Huicai Guo

Na+/K+-ATPase-mediated signal transduction and Na+/K+-ATPase regulation

A number of studies suggest that Na+/K+‐ATPase in caveolae interacts with neighboring membrane proteins and organizes cytosolic cascades of signaling proteins to send messages to intracellular organelles in different tissues, mostly in cardiac myocytes. Low concentration of ouabain binding to Na+/K+‐ATPase activates Src/epidermal growth factor receptor complex to initiate multiple signal pathways, which include PLC/IP3/CICR, PI3K, reactive oxygen species (ROS), PLC/DG/PKC/Raf/MEK/ERK1/2, and Ras/Raf/MEK/ERK1/2 pathways. In cardiac myocytes, the resulting downstream events include the induction of some early response proto‐oncogenes, activation of transcription factors, activator protein‐1, and nuclear factor‐κB, the regulation of a number of cardiac growth‐related genes, and the stimulation of protein synthesis and myocyte hypertrophy and apoptosis. Conversely, several factors acting through signal pathways, such as protein kinases, Ca2+, ROS, etc., can modulate the activity of the Na+/K+‐ATPase.

Chronic intermittent hypobaric hypoxia protects the heart against ischemia/reperfusion injury through upregulation of antioxidant enzymes in adult guinea pigs

AbstractAim:To investigate the protection and the anti-oxidative mechanism afforded by chronic intermittent hypobaric hypoxia (CIHH) against ischemia/reperfusion (I/R) injury in guinea pig hearts.Methods:Adult male guinea pigs were exposed to CIHH by mimicking a 5000 m high altitude (pB=404 mmHg, pO2=84 mmHg) in a hypobaric chamber for 6 h/day for 28 days. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia and the reperfusion period. The activity and protein expression of antioxidant enzymes in the left myocardium were evaluated using biochemical methods and Western blotting, respectively. Intracellular reactive oxygen species (ROS) were assessed using ROS-sensitive fluorescence.Results:After 30 min of global no-flow ischemia followed by 60 min of reperfusion, myocardial function had better recovery rates in CIHH guinea pig hearts than in control hearts. The activity and protein expression of superoxide dismutase (SOD) and catalase (CAT) were significantly increased in the myocardium of CIHH guinea pigs. Pretreatment of control hearts with an antioxidant mixture containing SOD and CAT exerted cardioprotective effects similar to CIHH. The irreversible CAT inhibitor aminotriazole (ATZ) abolished the cardioprotection of CIHH. Cardiac contractile dysfunction and oxidative stress induced by exogenous hydrogen peroxide (H2O2) were attenuated by CIHH and CAT.Conclusions:These data suggest that CIHH protects the heart against I/R injury through upregulation of antioxidant enzymes in guinea pig.

Sesamin ameliorates doxorubicin-induced cardiotoxicity: Involvement of Sirt1 and Mn-SOD pathway

Oxidative stress caused by doxorubicin (DOX) is believed to be a major underlying molecular mechanism of DOX-induced cardiotoxicity. Sesamin (Ses), an active component extracted from sesame seeds, exhibits antioxidative and anti-inflammatory effects. In the present study, possible protective mechanisms of Ses on DOX-induced cardiotoxicity were investigated in rats and cultured H9C2 cells. We demonstrated that Ses exhibits a significant protective effect on cardiac tissue in animal and cell models of DOX-induced cardiac injury. Moreover, Ses can ameliorate DOX-induced oxidative stress and mitochondrial damage. Further studies suggested that Ses is able to up-regulate the protein expression of Mn-SOD in normal rats and to restore the decreased expression of Mn-SOD in DOX-induced cardiac injury rats. Exposure to Ses or DOX alone slightly increased the protein expression of Sirt1; however, a more remarkable increase in Sirt1 protein level was detected in the Ses+DOX group. Treatment with a pan-sirtuin inhibitor (nicotinamide) or a Sirt1-specific inhibitor (EX-527) partially antagonised the effect of Ses on DOX-induced mitochondrial damage and completely abolished the effect of Ses on Mn-SOD expression. These findings indicate that the protective mechanisms of Ses on DOX-induced cardiotoxicity are involved in the alleviation of oxidative stress injury and Mn-SOD dysfunction, partially via the activation of Sirt1.

Enhancement of Na/K pump activity by chronic intermittent hypobaric hypoxia protected against reperfusion injury

Chronic intermittent hypobaric hypoxia (CIHH) has been shown to attenuate intracellular Na(+) accumulation and Ca(2+) overload during ischemia and reperfusion (I/R), both of which are closely related to the outcome of myocardial damage. Na/K pump plays an essential role in maintaining the equilibrium of intracellular Na(+) and Ca(2+) during I/R. It has been shown that enhancement of Na/K pump activity by ischemic preconditioning may be involved in the cardiac protection. Therefore, we tested whether Na/K pump was involved in the cardioprotection by CIHH. We found that Na/K pump current in cardiac myocytes of guinea pigs exposed to CIHH increased 1.45-fold. The K(1) and f(1), which reflect the portion of α(1)-isoform of Na/K pump, dramatically decreased or increased, respectively, in CIHH myocytes. Western blot analysis revealed that CIHH increased the protein expression of the α(1)-isoform by 76%, whereas the protein expression of the α(2)-isoform was not changed significantly. Na/K pump current was significantly suppressed in simulated I/R, and CIHH preserved the Na/K pump current. CIHH significantly improved the recovery of cell length and contraction during reperfusion. Furthermore, inhibition of Na/K pump by ouabain attenuated the protective effect afforded by CIHH. Collectively, these data suggest that the increase of Na/K pump activity following CIHH is due to the upregulating α(1)-isoform of Na/K pump, which may be one of the mechanisms of CIHH against I/R-induced injury.

Reduction of DNA damage induced by titanium dioxide nanoparticles through Nrf2 in vitro and in vivo

Titanium dioxide nanoparticles (Nano-TiO2) are widely used to additives in cosmetics, pharmaceutical, paints and foods. Recent studies have demonstrated that Nano-TiO2 induces DNA damage and increased the risk of cancer and the mechanism might relate with oxidative stress. The aim of this study was to evaluate the effects of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), an anti-oxidative mediator, on DNA damage induced by Nano-TiO2. Wildtype, Nrf2 knockout (Nrf2(-/-)) and tert-butylhydroquinone (tBHQ) pre-treated HepG2 cells and mice were treated with Nano-TiO2. And then the oxidative stress and DNA damage were evaluated. Our data showed that DNA damage, reactive oxygen species (ROS) generation and MDA content in Nano-TiO2 exposed cells were significantly increased than those of control in dose dependent manners. Nrf2/ARE droved the downstream genes including NAD(P)H dehydrogenase [quinine] 1(NQO1), heme oxygenase 1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC) expression were significantly higher in wildtype HepG2 cells after Nano-TiO2 treatment. After treatment with Nano-TiO2, the DNA damages were significantly increased in Nrf(-/-) cells and mice whereas significantly decreased in tBHQ pre-treatment cells and mice, compared with the wildtype HepG2 cells and mice, respectively. Our results indicated that the acquired of Nrf2 leads to a decreased susceptibility to DNA damages induction by Nano-TiO2 and decreasing of risk of cancer which would provide a strategy for a more efficacious sensitization of against of Nano-TiO2 toxication.

Na+/K+‐ATPase inhibition upregulates NMDA‐evoked currents in rat hippocampal CA1 pyramidal neurons

Na+/K+‐ATPase and N‐methyl‐d‐aspartate (NMDA) receptor in hippocampus play very important roles in the regulation of learning and memory. Here, we showed that dihydroouabain (DHO, 10−5–10−3 m), a Na+/K+‐ATPase inhibitor, significantly potentiated NMDA current in rat hippocampal CA1 pyramidal neurons, which was blocked by PP2 (the selective Src tyrosine kinase inhibitor) and PD‐98059 [the selective inhibitor of the mitogen‐activated protein kinases (MAPK) cascade]. These findings reported here uncover that Src mediates the cross‐talk between Na+/K+‐ATPase and NMDA receptor to transduce the signals from Na+/K+‐ATPase to the MAPK cascade and provide new insights into therapeutic target for deeper understanding of the nature of cognitive disorder.

The Paradox of Dopamine and Angiotensin II-Mediated Na+, K+-ATPase Regulation in Renal Proximal Tubules

Accumulated studies reported that the natruretic dopamine (DA) and the anti-natruretic angiotensin II (Ang II) represent an important mechanism to regulate renal Na+ and water excretion through intracellular secondary messengers to inhibit or activate renal proximal tubule (PT) Na+, K+-ATPase (NKA). The antagonistic actions were mediated by the phosphorylation of different position of NKA α1-subunit and different Pals-associated tight junction protein (PATJ) PDZ domains, the different protein kinase C (PKC) isoforms (PKC-β, PKC-ζ), the common adenylyl cyclase (AC) pathway, and the crosstalk and balance between DA and Ang II to NKA regulation. Besides, Ang II-mediated NKA modulation has bi-phasic effects.

Development of a screening system for DNA damage and repair of potential carcinogens based on dual luciferase assay in human HepG2 cell.

At present, different methods are used for the detection of early biological effects of DNA-damaging agents in environment. Some sensitive testing methods employing DNA damage-inducing genes RNR3, RAD51, RAD54 or growth-arrested and DNA damage-inducible gene 153 (Gadd 153) are used to detect the DNA damage. The host cell reactivation (HCR) assay is a functional assay that is based on the independent transfection of cells with either damaged or undamaged plasmid DNA and allows the identification of the genes responsible for DNA repair-deficient syndromes. In this study, we combined the gadd153-luc test system and HCR assay to measure the DNA damage and DNA repair by dual luciferase assay. We used 16 DNA-damaging agents all of which were detected by a positive dual luciferase reporter test system. The sensitivity of the dual luciferase assay system to detect DNA damage/repair was same as the gadd153-luc test system and/or the HCR assay. Since DNA repair is important to maintain genetic stability, DNA damage and repair have been good biomarkers of early biological effects of DNA-damaging agents. Accordingly, the measurement of DNA repair capacity should be a valued tool in molecular epidemiology studies. The dual luciferase assay described in this study is rapid, convenient, stable and standard.

The H1–H2 domain of the α1 isoform of Na+–K+–ATPase is involved in ouabain toxicity in rat ventricular myocytes

The composition of different isoforms of Na+-K+-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H₁-H₂ domain of α₁ and α₂ isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α₁ or α₂ isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca²⁺](i)), attenuated mitochondrial Ca²⁺ overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 μM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca²⁺](i) and the contractility induced by 1 μM but not that induced by 1 mM OUA. These results indicate that the H₁-H₂ domain of the NKA α₁ isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease.

Isoform-specific regulation of the Na+-K+ pump by adenosine in guinea pig ventricular myocytes

AbstractAim:The present study investigated the effect of adenosine on Na+-K+ pumps in acutely isolated guinea pig (Cavia sp.) ventricular myocytes.Methods:The whole-cell, patch-clamp technique was used to record the Na+-K+ pump current (Ip) in acutely isolated guinea pig ventricular myocytes.Results:Adenosine inhibited the high DHO-affinity pump current (Ih) in a concentration-dependent manner, which was blocked by the selective adenosine A1 receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists staurosporine, GF 109203X or the specific δ isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A1 receptor agonist CCPA and a specific activator peptide of PKC-δ, PP114. In contrast, the selective A2A receptor agonist CGS21680 and A3 receptor agonist Cl-IB-MECA did not affect Ih. Application of the selective A2A receptor antagonist SCH58261 and A3 receptor antagonist MRS1191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced Ih inhibition.Conclusion:The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of Ih via adenosine A1 receptors and the PKC-δ isoform.