Zhi-Ren Zhang

Hydrogen Sulfide Prevents Hydrogen Peroxide-Induced Activation of Epithelial Sodium Channel through a PTEN/PI(3,4,5)P3 Dependent Pathway

Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC PO; however, in these cells H2O2 failed to elevate ENaC PO. Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.

Single Cl− Channels Activated by Ca2+ in Drosophila S2 Cells Are Mediated By Bestrophins

Mutations in human bestrophin-1 (VMD2) are genetically linked to several forms of retinal degeneration but the underlying mechanisms are unknown. Bestrophin-1 (hBest1) has been proposed to be a Cl− channel involved in ion and fluid transport by the retinal pigment epithelium (RPE). To date, however, bestrophin currents have only been described in overexpression systems and not in any native cells. To test whether bestrophins function as Ca2+-activated Cl− (CaC) channels physiologically, we used interfering RNA (RNAi) in the Drosophila S2 cell line. S2 cells express four bestrophins (dbest1–4) and have an endogenous CaC current. The CaC current is abolished by several RNAi constructs to dbest1 and dbest2, but not dbest3 or dbest4. The endogenous CaC current was mimicked by expression of dbest1 in HEK cells, and the rectification and relative permeability of the current were altered by replacing F81 with cysteine. Single channel analysis of the S2 bestrophin currents revealed an ∼2-pS single channel with fast gating kinetics and linear current–voltage relationship. A similar channel was observed in CHO cells transfected with dbest1, but no such channel was seen in S2 cells treated with RNAi to dbest1. This provides definitive evidence that bestrophins are components of native CaC channels at the plasma membrane.

Mechanosensitive properties in the endothelium and their roles in the regulation of endothelial function.

Abstract: Vascular endothelial cells (ECs) line the luminal surface of blood vessels, which are exposed constantly to mechanical stimuli, such as fluid shear stress, cyclic strain, and blood pressure. In recent years, more and more evidence indicates that ECs sense these mechanical stimuli and subsequently convert mechanical stimuli into intracellular signals. The properties of ECs that sense the mechanical stimuli are defined as mechanosensors. There are a variety of mechanosensors that have been identified in ECs. These mechanosensors play an important role in regulating the function of the endothelium and vascular function, including blood pressure. This review focuses on the mechanosensors that have been identified in ECs and on the roles that mechanosensors play in the regulation of endothelium function, and in the regulation of vascular function.

β-elemene induces caspase-dependent apoptosis in human glioma cells in vitro through the upregulation of Bax and Fas/ FasL and downregulation of Bcl-2.

BACKGROUND β-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of β-elemene against glioma cells remains unclear. In the present study, we assessed effects of β-elemene on human glioma cells and explored the underlying mechanism. MATERIALS AND METHODS Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of β-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of β-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and β-elemne treatment group. RESULTS With increase in the concentration of β-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability (IC50) was 48.5 μg/mL for 24h. β-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with β-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by β-elemene in a time and does- dependent manner. CONCLUSIONS Our results indicate that β-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

Hypoxia augments the calcium-activated chloride current carried by anoctamin-1 in cardiac vascular endothelial cells of neonatal mice

The molecular identity of calcium‐activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin‐1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs).

Regulation of ASIC1 by Ca2+/calmodulin-dependent protein kinase II in human glioblastoma multiforme

Recent studies have implicated the acid-sensing ion channel 1 (ASIC1), a proton-gated cation channel that belongs to the epithelial sodium channel (ENaC)/Degenerin family, plays an important role in glioma cell migration. Among the ASIC subunits, only ASIC1a has been found be calcium permeable. However, it has not been determined whether Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates ASIC1 in glioblastoma multiforme (GBM). Herein, we report that ASIC1 and CaMKII assemble to form a functional complex at the plasma membrane of GBM cells. We found that migration ability was significantly attenuated in GBM cells that were pre-treated with autocamtide-2-related inhibitory peptide (AIP), a CaMKII-specific inhibitor, or psalmotoxin 1 (PcTX-1), a selective ASIC1 blocker. Furthermore, the inhibitory effect of AIP or PcTX-1 on migration was diminished when ASIC1 was knocked down in GBM cells; when ASIC1 knockdown GBM cells were concurrently treated with these two inhibitors, cell migration was slightly but significantly decreased. Using whole-cell patch-clamp recordings, we detected an amiloride-sensitive current in GBM cells, and this current was significantly inhibited by both PcTX-1 and AIP. Moreover, the magnitude of this current was dramatically decreased when ASIC1 was knocked down in GBM cells. The addition of AIP failed to further decrease the amplitude of this current. Taken together, these data suggest that ASIC1 and CaMKII form a functional complex in GBM cells. Furthermore, it can be concluded that CaMKII regulates the activity of ASIC1, which is associated with the ability of GBM cells to migrate.

Dietary salt regulates epithelial sodium channels in rat endothelial cells: adaptation of vasculature to salt

The epithelial sodium channel (ENaC) is expressed in vascular endothelial cells and is a negative modulator of vasodilation. However, the role of endothelial ENaCs in salt‐sensitive hypertension remains unclear. Here, we have investigated how endothelial ENaCs in Sprague‐Dawley (SD) rats respond to high‐salt (HS) challenge.

Characterization of Cardiac Anoctamin1 Ca2+‐Activated Chloride Channels and Functional Role in Ischemia‐Induced Arrhythmias

Anoctamin1 (ANO1) encodes a Ca2+‐activated chloride (Cl−) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1‐encoded CaCCs in ischemia‐induced arrhythmias in the heart. Quantitative real‐time RT‐PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch‐clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca2+‐activated Cl− current (ICl.Ca) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of ICl.Ca, which was inhibited by a specific ANO1 inhibitor, T16Ainh‐A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia‐induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper “spike and dome” compared to control cardiomyocytes from non‐ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia‐induced early phase 1 repolarization acceleration and caused a much shallower “spike and dome”. We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia‐induced increase in ANO1 expression may be responsible for the increased density of ICl.Ca in the ischemic heart and may contribute, at least in part, to ischemia‐induced arrhythmias. J. Cell. Physiol. 230: 337–346, 2015.

Hydrogen Sulfide Prevents Advanced Glycation End-Products Induced Activation of the Epithelial Sodium Channel

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds implicated in diabetes. Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal nephron plays an important role in diabetic hypertension. Here, we report that H2S antagonizes AGEs-induced ENaC activation in A6 cells. ENaC open probability (P O) in A6 cells was significantly increased by exogenous AGEs and that this AGEs-induced ENaC activity was abolished by NaHS (a donor of H2S) and TEMPOL. Incubating A6 cells with the catalase inhibitor 3-aminotriazole (3-AT) mimicked the effects of AGEs on ENaC activity, but did not induce any additive effect. We found that the expression levels of catalase were significantly reduced by AGEs and both AGEs and 3-AT facilitated ROS uptake in A6 cells, which were significantly inhibited by NaHS. The specific PTEN and PI3K inhibitors, BPV(pic) and LY294002, influence ENaC activity in AGEs-pretreated A6 cells. Moreover, after removal of AGEs from AGEs-pretreated A6 cells for 72 hours, ENaC P O remained at a high level, suggesting that an AGEs-related “metabolic memory” may be involved in sodium homeostasis. Our data, for the first time, show that H2S prevents AGEs-induced ENaC activation by targeting the ROS/PI3K/PTEN pathway.

Dietary salt blunts vasodilation by stimulating epithelial sodium channels in endothelial cells from salt‐sensitive Dahl rats

Our recent studies show that the reduced activity of epithelial sodium channels (ENaC) in endothelial cells accounts for the adaptation of vasculature to salt in Sprague–Dawley rats. The present study examines a hypothesis that enhanced ENaC activity mediates the loss of vasorelaxation in Dahl salt‐sensitive (SS) rats.