The extent of involvement of ouabain, hippocampal expression of Na+/K+-ATPase, and corticosterone/melatonin receptors ratio in modifying stress-induced behavior differs according to the stressor in context

The aim of this study was to assess the effect of two types of stressors, regarding the extent of involvement of ouabain (OUA), hippocampal sodium/potassium ATPase (NKA) expression, and the hippocampal corticosterone receptors (CR)/melatonin receptors (MR) expression ratio, on the behavioral and cardiovascular responses and on the hippocampal cornu ammonis zone 3 (CA3) and dentate gyrus (DG). Thirty adult male Wistar albino rats aged 7-8 months were exposed to either chronic immobilization or a disturbed dark/light cycle and treated with either ouabain or vehicle. In the immobilized group, in the absence of hippocampal corticosterone (CORT) changes, rats were non-responsive to stress, despite experiencing increased pulse rate, downregulated hippocampal sodium/potassium pump, and enhanced hippocampal CR/MR expression ratio. Prolonged darkness precipitated a reduced upright attack posture, with elevated CORT against hippocampal MR downregulation. Both immobilization and, to a lesser extent, prolonged darkness stress resulted in histopathological and ultrastructural neurodegenerative changes in the hippocampus. OUA administration did not change the behavioral resilience in restrained rats, despite persistence of the underlying biochemical derangements, added to decreased CORT. On the contrary, with exposure to short photoperiods, OUA reverted the behavior towards a combative reduction of inactivity, with unvaried CR/MR and CORT, while ameliorating hippocampal neuro-regeneration, with co-existing NKA and MR repressions. Therefore, the extent of OUA, hippocampal NKA expression, and CR/MR expression, and subsequent behavioral and cardiac responses and hippocampal histopathology, differ according to the type of stressor, whether immobilization or prolonged darkness.

The Effects of Ouabain on the Inner Ear Glial Cells in Mouse / 听力学及言语疾病杂志

To study the effects of ouabain on the inner ear glial cells, and to lay the foundation for the study of stem cell transplantation in the treatment of sensorineural hearing loss.Methods Sixty adult female SPF grade CBA / J mice were randomly divided into experimental group and control group, with 30 mice in each group.Animals in the experimental group received 3mM ouabain via the round window membrane, while mice in control group received normal saline.The mice were sacrificed at 7 days, 14 days and 30 days after the administration,respectively.Immunofluorescence histochemical staining was used to detect the inner ear glial cells in spiral ganglion.Results Some inner ear glial cells survived in the spiral ganglion of the experimental group, while with decreased numbers and disorganized structure compared to those of in the control group.Comparing to those of in the control group, the number and density of inner ear glial cells in the experimental group were significantly decreased from 7 days afterouabain administration,further decreased at 14 days and reduced to the lowest at 30 days after ouabain administration, the differences between the 2 groups were statistically significant (P < 0.05).Among the experimental group, the number of inner ear glial cells at 30 days was significantly decreased when compared to those of at 7 days and 14 days, respectively.Conclusion Application of ouabain to mouse inner ear via the round window membrane leads to an acute and progressive direct damage to the inner ear glial cells in the spiral ganglion.

Src/ROS signal transduction mechanism of low concentration ouabain on contraction in rat cardiocytes / 中国药学杂志

OBJECTIVE: To observe whether low concentration (1×10-8 mol·L-1) of ouabain (OUA) can increase the contractility in rat cardiocytes and investigate the Na/K pump signal transduction pathways related to positive inotropic action following the low concentration of OUA. METHODS: On enzymatic isolation of rats ventricular myocytes, the Na+/K+ pump current (Ip) was by whole-cell patch-clamp, in order to observe the low concentration of OUA on Ip. The contraction of a single myocyte was assessed by a video-based motion edge-detection system. (1) To detect and compare the potentiations of 1×10-8-1×10-3 mol·L-1 OUA on the contractility in rat cardiocytes. (2) The cardiocytes were pre-treated with PP2 (1 μmol·L-1), NAC (100 μmol·L-1), PD98059(50 μmol·L-1) for 5 min, and the effects of the signals transduction inhibitors on the positive inotropic effect of 1×10-8 mol·L-1 OUA was recorded. RESULTS: The 1×10-8-1×10-3 mol·L-1 OUA increased the contractility of rat cardiocytes (P0.05). CONCLUSION: The 1×10-8-1×10-3 mol·L-1 OUA could increase the contraction amplitude of cardiocytes in rats in concentration-dependent manner. Positive inotropic effect of OUA in low concentration is related to Na/K pump signal transduction. Multiple signal pathways regulate the positive inotropic effect of 1×10-8 mol·L-1 of OUA, including the Src/ROS signal pathway.

cAMP induction by ouabain promotes endothelin-1 secretion via MAPK/ERK signaling in beating rabbit atria

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na(+)-K(+)-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 micromol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 micromol/L), an inhibitor for reverse mode of Na(+)-Ca(2+) exchangers (NCX), but did not by L-type Ca2+ channel blocker nifedipine (1.0 micromol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 micromol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 micromol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 micromol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 micromol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

Three Mood Stabilizers for Inhibition of Ouabain-induced ERK1/2 Phosphorylation in Astrocytes / 中国医科大学学报

Objective To investigate the effets of three mood stabilizers on ouabain?induced ERK1/2 phosphorylation in astrocytes. Methods As?trocytes were treated with different agents and divided into different groups accordingly,namely,the control group with saline,the group with oua?bain,the group with mood stabilizers(lithium carbonate,carbamazepine,sodium valproate)and the group with ouabain+mood stabilizers. The phosphorylation of ERK1/2 in each group was analyzed by Western blot. Results Compared with saline and mood stabilizer groups,the phosphoryla?tion of ERK1/2 was increased in the ouabain group,with statistical significance(P<0.05). There was no significant difference in ERK1/2 phosphoryla?tion between the group with ouabain+mood stabilizers and the control or mood stabilizer group. Conclusion The three kinds of mood stabilizers can inhibit ouabain?induced ERK1/2 phosphorylation in astrocytes.

Ouabain and Endogenous Ouabain -Dr. Jekyll and Mr. Hyde of Cardiac Glycosides.

In current research reports several disease states are claimed to be associated with elevated levels of endogenous ouabain. These include hypertension, cardiac enlargement, cardiac and renal failure, and a variety of terminal disorders. It has been suggested that endogenous ouabain should be considered as a major contributor to increased blood pressure and overall cardiovascular risk. These hypotheses stand in stark contrast to decades of clinical experiences with ouabain in humans, which indicate that plant-derived ouabain is effective in treating heart failure. In patients with hypertension ouabain lowers blood pressure. These conclusive clinical experiences refute the hypothesis that ouabain in humans is a major contributor to increased blood pressure and overall cardiovascular risk. The pronounced dependence of the ouabain effects on the dose, the state of the autonomic nervous system and species-specific characteristics in pharmacokinetics may explain many of the conflicting research results that have been published on experiments with ouabain. Rodents have different sensitivities to cardiac glycosides than humans. Pharmacokinetics of cardiac glycosides in rodents are also different from humans. Therefore, caution is advised when extrapolating experimental results in rodents to humans. The mutually exclusive effects of ouabain and the endogenous inhibitor of the Na/K-ATPase observed in mammalian tissues do not support the hypothesis that this inhibitor is identical with ouabain, but favour the interpretation that De Wardener’s “third factor” is something different, which also reacts to ouabain antibodies.

Effect and Mechanism of Ouabain in Regulating Proliferation of Human Esophageal Carcinoma Cells / 胃肠病学

Background:Esophageal cancer is a common gastrointestinal cancer with poor prognosis,and effective chemotherapy is lacking currently. Studies have shown that cardiac glycosides can inhibit tumor cells growth,but its mechanism has not been fully clarified. Aims:To investigate the effect and mechanism of ouabain in regulating proliferation of human esophageal carcinoma cells. Methods:OE19 human esophageal carcinoma cells were treated with ouabain,and cells in control group were treated with DMSO. Cell proliferation was assessed by cell counting method. mRNA expressions of Sox2,Sox4,Sox7,Sox9 and Sox10 were determined by real-time PCR. Protein expression of Sox4 was determined by Western blotting. Gene expressions of phospho-histone3( ph3),a cell proliferation marker and Sox4 were detected by immunofluorescence staining. Results:Ouabain( ≥ 40 nmol/ L)could significantly inhibit OE19 cells proliferation. mRNA and protein expressions of Sox4 were significantly decreased in OE19 cells in ouabain(40 nmol/ L)group than those in control group(P 0. 05). Gene expressions of ph3 and Sox4 in nucleus of OE19 cells were decreased in ouabain (40 nmol/ L)group than those in control group. Conclusions:Ouabain is effective in inhibiting human esophageal carcinoma cells proliferation,the underlying mechanism might be related with down-regulation of Sox4 expression and the subsequent cell cycle modulation.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Mechanism of ouabain inducing the apoptosis of T lymphoblastic leukemia Jurkat cells / 医学研究生学报

Objective Ouabain is a cardiotonic steroid that can induce the apoptosis of many tumorous cells .This study was to investigate the anti-tumor mechanisms of ouabain by observing its effects on the apoptosis of T lymphoblastic leukemia Jurkat cells and the expressions of hTERT and c-myc mRNA and protein . Methods Jurkat cells were treated with ouabain at the concentrations of 50 and 100 nmol/L for 24 and 48 hours, and those treated with 1 ×PBS served as the control .Then the apoptosis rate of the cells was detected by flow cytometry after Annexin V/PI staining, the expressions of hTERT and c-myc mRNA determined by RT-PCR, and those of hTERT and c-myc protein by Western blot . Results The apoptosis rates of the Jurkat cells in the 50 and 100 nmol/L oua-bain groups were (5.67 ±3.71)%and (9.63 ±4.83)%respectively at 24 hours, and (19.67 ±4.55)%and (37.60 ±11.89)%at 48 hours, significantly higher than (4.23 ±1.01)%in the PBS control group at 48 hours (P<0.05).Compared with the control, the expressions of hTERT and c-myc mRNA were decreased by 200%and those of hTERT and c-myc protein by 224%and 400%, re-spectively, at 48 hours (P<0.05).There was a positive correlation between the reduction of the mRNA levels and that of the protein levels of hTERT and c-myc (P<0.05). Conclusion Ouabain can down-regulate the mRNA and protein expressions of hTERT and c-myc, which may be one of the mechanisms of its induction of the apoptosis of Jurkat T lymphocyte leukemia cells .

Damage of Spiral Ganglion Cell Induced by Ouabain Application in Cat / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Auditory neuropathy is a hearing disorder characterized by the absence or the marked impairment of the auditory brainstem responses with the preservation of the cochlear microphonics (CMs) and otoacoustic emissions. This suggests that outer hair cell (OHC) function is normal but proximal auditory function to OHCs is impaired. It is assumed that the lesion is localized at the level of the inner hair cells (IHCs), auditory nerve fibers, or the synapse between them. This study was aimed to observe the change of hearing threshold and pathology of spiral ganglion cell induced by ouabain application, and present basic data to explain the auditory neuropathy. MATERIALS AND METHOD: Twenty ears of twenty normal hearing cats were used in this study. Cats were treated with 100 microL ouabain (1 mM) applied on the round window. After three days, compound action potential (CAP) and CM were measured and the cochlea was obtained. Pathologic change of spiral ganglion cell was evaluated under light microscope after H&E stain. Normal saline was injected for the control group. RESULTS: In the ouabain group, CAP threshold was increased in all tested frequencies (p0.05). There was significant difference between CAP and CM threshold shift (p<0.001). In the control group, there was no significant difference in CAP and CM thresholds. Light microscopic findings show that the condensed chromatin and nuclear fragments of spiral ganglion cells of an ear was exposed to ouabain, and outer hair cell and inner hair cell were not damaged. CONCLUSION: This study shows that the CAP threshold was significantly increased but the CM threshold was not changed in the ouabain group. Ouabain induced damage of spiral ganglion cells. This study is not sufficient to explain auditory neuropathy because threshold shift of CAP is not obvious, but it would be helpful to explain that selective damage of spiral ganglion cell would be the mechanism of auditory neuropathy.

Effect of m-calpain in PKCα-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain.

There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate, PKC activity using an assay kit and assay of Na+/K+ATPase activity spectrophotometrically. We purified m-calpain and PKCα by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCα by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCα (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCα in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCα increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2+]i-mCalpain-PKCα signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.

The protective effect of grape seed proanthocyanidin extract on vascular remodeling in ouabain-induced hypertensive rats / 中华老年医学杂志

Objective To observe the effect of grape seed proanthocyanidin extract (GSPE) on vascular remodeling in ouabain-induced hypertensive rats.Methods A total of 30 male SpragueDawley rats were randomized into 3 groups:control group (received 0.9％ 1 ml normal saline by intraperitoneal injection and oral gavage in the morning),ouabain treatment group (received 2 mg/kg ouabain by intraperitoneal injection and 0.9 ％ 1 ml normal saline by oral gavage in the morning),and GSPE treatment group (received 2 mg/kg ouabain by intraperitoneal injection and 250 mg Kg 1 d-1 GSPE by oral gavage in the morning).Blood pressure was determined before and 5 weeks after the treatment.The aortas were observed 5 weeks after the treatment.The mRNA and protein levels of nuclear factor-κB (NF-κB p65) and transforming growth factor-β1 (TGF-β1) in rat aorta were detected using real-time PCR and Western blot,respectively.Morphological observations were obtained by Hemotoxylin and Eosin staining and Electron microscope.Results The systolic blood pressure was significantly lower in GSPE treatment group than in ouabain treatment group [(133.6±6.0) mm Hg vs.(146.5±7.9) mm Hg,1 mm Hg=0.133 kPa,P＜0.01].Morphological observation showed that the thickening aortic intimal and structural disorder were found in the ouabain treatment group,and aortic intimal structural integrity were normal in the other two groups.The mRNA and protein levels of NF-κB p65 and TGF-β1 were significantly lower in GSPE treatment group than in the ouabain treatment group (NF-κBp65:2.77±0.58 vs.3.14±0.64,0.73±0.20 vs.0.93±0.21,both P＜0.05; TGF-β1:5.80±0.67 vs.6.09±0.95,0.42±0.14 vs.0.69±0.16,both P＜0.05).Conclusions GSPE may inhibit endogenous ouabain,and delay the process of elevated blood pressure and vascular remodeling by inhibiting NF-κ B p65 and (or) TGF-β3 1 pathways.

Decreased BDNF levels in amygdala and hippocampus after intracerebroventricular administration of ouabain / Diminuição dos níveis de BDNF em amígdala e hipocampo após a administração intracerebroventricular de ouabaína

OBJECTIVE: The present study aims to investigate the effects of ouabain intracerebroventricular injection on BDNF levels in the amygdala and hippocampus of Wistar rats.METHODS: Animals received a single intracerebroventricular injection of ouabain (10-3 and 10-2 M) or artificial cerebrospinal fluid and immediately, 1h, 24h, or seven days after injection, BDNF levels were measured in the rat's amygdala and hippocampus by sandwich-ELISA (n = 8 animals per group).RESULTS: When evaluated immediately, 3h, or 24h after injection, ouabain in doses of 10-2 and 10-3 M does not alter BDNF levels in the amygdala and hippocampus. However, when evaluated seven days after injection, ouabain in 10-2 and 10-3 M, showed a significant reduction in BDNF levels in both brain regions evaluated.DISCUSSION: In conclusion, we propose that the ouabain decreased BDNF levels in the hippocampus and amygdala when assessed seven days after administration, supporting the Na/K ATPase hypothesis for bipolar illness.

OBJETIVO: O presente estudo tem como objetivo investigar os efeitos da injeção intracerebroventricular de ouabaína sobre os níveis de BDNF na amígdala e no hipocampo de ratos Wistar.MÉTODOS: Os animais receberam uma única injeção intracerebroventricular de ouabaína (10-3 and 10-2 M) ou fluido cerebroespinhal artificial e, imediatamente, 3h, 24h ou sete dias após a injeção, os níveis de BDNF foram mensurados na amígdala e hipocampo dos ratos por ELISA sandwich (n = 8 animais por grupo).RESULTADOS: Quando avaliados imediatamente após a injeção, 3h ou 24h, ouabaína nas doses 10-2 e 10-3 M não alterou os níveis de BDNF em ambas as estruturas avaliadas. Entretanto, quando avaliados sete dias após a injeção, ouabaína nas doses 10-2 e 10-3 M mostrou uma significante redução nos níveis de BDNF em amígdala e hipocampo.CONCLUSÃO: Em conclusão, propõe-se que a administração de ouabaína diminuiu os níveis de BDNF em amígdala e hipocampo quando avaliados sete dias após a injeção, suportando a hipótese da participação da Na/K ATPase no transtorno bipolar.

Hypertensive effects of the iv administration of picomoles of ouabain

Ouabain, an endogenous digitalis compound, has been detected in nanomolar concentrations in the plasma of several mammals and is associated with the development of hypertension. In addition, plasma ouabain is increased in several hypertension models, and the acute or chronic administration of ouabain increases blood pressure in rodents. These results suggest a possible association between ouabain and the genesis or development and maintenance of arterial hypertension. One explanation for this association is that ouabain binds to the α-subunit of the Na+ pump, inhibiting its activity. Inhibition of this pump increases intracellular Na+, which reduces the activity of the sarcolemmal Na+/Ca2+ exchanger and thereby reduces Ca2+ extrusion. Consequently, intracellular Ca2+ increases and is taken up by the sarcoplasmic reticulum, which, upon activation, releases more calcium and increases the vascular smooth muscle tone. In fact, acute treatment with ouabain enhances the vascular reactivity to vasopressor agents, increases the release of norepinephrine from the perivascular adrenergic nerve endings and promotes increases in the activity of endothelial angiotensin-converting enzyme and the local synthesis of angiotensin II in the tail vascular bed. Additionally, the hypertension induced by ouabain has been associated with central mechanisms that increase sympathetic tone, subsequent to the activation of the cerebral renin-angiotensin system. Thus, the association with peripheral mechanisms and central mechanisms, mainly involving the renin-angiotensin system, may contribute to the acute effects of ouabain-induced elevation of arterial blood pressure.

A low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 ìg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 ± 5.1 to 150 ± 4.7; DAP: 93.7 ± 7.7 to 116 ± 3.5 mmHg; P<0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

Low concentration of ouabain elevates intracellular free calcium in guinea pig ventricular myocytes: The possible signal transduction pathway / 第二军医大学学报

Objective: To study the effect of low concentration of ouabain MUM on intracellular calcium concentration ([Ca2+]i) in guinea pig ventricular myocytes and to understand whether low concentration of OUA can increase [Ca2+]i through Na+, K+-ATPase channel. Methods: The guinea pig ventricular myocytes were obtained by enzymatic digestion and the [Ca2+]i fluorescent density of individual myocytes was observed under confocal laser scanning microscope. The isolated ventricular myocytes were then incubated with different concentrations of ouabain(10-9, 10-8, 10-7, 10-6 mol · L-1). The sediment was subjected to Western blot analysis to assess the phosphorylation of Src by OUA. Results: In normal Tyrode' s solution and Ca2+-free Tyrode's solution, OUA (1 × 10-9 -1 × 10-6) mol · L-1 elevated [Ca2+]i in a concentration-dependent manner, with the elevation in normal Tyrode's solution more obvious (P < 0.05). Genistein (GST) (1, 10, 50, and 100 μmol - L-1) abolished the OUA-induced increases of [Ca2+]i in a concentration-dependent manner. There were two tyrosine-phosphorylated bands, with the molecular weights being 120 000 and 70 000. Compared with control group, the densities of the 2 bands in all OUA groups were significantly higher (P<0.05) and GST could obviously inhibit the elevating effect of OUA. Conclusion: Low concentration of OUA may promote opening of Ca2+ channel and release of intracellular Ca2+, and subsequently elevate intracellular free calcuim through phosphorylation of tyrosine and activition of OUA/Na+, k+-ATPase/Src signal transduction pathway.

A key role for Na+/K+-ATPase in the endothelium-dependent oscillatory activity of mouse small mesenteric arteries

Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group) underwent left coronary artery ligation (infarcted, Inf) or sham-operation (Sham). Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e) and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05). The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05), while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

Protective effects of ouabain conjugated peptide from Ph. D.-7 Library on vascular endothelial cell / 细胞与分子免疫学杂志

AIM: To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension. METHODS: In this study, ouabain was used as a target to screen ouabain conjugated peptide (OCP) from Ph. D. -7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na~+-K~+-ATPase α1-subunit and β1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na~+ in EAhy926 cells were measured by laser confocal microscope. RESULTS: The ouabain conjugated peptide was found out, and it was occupied in 0.64(9/14). The analysis of protein showed that the obtained peptides had no homology with Na~+-K~+-ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and ~3H-ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na~+-K~+-ATPase α1-subunit and down-regulated expression of Na~+-K~+-ATPase β1-subunit induced by ouabain in EAhy926 cells. CONCLUSION: The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na~+-K~+-ATPase and explore novel anti-ouabain agents.