Vanadate-induced inhibition of renin secretion is unrelated to inhibition Na,K-ATPase activity

There is evidence that three inhibitors of Na,K-ATPase activity--ouabain, K-free extracellular fluid, and vanadate--inhibit renin secretion by increasing Ca2+ concentration in juxtaglomerular cells, but in the case of vanadate, it is uncertain whether the increase in Ca2+ is due to a decrease in Ca2+ efflux (inhibition of Ca-ATPase activity, or inhibition of Na,K-ATPase activity, followed by an increase in intracellular Na+ and a decrease in Na-Ca exchange) or to an increase in Ca2+ influx through potential operated Ca channels (inhibition of electrogenic Na,K transport, followed by membrane depolarization and activation of Ca channels). In the present experiments, the rat renal cortical slice preparation was used to compare and contrast the effects of ouabain, of K-free fluid, and of vanadate on renin secretion, in the absence and presence of methoxyverapamil, a Ca channel blocker. Basal renin secretory rate averaged 7.7 +/- 0.3 GU/g/60 min, and secretory rate was reduced to nearly zero by 1 mM ouabain, by K-free fluid, by 0.5 mM vanadate, and by K-depolarization (increasing extracellular K+ to 60 mM). Although 0.5 microM methoxyverapamil completely blocked the inhibitory effect of K-depolarization, it failed to antagonize the inhibitory effects of ouabain, of K-free fluid, and of vanadate. A concentration of methoxyverapamil two hundred times higher (100 microM) completely blocked the inhibitory effects of vanadate, but still failed to antagonize the effects of ouabain and of K-free fluid. Collectively, these observations demonstrate that vanadate-induced inhibition of renin secretion cannot be attributed entirely to Na,K-ATPase inhibition, since in the presence of methoxyverapamil, the effect of vanadate differed from the effects of either ouabain (a specific Na,K-ATPase inhibitor) or K-free fluid. Moreover, it cannot be attributed entirely to a depolarization-induced influx of Ca2+ through potential-operated Ca channels, since methoxyverapamil antagonized K-depolarization-induced inhibition of renin secretion much more effectively than it antagonized vanadate-induced inhibition.     

The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific alpha isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the alpha2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive alpha2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the alpha2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.     

Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart

The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.     

On the lack of inotropy of cardiac glycosides on skeletal muscle: a comparison of Na+, K+-ATPases from skeletal and cardiac muscle.

Comparison of Na,K-ATPase from skeletal and cardiac muscle revealed that, although the skeletal muscle enzyme was only slightly less sensitive to inhibition by ouabain, the rates of [³H]ouabain binding to, and dissociation from, the skeletal enzyme were much faster than the corresponding rates for the cardiac enzyme. The skeletal muscle enzyme required higher concentrations of potassium to stabilize the ouabainenzyme complex and to stimulate the K⁺-phosphatase activity. The K⁺-phosphatase activity was only 8% of the Na,K-ATPase activity of the skeletal muscle enzyme, compared to 22% for the cardiac preparation. The glycoprotein subunit found in Na,K-ATPases from cardiac and many other tissues appeared to be absent in the enzyme from skeletal muscle. The differences in binding and dissociation rates for ouabain suggest that there may be significant differences in the structure of the digitalis receptor in the two enzymes. The I₅₀ for ouabain inhibition of the skeletal muscle Na,K-ATPase was, however, only slightly higher than for the cardiac enzyme, suggesting that the lack of an inotropic effect of cardiac glycosides on skeletal muscle could not be due to failure of the digitalis drugs to bind to and inhibit the membrane-linked sodium pump.     

Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma-enriched fraction from vascular smooth muscle

Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma-enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma.     

Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity.

The cardiac glycoside sensitivity of the rat heart changes during postnatal maturation and in response to certain pathological conditions. The Na,K-ATPase is thought to be the receptor for cardiac glycosides, and there are three isozymes of its catalytic (alpha) subunit with different cardiac glycoside affinities: alpha 1 (low affinity) and alpha 2 and alpha 3 (high affinity). We examined the developmental expression of the alpha subunit isozymes in rat ventricular membrane preparations by immunoblotting with isozyme-specific antibodies. The alpha 1 isozyme was present throughout all stages of maturation. A developmental switch from alpha 3 to alpha 2 occurred between 14 and 21 days after birth. Measurements of [3H]ouabain binding and inhibition of Na,K-ATPase activity indicated that alpha 2 and alpha 3 should make equivalent contributions to ion pump capacity; in both neonatal natal and adult preparations, ouabain interacted with a single class of high-affinity binding sites (KD = 15 or 40 nM, respectively; Bmax = 4-5 pmol/mg protein), and at low concentrations produced a similar degree of Na,K-ATPase inhibition (25%). The results indicate that the developmental difference in cardiac glycoside sensitivity cannot be explained by quantitative differences in the proportion of high-affinity isozymes of the Na,K-ATPase. The switch from alpha 3 to alpha 2 coincides with other major changes in cardiac electrophysiology and calcium metabolism.     

Ouabain sensitivity of the alpha 3 isozyme of rat Na,K-ATPase

The Na,K-ATPase of rat brainstem axolemma membranes contains two isozymes of its catalytic subunit, alpha 2 and alpha 3. To isolate the alpha 3 isozyme functionally, purified axolemma Na,K-ATPase was treated with trypsin. Immunoblot analysis of trypsin-treated Na,K-ATPase using isozyme-specific antibodies showed that alpha 3 was significantly more resistant to digestion than alpha 2. The trypsin-resistant alpha 3 isozyme fraction, devoid of alpha 2, contained 50-60% of the ATPase activity, and was inhibited by ouabain half-maximally at 0.13 microM. This indicates that the alpha 3 Na,K-ATPase isozyme has a high sensitivity to cardiac glycosides.     

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.     

Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I(P)) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I(P) because of the following: (1) it was absent in 0 mM [K(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.     

The Na,K-ATPase of nervous tissue

The Na,K-ATPase has been only partially purified from nervous tissue, yet it is clear that two forms (and +) of the catalytic subunit are present. is a component subunit of the glial Na,K-ATPase, which has a relatively low affinity for binding cardiac glycosides and + has been identified as a subunit of the Na,K-ATPase which has relatively high affinity for cardiac glycosides. The + form may also be sensitive to indirect modulation by neurotransmitters or hormones. The ratio of + / changes in the nervous system during development, and + appears to be the predominant species in adult neurones. Changes in Na,K-ATPase activity have been associated with several abnormalities in the nervous system, including epilepsy and altered nerve conduction velocity, but a causal relationship has not been definitively established. Although the Na,K-ATPase has a pivotal role in Na⁺ and K⁺ transport in the nervous system, a special role for the glial Na,K-ATPase in clearing extracellular K⁺ remains controversial.     

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.     

Ouabain-induced blockade of alpha2 isoform of the Na,K-ATPase on electrophysiological and contractile characteristics of the rat diaphragm

In experiments with isolated neuromuscular preparation of the rat diaphragm, selective blockade of alpha2 isoform of the Na,K-ATPase with ouabain (1 mcmol/L) induced steady depolarization of muscle fibers that reached a maximum of 4 mV, a decrease in amplitude of muscle fiber action potential, and prolonged raising and decline phases of the action potential. At the same time, the force, time to peak, and half relaxation time of the isometric muscle twitch were increased, as well as the area under the contraction curve. During continuous fatiguing stimulation (2/s), a more pronounced decline of contraction speed was observed in presence of ouabain; dynamics of the half-relaxation time remaining unchanged. It is suggested that blockade of alpha2 isoform of the Na,K-ATPase impairs excitation-contraction coupling resulting in a delay of Ca2+ release from sarcoplasmic reticulum. The increase in contraction force seems to result from a mechanism similar to that of positive inotropic effect of cardiac glycosides in heart muscle. Physiological significance of the skeletal muscle alpha2 isoform of the Na,K-ATPase in regulation of Ca2+ and Na+ concentrations near triadic junctions and in regulatory processes involving the Na,K-ATPase endogenous modulators or transmitter acetylcholine is discussed.     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.

Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations.     

Studies on ouabain-complexed (Na+ +K+)-ATPase carried out with vanadate

Vanadate is able to promote the binding of ouabain to (Na+ +K+)-ATPase and it is shown that vanadate is trapped in the enzyme-ouabain complex. Also ouabain-bound enzyme, the formation of which was facilitated by (Mg2+ +Na+ +ATP) or (Mg2+ +Pi), is accessible to vanadate when washed free of competing ligands used for the promotion of ouabain binding. For vanadate binding to (Na+ +K+)-ATPase and to enzyme-ouabain complexes a divalent cation (Mg2+ or Mn2+) is indispensable, indicating that the cation does not remain attached to the ouabain-bound enzyme. K+ further increases vanadate binding in the absence of ouabain, but seems to have no additional role in case of vanadate binding to enzyme-ouabain complexes. Mn2+ is more efficient than Mg2+ in promoting binding of vanadate and ouabain to (Na+ +K+)-ATPase. That K+ in combination with Mn2+, in analogy with the effect in combination with Mg2+, increases the equilibrium binding level of vanadate and decreases that of ouabain does not seem to favour the hypothesis of selection of a special E2-subconformation by Mn2+. The vanadate-trapped enzyme-ouabain complex was examined for simultaneous nucleotide binding which could demonstrate a two-substrate mechanism per functional unit of the enzyme. The acceleration by (Na+ +ATP) of ouabain release from the (Mg2+ +Pi)-facilitated enzyme-ouabain complex does not, as anticipated, support such a mechanism. On the other hand, the deceleration of vanadate release as well as of ouabain release from a (Mg2+ +vanadate)-promoted complex could be consistent with a two-substrate mechanism working out-of-phase.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.     

Cardiac glycosides stimulate phospholipase C activity in rat pinealocytes

Ouabain and related cardiac glycosides stimulate phospholipase C activity 5-fold in rat pinealocytes. The combined treatment of ouabain and norepinephrine, which also stimulates phospholipase C, produces an additive effect. The effects of either ouabain or norepinephrine are blocked by EGTA. However, there are notable differences. The stimulatory effect of ouabain is lost when extracellular Na+ is reduced to 20 mM and is not blocked by prazosin. In contrast, the stimulatory effect of norepinephrine is not blocked when extracellular Na+ is reduced to 20 mM but is blocked by prazosin. Ouabain appears to increase phospholipase C activity through a mechanism involving inhibition of Na+,K+-ATPase, and an accumulation of intracellular Na+ and Ca2+, not involving alpha 1-adrenoceptors. These findings raise the possibility that activation of phospholipase C might be a more general effect of cardiac glycosides.     

Human Breast Tumor Cells Are More Resistant to Cardiac Glycoside Toxicity Than Non-Tumorigenic Breast Cells

Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.