Ouabaine

FIGURE 7.12 Several deoxy sugars and ouabain, which contains a-L-rhamnose (Rlia). Hydrogen atoms highlighted in red are deoxy positions. 

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 ... 

Cardiac Glycosides. Figure 2 Cardiac glycosides - molecular structure of digoxin and ouabain. Digoxin contains an additional hydroxy group in position 12 compared to digitoxin. 

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... 

Sirianni SR, Huang CC. 1980. Comparison of induction of sister chromatid exchange, 8-azaguanine- and ouabain-resistant mutants by cyclophosphamide, ifosfamide and l-(pyridyl-3)-3,3-dimethyltriazene in Chinese hamster cells cultured in diffusion chambers in mice. Carcinogenesis 1 353-355. 

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.)...

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

Tamplin et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with TTX-like biological activity in tissue culture assay, counteracting the lethal effect of veratridine on ouabain-treated mouse neuroblastoma cells. Concentrations of TTX-like activity ranged from 5 to 100 ng/L of culture when compared to standard TTX. The same bacterial extracts also displaced radiolabelled STX from rat brain membrane sodium channel receptors and inhibited the compound action potential of frog sciatic nerve. However, the same extracts did not show TTX-like blocking events of sodium current when applied to rat sarcolemmal sodium channels in planar lipid bilayers. 

Palytoxin (PTX), isolated from the zanthid Palythoa species, caused a rapid contraction followed by a slow phasic contraction of the guinea pig vas deferens. The second component of PTX-induced contraction was markedly inhibited by adrenergic blocking agents, whereas the first component was blocked by ouabain. In pheochromocytoma cells,... 

Palytoxin (PTX) is one of the most potent marine toxins known and the lethal dose (LD q) of the toxin in mice is 0.5 Mg/kg when injected i.v. The molecular structure of the toxin has been determined fully (1,2). PTX causes contractions in smooth muscle (i) and has a positive inotropic action in cardiac muscle (4-6). PTX also induces membrane depolarization in intestinal smooth (i), skeletal (4), and heart muscles (5-7), myelinated fibers (8), spinal cord (9), and squid axons (10). PTX has been demonstrated to cause NE release from adrenergic neurons (11,12). Biochemical studies have indicated that PTX causes a release of K from erythrocytes, which is followed by hemolysis (13-15). The PTX-induced release of K from erythrocytes is depress by ouabain and that the binding of ouabain to the membrane fragments is inhibited by PTX (15). 

Mechanical Response. PTX (5 x 10" to 3 x 10 M) caused a concentration-dependent contraction of the guinea pig vas deferens. The configuration of contractile response indicated the presence of two components, an initial rapid component followed by a second slow component. The first component of the response to PTX was abolished after treatment with Mg (10 mM), Ca -free medium, or ouabain (10 M), but remained almost unaffected by phentolamine (10 M), reserpine, 6-OHDA, atropine, or mecamylamine (10" M). The second component of the response to PTX was also completely inhibited after the incubation in the high or Ca -free medium. Phentolamine, reserpine, or 6-... 

OHDA markedly reduced only the second component. The second component also was inhibited by verapamil or low Na medium, but was potentiated after treatment with ouabain (10 M). 

The initial component of the PTX-induced contraction is selectively inhibited by ouabain, whereas the second component is rather potentiated by it (77). A similar inhibitory effect of ouabain on the contractile response to PTX is observed in the umbilical artery, which is devoid of adrenergic innervation 18). 

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