Vanadyl (IV) and vanadate (V) binding to selected endogenous phosphate, carboxyl, and amino ligands; calculations of cellular vanadium species distribution.

Vanadium enters cells as vanadate (V) where it is reduced to vanadyl (IV), VO2+. Vanadate species at plasma pH, H2VO4-, and HVO4(2-) are referred to as VO3-. To gain an insight into the subcellular vanadium distribution we measured the binding of VO3- and VO2+ to extra- and intracellular ligands, and calculated free and bound fractions of these ions for expected in vivo conditions. The association constants (K) were determined by the pH shift caused by an addition of VOSO4 or NaVO3 to individual ligand solutions at 20 degrees C and a pH equal to the pK of the reactive groups. The pk's for binding of VO2+ were ATP, 5.9; ADP, 5.5; AMP, 5.1; Pi 4.3; creatine phosphate (CP), 3.6; glutamic acid, 3.4; aspartic acid, 3.1; human serum albumin, 3.1; glutathione, 2.7; ascorbic acid, 3.3; citric acid, 4.0. The pk of VO3- and human serum albumin was 3.3 and of that VO3- and glutathione was 4.2. VO3- did not bind to ATP, even via Mg2+ or Ca2+ bridges. We calculated that in cells approximately 1% of total VO2+ is unbound, which is 10(-10)-10(-9) M since published values for total vanadium (mainly VO2+) concentrations in tissues are on the order of 10(-8)-10(-7) M. Free VO2+ may be even less because of binding to additional ligands not considered and due to spontaneous hydrolysis to VOOH+ and VO(OH)2(2+) at intracellular pH. The binding of VO2+ to each ligand was corrected for presence of multiple ligands and competition by H+, K+, and Mg2+. In cells with no CP, up to 70% of VO2+ is bound to phosphates and up to 29% to proteins; in cells with 30 mM CP (as in muscle), approximately 95% is bound to phosphates (CP binds up to 61% of total VO2+) and approximately 4% to proteins; in cells with 2 mM ascorbic acid (as in brain), the vitamin binds approximately 3% of total VO2+. These binding values apply for the total VO2+ concentration range of 10(-8)-10(-5) M. The intracellular binding and a reducing environment protect the freshly reduced VO2+ from oxidation to VO3- that would otherwise occur at neutral pH. This strong affinity of VO2+ primarily for phosphates also explains the mechanism for the intracellular accumulation of vanadium which is a factor in previously observed transport of VO3- into cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship of cardiac muscle tension to Na+,K+-adenosine triphosphatase activity after chronic digitoxin administration in cats.

To obtain a better understanding of the mechanism of action of the cardiac glycosides, we examined inotropic and biochemical effects of digitoxin in myocardium from cats chronically exposed to the drug. The mechanical function of papillary muscles was tested isometrically and left ventricular tissue was analyzed for Na+,K+-dependent adenosine triphosphatase ATPase activity. Muscles from control cat hearts developed tension at 2.5 +/- 0.7 g/mm2; muscles from cats that received subcutaneous digitoxin--100 micrograms/kg on day 1, followed by 40 micrograms/kg/day for 4 days (group A), and 75 micrograms/kg on day 1, followed by 25 micrograms/kg/day for 9 days (group B)--developed significantly greater (p less than 0.05) tension of 4.8 +/- 0.3 and 3.6 +/- 0.6 g/mm2, respectively. Further, in vitro maximal responsiveness to digitoxin was greater in the muscles from digitalized groups than in controls (p less than 0.05): Muscles from control cats had a maximal response to in vitro addition of digitoxin of 3.5 +/- 0.1 g/mm2; muscles from cats in group A reached 4.9 +/- 0.3 g/mm2, and those from group B, 4.5 +/- 0.7 g/mm2. Specific activity of microsomal Na+,K+-ATPase from hearts of digitalized groups A and B was inhibited by 50-70% (p less than 0.01). Developed tension, specific Na+,K+-ATPase activity, and in vitro maximal responsiveness to digitoxin in a third group (C) of cats receiving the least daily digitoxin (75 micrograms/kg on day 1, followed by 15 micrograms/kg/day for 29 days) were not different from controls. Mean plasma digitoxin concentrations were 33, 16, and 3 ng/ml in groups A, B, and C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of inhibition of human renal adenosine triphosphatases by cisplatin and chloroplatinic acid.

Cisplatin and chloroplatinic acid were examined for in vitro inhibition of human renal microsomal adenosine triphosphatases activated by Na+ + K+ + Mg2+, Mg2+, and Ca2+. The concentrations of cisplatin to inhibit 50% of activity (I50) were approximately 7 X 10(-4) M for all enzymes studied; I50s of chloroplatinic acid were on the order of 10(-5) M for Na+ + K+ + Mg2+ ATPase and Ca2+ ATPase and 10(-7) M for Mg2+ ATPase in the presence of Na+ + K+ + ouabain. Inhibition of Na+ + K+ + Mg2+ ATPase by cisplatin or chloroplatinic acid was reversible and was not altered by varying Na+, K+, or Mg2+ concentrations; ATP or MgATP increased inhibition by cisplatin but not by chloroplatinic acid; acidic pH of 6.8 lowered inhibition by chloroplatinic acid but not by cisplatin. Cysteine, glutathione (-SH reagents), and ascorbic acid greatly reduced inhibition of all enzymes studied by chloroplatinic acid; in the case of cisplatin, -SH reagents had only a minimal protective effect but ascorbic acid somewhat increased inhibition. Methionine greatly increased inhibition by cisplatin but provided minimal protection in the case of chloroplatinic acid. In view of the hypothesis that inhibition of renal Na+ + K+ ATPase may be associated with tubular damage, the inhibition of Na+ + K+ ATPase may be relevant to the mechanism of platinum toxicity.

Inhibition of adenosine triphosphatase in vitro by silver nitrate and silver sulfadiazine.

Inhibition of adenosine triphosphatase (ATPase) by silver nitrate (AgNO3) in vitro was studied in microsomal fractions or tissue homogenates of canine brain and kidney, and human kidney. In microsomal fractions, AgNO3 was an indiscriminate inhibitor of ouabain-sensitive (Na+ + K+ ATPase) and ouabain-insensitive (Mg2+ ATPase) activities with 50% inhibition obtaining at concentrations on the order of 10(-7) to 10(-6)M. The enzyme was protected by cysteine. Changing the concentrations of Na+, K+, H+, Mg2+ and ATP did not alter the fractional inhibition of Na+ + K+ ATPase by a constant concentration of AgNO3. An aqueous suspension of silver sulfadiazine had an inhibitory potency similar to AgNO3. It was concluded that silver gives a different pattern of Na+ + K+ ATPase inhibition than other metallic inhibitors of the enzyme so far examined.

Vanadium: chemistry and the kidney.

Vanadium (V), a metallic element of the first transition series, is widely distributed in the environment. Although an essential trace element in higher animals, chronic exposure to V is of concern because of its increased concentration near industrial operations, its occurrence in the ash of combustion products of petroleum and coal, and its subsequent biomagnification in the environment. V is found in trace amounts in both terrestrial and aquatic animals and in solution can form inorganic orthovanadate oxyanions that, if absorbed, are eliminated primarily by the kidneys. Because V is often found concentrated in renal tissue to the largest extent in the body, the kidneys may represent a major site of action. Moreover, V in the vanadate configuration increases the urinary excretion of solutes and water in the rat, and inhibits renal organic ion accumulation and renal Na+, K+-ATPase in vitro and in vivo. Furthermore, as a nutritionally required element, V may play a regulatory role in salt and water excretion by modification of the Na+ pump in the kidney.

Vanadium-induced inhibition of renal Na+, K+-adenosinetriphosphatase in the chicken after chronic dietary exposure.

Recent work has shown that V accumulates in the kidney and is a potent inhibitor of Na+, K+-adenosinetriphosphatase (ATPase) in vitro. Thus, as a nutritionally required element, V may regulate cation transport. The effect of chronic intake of the metal on Na+, K+-ATPase in vivo has not been reported. In this study laying strain chickens were fed calcium orthovanadate for 15 mo from d 1 of age at levels of 0, 25, 50, and 100 ppm in the diet. Whole tissue homogenates and 13,000 X g fractions were analyzed for ATPase activities. Concentrations of V producing 50% inhibition of Na+, K+-ATPase activity ranged from 1.0 X 10(-5) M in liver to 1.8 X 10(-6) M in kidney, which was the most sensitive tissue tested in vitro. Mg2+ -ATPase was more resistant to V than Na+, K+-ATPase. Studies in vivo suggested a V-dependent inhibition of renal Na+, K+-ATPase. Correlation of enzyme specific activity and levels of V in kidneys suggested V-ATPase mediated alteration in renal function.

Effects of chronic digitalization on cardiac and renal Na+ + K+-dependent adenosine triphosphate activity and circulating catecholamines in the dog.

To extend our understanding of the mechanism of action of digitalis drugs, we studied electrocardiograms (ECGs), renal function, plasma concentrations of catecholamines, and myocardial and renal Na+ + K+-dependent adenosine triphosphate (Na+ + K+ ATPase) activity in chronically digitalized dogs. Five healthy, male, mongrel dogs received a therapeutic regimen of digoxin (0.1 mg/kg on day 1 in three divided doses followed by 0.025 mg/kg per day) orally for 2-4 months. This resulted in plasma digoxin concentrations of 1.1 to 4.7 ng/ml as determined by radioimmunoassay. Six control dogs received daily gelatin capsules by mouth. ECGs monitored throughout the study showed no changes. Digitalized dogs had elevated plasma norepinephrine concentrations (347 vs. 137 pg/ml in controls) and no change in plasma epinephrine concentrations. Digitalized dogs had elevated glomerular filtration rates (0.74 vs. 0.94 ml/min per g of kidney) without significant changes in renal handling of electrolytes and water. All of the above studies were done without the aid of restraining drugs or infusions. The animals were killed with an overdose of pentobarbital for in vitro studies. In digitalized dogs, microsomal Na+ + K+ ATPase-specific activity was 26 to 33% lower in the renal cortex, medulla, and papilla, and 46% lower in the cardiac left ventricle than in control dogs. Digitalization did not alter the osmolalities of renal tissues. We conclude that chronic reduction Na+ + K+ ATPase activity by one-third dose does not cause abnormalities in renal handling of electrolytes and water, and inhibition of Na+ + K+ ATPase in the left ventricular muscle by one-half is associated with no obvious ECG changes in the dog. Further, elevated plasma norepinephrine concentrations may contribute to both the therapeutic and the toxic effects of digitalis.

Inhibition by metals of a canine renal calcium, magnesium-activated adenosinetriphosphatase.

A number of metals were examined for inhibition of a canine renal calcium, magnesium-activated adenosinetriphosphatase (Ca2+, Mg2+-ATPase). Of the 27 metals investigated, only compounds of mercury, silver, gold, and uranium demonstrated 50% inhibition of the enzyme at concentrations lower than 10(-4) M. The order of inhibitory potency was Hg greater than Ag greater than U greater than Au. Organic mercury (chlormerodrin, mersalyl, p-chloromercuribenzoate) was less potent than inorganic mercuric chloride, but organic gold sodium thiomalate was equipotent with inorganic gold chloride. The inhibition produced by each metal decreased parallel to the decrease in enzyme activity, seen as the source of enzyme moved from the outer cortex inward to the papilla of the kidney. The regions of highest activity showed the greatest inhibition by each metal, and inhibition decreased as the control activity of the tissue decreased. This variability of inhibition was not related to the protein content of the enzyme preparation. As the ATP concentration increased, the inhibition produced by U was reduced; if the Mg (but not the Ca concentration) was increased while the ATP concentration remained constant, the inhibition increased. Changes in the Ca, Mg, and ATP concentrations did not alter the inhibition produced by Hg, Ag, and Au.

Properties and drug sensitivity of adenosine triphosphatases from Schistosoma mansoni.

The hydrolysis of ATP was measured in the presence of schistosome homogenates and various cations. The enzyme was stimulated strongly by either Ca2+ or Mg2+. Na+ added to the activation by Ca2+. A minor (17%) component was Na+ + K+ + Mg2+-dependent and ouabain-sensitive. Praziquantel, niridazole, oxamniquine, and hycanthone had no direct effect on the ATPase activity of schistosome homogenates. When schistosomes were pretreated with these drugs in vitro, washed thoroughly, and then homogenized, hycanthone, praziquantel, and oxamniquine caused a reduction in ATPase content of the worms. Niridazole did not share this effect. These results suggest that antischistosomal drugs did not directly inhibit ATPase, but did reduce ATPase in whole worms, possibly by removing or damaging the tegument, which is thought to contain most of the ATPase activity. In vitro ATPase measurements may be a useful indicator of pharmacologic activity of some types of drugs.

Inhibition of adenosine triphosphatases by gold.

Inhibition of adenosine triphosphatase (ATPase) by chlorauric acid (Au3+) and gold sodium thiomalate (Au+) was studied in dog brain and kidney and in human kidney enzyme preparations. Au3+ indiscriminately affected ouabain-sensitive (Na+ + K+-dependent) ATPase and ouabain-insensitive (Mg2+-dependent) ATPase with concentrations for 50% inhibition (I50) approximately 10(-6) M. The I50 of Au3+ for Na+ + K+ ATPase was several-fold higher in homogenates than in microsomal fractions. The enzyme was protected by bovine serum albumin. Although Au3+ and Au+ were equipotent against Mg2+ ATPase, Au+ inhibited Na+ + K+ ATPase 2 to 3 times more effectively than did Au3+. The inhibitory action of Au3+ (but not Au+) was potentiated by ascorbic acid, suggesting reduction of Au3+ to Au+ by ascorbic acid. The fractional inhibition of Na+ + K+ ATPase by Au3+ or Au+ was not affected by changing concentrations of NaCl, KCl, MgCl2, ATP, and MgATP. Decreasing pH from 8.0 to 6.8 enhanced both Au+ and Au3+ inhibition. We conclude that gold is one of the most potent nonspecific of Na+ + K+ ATPase, with characteristics differing from other metallic inhibitors of this enzyme system.

Inhibition by vanadium of sodium and potassium dependent adenosinetriphosphatase derived from animal and human tissues.

Inhibition of adenosinetriphosphatase (ATPase) by vanadium pentoxide (dissolved in water or in sodium hydroxide solution) was studied in microsomal fractions and tissue homogenates of kidney, brain, and heart of several species, including humans (kidney only). In some preparations vanadium was found to be the most potent inhibitor of Na+ + K+ATPase activity so far reported. Concentrations of vanadium causing 50 percent inhibition of Na+ + K+ATPase activity ranged from 6 x 10(-8) to 5 x 10(-7) M in microsomal fractions and from 2 x 10(-7) to 1 x 10(-6) M in tissue homogenates. Renal and cardiac enzymes were more sensitive to vanadium than the brain enzyme, a phenomenon independent of enzyme specific activity. The enzyme in tissue homogenates was more resistant to vanadium than the microsomal enzyme derived from the same tissues, suggesting a presence in tissues of protective agents. Mg2+ ATPase, which contaminated the enzyme preparations to a variable degree, was 1,000-10,000 times more resistant to vanadium than was Na+ + K+ATPase. More detailed studies on the mechanism of inhibition were performed with dog and human kidney enzymes. The reversible nature of the inhibition was suggested by the fact that fractional inactivation of Na+ + K+ATPase by vanadium was independent of enzyme protein concentrations. The inhibitory effect was reduced by Na+ and increased by K+ or Mg2+. ATP alone, but not MgATP, antagonized the inhibition. This could mean that vanadium inhibits the Na+ + K+ATPase at the site activated by Na+, and that ATP protects the enzyme either by binding vanadium or by competing for a mutual receptor on the enzyme. The inhibition was reduced by bovine serum albumin, probably binding vanadium. The inhibition was also diminished by reducing agents, ascorbic acid and citric acid.

Inhibitory characteristics of lead chloride in sodium- and potassium- dependent adenosinetriphosphatase preparations derived from kidney, brain, and heart of several species.

Inhibition of adenosinetriphosphatase (ATPase) by lead chloride (PbCl2) was studied in microsomal fractions or tissue homogenates of kidney, brain, and heart of several species, including humans. The concentration of PbCl2 causing 50% inhibition (I50) of Na+ + K+ ATPase activity varied from 8 X 10(-6) to 8 X 10(-5) M, depending on the species and organ of origin of the enzyme. The enzyme preparations derived from various parts of the kidney showed no differential sensitivity to PbCl2. These differences in sensitivity to lead were not related to specific activity of the enzyme or to the protein content of the preparations studied. Mg2+ ATPase, which contaminated the enzyme preparations to a variable degree, was 10--100 times more resistant to PbCl2 than was Na+ + K+-activated ATPase. The following more detailed studies were performed on the dog brain and/or kidney enzyme. The inhibition of microsomal Na+ + K+ ATPase was characterized by reversible kinetics. The inhibitory effect was antagonized by Na+, increased by Mg2+, and not altered by K+. ATP alone, or together with Mg2+, antagonized the inhibition. Disodium edetate prevented or reversed the inhibition. These inhibitory characteristics suggest that Pb2+ inhibits Na+ + K+ ATPase at the Na+-dependent phosphorylation site, and that ATP chelates Pb2+ in competition with Mg2+. Combining Pb2+ with ATP may not only result in a reduction of ATPase activity but also cause a relative ATP deficiency if lead is present in sufficiently high concentration.

Inhibition of renal adenosine triphosphatase by cadmium.

The effects of CdCl2 on adenosine triphosphatase (ATPase) were studied microsomal fractions or tissue homogenates of outer cortex, inner cortex and outer medulla of dog kidney. Cd was found to be an inhibitor of Na+ +K+ Atpase with 150 value of 2.1 to 3.2 X 10(-4) M regardless of type or source of the enzyme preparation tested. Mg++ ATPase was about 10-fold less sensitive to inhibition by Cd than Na+ +K+ ATPase. The inhibition of microsomal NA+ +K+ ATPase from outer medulla was characterized by irreversible kinetics. The inhibitory effect was not altered by varying Na+ or K+ concentrations, but was decreased by disodium ethylenediaminetetracetic acid (EDTA). EDTA was more effective in preventing than in reversing the inhibition. Na+ +K+ ATPase from kidneys of several other mammalian species showed a similar sensitivity to Cd.