Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.

Development of a new ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay.

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC-MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid-liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC-MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.

Visual monitoring and optical recognition of digoxin by functionalized AuNPs and triangular AgNPs as efficient optical nano-probes.

In this study, we presented elective, sensitive, and rapid UV-Vis spectrophotometry and calorimetric assay for the recognition of digoxin. Therefore, cysteamine-gold nanoparticles (Cys A-AuNPs) in the presence of cysteine acid amine and Silver nanoparticles in the presence of tetramethyl benzidine and hydrogen peroxide (AgNPs-TMB [3,3',5,5'-tetramethylbenzidine]-H2 O2 ) were synthesized and utilized as the desired probe. Finally, color variation of probes was observed in the absence and presence of digoxin. Obtained results indicate that the color of Cys A-AuNPs changed from dark pink to light in the absence and the presence of digoxin, respectively. Also, the color of AgNPs-TMB-H2 O2 changed from dark blue to light blue, in the absence and the presence of digoxin, respectively. Moreover, UV-Vis spectroscopies results indicate digoxin with a low limit of quantification of 0.125 ppm in human plasma samples which linear range was 0.125 to 11 ppm.

Structural Insights into the Interactions of Digoxin and Na+/K+-ATPase and Other Targets for the Inhibition of Cancer Cell Proliferation.

Digoxin is a cardiac glycoside long used to treat congestive heart failure and found recently to show antitumor potential. The hydroxy groups connected at the C-12, C-14, and C-3'a positions; the C-17 unsaturated lactone unit; the conformation of the steroid core; and the C-3 saccharide moiety have been demonstrated as being important for digoxin's cytotoxicity and interactions with Na+/K+-ATPase. The docking profiles for digoxin and several derivatives and Na+/K+-ATPase were investigated; an additional small Asn130 side pocket was revealed, which could be useful in the design of novel digoxin-like antitumor agents. In addition, the docking scores for digoxin and its derivatives were found to correlate with their cytotoxicity, indicating a potential use of these values in the prediction of the cancer cell cytotoxicity of other cardiac glycosides. Moreover, in these docking studies, digoxin was found to bind to FIH-1 and NF-κB but not HDAC, IAP, and PI3K, suggesting that this cardiac glycoside directly targets FIH-1, Na+/K+-ATPase, and NF-κB to mediate its antitumor potential. Differentially, digoxigenin, the aglycon of digoxin, binds to HDAC and PI3K, but not FIH-1, IAP, Na+/K+-ATPase, and NF-κB, indicating that this compound may target tumor autophagy and metabolism to mediate its antitumor propensity.

Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation.

Maintenance of Na+ and K+ gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.

Cardiotonic steroids as potential Na+/K+-ATPase inhibitors - a computational study.

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na+/K+-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na+/K+-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470 kcal/mol to 7.234 kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470 kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na+/K+-ATPase-ouabain and Na+/K+-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100 ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.

Probe Cocktail to Assess Transporter Function in Sandwich-Cultured Human Hepatocytes.

PURPOSE: Probe substrates are used routinely to assess transporter function in vitro. Administration of multiple probe substrates together as a "cocktail" in sandwich-cultured human hepatocytes (SCHH) could increase the throughput of transporter function assessment in a physiologically-relevant in vitro system. This study was designed to compare transporter function between cocktail and single agent administration in SCHH. METHODS: Rosuvastatin, digoxin, and metformin were selected as probe substrates of hepatic transporters OATP1B1, OATP1B3, BCRP, P-gp, and OCT1. Total accumulation (Cells+Bile) and biliary excretion index (BEI) values derived from administration of the cocktail were compared to values obtained after administration of single agents in the absence and presence of a model inhibitor, erythromycin estolate. RESULTS: For rosuvastatin and metformin accumulation, the ratio of means [90% confidence interval (CI)] for cocktail to single agent administration was 100% [94%, 106%] and 90% [82%, 99%], respectively. Therefore, the cocktail and single-agent mode of administration were deemed equivalent per standard equivalence criterion of 80-120% for rosuvastatin and metformin accumulation, but not for digoxin accumulation (77% [62%, 92%]). The ratio of means [90% CI] for rosuvastatin BEI values between the two administration modes (105% [97%, 114%]) also was deemed equivalent. The ratio for digoxin BEI values between the two administration modes was 99% [78%, 120%]. In the presence of erythromycin estolate, the two administration modes were deemed equivalent for evaluation of rosuvastatin, digoxin, and metformin accumulation; the ratio of means [90% CI] was 104% [94%, 115%], 94% [82%, 105%], and 100% [88%, 111%], respectively. However, rosuvastatin and digoxin BEI values were low and quite variable in the presence of the inhibitor, so the BEI results were inconclusive. CONCLUSIONS: These data suggest that rosuvastatin and metformin can be administered as a cocktail to evaluate the function of OATP1B1, OATP1B3, BCRP, and OCT1 in SCHH, and that digoxin may not be an ideal component of such a cocktail.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Convallatoxin, the active cardiac glycoside of lily of the valley, minimally affects the ADVIA Centaur digoxin assay.

OBJECTIVE: Lily of the valley is a poisonous plant due to the presence of the cardiac glycoside convallatoxin which is known to interfere with serum digoxin measurement using the LOCI digoxin assay and other digoxin assays. We evaluated potential interference of convallatoxin as well as extract of lily of the valley with the ADVIA Centaur digoxin assay by comparing results obtained using the LOCI digoxin assay. MATERIALS AND METHODS: Aliquots of a drug-free serum pool and a digoxin serum pool were supplemented with nanograms to 1 µg quantities of convallatoxin or 1.0 and 2.5 µL of lily of the valley extract per milliliter of serum followed by measurement of digoxin concentrations using the LOCI and ADVIA Centaur digoxin assays. RESULTS: Apparent digoxin concentrations were minimal using the ADVIA Centaur digoxin assay when aliquots of drug-free serum were supplemented with convallatoxin or extract of lily of the valley but apparent digoxin levels were very high using the LOCI digoxin assay. Moreover, minimal interference in serum digoxin measurement using the ADVIA Centaur digoxin assay was observed when aliquots of serum digoxin pool were further supplemented with lily of the valley extract. As expected, the LOCI digoxin assay showed significant interference of convallatoxin in serum digoxin measurement. CONCLUSIONS: Significant interference of convallatoxin in serum digoxin measurement using the LOCI digoxin assay could be minimized using the ADVIA Centaur digoxin assay.

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach.

Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex.

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.

The inhibition effects of some natural products on lactoperoxidase purified from bovine milk.

In this study, inhibition profiles of some natural products, which are digoxin, L-Dopa, dopamine, isoliquiritigenin, and 1,1,2,2-tetrakis(p-hydroxyphenyl)ethane (Tetrakis), were investigated against bovine lactoperoxidase (LPO) enzyme. Digoxin, L-Dopa, and dopamine are active ingredients of some drugs, which have important functions in our body, especially in cases of heart failure. Isoliquiritigenin and tetrakis are types of natural phenolic compounds, which play an important role in cancer prevention and treatment. LPO enzyme was purified from bovine milk using sepharose-4B-l-tyrosine sulfonamide affinity column chromatography. LPO is responsible for the nonimmune biological defense system and has antibacterial activity so selection of these active substances is important. The inhibition studies are performed with the ABTS substrate. Bovine LPO enzyme was effectively inhibited by phenolic molecules. Ki values of these natural products were found as 0.20 ± 0.09, 0.22 ± 0.17, 0.49 ± 0.11, 0.49 ± 0.27, and 1.20 ± 0.25 µM, respectively. Tetrakis and digoxin exhibited noncompetitive inhibition, and other molecules showed competitive inhibition.

Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity.

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

Implants composed of digoxin and poly(ε-caprolactone): development, characterization, anti-proliferative and anti-angiogenic activities.

Drug delivery systems could be applied to locally treat cervical cancer, thus preventing the drawbacks of conventional therapy. In this study, anti-proliferative and anti-angiogenic effects of digoxin incorporated into poly(ε-caprolactone) implants were evaluated, aiming at the local treatment of cervical cancer. Implants were characterized, and the in vitro release profile of digoxin was demonstrated. Anti-proliferative and anti-angiogenic activities of digoxin were investigated by using chorioallantoic membrane and human cervix carcinoma (HeLa) cells, respectively. The chemical structure of digoxin and the semi-crystalline nature of poly(ε-caprolactone) were preserved after designing implants. The hydrophobicity of drug and polymer as well as the semi-crystalline structure provided a controlled diffusion of digoxin from implants. Digoxin released from implantable devices exhibited anti-proliferative activity against HeLa cells. The anti-angiogenic effect was also shown. Finally, implants composed of digoxin and poly(ε-caprolactone) could be applied as a therapeutic alternative to treat the early stage of cervical cancer, once they were able to locally control the release of this anti-angiogenic and anti-proliferative drug, minimizing its systemic side effects and toxicity.

Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.

Trantinterol, a novel ß2-adrenoceptor agonist, noncompetitively inhibits P-glycoprotein function in vitro and in vivo.

P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel ß2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bidirectional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system, where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 µM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a noncompetitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent noncompetitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs coadministered with it.

Quantitative evaluation of biological reaction kinetics in confined nanospaces.

Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis.

Evaluation of digoxin-boronate ester formation through in-capillary derivatisation-large volume sample stacking-capillary zone electrophoresis.

Determination of digoxin through in-capillary derivatisation based on the formation of o-tolyl- and 2-naphthyl-anionic boronate esters in combination with large volume sample stacking-capillary electrophoresis is proposed. The derivatisation reaction was performed at basic pH values to obtain compounds with a charge and chromophore group during the stacking process. After stacking, the species were separated and detected at 225 nm using p-nitrophenol as an internal standard. Stacking and derivatisation parameters such as pre-concentration time, preconcentration voltage and injection time (relation between the analyte and the derivatisation agent) were evaluated using a Box-Behnken design. Under optimal conditions, the proposed method exhibits a linear range of 1.08-50.00 µM with a limit of detection of 0.36 µM; additionally, adequate repeatability and reproducibility was obtained (%RSD ≤ 5.0%). The methodology was validated by comparing it to an HPLC-UV established methodology and was successfully applied for the determination of digoxin in pharmaceutical tablets and blood serum samples, showing a positive performance for these matrices.

Na,K-ATPase activity modulates Src activation: a role for ATP/ADP ratio.

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase.

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.