Ouabain Sensitivity Research Articles

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status

BackgroundAcute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.MethodsDigoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.ResultsPrimitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.ConclusionsOur data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery.53GxHmRcFt-KhVdMmSCJrsVideo

Na,K-ATPase α4, and Not Na,K-ATPase α1, is the Main Contributor to Sperm Motility, But its High Ouabain Binding Affinity Site is Not Required for Male Fertility in Mice.

Mammalian sperm express two Na,K-ATPase (NKA) isoforms, Na,K-ATPase α4 (NKAα4) and Na,K-ATPase α1 (NKAα1). While NKAα4 is critical to sperm motility, the role of NKAα1 in sperm movement remains unknown. We determined this here using a genetic and pharmacological approach, modifying the affinity of NKAα1 and NKAα4 for the inhibitor ouabain to selectively block the function of each isoform. Sperm from wild-type (WT) mice (naturally containing ouabain-resistant NKAα1 and ouabain-sensitive NKAα4) and three newly generated mouse lines, expressing both NKAα1 and NKAα4 ouabain resistant (OR), ouabain sensitive (OS), and with their ouabain affinity switched (SW) were used. All mouse lines produced normal sperm numbers and were fertile. All sperm types showed NKAα isoform expression levels and activity comparable to WT, and kinetics for ouabain inhibition confirming the expected changes in ouabain affinity for each NKA isoform. Ouabain at 1μM, which only block ouabain-sensitive NKA, significantly inhibited total, progressive, and hyperactivated sperm motility in WT and OS, but had no significant effect on OR or SW sperm. Higher ouabain (1mM), which inhibits both ouabain-sensitive and ouabain-resistant NKA, had little additional effect on sperm motility in all mouse lines, including the OR and SW. A similar pattern was found for the effect of ouabain on sperm intracellular sodium ([Na+]i). These results indicate that NKAα4, but not NKAα1 is the main contributor to sperm motility and that the ouabain affinity site in NKA is not an essential requirement for male fertility.

The electrophysiological analysis of ouabain sensitivity of mouse and guinea pig sinoauricular node cells

IntroductionIt is known that Na+/K+‐ATPase isoforms differ significantly in their response to the cardiac glycosides. Thus, the organism reactions to glycoside are species‐dependent.The aim of the present study was to assess the impact of the Na+/K+‐ATPase current on electrical activity of mice and guinea pigs sinoauricular (SA) node.MethodsThe experiments were conducted on male albino mice (age 8–10 weeks, body mass 30±5 g, n=14) and male guinea pigs (age 4–5 months, body mass 550±50 g, n=7). The action potentials (APs) were registered in the isolated SA region strips from the side of subendocardial side along the SA artery using standard microelectrode method.ResultsThe cells with spontaneous APs generation and the slowest maximal upstroke velocity in phase 0 (dV/dtmax) – 3.2±0.7 and 4.1±2.7 V/s, in mouse and guinea pig respectively (Table 1.) – and with the diastolic depolarization (DD) phase (phase 4, diastole) were identified in control Tyrode's solution. The spontaneous APs generation frequencies at 31°C were 286±16 min−1 in mice myocardium strips and 103±30 min−1 in guinea pigs strips. The exposition of mice SA myocardium strips to ouabain (1 μmol/l, n=5) decreased the APs generation frequency by 10% as a result of the 17% increase of the AP duration at 90% repolarization (APD90) (Table 1.). These changes were accompanied by the 28% decrease of dV/dtmax. The exposition of guinea pig SA node cells to ouabain (1 μmol/l, n=4) decreased the DD phase duration by 30% and therefore increased the APs generation frequency by 23% (Table 1.) The higher concentrations of ouabain (10 μmol/l instead of 1 μmol/l) decreased the APs generation frequency in the mouse SA cells (n=9) by 14% due to the 20–25% increase of both APD90 и DD. The diastolic depolarization rate (V4) и dV/dtmax decreased by one third (Table 1.). The higher concentrations of ouabain (10 μmol/l) blocked the electrical activity in guinea pig SA strips (n=3) after 11±2 min of compound treatment. At the same time, the sarcolemma depolarization, decreased AP amplitude and dV/dtmax as well increased APs generation frequency by 35% (on average) due to shorter DD phase were registered (Figure 1). In mouse SA strips (n=3) ouabain in concentration 100 μmol/l at first decreased APs generation frequency by 24%, and then totally suppressed the electrical activity in the cells.We assumed that the observed effects of ouabain were not only a result of different sensitivity of Na+/K+‐ATPase isoforms, but also of higher intracellular concentration of ([Na+]i) in mice compared to guinea pigs. Our findings suggest that differences in pacemaker activity mechanisms in these two species should be taken into account when testing the prospective pharmacological compounds for treatment of dysrhythmias of different origin and developing mathematical models of APs generation.Support or Funding InformationThe study was supported by project № AAAA‐A17‐117012310152‐2 and № AAAA‐A18‐ 118012290365‐2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gestation changes sodium pump isoform expression, leading to changes in ouabain sensitivity, contractility, and intracellular calcium in rat uterus.

Developmental and tissue‐specific differences in isoforms allow Na+, K+‐ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na+, K+ ATPase, which creates ionic gradients that drive excitation‐contraction coupling. It is unknown whether Na+, K+ ATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation‐dependent differential expression of isoforms would affect contractile responses to Na+, K+ ATPase α subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation‐dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na+, K+ ATPase isoforms in myometrium; (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity; and (3) if changes in contractile responses can be explained by changes in intracellular [Ca2+]. Changes in abundance and distribution of the Na+, K+ ATPase α, β and FXYD1 and 2 isoforms, were studied in rat uterus from nonpregnant, and early, mid‐, and term gestation. All α, β subunit isoforms (1,2,3) and FXYD1 were detected but FXYD2 was absent. The α1 and β1 isoforms were unchanged throughout pregnancy, whereas α2 and α3 significant decreased at term while β2 and FXYD1 significantly increased from mid‐term onwards. These changes in expression correlated with increased functional sensitivity to ouabain, and parallel changes in intracellular Ca2+, measured with Indo‐1. In conclusion, gestation induces specific regulatory changes in expression of Na+, K+ ATPase isoforms in the uterus which influence contractility and may be related to the physiological requirements for successful pregnancy and delivery.

Ouabain targets the Na+/K+-ATPase α3 isoform to inhibit cancer cell proliferation and induce apoptosis.

Ouabain has been used for the treatment of heart failure and atrial fibrillation. Its potential anticancer effect has also attracted great interest. The aim of the present study was to evaluate the anticancer effect of ouabain and investigate its molecular target. The effects of ouabain on the viability of and induction of cellular death on OS-RC-2 renal cancer cells were examined using the MTT assay and acridine orange/ethidium bromide staining. The levels of Ca2+ and reactive oxygen species were determined using Fura-3-acetoxymethyl ester and dichloro-dihydro-fluorescein diacetate probes, respectively. Apoptosis was examined using annexin V-fluorescein isothiocyanate/propidium iodide staining and western blotting. The expression profile of the different Na+/K+-ATPase (NKA) isoforms in NCI-H446 small cell lung cancer cells was determined using immunocytochemistry and reverse transcription polymerase chain reaction analysis. In the present study, it was demonstrated that ouabain inhibited cancer cell proliferation and induced apoptosis while no significant difference in the expression of NKA α1 and α3 isoforms was detected following 48 h of ouabain treatment. Furthermore, expression of NKA α3 but not the α1 isoform was associated with ouabain sensitivity. The results of the present study indicated that ouabain targets the NKA α3 isoform, inhibits cancer cell proliferation and induces apoptosis.

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC50 for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC50 550 μM) and Q111T-N122H (IC50 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.

Overexpression of the Polycystin-1 C-Tail Enhances Sensitivity of M-1 Cells to Ouabain

Cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) are abnormally sensitive to ouabain, responding to physiological ouabain concentrations with enhanced proliferation and increased forskolin-induced transepithelial fluid secretion. This requires activation of the epidermal growth factor receptor (EGFR), Src kinase and the extracellular signal-regulated kinases MEK and ERK. Here, we have determined if the ADPKD phenotype obtained in mouse cortical collecting duct cells by stable overexpression of the C-terminal domain of polycystin-1 (PC-1 C-tail) also elicits the ADPKD-like response to ouabain in the cells. M-1 C20 cells expressing the PC-1 C-tail and M-1 C17 cells lacking expression of this construct were treated with physiological concentrations of ouabain, and cell proliferation, activation of the EGFR-Src-MEK-ERK pathway, forskolin-induced transepithelial Cl(-) secretion and the sensitivity of Na,K-ATPase to ouabain were explored. M-1 C20 cells responded to ouabain with increased cell proliferation and ERK phosphorylation. Ouabain also augmented forskolin-induced and cystic fibrosis transmembrane conductance regulator-mediated apical secretion of Cl(-) in M-1 C20 cells. These effects required activation of EGFR, Src and MEK. In contrast, ouabain had no significant effects on M-1 C17 cells. Interestingly, approximately 20% of the Na,K-ATPase from M-1 C20 cells presented an abnormally increased sensitivity to ouabain. Overexpression of PC-1 C-tail in M-1 C20 cells is associated with an ouabain-sensitive phenotype and an increased ability of the cells to proliferate and secrete anions upon ouabain stimulation. This phenotype mimics the ouabain sensitivity of ADPKD cells and may help promote their cystogenic potential.

Expression of organic cation transporter 3 (SLC22A3) and plasma membrane monoamine transporter (SLC29A4) in human umbilical vein endothelial cells and their relevance for histamine uptake

Background Increased plasma histamine levels lead to pathological events. Endothelial cells actively participate in histamine clearance by promoting its uptake via yet unidentified carriers, thus limiting histamine effects. The organic cation transporter 3 (OCT3) and plasma membrane monoamine transporter (PMAT) are the two most prominent transporters for endogenous monoamines. OCT3 and PMAT show Na/K-ATPase independency. Both are highly sensitive to inhibition by the isocyanine compound, decynium-22. However, OCT3 is highly sensitive to corticosteron, whereas PMAT is not. We showed in the past that decynium-22 inhibits histamine uptake in cultured human umbilical vascular endothelial cells (HUVEC). In the present study we identified the expression of OCT3 and PMAT in freshly isolated and cultured HUVEC as well as some characteristics of histamine uptake in cultured HUVEC such as ouabain and corticosteron sensitivity.

High-affinity ouabain receptors: primary membrane sensors for ionizing radiation

Although the high sensitivity of the Na/K pump in cell membrane to ionizing radiation is well known in literature, the individual role of different isoforms of pump in determination of its radio-sensitivity is not clear yet. This is the subject of the present investigation. Using isotope, electro-physiological and enzymological methods, the effect of γ-ionizing radiation on cell membrane voltage-current characteristics, acetylcholine-induced membrane current, 22Na+ and 45Ca2+ exchange between cells and bathing solution, Na+K+-ATPase activity, dose-dependent ouabain binding with cell membrane, intracellular cAMP and membrane phosphorylation in snail neurons were studied. The changes in neurons as a result of 30-min γ-radiation exposure of snails to 5.16 Ci/kg at the end of the first 30 min of post-radiation period were as follows: the increase in membrane ionic conductance reversed the ouabain sensitivity of acetylcholine-induced currents, stimulation of 22Na+ and 45Ca2+ uptakes, inhibition of Na/K pump, activation Na/Ca exchange in reversed mode, increase in ouabain binding with high-affinity α3 and decrease with α2 middle-affinity receptors, decrease in intracellular cAMP content and membrane dephosphorylation. On the basis of the obtained data, it is suggested that both α3 and α2 catalytic subunits of Na++K+-ATPase serve primary membrane sensors through the activation of which the biological effect of γ-radiation on neurons is realized. The IR has activation effects on α3-dependent Na+/Ca2+ exchange in forward and its inactivation on α2-dependent reverse modes.

Age-Dependent Brain Tissue Hydration, Ca Exchange and their Dose- Dependent Ouabain Sensitivity

Tissue hydration, dose-dependent 3H-ouabain binding, 45Ca2+ exchange in rati?½s brain cortex, subcortex and cerebellum were studied in three age groups. Age-dependent tissue dehydration in all three zones of brain was due to inhibition of Na+/K+ pump. The age-dependence of cell hydration in cortex was more expressed. The curve of dose-dependent ouabain binding consists of three components corresponding to Na+/K+ pump isoforms (α1, α2, α3). Age-dependency of these isoforms was more expressed in cortex than in subcortex and cerebellum. High affinity receptors were depressed in old ratsi?½ brain tissues. Initial 45Ca2+ uptake in three brain zones of old rats was depressed as compared to that of young animals. Ouabain at 10-9 M has activation effect on 45Ca2+ uptake, which was also age dependent. Initial 45Ca2+ efflux in cortex and subcortex tissue in old rats was significantly depressed as compared to young ones while in cerebellum the opposite age-dependence was observed. The curves of dose-dependent ouabain effect on 45Ca2+ efflux and cell hydration consist of 6 components. However, close correlation between kinetics of 45Ca2+ efflux and cell hydration was not observed. It is suggested that brain tissue dehydration in aged animals is a consequence of Na+/K+ pump dysfunction induced intracellular calcium elevation. It is suggested that α3 receptors are functionally connected with intracellular Ca2+ buffering systems through intracellular signaling systems and their dysfunction in aged brain is a consequence of [Ca2+]i increase. Obtained data allow us to conclude that endogen nanomolar ouabain-like species circulating in mammalsi?½ blood removing Ca2+ from cells could have a beneficial effect on brain of old animals.

Ouabain-Sensitive α1 Na,K-ATPase Enhances Natriuretic Response to Saline Load

Na,K-ATPase is ubiquitously expressed and is essential for maintaining electrochemical and osmotic gradients. The alpha subunit of Na,K-ATPase is the receptor for cardiotonic steroids, which act through the ouabain-binding site and are important in cardiovascular regulation. Interestingly, the presence of endogenous Na,K-ATPase ligands has been implicated in the natriuretic response to perturbations such as hypertension and salt loading; therefore, it is important to characterize the role of the ouabain-binding sites in this context. Because the alpha1 isoform of mice and rats is relatively ouabain resistant, gene-targeting strategies were used to produce mice with reversed responses of the alpha1 and/or alpha2 isoforms to ouabain to assess for altered natriuretic responses to acute salt loading. Regardless of the sensitivity of the alpha2 isoform to ouabain, conferring ouabain sensitivity to alpha1 augmented the natriuretic response to an acute salt load. In addition, when endogenous Na,K-ATPase inhibitors were sequestered with an anti-digoxin antibody fragment, the sodium excretion rates in the ouabain-sensitive alpha1 isoform mice were equivalent to the ouabain-resistant alpha1 isoform mice. These data suggest that the ouabain-binding site of the alpha1 Na,K-ATPase can participate in the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands.

ACTH-induced hypertension is dependent on the ouabain-binding site of the α2-Na+-K+-ATPase subunit

ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant alpha(2)-Na(+)-K(+)-ATPase subunits (alpha2(R/R) mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant alpha2(R/R) mice vs. wild-type mice (ouabain-sensitive alpha(2)-Na(+)-K(+)-ATPase, alpha2(S/S) mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in alpha2(S/S) (104.0 +/- 2.6 to 117.7 +/- 3.0 mmHg) but not in alpha2(R/R) mice (108.2 +/- 3.2 to 111.5 +/- 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of alpha(2)-Na(+)-K(+)-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the alpha(2)-Na(+)-K(+)-ATPase. Baseline cardiac performance was elevated in alpha2(S/S) but not alpha2(R/R) mice. Overall, the data establish that the alpha(2)-Na(+)-K(+)-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K(+) (ATP) channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na(+)/K(+)-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na(+)/K(+)-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na(+)/K(+)-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K(+) (ATP) channels. 5-HD did not affect the ouabain sensitivity of Na(+)/K(+)-ATPase. Glyburide had both activating and inhibitory effects on Na(+)/K(+)-ATPase at concentrations used to block plasma membrane K(+) (ATP) channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na(+)/K(+)-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K(+) (ATP) channels, when the relation of this channel to Na(+)/K(+)-ATPase is being studied.

Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

The Na(+)/K(+)-ATPase (NKA) is the main route for Na(+) extrusion from cardiac myocytes. Different NKA alpha-subunit isoforms are present in the heart. NKA-alpha1 is predominant, although there is a variable amount of NKA-alpha2 in adult ventricular myocytes of most species. It has been proposed that NKA-alpha2 is localized mainly in T-tubules (TT), where it could regulate local Na(+)/Ca(2+) exchange and thus cardiac myocyte Ca(2+). However, there is controversy as to where NKA-alpha1 vs. NKA-alpha2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I(Pump)) measurements and the different ouabain sensitivity of NKA-alpha1 (low) and NKA-alpha2 (high) in rat heart. Ouabain-dependent I(Pump) inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-alpha2, K(1/2) = 0.38 +/- 0.16 microM) that accounts for 29.5 +/- 1.3% of I(Pump) and a low-affinity isoform (NKA-alpha1, K(1/2) = 141 +/- 17 microM) that accounts for 70.5% of I(Pump). Detubulation decreased cell capacitance from 164 +/- 6 to 120 +/- 8 pF and reduced I(Pump) density from 1.24 +/- 0.05 to 1.02 +/- 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-alpha2 accounted for only 18.2 +/- 1.1% of I(Pump). Thus, approximately 63% of I(Pump) generated by NKA-alpha2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-alpha2/NKA-alpha1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-alpha2 is approximately 4.5 times higher in the T-tubules vs. ESL, whereas NKA-alpha1 is almost uniformly distributed between the TT and ESL.

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

Fluorometric Measurements of Intermolecular Distances between the α- and β-Subunits of the Na+/K+-ATPase

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.

Ouabain Inhibits Tubuloglomerular Feedback in Mutant Mice with Ouabain-Sensitive α1 Na,K-ATPase

Initiation of tubuloglomerular feedback (TGF) depends on Na-K-2Cl co-transport in the macula densa (MD), but it is less clear whether Na,K-ATPase is responsible for establishing the inward Na+ gradient. It has been proposed that apical colonic H,K-ATPase, perhaps in concert with the Na/H exchanger (NHE2), may account for MD Na+ exit in these cells. This study evaluated TGF responses by micropuncture in mutant mice with altered ouabain sensitivity of the alpha1 and alpha2 Na,K-ATPase isoforms. TGF responses in alpha1-sensitive/alpha2-resistant mice were inhibited by intravenous ouabain (control stop-flow pressure = 9.7 +/- 0.9 versus 1.6 +/- 0.5 mmHg with intravenous ouabain). Subsequent inclusion of cyclohexyladenosine (10 microM) in the tubule perfusate confirmed the ability of the afferent arteriole to contract in the presence of ouabain. In alpha1-resistant/alpha2-resistant mice, ouabain infusion had no effect on TGF responses. In separate experiments, loop of Henle perfusion with 50 microM ouabain decreased TGF responses (control stop-flow pressure) from 10.5 +/- 1.1 to 3.9 +/- 1.0 mmHg in alpha1-sensitive/alpha2-resistant mice but had no effect in alpha1-resistant/alpha2-resistant mice, and afferent arteriole responsiveness again was confirmed by cyclohexyladenosine. TGF responses in NHE2 and colonic H,K-ATPase knockout mice were not different from those of wild-type mice. These data indicate that TGF requires activity of the alpha1 Na,K-ATPase, presumably in the MD. Furthermore, the data show that neither NHE2 nor colonic H,K-ATPase is essential for initiation of TGF responses.

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.

Inhibition of α1 Na/K‐ATPase blocks tubuloglomerular feedback (TGF) in mutant mice

Na-K-2Cl cotransport is responsible for NaCl entry and initiation of TGF in the macula densa (MD), but it is less clear whether Na-K-ATPase (NKA) is responsible for Na+ exit from MD cells. Expression of Na-K-ATPase is low in MD and it has been proposed that H-K-ATPase may account for Na+ exit and maintenance of the Na+ gradient. Functional analyses have been limited by the fact that the α1 isoform of NKA – the isoform expressed in renal epithelium – is largely insensitive to ouabain in rodents and therefore resistant to pharmacological manipulation. We therefore evaluated TGF responses in gene knock-in mice in which the ouabain sensitivity of NKA isoforms was altered: α1-sensitive/α2-resistant mice (α1s/α2r), and α1-resistant/α2-resistant mice (α1r/α2r). In micropuncture experiments, TGF responses were evaluated: 1) before and during i.v. infusion of ouabain (200 ng/min) and 2) before and during luminal application of ouabain. As shown in the figure, i.v. ouabain inhibited TGF in α1s/α2r mice, but not in α1r/α2r mice. Responses were restored by cyclohexyladenosine (CHA, 10μM) in the loop of Henle (LH) perfusate. In the second series, LH perfusion with 50μM ouabain decreased maximal TGF responses from 10.5±1.1 to 4.8±1.3 mmHg in α1s/α2r mice, but had no effect in α1r/α2r mice (10.8±1.3 to 10.7±1.3 mmHg). Responses were restored by CHA in the perfusate. These data indicate that TGF requires activity of the α1NKA. Since TGF could be restored by the adenosine analog CHA – a downstream effector in the TGF signaling cascade – the data also suggest that ouabain exerts its effect at the MD rather than at the afferent arteriole. Supported by NIH-DK57552 & HL66062.

Steroid signaling at the cell surface: Progesterone binding to the N‐terminal extracellular loop of the Rana pipiens oocyte Na/K‐ATPase alpha subunit.

Progesterone (P) activates the amphibian oocyte through a non-transcriptional signaling system in the plasma membrane that is thought to induce two parallel pathways that converge on the activation of cyclin B-cdc2. The initial P binding site is a −110 kDa membrane peptide that results in release of a cascade of lipid second messengers and a fall in [cAMP]i. Our analysis of [3H]ouabain binding to the plasma membrane of the Rana pipiens oocyte indicates a high affinity ouabain site linked to pump inhibition and a low affinity ouabain site not associated with the pump. P-ouabain competition studies indicate that binding of P at the oocyte plasma membrane is at the low affinity ouabain binding site located in the external loop between the H1-H2 region of the 112 kDa alpha1-subunit of the Na/K-ATPase. N and C termini of the alpha-subunit are located intracellularly and the alpha-subunit has 10 transmembrane domains. Based on X-ray crystallographic studies of the P-cytosol receptor (Nature 393:392, 1998) and site-directed mutagenesis studies of ouabain binding (Acta Physiol Scand. 163:69, 1998), we predict that the 3-keto group of P may bind to its peptide receptor via hydrogen bond donors in the external loop between H1-H2: Gln-120, pro-127, Asp-130 and Asn-131. Each residue is known to be essential for ouabain inhibition, or identified as determinants of ouabain sensitivity. This may represent the first description of a steroid binding receptor at the cell surface. (Supported by HD 10463).