed the peptidic nature (M.W. less than 1000) of the natriuretic factor(s). However, the two biological activities, namely natriuretic and ouabain-like activities, reside in distinct and chemically different compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Chicken growth hormone: cDNA-synthesis and base sequence.

1. Growth hormone (GH)-cDNA was synthesized from poly A(+)-mRNA extracts of chicken pituitary glands. 2. Chicken-cDNA library was cloned into E. coli. 3. Base sequence analysis of chicken GH-cDNA revealed only 70% similarity compared with duck GH-cDNA, and 97% similarity with a previously published chicken GH-cDNA sequence. 4. Dissimilarities in base sequences are primarily observed in the 3'-non-coding region of GH-cDNAs (chicken and duck). 5. Comparisons of amino acid sequences of chicken and duck GH exhibit only three substitutions, while the amino acid sequences of GHs of chicken are identical.

Experimental determination of the kinetics of calcium-binding with chondroitin sulphate and the effects of uric acid on this process.

The calcium-binding kinetics of chondroitin sulphate C (CS) have been determined using equilibrium analysis including 45Ca. There is a linear relationship between the extent of the Ca binding and the concentration of CS present. 1 mumol CS disaccharide unit binds 0.757 mumol Ca. Scatchard plots of the data have revealed a single constant of dissociation (KD = 0.1429). In the presence of urate ions, and dependent on the pH value, the ability of CS to bind Ca may be impaired by as much as 31%. These measurements have supported the theory that urate ions interact with the GAGs in urine.

[Analysis of the biological effect of city smog extract. VI. Comparative investigations on the effect of city smog extracts and their fractions on DNA synthesis of hamster kidney and embryonic cells and kidney cells of the primate Cercopithecus aethiops (author's transl)]. / Untersuchung der biologischen Wirkung von atmosphärischen Feinstaubextrakten. VI. Vergleichende Untersuchungen über die Wirkung atmosphärischer Feinstaubextrakte und ihrer Teilfraktionen auf die DNS-Synthese von Hamsternierenzellen, Hamsterembryonalzellen und Affennierenzellen in vitro.

We analyzed the effect of two samples of city smog extracts and their fractions from Duisburg on the rate of DNA synthesis of mammalian cell cultures. - We employed tissue cultures of kidney and embryonic cells from the Syrian golden hamster (Cricetus aureatus) and kidney cells from the African green monkey (Cercopithecus aethiops). The DNA synthesis was determined autoradiographically after incubation with 3H-thymidine. We found that both city smog extracts exerted a dose-dependent suppression of cellular DNA synthesis. Monkey kidney cells were more sensitive than rodent cells. Analysis of single fractions indicate that the inhibition of DNA synthesis is the result of combined effects of all fractions. At present time it is not possible to correlate the toxic effect of the complete extract special to a single fraction or compound group.

[Autoradiographic investigations on the effect of city smog extract on DNA synthesis and cell cycle of mammalian cells in vitro. II. Alterations of the cell cycle of hamsters kidney cells and hamster embryonic cells in presence of city smog extract (author's transl)]. / Autoradiographische Untersuchungen über den Einfluss von atmosphärischem Feinstaubextrakt auf die DNS-Synthese und den Zellzyklus von Säugetierzellen in vitro. II. Veränderungen des Zellzyklus von Hamsternierenzellen und Hamsterembryonalzellen in Gegenwart atmosphärischen Feinstaubextraktes.

We used the autoradiographic method according to Quastler and Sherman to analyse alterations in the cell cycle under the influence of city smog extracts. Investigations were performed on logarithmically growing cultures of kidney and embryonic cells of the Syrian golden hamster. Low concentrations of city smog extracts (0.125 micrograms/ml Benzo(a)pyren-equivalent) induced a remarkable delay of cell entrance into DNA-synthesis. Furthermore a considerable prolongation of generation time and phase of DNA-synthesis was detected. The number of mitosis was strongly reduced. Already a doubling of concentration of city smog extract caused an almost complete breakdown of the cell cycle and a disappearing of mitosis for a time period of 10 hours. Our results strongly indicate that city smog extracts lead to a severe alteration of the molecular biology of the cell. Taking this in consideration, it can be assumed that a long term exposure of human beings to the city smog could induce an injury of health.

[Autoradiographic investigations on the effect of city smog extract on DNA synthesis and cell cycle of mammalian cells in vitro. I. Effect of city smog extract on DNA synthesis of kidney- and embryonic cells of the golden hamster in vitro (author's transl)]. / Autoradiographische Untersuchungen über den Einfluss von atmosphärischem Feinstaubextrakt auf die DNS-Synthese und den Zellzyklus von Säugetieren in vitro. I. Wirkung von atmosphärischem Feinstaubextrakt auf die DNS-Synthese von Hamsternierenzellen und Hamsterembryonalzellen in vitro.

We analysed the effect of city smog extract from Düsseldorf on DNA synthesis of mammalian cells in vitro. Airborne dust was extracted with aceton and thereafter transferred to dimethylsulfoxide. Dosage was calculated according to the benzo(a)pyrene content. We used logarithmically growing cultures of hamster kidney and embryonic cells. DNA synthesis was determined autoradiographically by incorporation of 3H-Thymidine. We found that city smog extract exerted a dose-dependent cytotoxic effect leading to a decrease of DNA synthesizing cells. High concentrations of city smog extract induced cell necrosis and suppressed DNA synthesis completely. Moderate doses of extract caused a dose-dependent, but temporary cessation of DNA synthesis. Cells resumed DNA synthesis after a certain delay. Low concentrations of city smog extract induced no detectable effects. Inhibition of DNA synthesis was evident already one hour after addition of extract. Therefore a direct effect on DNA metabolism could be supposed. Furthermore, exposed cultures demonstrated a delay in entrance of new cells into the DNA synthesis. Alterations in DNA synthesis could be of great importance for carcinogenesis, especially if we take in consideration the content of carcinogens in city smog extract.

[Analysis of the biological effect of city smog extract. V. Comparative investigations on the effect of city smog extracts on DNA synthesis of Syrian hamster kidney and embryonic cells and of African green monkey kidney cells in vitro (author's transl)]. / Untersuchung der biologischen Wirkung von atmosphärischen Feinstaubextrakten. V. Vergleichende Untersuchungen über die Wirkung atmosphärischer Feinstaubextrakte auf die DNS-Synthese von Hamsternierenzellen, Hamsterembryonalzellen und Affennierenzellen in vitro.

We analysed the effect of two samples of city smog extract from Bochum and Duisburg on DNA synthesis of mammalian cells in vitro. As a test system we used tissue cultures of kidney and embryonic cells from the Syrian golden hamster and monkey kidney cells from Cercopithecus aethiops. DNA synthesis of cells was measured by autoradiography using 3H-Thymidine. Both samples of city smog extract exerted a dose-dependent decrease of the rate of DNA synthesis in tissue culture cells. These alterations of nucleic acid metabolism were expressed by a reduction of DNA-synthesizing cells and by a delay of entrance of cells in DNA synthesis. High concentrations of city smog extracts induced a large number of cell necroses. Monkey kidney cells were more sensitive to the toxic action than hamster cells. Furthermore the city smog extract from Duisburg showed a stronger toxic effect than the extract from Bochum.

[Analysis of the biological effect of city smog extract. II. Effect of a city smog extract on cell growth and DNA synthesis of hamster kidney cells in vitro (author's transl)]. / Untersuchung der biologischen Wirkung von atmosphärischen Feinstaubextrakten. II. Einfluss eines atmosphärischen Feinstaubextraktes auf die Zellvermehrung und die DNS-Synthese von Hamsternierenzellen in vitro.

A city smog extract from an urban area inhibits the cell growth of hamster kidney cells in vitro. Parallel to an inhibition of cell multiplication a diminished rate of total DNA synthesis appeared. The number of cells in DNA synthesis is depressed in presence of city smog extract. These phenomena revealed a dose-response relationship. The biological effect of city smog extract is discussed.