ID clarify the effects of ouabain on the ERG c-wave, isolated chick retinas were exposed to different concentrations of ouabain and the results noted. Although the c-wave was abolished at a highe. dose of ouabain (10(-4)M), its amp1itude increased in the presence of ouabain at a concentration of 10(-7)M, which was within the range of clinical use of the cardiac glycoside. On the other hand, the standing potential of the retina did not change appreciably until 10(-6)M and then decreased gradually at higher concentrations.In the presence of 10(-4)M ouabain, the concentration which completely blorked Na-K-ATPase, both the c-wave and the standing potential were almost abolished. These phenomena were more conspicuous when ouabain was applied to the vitreous side rather than the choroidal side. In the presence of 10(-7)M ouabain, the light sensitivity of the retina was elevated to 0.5 log unit and the maximum response increased about 30%. This may be a sign of visual complications of ouabain, such as metachromatopsia.
Animals ; Chickens ; Electroretinography ; Ouabain/*pharmacology ; Photic Stimulation ; Retina/*drug effects

The purpose of this study was to investigate the effects of phytoestrogen genistein (GST) on delayed after depolarization (DAD) and triggered activity (TA) induced by ouabain in guinea pig papillary muscles. Action potentials (APs) were recorded from the guinea pig papillary muscles with standard glass microelectrode technique. The results are as follows: (1) DAD and TA induced by ouabain (1 micromol/L) were markedly inhibited by pretreatment with GST (10, 50, 100 micromol/L) in a concentration-dependent manner. (2) NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), an NO synthase inhibitor, failed to affect the above effects of GST. (3) 5 micromol/L 17beta-estradiol (E2) or 10 micromol/L GST alone showed no effects on DAD and TA, whereas GST combined with E(2) at the same doses exerted significant inhibitory effects on DAD and TA. Since GST is known to reduce the calcium influx, it is suggested that GST might have antiarrhythmic effects, possibly by reducing calcium influx. The antiarrhythmic effects of GST may contribute to its cardioprotective action.
Action Potentials ; drug effects ; Animals ; Genistein ; pharmacology ; Guinea Pigs ; Male ; Neuromuscular Depolarizing Agents ; pharmacology ; Ouabain ; pharmacology ; Papillary Muscles ; drug effects ; physiology ; Phytoestrogens ; pharmacology

Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Gene Expression Profiling ; Humans ; Ouabain ; pharmacology ; Umbilical Veins ; cytology

AIMTo study the effect of Na+, K(+)-ATPase inhibition by ouabain on growth and death of vascular endothelial cells ECV304 and involved mechanisms.

METHODSGrowth inhibition of ouabain on ECV304 cells was analyzed using MTT assay. The feature of cell death was studied by Hoechst 33342/PI staining, transmission electron microscopy and DNA agarose gel electrophoresis in ECV304 cells treated with ouabain. The mRNA expression of Na+, K(+)-ATPase alpha1, beta1-subunit was examined by reverse transcription PCR (RT-PCR).

RESULTSOuabain inhibited the growth of ECV304 cells in a dose and time-dependent manner. 10 micromol/L ouabain treated for 24 hours could stimulate the necrosis of ECV304 cells; When treated with 0.1 micromol/L ouabain for 24-48 hours, the cells showed obviously defluxion, the loss of cell-cell contacts, nuclear chromatin condensation, chromatin margination and DNA fragmentation. Na+, K(+)-ATPase alpha1-subunit mRNA expression was significantly up-regulated in ECV304 cells treated with ouabain while the beta1-subunit expression conversely showed a significant decrease.

CONCLUSIONOuabain could up-regulate Na+, K(+)-ATPase alpha1-Subunit expression and reduce beta1-Subunit expression which mediated signal transduction and decreased cell-cell adhesions and induced ECV304 cells death.

Cell Death ; drug effects ; Cell Line ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Ouabain ; pharmacology ; Sodium-Potassium-Exchanging ATPase ; metabolism

AIMTo observe the effects of ouabain on vascular smooth muscle (VSM) of the guinea pig and its interactions with Ca2+ and norepinephrine (NE).

METHODSUsing isolated thoracic aortic ring of the guinea pig, the degrees of contractile activity of drugs were recorded.

RESULTSOuabain showed a direct contractile effect in a concentration-dependent manner on thoracic aortic ring of guinea pig. Ouabain shifted the NE dose-response curve to the left without changing in the maxium response. Ouabain shifted the CaCl2 dose-response curve to the left and upward, increased the maximum response to Ca2+; In Ca(2+)-free medium, the ouabain induced contraction was abolished, an increase in extracellular Ca2+ restored the response; nifedipine and verapamil abolished the ouabain induced contraction.

CONCLUSIONThe ouabain induced contraction is mainly dependent on the extracellular Ca2+ concentration, independent on the presence of endothelia of aorta, suggesting that Ca2+ antagonist may treat the hypertension induced by ouabain. Ouabain, NE and CaCl2 have synergetic contractile effects, suggesting that the synergetic contractile effects of ouabain and NE may contribute to the generation and development of hypertension.

Animals ; Aorta, Thoracic ; drug effects ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Chloride ; pharmacology ; Drug Synergism ; Female ; Guinea Pigs ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth, Vascular ; drug effects ; Nifedipine ; pharmacology ; Norepinephrine ; pharmacology ; Ouabain ; pharmacology ; Verapamil ; pharmacology

OBJECTIVETo investigate the changes in rat cardiac function and myocardium ultrastructure in response to ouabain treatment.

METHODSTwenty-four male SD rats were randomized into two equal groups to receive daily intraperitoneal injection of ouabain or saline for 4 consecutive weeks, and their systolic blood pressure (SBP) was recorded weekly. After 4 weeks of injection, echocardiography was performed and the hemodynamic parameters were measured by invasive cardiac catheterization, and the changes in myocardium ultrastructure observed using transmission electron microscopy.

RESULTSAfter 4 weeks of ouabain injection, no significant changes in the mean SBP occurred in comparison with the saline group, but echocardiographic examination showed significant increases in the left ventricular end-diastolic and end-systolic diameters, septum thickness, posterior wall thickness, left ventricular mass and isovolumetric relaxation time but significantly lowered E/A ratio, ejection fraction and fractional shortening after ouabain treatment (P<0.05). Invasive monitoring revealed significant attenuation of the left ventricular developed pressure, rate of pressure development (+dp/dt) and rate of pressure decay (-dp/dt), and increment of the left ventricular end diastolic pressure. Myofibrillar fragmentation, swelling of the cardiac myocytes, absence of the Z line, increases of the mitochondria and collagen fibers were found in ouabain group by transmission electron microscopy.

CONCLUSIONOuabain can induce left ventricular enlargement, cardiac wall thickening, myocardial ultrastructural alterations, systolic and diastolic dysfunction in rats before blood pressure elevation is detected, indicating that ouabain can directly cause cardiac damage in rats.

Animals ; Echocardiography ; Heart ; drug effects ; physiopathology ; Male ; Microscopy, Electron, Transmission ; Myocardium ; pathology ; ultrastructure ; Ouabain ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The study was aimed to explore the apoptosis effect of ouabain on Jurkat cells and its mechanism. MTT method was used to observe the inhibitory effect of ouabain on Jurkat cell proliferation. Apoptosis was detected by using flow cytometry (FCM) and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method (TUNEL). The protein expressions of Bax, Bcl-2 and active subunits of caspase-3 were measured by Western blot. Activities of caspase-3 were determined by colorimetry method. The results showed that ouabain could induce apoptosis of Jurkat cells, the expression of Bax protein in process of cell apoptosis, caspase-3 activity of Jurkat cells were remarkably enhanced after ouabain treatment. It is concluded that ouabain may induce apoptosis of Jurkat cells due to the activation of caspase-3 resulting from regulation of Bax protein and Bcl-2 gene expressions.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Genes, bcl-2 ; genetics ; Humans ; Jurkat Cells ; Ouabain ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics

OBJECTIVETo assess the binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment (HES1 derivative).

METHODSHES1 derivative was synthesized by Fmoc method and purified by high-performance liquid chromatography-mass spectrometry, and its binding activity was identified by radioligand binding assay.

RESULTS3H-ouabain and synthetic HES1 derivative showed some binding activity with the equilibrium dissociation constant (KD) of 24.58 nmol/L, with the the receptor density of 492.43 fmol x mg(-1) pro. and IC50 of 3.078 x 10(-7) mol/L.

CONCLUSIONHES1 derivative can bind to ouabain and has the potential of becoming an effective therapeutic agent.

Binding Sites ; drug effects ; Extracellular Space ; metabolism ; Humans ; Ouabain ; chemistry ; pharmacology ; Peptides ; chemistry ; Protein Binding ; Sodium-Potassium-Exchanging ATPase ; chemistry ; genetics ; metabolism

OBJECTIVE: To evaluate the protective effect of low dose ouabain on injury of cultured spiral ganglion neurons evoked by trophic factors deprived and to explore the mechanism. METHOD: Spiral ganglion neurons were cultured in vitro for 7 days, and then exposed to Neurobasal medium + B27 supplement, Neurobasal medium only or Neurobasal medium + 10 nmol/L ouabain, respectively. After 48 h exposed to different medium, spiral ganglion neurons were stained by FITC labeled Annexin-V and PI, then apoptosis index were calculated using fluorescent microscope and flow cytometry, respectively. In addition, spiral ganglion tissues were cultured for 48 h to evaluate dendrite growth of spiral ganglion neurons in each group. Immunocytochemistry were performed to detect the level of Bcl-2 in each group at 6 h and 12 h. RESULT: Spiral ganglion neurons exposed to Neurobasal medium +10 nmol/L ouabain have a similar apoptosis index compare with that of Neurobasal medium + B27 supplement, but a much lower apoptosis index than that of Neurobasal medium only. In addition, dendrite growth of spiral ganglion neurons exposed to Neurobasal medium +10 nmol/L ouabain was much longer than that of Neurobasal medium only. Bcl-2 level increased in spiral ganglion neurons exposed to Neurobasal medium + 10 nmol/L ouabain at 6 h. CONCLUSION Low dose of ouabain protects injury of spiral ganglion neurons evoked by trophic factors deprived in vitro. This effect may mediated by increasing the level of Bcl-2.
Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Ouabain ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; drug effects ; pathology

In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.
Amino Acid Sequence ; Animals ; Chromatography, High Pressure Liquid ; Erythrocyte Membrane ; drug effects ; Ouabain ; pharmacology ; Peptide Fragments ; metabolism ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism