Regulation of the epithelial Na+ channel by the RH domain of G protein-coupled receptor kinase, GRK2, and Galphaq/11.

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na(+) absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ((K220R)GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 ((D110A)GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.

Adaptive logic circuits with doping-free ambipolar carbon nanotube transistors.

A CMOS-like inverter was integrated by using ambipolar carbon nanotube (CNT) transistors without doping. The ambipolar CNT transistors automatically configure themselves to play a role as an n-type or p-type transistor in a logic circuit depending on the supply voltage (V(DD)) and ground. A NOR (NAND) gate is adaptively converted to a NAND (NOR) gate. This adaptiveness of logic gates exhibiting two logic gate functions in a single logic circuit offers a new opportunity for designing logic circuits with high integration density for next generation applications.

Reduction-controlled viologen in bisolvent as an environmentally stable n-type dopant for carbon nanotubes.

Various viologens have been used to control the doping of single-walled carbon nanotubes (SWCNTs) via direct redox reactions. A new method of extracting neutral viologen (V(0)) was introduced using a biphase of toluene and viologen-dissolved water. A reductant of sodium borohydride transferred positively charged viologen (V(2+)) into V(0), where the reduced V(0) was separated into toluene with high separation yield. This separated V(0) solution was dropped on carbon nanotube transistors to investigate the doping effect of CNTs. With a viologen concentration of 3 mM, all the p-type CNT transistors were converted to n-type with improved on/off ratios. This was achieved by donating electrons spontaneously to CNTs from neutral V(0), leaving energetically stable V(2+) on the nanotube surface again. The doped CNTs were stable in water due to the presence of hydrophobic V(0) at the outermost CNT transistors, which may act as a protecting layer to prevent further oxidation from water.

Regulation of epithelial Na+ channels by aldosterone: role of Sgk1.

1. The epithelial sodium channel (ENaC) is tightly regulated by hormonal and humoral factors, including cytosolic ion concentration and glucocorticoid and mineralocorticoid hormones. Many of these regulators of ENaC control its activity by regulating its surface expression via neural precursor cell-expressed developmentally downregulated (gene 4) protein (Nedd4-2). 2. During the early phase of aldosterone action, Nedd4-2-dependent downregulation of ENaC is inhibited by the serum- and glucocorticoid-induced kinase 1 (Sgk1). 3. Sgk1 phosphorylates Nedd4-2. Subsequently, phosphorylated Nedd4-2 binds to the 14-3-3 protein and, hence, reduces binding of Nedd4-2 to ENaC. 4. Nedd4-2 is also phosphorylated by protein kinase B (Akt1). Both Sgk1 and Akt1 are part of the insulin signalling pathway that increases transepithelial Na(+) absorption by inhibiting Nedd4-2 and activating ENaC.

P2Y6 receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 & CXCL8 mRNA expression in respiratory epithelium.

One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-ß, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (ß-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y6R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca2+ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y6 purinergic receptors.

Zinc transport and the inhibition of the L-type calcium channel are two separable functions of ZnT-1.

Traditionally, proteins are considered to perform a single role, be it as an enzyme, a channel, a transporter or as a structural scaffold. However, recent studies have described moonlighting proteins that perform distinct and independent functions; for example, TRPM7 is both an ion channel and a kinase. ZnT-1 is a member of the Carrier Diffusion Facilitator family that is expressed throughout the phylogenetic tree from bacteria to humans. Since its cloning in 1995, ZnT-1 is considered a major extruder of Zn2+ based on its capability to protect cells against zinc toxicity. Recently, we reported that ZnT-1 inhibits the L-type calcium channel (LTCC), a major Zn2+ and Ca2+ entry pathway. Here we show that ZnT-1 is a dual-function protein by demonstrating that its abilities to exchange Zn2+/H+ and to inhibit the LTCC are independent of each other and are mediated by different parts of the protein. Specifically, mutations in the membrane-spanning helices that render ZnT-1 unable to transport zinc do not prevent it from inhibiting the LTCC. Moreover, a fragment consisting of the intracellular ZnT-1 C-terminal, which lacks all ion-transfer segments, inhibits the LTCC as efficiently as wild-type ZnT-1. Our data therefore indicates that ZnT-1 performs two structurally independent functions related to zinc homeostasis.

H-Ras mediates the inhibitory effect of epidermal growth factor on the epithelial Na+ channel.

The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development.

Upregulation of haptoglobin in reactive astrocytes after transient forebrain ischemia in rats.

Immunohistochemistry for haptoglobin (Hp) in the postischemic hippocampus demonstrated an immunoreactivity visible one day after reperfusion and continuing to increase until 14 days after ischemia. The immunoreactivity was most prominent in CA1 and the dentate hilar region, especially in cells with astroglial morphology. Double immunofluorescence histochemistry confirmed colocalization of the Hp and glial fibrillary acidic protein. Furthermore, a reverse transcription-polymerase chain reaction study confirmed an elevated Hp mRNA level in the postischemic hippocampus. The Hp gene expression was also upregulated in C6 and A-172 glioblastoma cell lines after H O treatment. These findings suggest that Hp is synthesized in reactive astrocytes in response to ischemia-reperfusion injury.

Effects of Val34Leu and Val35Leu polymorphism on the enzyme activity of the coagulation factor XIII-A.

Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (gammaFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these gammaFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated gammaFXIII-A were characterized. gammaFXIII-A (V34L) and gammaFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type gammagFXIII-A and V35L variant. However, the activated gammaFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2-plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.

ZnT-1 extrudes zinc from mammalian cells functioning as a Zn(2+)/H(+) exchanger.

ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protecting cells against Zn(2+) toxicity. Our results demonstrate that ZnT-1 extrudes zinc from mammalian cells by functioning as a Zn(2+)/H(+) exchanger.

Thermal conversion of electronic and electrical properties of AuCl3-doped single-walled carbon nanotubes.

By using carbon-free inorganic atomic layer involving heat treatment from 150 to 300 °C, environmentally stable and permanent modulation of the electronic and electrical properties of single-walled carbon nanotubes (SWCNTs) from p-type to ambi-polar and possibly to n-type has been demonstrated. At low heat treatment temperature, a strong p-doping effect from Au(3+) ions to CNTs due to a large difference in reduction potential between them is dominant. However at higher temperature, the gold species are thermally reduced, and thermally induced CNT-Cl finally occurs by the decomposition reaction of AuCl(3). Thus, in the AuCl(3)-doped SWCNTs treated at higher temperature, the p-type doping effect is suppressed and an n-type property from CNT-Cl is thermally induced. Thermal conversion of the majority carrier type of AuCl(3)-doped SWNTs is systematically investigated by combining various optical and electrical tools.

Laser thinning for monolayer graphene formation: heat sink and interference effect.

Despite the availability of large-area graphene synthesized by chemical vapor deposition (CVD), the control of a uniform monolayer graphene remained challenging. Here, we report a method of acquiring monolayer graphene by laser irradiation. The accumulation of heat on graphene by absorbing light, followed by oxidative burning of upper graphene layers, which strongly relies on the wavelength of light and optical parameters of the substrate, was in situ measured by the G-band shift in Raman spectroscopy. The substrate plays a crucial role as a heat sink for the bottom monolayer graphene, resulting in no burning or etching. Oscillatory thinning behavior dependent on the substrate oxide thickness was evaluated by adopting a simple Fresnel's equation. This paves the way for future research in utilizing monolayer graphene for high-speed electronic devices.

Graphene/carbon nanotube hybrid-based transparent 2D optical array.

Graphene/carbon nanotube (CNT) hybrid structures are fabricated for use as optical arrays. Vertically aligned CNTs are directly synthesized on a graphene/quartz substrate using plasma-enhanced chemical vapor deposition (PECVD). Graphene preserves the transparency and resistance during CNT growth. Highly aligned single-walled CNTs show a better performance for the diffraction intensity.

Carbon nanotube doping mechanism in a salt solution and hygroscopic effect: density functional theory.

The mechanism of doping carbon nanotubes (CNTs) with a salt solution was investigated using the density functional theory. We propose that the anion-CNT complex is a key component in doping CNTs. Although the cations play an important role in ionizing CNTs as an intermediate precursor, the ionized CNTs are neutralized further by forming a stable anion-CNT complex as a final reactant. The anion-CNT bond has a strong ionic bonding character and clearly shows p-type behavior by shifting the Fermi level toward the valence band. The midgap state is introduced by the strong binding of carbon and anion atoms. These localized charged anion sites are highly hygroscopic and induce the adsorption of water molecules. This behavior provides a new possibility for using anion-functionalized CNTs as humidity sensors.

The activity of the epithelial sodium channels is regulated by caveolin-1 via a Nedd4-2-dependent mechanism.

It has recently been shown that the epithelial Na(+) channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the beta- and gamma-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.

Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2.

The epithelial sodium channel (ENaC) plays an important role in transepithelial Na(+) absorption; hence its function is essential for maintaining Na(+) and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1(K179M), decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser(342) and Ser(428), completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na(+) absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

(NDRG2) stimulates amiloride-sensitive Na+ currents in Xenopus laevis oocytes and fisher rat thyroid cells.

Regulation of the epithelial sodium channel (ENaC) is highly complex and may involve several aldosterone-induced regulatory proteins. The N-Myc downstream-regulated gene 2 (NDRG2) has been identified as an early aldosterone-induced gene. Therefore, we hypothesized that NDRG2 may affect ENaC function. To test this hypothesis we measured the amiloride-sensitive (2 microm) whole cell current (DeltaI(ami)) in Xenopus laevis oocytes expressing ENaC alone or co-expressing ENaC and NDRG2. Co-expression of NDRG2 significantly increased DeltaI(ami) in some, but not, all batches of oocytes tested. An inhibitory effect of NDRG2 was never observed. Using a chemiluminescence assay we demonstrated that the NDRG2-induced increase in ENaC currents was accompanied by a similar increase in channel surface expression. The stimulatory effect of NDRG2 was preserved in oocytes maintained in a low sodium bath solution to prevent sodium feedback inhibition. These findings suggest that the stimulatory effect of NDRG2 is independent of sodium feedback regulation. Furthermore, the stimulatory effect of NDRG2 on ENaC was at least in part additive to that of Sgk1. A short isoform of NDRG2 also stimulated DeltaI(ami). Overexpression of NDRG2 and ENaC in Fisher rat thyroid cells confirmed the stimulatory effect of NDRG2 on ENaC-mediated short-circuit current (I(SC-ami)). In addition, small interference RNA against NDRG2 largely reduced I(SC-ami) in Fisher rat thyroid cells. Our results indicate that NDRG2 is a likely candidate to contribute to aldosterone-mediated ENaC regulation.

The kringle domain of tissue-type plasminogen activator inhibits in vivo tumor growth.

The two-kringle domain of tissue-type plasminogen activator (t-PA) has previously been shown to contain anti-angiogenesis activity. In this study, we explored the potential in vivo anti-tumor effects of the recombinant kringle domain (TK1-2) of human t-PA. Anti-tumor effects of purified Pichia-driven TK1-2 were examined in nude mice models by subcutaneous implantation of human lung (A-549) and colon (DLD-1, HCT-116) cancer cell lines. Mice bearing the tumors were injected with PBS or purified TK1-2 (30 mg/kg) i.p. every day for 22 days. TK1-2 treatment suppressed the A-549, DLD-1, and HCT-116 tumor growth by 85.3%, 52.4%, and 62.5%, respectively. Immunohistological examination of the tumor tissues showed that TK1-2 treatment decreased the vessel density and also the expression of angiogenesis-related factors including angiogenin, VEGF, alpha-SMA, vWF, and TNF-alpha, and increased the apoptotic fraction of cells. TK1-2 neither inhibited in vitro growth of these cancer cells nor affected t-PA-mediated fibrin clot lysis. These results suggest that TK1-2 inhibits the tumor growth by suppression of angiogenesis without interfering with fibrinolysis.

Involvement of CCAAT/enhancer-binding protein alpha in haptoglobin gene expression by all-trans-retinoic acid.

Several acute-phase plasma proteins, including haptoglobin (Hp), are induced in the liver in response to inflammation. Recently, we found that Hp gene expression is up-regulated by all-trans-retinoic acid (ATRA) in the extrahepatic monocytic cell line, THP-1. To investigate the molecular mechanism underlying ATRA-induced Hp gene expression, we analyzed the induction of transcription factor CCAAT/enhancer-binding protein (C/EBP) isoforms in ATRA-stimulated THP-1 cells and their binding to the Hp promoter. Western blot analysis showed that treatment with ATRA increased C/EBPalpha and beta expression, but decreased that of C/EBPdelta. Electrophoretic mobility shift and supershift assays demonstrated that only C/EBPalpha of the C/EBP isoforms bound to the C/EBP DNA-binding sites in the Hp promoter. Furthermore, when ATRA-dependent Hp induction was inhibited by sodium butyrate or auranofin, induction of C/EBPalpha, but not C/EBPbeta, was also diminished. These results suggest that C/EBPalpha is involved in the activation of Hp gene expression by ATRA in human monocytic cells.