Ouabain ATPase

FIGURE 10.8 A schematic diagram of the Na, K -ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ions are transported out of the cell, and ions are transported in. The transport stoichiometry is 3 Na out and 2 in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na, K -ATPase by binding on the extracellular surface of the pump protein. 

ATPase inhibitor. In such patients, inhibition of the sodium pump in the cells lining the blood vessel wall results in accumulation of sodium and calcium in these cells and the narrowing of the vessels to create hypertension. An 8-year study aimed at the isolation and identification of the agent responsible for these effects by researchers at the University of Maryland Medical School and the Upjohn Laboratories in Michigan recently yielded a surprising result. Mass spectrometric analysis of compounds isolated from many hundreds of gallons of blood plasma has revealed that the hypertensive agent is ouabain itself or a closely related molecule ... 

The reactivity of the sodium pump isoforms toward ouabain can significantly differ between certain isoenzymes. The al-isoform from rat is reported to be 100-fold more resistant to ouabain than a2- or a3-isoform. In other species like rabbit, pig, dog or human these differences are not as distinct as in rat, due to a more sensitive al-isoform. At least two different types of ouabain binding sites are reported for the Na+/fC+-ATPase [1]. 

The Na+/K+-ATPase is the only enzyme known to interact with CTS, which reversibly bind to the extracellular side of the Na+/K+-ATPase at the E2-P conformational state [E2-P ouabain] and inhibit ATP hydrolysis and ion transport (Fig. lb, step 4). 

If 50% of Europeans with essential hypertension are affected by this disease because of an elevated secretion of endogenous ouabain, then there might be a chance to block its interaction at the cardiac glycoside binding site of Na+/K+-ATPase and thus lower blood pressure. This therapeutic approach seems to be successfiil. Recent studies provide evidence that the cardenolide analogue Rostafuroxin (PST 2238 Fig. 4) at very low concentrations can overcome the ouabain-induced tise of hypertension in experimental animals [6]. This compound has recently entered the phase I of clinical trials and is certainly a prototype of a new class of antihypertensive drugs. 

Ferrari P, Ferrandi M, Valentini G et al (2006) Rostafuroxin an ouabain antagonist that corrects renal and vascular Na+-K+-ATPase alterations in ouabain and adducin-dependent hypertension. Am J Physiol Regul Integr Comp Physiol 290 R529-R535... 

Cells are normally kept at osmotic (water activity) equilibrium by the action of the Na-pump. Inhibition of the pump with the specific Na -K -ATPase inhibitor, ouabain, causes cell swelling as does inhibition of it by hypothermia. The intracellular environment contains a high concentration of K (100 to 120 mM, in most mammalian cells), lower concentrations of Na (about 10 to 30 mM), and high... 

Figure 41-13. Stoichiometry of the Na+-K ATPase pump. This pump moves three Na ions from inside the cell to the outside and brings two K+ ions from the outside to the inside for every molecule of ATP hydrolyzed to ADP by the membrane-associated ATPase. Ouabain and other cardiac glycosides inhibit this pump by acting on the extracellular surface of the membrane. (Courtesy of R Post.)...

Furthermore, it has been found that ouabain, convallatoxin, and cymarin inhibit the PTX-induced contraction of rabbit aortic vascular smooth muscle (79). These observations have also provided evidence for the involvement of Na ,K -ATPase on the contractile effect of PTX in smooth muscle. 

In an ideal pure preparation of Na,K-ATPase from outer renal medulla, the al subunit forms 65 70% of the total protein and the molar ratio of a to is 1 1, corresponding to a mass ratio of about 3 1 [1,5]. Functionally the preparation should be fully active in the sense that each a/ unit binds ATP, Pj, cations and the inhibitors vanadate and ouabain. The molecular activity should be close to a maximum value of 7 000-8 000 Pj/min. The highest reported binding capacities for ATP and phosphate are in the range 5-6 nmol/mg protein and close to one ligand per otjS unit [29], when fractions with maximum specific activities of Na,K-ATPase [40 50 pmo Pj/min mg protein) are selected for assay. 

Fig. 5. Occlusion of ouabain complexes with ( ) - Na or (A) and unliganded Na,K-ATPase with (A) Rb. Procedure as described before [89] using incubation at increasing eoncentrations of Na" or Rb for 15 min at 20 C with 1 mM ouabain, 1 mM MgCl2 and 1 mM Pj-Tris.

Although it is widely accepted that ischaemia/ reperfusion-induced oxidant stress is associated with a reduction of Na/K ATPase activity, it is difficult to determine which features of this process are responsible for this effect. A classical approach to this type of problem has been to determine the effect of the application of selected metabolites or agents on the activity of the enzyme of interest, an approach that has been exploited for the sarcolemmal Na/K ATPase and glutathione (Haddock et al., 1990). The application of GSH (O.l-l.OmM) induces a concentration-dependent increase in the activity of a bovine isolated ventricular Na/K ATPase preparation (determined by the ouabain-sensitive hydrolysis of ATP to release inorganic phosphate). In the presence of 1 mM GSH there was a 38% stimulation of activity compared to untreated control... 

Figure 4.9 Effect of reduced glutathione (GSH) (0.25-1.0 ihm) and oxidized glutathione (GSSG) (0.25-1.0 mM) on ouabain-sensitive Na/K ATPase activity. An isolated Na/K ATPase preparation was prepared from fresh bovine ventricular tissue. Na/K ATPase activity was determined and quantified by the ouabain-sensitive hydrolysis of ATP to yield Inorganic phosphate. The rate of inorganic phosphate production was compared prior to and following the addition of either GSH or GSSG to the Incubation mixture. The data are presented as...

The cytochemical quantification of ouabain-sensitive Na/K ATPase activity in 10 /im thick whole heart slices. 

Na/K ATPase ICB natural product database (37 K), QSAR, Chemfinder similarity search based on ouabain 4 hits/10 tested [70]... 

Peng, M., Huang, L., Xie, Z., Huang, W. H. and Askari, A. Partial inhibition of Na+/K+-ATPase by ouabain induces the Ca2+-dependent expressions of early-response genes in cardiac myocytes. /. Biol. Chem. 271 10372-10378,1996. 

The effects of Li+ upon hematopoiesis have been proposed to be due to two different systems modification of the activity of the membrane Na+/K+-ATPase, and the inhibition of adenylate cyclase. Monovalent cation flux, in particular Na+ transport, is known to influence the differentiation and proliferation of hematopoietic stem cells. For instance, ouabain, an effective inhibitor of the membrane Na+/K+-ATPase, blocks the proliferation of lymphocytes and has been shown to attenuate the Li+-induced proliferation of granulocyte precursors [208]. Conversely, Li+ can reverse the actions of amphotericin and monensin, which mediate Na+ transport and which inhibit CFU-GM, CFU-E, and CFU-MK colony formation in the absence of Li+ [209]. Therefore, the influence of Li+ upon normal physiological cation transport—for example, its influence upon Na+/K+-ATPase activity—may be partly responsible for the observed interference in hematopoiesis. 

Burdyga I was involved in these experiments, and we were also working on the Na+/Ca2+ exchanger. When we used ouabain to block the Na+ pump, there was a limited rise of Na+ and then the Na+/Ca2+ exchanger drives Na+ out at the expense of Ca-ATPase activity. What I found later is that there is a massive load of the Ca2+... 

Desaiah D, Gilliland IK, Ho IK, et al. 1980a. Inhibition of mouse brain synaptosomal ATPases and ouabain binding by chlordecone. Toxicol Lett 6 275-285. 

The experiments were continued by Hoffman and Whittam, who concluded that a protein, an ATPase, in the membrane was necessary for active transport and was vectorially organized, with ATP and Na+ being required internally and K+ externally where ouabain was inhibitory. The ATPase was finally identified as the sodium pump by Skou (1957) it vectorially translocated Na+ and K+ across the membrane, and was phosphorylated transiently in the process. 

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