OBJECTIVETo explore the effect of ouabain on intracellular Ca(2+) concentration ([Ca(2+)]i) in thoracic aorta vascular smooth muscle cells (VSMCs) in vitro.

METHODSPrimary SD rat thoracic aorta VSMCs were cultured by tissue adherent method and identified by immunochemistry. The binding ability between ouabain and VSMCs was detected by autoradiography, and fluo 3-AM (a Ca(2+) fluorescent probe) was employed to investigate whether ouabain affected VSMCs within a short period of time. The effect of a truncated fragment of the sodium pump α2 subunit was assayed in antagonizing the effect of ouabain on [Ca(2+)]i in the VSMCs.

RESULTSWithin the concentration range of 0.1-100 nmol/L, ouabain was found to dose-dependently bind to the VSMCs. Different concentrations of ouabain (0-3200 nmol/L) caused a transient, dose-dependent increase in [Ca(2+)]i in the VSMCs, which was antagonized by the application of the truncated fragment of sodium pump α2 subunit.

CONCLUSIONSElevations in [Ca(2+)]i in the VSMCs can be the cytological basis of high ouabain-induced hypertension. The truncated fragment of the sodium pump α2 subunit can antagonize ouabain-induced increase of [Ca(2+)]i in the VSMCs, which provides a clue for understanding the pathogenesis of and devising a therapeutic strategy for high ouabain-induced hypertension.

Animals ; Aorta, Thoracic ; cytology ; Calcium ; metabolism ; Cells, Cultured ; Cytoplasm ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Ouabain ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase

BACKGROUNDOuabain and digoxin are important cardiac glycoside and related to many cardiovascular diseases. The purpose of this study was to investigate the changes of sodium pump α-subunit expression in rats and compare the effects of ouabain (OUA) and digoxin (DIG) on the development of hypertension.

METHODSIn situ hybridization was performed. Specific sequence oligonucleotide probe tailing with a Dig-dUTP hybrid to target nucleic acids of the sodium pump α-subunit. According to counting positive particles sodium pump subunit expression was analyzed with statistical methods.

RESULTSOn day 16 of drug administration, the blood pressure of rats increased significantly in the OUA group. In the DIG group, the blood pressure revealed no significant difference when compared to the control group. In addition, the effects of OUA and DIG on sodium pump α-subunit RNA expression in tissues differed.

CONCLUSIONSOUA and DIG can not only change the configuration of the sodium pump to depress their activity, but also influence their gene expression which is important in the mechanism of hypertension. This may be a key point in the pathogenesis of hypertension in the manner in which OUA differs from DIG and changes the sodium pump gene expression in the arteries and kidneys of rats.

Animals ; Blood Pressure ; drug effects ; Digoxin ; therapeutic use ; Hypertension ; drug therapy ; In Situ Hybridization ; Male ; Ouabain ; therapeutic use ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase ; metabolism

A growing number of researches have shown that ouabain can regulate mammalian sperm function and male reproduction by modulating the sperm motility, capacitation and acrosome reaction in vitro. This study further examined the relationship between ouabain and asthenozoospermia. In this study, the rat was intraperitoneally injected with ouabain at different concentrations (low-dose ouabain group: 12.5 μg/kg body weight per day, and high-dose ouabain group: 25 μg/kg body weight per day) for 30 days to establish the asthenozoospermia model. The sperms from 60 males with normal fertility were incubated with ouabain of gradient concentrations (10(-7)-10(-2) mol/L) for 4 h. The sperm motility was evaluated under a microscope. Moreover, the endogenous ouabain (EO) level was determined in seminal plasma of mild or severe asthenozoospermia patients and males with normal fertility by competitive inhibition ELISA. The results showed that the sperm motility was significantly diminished in the rats treated with different concentrations of ouabain. The number of motile sperms (grades a and b) was decreased greatly in a time- and dose-dependent manner in 10(-5)-10(-2) mol/L ouabain groups (P<0.01), while no obvious change in sperm motility was observed in 10(-7)-10(-6)mol/L groups even for 4-h incubation (P>0.05). Furthermore, the EO level was significantly increased in asthenozoospermia patients as compared with that in males with normal fertility (25.27±1.71 μg/L in mild asthenozoospermia patients, 26.52±1.82 μg/L in severe asthenozoospermia patients, 19.31±1.45 μg/L in normal fertility men) (P<0.01). In conclusion, rat asthenozoospermia was successfully established by intraperitoneal injection of ouabain, and 10(-5) mol/L ouabain was sufficient enough to inhibit sperm motility in vitro. Moreover, EO, a normal constituent of seminal plasma, was highly expressed in asthenozoospermia males as compared with normal fertility ones.
Animals ; Asthenozoospermia ; chemically induced ; metabolism ; physiopathology ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Humans ; Injections, Intraperitoneal ; Male ; Ouabain ; metabolism ; pharmacology ; toxicity ; Rats ; Rats, Sprague-Dawley ; Semen ; metabolism ; Sperm Motility ; drug effects ; physiology ; Spermatozoa ; drug effects ; physiology ; Time Factors

BACKGROUNDOuabain is a mammalian adrenocortical hormone that is involved in the pathogenesis of hypertension by inhibiting Na-K ATPase activity. It also participates in a variety of kinase-mediated signaling pathways associated with Na-K ATPase. Previous studies have shown that ouabain can cause cardiac remodeling independent of elevated blood pressure and that proliferating cell nuclear antigen (PCNA) plays a coordinating role for numerous proteins involved in multiple processes associated with DNA synthesis. Therefore, we hypothesized that ouabain might play a role in the cerebral cortex through signaling pathways independent of hypertension. And PCNA might be involved in this process.

METHODSMale Sprague-Dawley rats were treated with ouabain or with 0.9% nitric sodium as the control group. Systolic blood pressure was recorded weekly. After four weeks of treatment, morphological changes in the cerebral cortex were analyzed using light and transmission electron microscopy. The expression of PCNA in the cerebral cortex was evaluated by immunohistochemistry, real time quantitative PCR, and Western blotting.

RESULTSAfter 4-week treatment, there was no significant difference in systolic blood pressure compared with the control group, but both structural deterioration and up-regulated expression of PCNA in the brain was induced by ouabain treatment.

CONCLUSIONSThese results suggest that ouabain induces alterations in the brain structure, and this effect is independent of blood pressure. PCNA might be involved in the repair process of ouabain-induced brain damage.

Animals ; Blood Pressure ; drug effects ; Hypertension ; drug therapy ; metabolism ; Immunohistochemistry ; Male ; Ouabain ; therapeutic use ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Sodium-Potassium-Exchanging ATPase ; metabolism

OBJECTIVEPeriplocin is an active digitalis-like component from Cortex Periplocae, which has been widely used in the treatment of heart diseases in China for many years. According to the recommendations on the cardiovascular effect of periplocin from in vivo experiments, subsequent in vitro experiments are greatly needed for the global assessment of periplocin. The objective of this study is to investigate the cell proliferation effect and the mechanism of periplocin on endothelial cells.

METHODSThe proliferative activity of periplocin (0.4, 2, 10, 50, 250 micromol/L; 6, 12, 24, 48, 72 h) was investigated by a comparison with the well-reported cardiac glycoside, ouabain, on mouse cardiac microvascular endothelial cells (CMEC). 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) and 5-bromo-2-deoxyuridine (BrdU) assays were used to evaluate cell proliferation and viability. Subsequently, cDNA microarray experiments were performed on periplocin- (50 micromol/L) and ouabain- (50 micromol/L) treated cells, and data was analyzed by ArrayTrack software.

RESULTSPeriplocin could increase cell viability to a level lower than ouabain in the MTT analysis, but decrease LDH release simultaneously. The BrdU incorporation assay showed an increase in cell proliferation with 2-50 micromol/L periplocin. Genes related to protein serine/threonine kinase were the most significantly enriched in the 160 genes identified in periplocin versus the control. In the 165 genes regulated by periplocin versus ouabain, GTP-binding was the most altered term.

CONCLUSIONSThe results demonstrated the proliferation action of periplocin on CMEC. Meanwhile, its lower cytotoxicity compared to ouabain provides a new insight into the treatment of heart failure.

Animals ; Animals, Newborn ; Cardiac Glycosides ; pharmacology ; Cardiotonic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; Cells, Cultured ; Coronary Vessels ; drug effects ; metabolism ; physiology ; Drug Evaluation, Preclinical ; Endothelial Cells ; drug effects ; metabolism ; physiology ; Gene Expression Profiling ; Gene Expression Regulation ; drug effects ; Mice ; Microvessels ; drug effects ; metabolism ; physiology ; Models, Biological ; Myocardium ; metabolism ; Oligonucleotide Array Sequence Analysis ; Ouabain ; pharmacology ; Saponins ; pharmacology

OBJECTIVETo investigate the role of renal sympathetic nerves in renal sodium transport in ouabain-hypertensive rats (OHR).

METHODSSixteen male SD rats with sham renal denervation (Sham-RDNX) and 16 with renal denervation (RDNX) were randomly into normal control group and ouabain group to receive intraperitoneal injection of normal saline (1 ml/kg) and ouabain (27.8 µg/kg) once a day, respectively. Systolic blood pressure (SBP), heart rate and body weight were recorded weekly. Food consumption of the rats was determined twice a week. After a 4-week treatment, blood and 24 h urine samples were collected to measure the serum and urinary concentration of sodium, trace lithium and creatinine. Endogenous creatinine clearance rate (Ccr), fractional excretions of sodium (FENa), fractional excretions of lithium (FELi) and fractional reabsorption of sodium in the postproximal tubules (FDRNa) were calculated. Plasma renin activity was determined by radioimmunoassay. Norepinephrine was extracted from the renal tissue and assayed for norepinephrine content by HPLC.

RESULTSThe body weight, food intake and heart rate showed no significant difference among the 4 groups (P > 0.05). After 4 weeks, the SBP of control RDNX group (CDNX) was significantly lower than that of the control Sham-DNX group (Csham)(P < 0.05); the SBP of ouabain RDNX group (ODNX) was also significantly lower than that of ouabain Sham-DNX group (Osham) (P < 0.05); RNDX lowered SBP by about 10 mmHg in both ouabain groups and control groups. The SBP was significantly higher in Osham and ODNX groups than in the corresponding control groups (P < 0.01), also significantly higher in ODNX group than in Csham group (P < 0.01). Ccr showed no significant difference among the 4 groups(P > 0.05). FENa, FELi and FDRNa were significantly lower in ouabain groups than in the corresponding control groups (P < 0.05, P < 0.01, P < 0.05), but FENa, FELi and FDRNa of ODNX group were similar with those of Osham group (P > 0.05); FENa , FELi and FDRNa were similar between CDNX and Csham groups (P > 0.05). The plasma renin activity was comparable between the 4 groups (P > 0.05). Renal norepinephrine level was markedly reduced in RDNX group compared with that in Sham-RDNX group in both ouabain and control groups (P < 0.01).

CONCLUSIONThe increase of proximal tubule sodium reabsorption in OHR is not dependent on the renal sympathetic nerve.

Animals ; Hypertension ; chemically induced ; metabolism ; Kidney ; innervation ; Male ; Ouabain ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism ; Sodium Channels ; metabolism ; Sympathetic Nervous System ; physiology

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

The aim of this study was to investigate the effects of ouabain and some specific signal pathway inhibitors on growth regulation in various kinds of leukemia cell lines and to explore the role of signal pathways participating in proliferation or apoptosis of leukemia cells induced by ouabain. By using MTT, the survival rates of leukemia cell lines were observed after utilizing ouabain and the specific signal pathway inhibitors. The expressions of Na(+), K(+)-ATPase alpha1 subunit of leukemia cells were evaluated by RT-PCR and Western blot. The results showed that low concentration of ouabain (10 nmol/L) could increase the survival rates of lymphocytic leukemia Jhhan cell line and megakaryocytic leukemia M07e cell line, and could up-regulate the expression of Na(+), K(+)-ATPase alpha1 subunit. Proliferation of these leukemia cells induced by ouabain could be inhibited by PP2 and PD98059 with different extents. It is concluded that Na(+), K(+)-ATPase plays an important role in signal transductions through binding to CTS (ouabain), and they can activate complex signal pathways regulating the growth of leukemia cells. The proliferation effects of cells promoted by ouabain are mediated by activation of Src kinase and ERK1/2 dependent signaling pathway.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; Ouabain ; pharmacology ; Signal Transduction ; drug effects ; Sodium-Potassium-Exchanging ATPase ; metabolism

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

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