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

OBJECTIVECardiotonic steroids (CTS) can bind to Na+, K+ -ATPase to activate complex intracellular signaling cascades regulating the proliferation and apoptosis of cells. The aim of this study was to investigate the effects of ouabain at different concentrations on growth regulation in various kinds of leukemia cell lines and explore the pathogenesis of leukemia, the functions of Na+, K+ -ATPase as a signal transduction conductor and its effects on cell growth.

METHODSUsing the MTT assay, the survival rates of leukemia cell lines were observed 6, 12 and 24 hrs after treatment with 1 or 10 nmol/L ouabain. The expression of Na+, K+ -ATPase alpha1 subunit of leukemia cells was detected by Western blot.

RESULTSThe MTT results showed that ouabain at 1 nmol/L or 10 nmol/L induced proliferation of lymphocytic leukemia B95 and Jhhan cell lines, as well as megakaryocytic leukemia M07e and Meg01 cell lines. Ouabain at 1 nmol/L or 10 nmol/L increased the expression of Na+, K+ -ATPase alpha1 subunit. There were significant differences in the proliferation and the expression of Na+, K+ -ATPase alpha1 subunit of the leukemia cell lines between the ouabain treatment and the blank control groups 24 hrs after ouabain treatment (P<0.05). The proliferation effect of leukemia cell lines was in a direct proportion with the ouabain concentration and incubation time.

CONCLUSIONSNa+, K+ -ATPase plays an important role in signal transductions. Through binding to ouabain, Na+, K+ -ATPase may regulate proliferation of leukemia cell lines of different origins. Ouabain at 1 nmol/L or 10 nmol/L may induce proliferation of lymphocytic leukemia cell lines (B95, Jhhan) and megakaryocytic leukemia cell lines (M07e, Meg01), and the proliferation effect was in a direct proportion with the concentration and incubation time of ouabain.

Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Leukemia ; pathology ; Ouabain ; pharmacology ; Signal Transduction ; Sodium-Potassium-Exchanging ATPase ; analysis ; genetics ; physiology

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

This study was aimed to investigate the effects of ouabain at low concentrations on growth regulation in various leukemia cell lines and to determine the therapeutic potential of ouabain in leukemia. By using MTT, flow cytometry (FCM), the changes in cell growth and cell cycle of leukemia cell lines were observed after treating with ouabain at low concentrations (ouabain (ouabain induced the proliferation of lymphocytic leukemia B95 and Jhhan cell lines, increased the percentage of cells at S phase and G2/M phase and up-regulated the expression of sodium pump alpha1 subunit. The basic expression of the sodium pump alpha1 subunit in M07e and Meg-01 was lower than that in B95 and Jhhan. It is concluded that the low concentrations of ouabain inhibit the cell growth of the megakaryocytic leukemia cell lines M07e and Meg-0, which may be related with the low expression of sodium pump alpha1 subunit of that cell lines and the negative regulation of the protein induced by ouabain.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Leukemia ; pathology ; Leukemia, Megakaryoblastic, Acute ; pathology ; Ouabain ; pharmacology ; Sodium-Potassium-Exchanging ATPase ; metabolism

Endogenous ouabain (EO) is a recently found hormone that may be secreted from adrenal cortex. As an endogenous mammalian analogue of cardiac glycosides and an inhibitor of the sodium pump, it regulates the body fluid balance, urine sodium extraction and vasoconstrictive tone, and thus plays an important role in the pathogenesis of hypertension and some other cardiovascular disorders. This articke reviews its biological features, receptor and antibody, detection, effects on diseases and relationship with endothelial cells.
Adrenal Cortex ; metabolism ; Animals ; Antibodies ; therapeutic use ; Humans ; Hypertension ; etiology ; Nephrotic Syndrome ; etiology ; Ouabain ; immunology ; pharmacology ; Sodium-Potassium-Exchanging ATPase ; immunology ; physiology

The effects of low concentration of dihydroouabain (DHO) on intracellular calcium concentration ([Ca(2+)](i)) were investigated in guinea pig ventricular myocytes. [Ca(2+)](i) was detected by confocal microscopy and represented by fluorescent intensity. DHO (1 fmol/L~1 mmol/L) increased [Ca(2+)](i), especially at 10 pmol/L. Nisoldipine, egtazic acid, or tetrodotoxin partially inhibited the effect of 10 pmol/L DHO on [Ca(2+)](i). The effects of DHO remained in the absence of extracellular K(+) and Na(+). These results suggest that low concentration of DHO might increase [Ca(2+)](i) via the receptor-operated Ca(2+) channels, TTX-sensitive Na(+) channels or/and triggering of intracellular calcium release; Na(+)/K(+) pump and Na(+)/Ca(2+) exchange seem not involved in the effect of DHO.
Animals ; Calcium ; metabolism ; Guinea Pigs ; Heart Ventricles ; cytology ; Male ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Ouabain ; analogs & derivatives ; pharmacology ; Patch-Clamp Techniques