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.
Medical Policlinic/Department of Medicine, University of Bonn, Germany.
H J
HJ
Kramer
G
G
Krampitz
A
A
Bäcker
H
H
Meyer-Lehnert
D000328:Adult; D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology / Q000652:urine; D001685:Biological Factors / Q000737:chemistry / Q000494:pharmacology / Q000652:urine*; D002118:Calcium / Q000378:metabolism; D002298:Cardenolides; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004077:Digoxin; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology / Q000652:urine*; D005260:Female; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D009942:Organometallic Compounds / Q000737:chemistry / Q000494:pharmacology / Q000652:urine*; D051381:Rats; D012503:Saponins; D012964:Sodium / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013552:Swine; D014639:Vanadium / Q000652:urine; D014661:Vasoconstriction / Q000187:drug effects
D001685:Biological Factors; D002298:Cardenolides; D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; D012503:Saponins; C034504:digoxin-like factors; C095018:vanadium diascorbate; D014639:Vanadium; D012965:Sodium Chloride; D004077:Digoxin; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D001205:Ascorbic Acid; D002118:Calcium