bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular signal transduction during gastrin-induced histamine secretion in rat gastric ECL cells
Authors: Zanner Robert , Hapfelmeier Gerhard , Gratzl Manfred , Prinz Christian
Published: May 2013

Activation of Gqprotein-coupled receptors usually causes a biphasic increase in intracellular calcium concentration ([Ca2+]i) that is crucial for secretion in nonexcitable cells. In gastric enterochromaffin-like (ECL) cells, stimulation with gastrin leads to a prompt biphasic calcium response followed by histamine secretion. This study investigates the underlying signaling events in this neuroendocrine cell type. In ECL cells, RT-PCR suggested the presence of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes 1–3. The IP3R antagonist 2-aminoethoxydiphenyl borate abolished both gastrin-induced elevation of [Ca2+]i and histamine release. Thapsigargin increased [Ca2+]i, however, without inducing histamine secretion. In thapsigargin-pretreated cells, gastrin increased [Ca2+]i through calcium influx across the plasma membrane. Both nimodipine and SKF-96365 inhibited gastrin-induced histamine release. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate induced histamine secretion, an effect that was prevented by nimodipine. In summary, gastrin-stimulated histamine release depends on IP3R activation and plasmalemmal calcium entry. Gastrin-induced calcium influx was mediated by dihydropyridine-sensitive calcium channels that appear to be L-type channels activated through a pathway involving activation of PKC.
Acid secretion and proton conductance in human airway epithelium
Authors: Fischer Horst , Widdicombe Jonathan H. , Illek Beate
Published: May 2013

Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 ± 0.04 μmol · h−1 · cm−2, and mucosal pH equilibrated at 6.85 ± 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 ± 0.09 and 0.24 ± 0.09 μmol · h−1 · cm−2, respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl2 and was unaffected by mucosal bafilomycin A1, Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 ± 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.
Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion, spreading, and migration
Authors: Robinson Paul A. , Brown Susan , McGrath Meagan J. , Coghill Imogen D. , Gurung Rajendra , Mitchell Christina A.
Published: May 2013

The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and cardiac muscle, and its expression is downregulated significantly in dilated human cardiomyopathy. However, the function of SLIM1 is unknown. In this study, we investigated the intracellular localization of SLIM1. Endogenous and recombinant SLIM1 localized to the nucleus, stress fibers, and focal adhesions in skeletal myoblasts plated on fibronectin, collagen, or laminin. However, after inhibition of integrin signaling either by plating on poly-l-lysine or by soluble RGD peptide, SLIM1 localized diffusely in the cytosol, with decreased nuclear expression. Disruption of the actin cytoskeleton by cytochalasin D did not inhibit nuclear localization of SLIM1 in integrin-activated cells. Green fluorescent protein-tagged SLIM1 shuttled in the nucleus of untransfected NIH 3T3 cells, in a heterokaryon fusion assay. Overexpression of SLIM1 in Sol8 myoblasts inhibited cell adhesion and promoted cell spreading and migration. These studies show SLIM1 localizes in an integrin-dependent manner to the nucleus and focal adhesions where it functions downstream of integrin activation to promote cell spreading and migration.
Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution
Authors: Rao Jaladanki N. , Guo Xin , Liu Lan , Zou Tongtong , Murthy Karnam S. , Yuan Jason X.-J. , Wang Jian-Ying
Published: May 2013

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca2+ concentration ([Ca2+]cyt) through controlling voltage-gated K+ channel expression and membrane potential ( E m) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca2+]cyt activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca2+]cyt, by either polyamine depletion or exposure to the Ca2+-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca2+]cyt also inhibited cell migration after wounding. Elevation of [Ca2+]cyt induced by the Ca2+ ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca2+]cytcan interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.
Myostatin is an inhibitor of myogenic differentiation
Authors: Rı́os Ramón , Carneiro Isabel , Arce Víctor M. , Devesa Jesús
Published: May 2013

Myostatin (MSTN), a transforming growth factor (TGF)-β superfamily member, has been shown to negatively regulate muscle growth by inhibiting muscle precursor cell proliferation. Here, we stably transfected C2C12 cells with mouse MSTN cDNA to investigate its possible role in myoblast differentiation. We found that MSTN cDNA overexpression reversibly inhibits the myogenic process by downregulating mRNA levels of the muscle regulatory factors myoD and myogenin, as well as the activity of their downstream target creatine kinase. Taking into consideration that MSTN expression during development is restricted to muscle, our results suggest that MSTN probably regulates myogenic differentiation by an autocrine mechanism.
Insulin-stimulated trafficking of ENaC in renal cells requires PI 3-kinase activity
Authors: Blazer-Yost Bonnie L. , Esterman Michail A. , Vlahos Chris J.
Published: May 2013

αENaC-EGFP (enhanced green fluorescent protein-tagged α-subunit of the epithelial Na+ channel) stably transfected clonal lines derived from the A6 parental cell line were used to study the physical mechanisms of insulin-stimulated Na+ transport. Within 1 min of insulin stimulation, ENaC migrates from a diffuse cytoplasmic localization to the apical and lateral membranes. Concurrently, after insulin stimulation, phosphatidylinositol 3-kinase (PI 3-kinase) is colocalized with ENaC on the lateral but not apical membrane. An inhibitor of PI 3-kinase, LY-294002, does not inhibit ENaC/PI 3-kinase colocalization but does alter the intracellular site of the colocalization, preventing the translocation of ENaC to the lateral and apical membranes. These data show that insulin stimulation causes the migration of ENaC to the lateral and apical cell membranes and that this trafficking is dependent on PI 3-kinase activity.
PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle
Authors: Murthy Karnam S. , Zhou Huiping , Makhlouf Gabriel M.
Published: May 2013

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca2+release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.
Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM
Authors: Isnard-Bagnis Corinne , Da Silva Nicolas , Beaulieu Valérie , Yu Alan S. L. , Brown Dennis , Breton Sylvie
Published: May 2013

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl−and H+. We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H+-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H+-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H+-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.
Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes
Authors: White Carl , McGeown J. Graham
Published: May 2013

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 μM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 μM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in ∼20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 μM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 μM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 μM), which sensitizes the IP3R to IP3. In cells voltage clamped at –40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.
Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture
Authors: Kubis Hans-Peter , Hanke Nina , Scheibe Renate J. , Meissner Joachim D. , Gros Gerolf
Published: May 2013

The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca2+ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca2+ concentration ([Ca2+]i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of ∼500 nM. Although 70 nM [Ca2+]i does not activate calcineurin-NFAT, we show by the use of Ca2+ ionophore that the system is fully activated when [Ca2+]i is ≥150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca2+ concentration.
Facilitated diffusion of urate in avian brush-border membrane vesicles
Authors: Grassl Steven M.
Published: May 2013

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward α-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.
A role for PKC-δ and PI 3-kinase in TNF-α-mediated antiapoptotic signaling in the human neutrophil
Authors: Kilpatrick Laurie E. , Lee Julia Y. , Haines Kathleen M. , Campbell Donald E. , Sullivan Kathleen E. , Korchak Helen M.
Published: May 2013

The proinflammatory cytokine tumor necrosis factor (TNF)-α has been implicated in the attenuation of neutrophil spontaneous apoptosis during sepsis. Antiapoptotic signaling is principally mediated through the p60TNF receptor (p60TNFR). In neutrophils, TNF-α is an incomplete secretagogue and requires input from a ligated integrin(s) for neutrophil activation. In adherent neutrophils, TNF-α triggers association of both protein kinase C (PKC)-δ and phosphatidylinositol (PI) 3-kinase with the p60TNFR. In this study, a role for PKC-δ and PI 3-kinase in TNF-α-mediated antiapoptotic signaling was examined. TNF-α inhibited spontaneous apoptosis in fibronectin-adherent neutrophils, and this antiapoptotic signaling was blocked by the PKC-δ inhibitor rottlerin, but not by an inhibitor of Ca2+-dependent PKC isotypes, Go-6976. Inhibition of PI 3-kinase by LY-294002 also inhibited TNF-α-mediated antiapoptotic signaling. Cycloheximide blocked TNF-α-mediated antiapoptotic signaling, suggesting protein synthesis is required. Inhibition of either PKC-δ or PI 3-kinase attenuated TNF-α-mediated activation of the antiapoptotic transcription factor NFκB. Thus both PKC-δ and PI 3-kinase have essential roles in TNF-α-mediated antiapoptotic signaling in adherent neutrophils.
The γ-subunit of ENaC is more important for channel surface expression than the β-subunit
Authors: Konstas Angelos-Aristeidis , Korbmacher Christoph
Published: May 2013

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and is composed of three homologous subunits: α, β, and γ. Only heteromultimeric channels made of αβγENaC are efficiently expressed at the cell surface, resulting in maximally amiloride-sensitive currents. To study the relative importance of various regions of the β- and γ-subunits for the expression of functional ENaC channels at the cell surface, we constructed hemagglutinin (HA)-tagged β-γ-chimeric subunits composed of β- and γ-subunit regions and coexpressed them with HA-tagged αβ- and αγ-subunits in Xenopus laevis oocytes. The whole cell amiloride-sensitive sodium current (Δ I ami) and surface expression of channels were assessed in parallel using the two-electrode voltage-clamp technique and a chemiluminescence assay. Because coexpression of αγENaC resulted in larger Δ I ami and surface expression compared with coexpression of αβENaC, we hypothesized that the γ-subunit is more important for ENaC trafficking than the β-subunit. Using chimeras, we demonstrated that channel activity is largely preserved when the highly conserved second cysteine rich domains (CRD2) of the β- and γ-subunits are exchanged. In contrast, exchanging the whole extracellular loops of the β- and the γ-subunits largely reduced ENaC currents and ENaC expression in the membrane. This indicates that there is limited interchangeability between molecular regions of the two subunits. Interestingly, our chimera studies demonstrated that the intracellular termini and the two transmembrane domains of γENaC are more important for the expression of functional channels at the cell surface than the corresponding regions of βENaC.
Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction
Authors: Mifflin Randy C. , Saada Jamal I. , Di Mari John F. , Adegboyega Patrick A. , Valentich John D. , Powell Don W.
Published: May 2013

Elevated mucosal interleukin-1 (IL-1) levels are frequently seen during acute and chronic intestinal inflammation, and IL-1 neutralization lessens the severity of inflammation. One major effect of IL-1 is the increased release of eicosanoid mediators via induction of cyclooxygenase-2 (COX-2). One site of COX-2-derived prostaglandin synthesis during acute and chronic intestinal inflammation is the intestinal myofibroblast. COX-2 expression has also been documented in these cells in colonic neoplasms. Thus an understanding of the regulation of COX-2 expression in human intestinal myofibroblasts is important. As an initial step toward this goal we have characterized IL-1α signaling pathways that induce COX-2 expression in cultured human intestinal myofibroblasts. IL-1 treatment resulted in a dramatic transcriptional induction of COX-2 gene expression. Activation of nuclear factor-κB (NF-κB), extracellular signal-regulated protein kinase (ERK), p38, and protein kinase C (PKC) signaling pathways was each necessary for optimal COX-2 induction. In contrast to what occurs in other cell types, including other myofibroblasts such as renal mesangial cells, PKC inhibition did not prevent IL-1-induced NF-κB or mitogen activated protein kinase/ stress-activated protein kinase activation, suggesting a novel role for PKC isoforms during this process. The stimulatory effects of PKC, NF-κB, ERK-1/2, and presumably c-Jun NH2-terminal kinase activation were exerted at the transcriptional level, whereas p38 activation resulted in increased stability of the COX-2 message. We conclude that, in intestinal myofibroblasts, IL-1-mediated induction of COX-2 expression is a complex process that requires input from multiple signaling pathways. Each parallel pathway acts in relative autonomy, the sum of their actions culminating in a dramatic increase in COX-2 transcription and message stability.
Influence of calcium on myosin thick filament formation in intact airway smooth muscle
Authors: Herrera Ana M. , Kuo Kuo-Hsing , Seow Chun Y.
Published: May 2013

Myosin thick filaments have been shown to be structurally labile in intact smooth muscles. Although the mechanism of thick filament assembly/disassembly for purified myosins in solution has been well described, regulation of thick filament formation in intact muscle is still poorly understood. The present study investigates the effect of resting calcium level on thick filament maintenance in intact airway smooth muscle and on thick filament formation during activation. Cross-sectional density of the thick filaments measured electron microscopically showed that the density increased substantially (144%) when the muscle was activated. The abundance of filamentous myosins in relaxed muscle was calcium sensitive; in the absence of calcium (with EGTA), the filament density deceased by 35%. Length oscillation imposed on the muscle under zero-calcium conditions produced no further reduction in the density. Isometric force and filament density recovered fully after reincubation of the muscle in normal physiological saline. The results suggest that in airway smooth muscle, filamentous myosins exist in equilibrium with monomeric myosins; muscle activation favors filament formation, and the resting calcium level is crucial for preservation of the filaments in the relaxed state.
Mechanotransduction through the cytoskeleton
Authors: Shafrir Yinon , Forgacs Gabor
Published: May 2013

We constructed a model cytoskeleton to investigate the proposal that this interconnected filamentous structure can act as a mechano- and signal transducer. The model cytoskeleton is composed of rigid rods representing actin filaments, which are connected with springs representing cross-linker molecules. The entire mesh is placed in viscous cytoplasm. The model eukaryotic cell is submitted to either shock wave-like or periodic mechanical perturbations at its membrane. We calculated the efficiency of this network to transmit energy to the nuclear wall as a function of cross-linker stiffness, cytoplasmic viscosity, and external stimulation frequency. We found that the cytoskeleton behaves as a tunable band filter: for given linker molecules, energy transmission peaks in a narrow range of stimulation frequencies. Most of the normal modes of the network are spread over the same frequency range. Outside this range, signals are practically unable to reach their destination. Changing the cellular ratios of linker molecules with different elastic characteristics can control the allowable frequency range and, with it, the efficiency of mechanotransduction.
Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor
Authors: Choi Bok Hee , Choi Jin-Sung , Rhie Duck-Joo , Yoon Shin Hee , Min Do Sik , Jo Yang-Hyeok , Kim Myung-Suk , Hahn Sang June
Published: May 2013

The action of tyrphostin AG-1478, a potent protein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels (Kv1.5) stably expressed in Chinese hamster ovary cells was investigated using the whole cell patch-clamp technique. AG-1478 rapidly and reversibly inhibited Kv1.5 currents at 50 mV in a concentration-dependent manner with an IC50 of 9.82 μM. AG-1478 accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. Pretreatment with the structurally dissimilar PTK inhibitors (genistein and lavendustin A) had no effect on the AG-1478-induced inhibition of Kv1.5 and did not modify the AG-1478-induced current kinetics. The rate constants for binding and unbinding of AG-1478 were 1.46 μM−1 · s−1 and 10.19 s−1, respectively. The AG-1478-induced inhibition of Kv1.5 channels was voltage dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG-1478 produced no significant effect on the steady-state activation or inactivation curves. AG-1478 slowed the deactivation time course, resulting in a tail crossover phenomenon. Inhibition of Kv1.5 by AG-1478 was use dependent. The present results suggest that AG-1478 acts directly on Kv1.5 currents as an open-channel blocker and independently of the effects of AG-1478 on PTK activity.
Exogenous eosinophil activation converts PSGL-1-dependent binding to CD18-dependent stable adhesion to platelets in shear flow
Authors: McCarty Owen J. T. , Tien Niven , Bochner Bruce S. , Konstantopoulos Konstantinos
Published: May 2013

This study examined the binding kinetics and molecular requirements of eosinophil adhesion to surface-anchored platelets in shear flow. P-selectin glycoprotein ligand-1 (PSGL-1) binding to platelet P-selectin initiates tethering and rolling of eosinophils to platelets under flow. These primary interacting cells assist in the capture of free-flowing eosinophils through homotypic tethering (secondary interactions) mediated via L-selectin-PSGL-1 interactions. Differences between eosinophils and neutrophils in PSGL-1 and L-selectin expression levels predict the pattern and relative extent of their adhesive interactions with immobilized platelets under shear, as well as the relative magnitude of their average rolling velocities. The majority of tethered eosinophils become rapidly stationary on the platelet layer, a process that is predominantly mediated via eosinophil PSGL-1 binding to platelet P-selectin and has an absolute requirement for intact cytoskeleton. Only a small fraction of these stationary eosinophils develop shear-resistant attachments mediated by CD18 integrins. However, stimulation of eosinophils with eotaxin-2 converts PSGL-1-P-selectin-dependent stationary adhesion to CD18-mediated shear-resistant stable attachment. These studies provide insights for designing strategies based on blocking of eosinophil-platelet interactions to combat thrombotic disorders in hypereosinophilic patients.
sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+transport
Authors: Faletti Carla J. , Perrotti Nicola , Taylor Simeon I. , Blazer-Yost Bonnie L.
Published: May 2013

To study the role of sgk (serum, glucocorticoid-induced kinase) in hormonal regulation of Na+ transport mediated by the epithelial Na+ channel (ENaC), clonal cell lines stably expressing human sgk, an S422A sgk mutant, or a D222A sgk mutant were created in the background of the A6 model renal epithelial cell line. Expression of normal sgkresults in a 3.5-fold enhancement of basal transport and potentiation of the natriferic response to antidiuretic hormone (ADH). Transfection of a S422A mutant form of sgk, which cannot be phosphorylated by phosphatidylinositol-dependent kinase (PDK)-2, results in a cell line that is indistinguishable from the parent line in basal and hormone-stimulated Na+ transport. The D222A sgk mutant, which lacks kinase activity, functions as a dominant-negative mutant inhibiting basal as well as peptide- and steroid hormone-stimulated Na+ transport. Thus sgk activity is necessary for ENaC-mediated Na+transport. Phosphorylation and activation by PDK-2 are necessary for sgk stimulation of ENaC. Expression of normal sgkover endogenous levels results in a potentiated natriferic response to ADH, suggesting that the enzyme is a rate-limiting step for the hormone response. In contrast, sgk does not appear to be the rate-limiting step for the cellular response to aldosterone or insulin.
PPARγ coactivator-1α expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations
Authors: Irrcher Isabella , Adhihetty Peter J. , Sheehan Treacey , Joseph Anna-Maria , Hood David A.
Published: May 2013

The transcriptional coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been identified as an important mediator of mitochondrial biogenesis based on its ability to interact with transcription factors that activate nuclear genes encoding mitochondrial proteins. The induction of PGC-1α protein expression under conditions that provoke mitochondrial biogenesis, such as contractile activity or thyroid hormone (T3) treatment, is not fully characterized. Thus we related PGC-1α protein expression to cytochrome c oxidase (COX) activity in 1) tissues of varying oxidative capacities, 2) tissues from animals treated with T3, and 3) skeletal muscle subject to contractile activity both in cell culture and in vivo. Our results demonstrate a strong positive correlation ( r = 0.74; P < 0.05) between changes in PGC-1α and COX activity, used as an index of mitochondrial adaptations. The highest constitutive levels of PGC-1α were found in the heart, whereas the lowest were measured in fast-twitch white muscle and liver. T3 increased PGC-1α content similarly in both fast- and slow-twitch muscle, as well as in the liver, but not in heart. T3 also induced early (6 h) increases in AMP-activated protein kinase (AMPKα) activity, as well as later (5 day) increases in p38 MAP kinase activity in slow-twitch, but not in fast-twitch, muscle. Contractile activity provoked early increases in PGC-1α, coincident with increases in mitochondrial transcription factor A (Tfam), and nuclear respiratory factor-1 (NRF-1) protein expression, suggesting that PGC-1α is physiologically important in coordinating the expression of the nuclear and mitochondrial genomes. Ca2+ ionophore treatment of muscle cells led to an approximately threefold increase in PGC-1α protein, and contractile activity induced rapid and marked increases in both p38 MAP kinase and AMPKα activities. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) treatment of muscle cells also led to parallel increases in AMPKα activity and PGC-1α protein levels. These data are consistent with observations that indicate that increases in PGC-1α protein are affected by Ca2+ signaling mechanisms, AMPKα activity, as well as posttranslational phosphorylation events that increase PGC-1α protein stability. Our data support a role for PGC-1α in the physiological regulation of mitochondrial content in a variety of tissues and suggest that increases in PGC-1α expression form part of a unifying pathway that promotes both T3- and contractile activity-induced mitochondrial adaptations.
Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells
Authors: Hollywood M. A. , Sergeant G. P. , McHale N. G. , Thornbury K. D.
Published: May 2013

Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca2+ current ( ICa), followed by a Ca2+-activated Cl- current, and, on stepping back to -80 mV, large Cl- tail currents were observed. Both currents were abolished when the cells were superfused with Ca2+-free bath solution, suggesting that Ca2+ influx was necessary for activation of the Cl- current. The Cl- current was also abolished when Ba2+ was substituted for Ca2+ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca2+-induced Ca2+ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation.
Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells
Authors: Schwake Lukas , Henkel Andreas W. , Riedel Hans D. , Schlenker Thorsten , Both Matthias , Migala Andrea , Hadaschik Boris , Henfling Nataly , Stremmel Wolfgang
Published: May 2013

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.
Prolonged force increase following a high-frequency burst is not due to a sustained elevation of [Ca2+]i
Authors: Abbate F. , Bruton J. D. , De Haan A. , Westerblad H.
Published: May 2013

A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly ( P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle ( n = 7). However, during this potentiation, [Ca2+]iwas not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.
Functional properties of a brain-specific NH2-terminally spliced modulator of Kv4 channels
Authors: Boland Linda M. , Jiang Min , Lee So Yeong , Fahrenkrug Scott C. , Harnett Mark T. , O'Grady Scott M.
Published: May 2013

Kv4/K channel-interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K channels in brain and heart. Considering the importance of alternative splicing to the quantitative features of KChIP gating modulation, a previously uncharacterized splice form of KChIP1 was functionally characterized. The KChIP1b splice variant differs from the previously characterized KChIP1a splice form by the inclusion of a novel amino-terminal region that is encoded by an alternative exon that is conserved in mouse, rat, and human genes. The expression of KChIP1b mRNA was high in brain but undetectable in heart or liver by RT-PCR. In cerebellar tissue, KChIP1b and KChIP1a transcripts were expressed at nearly equal levels. Coexpression of KChIP1b or KChIP1a with Kv4.2 channels in oocytes slowed K current decay and destabilized open-inactivated channel gating. Like other KChIP subunits, KChIP1b increased Kv4.2 current amplitude and KChIP1b also shifted Kv4.2 conductance-voltage curves by —10 mV. The development of Kv4.2 channel inactivation accessed from closed gating states was faster with KChIP1b coexpression. Deletion of the novel amino-terminal region in KChIP1b selectively altered the subunit's modulation of Kv4.2 closed inactivation gating. The role of the KChIP1b NH2-terminal region was further confirmed by direct comparison of the properties of the NH2-terminal deletion mutant and the KChIP1a subunit, which is encoded by a transcript that lacks the novel exon. The features of KChIP1b modulation of Kv4 channels are likely to be conserved in mammals and demonstrate a role for the KChIP1 NH2-terminal region in the regulation of closed inactivation gating.
Role of airway surface liquid and submucosal glands in cystic fibrosis lung disease
Authors: Verkman A. S. , Song Yuanlin , Thiagarajah Jay R.
Published: May 2013

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, an epithelial chloride channel expressed in the airways, pancreas, testis, and other tissues. A central question is how defective CFTR function in CF leads to chronic lung infection and deterioration of lung function. Several mechanisms have been proposed to explain lung disease in CF, including abnormal airway surface liquid (ASL) properties, defective airway submucosal gland function, altered inflammatory response, defective organellar acidification, loss of CFTR regulation of plasma membrane ion transporters, and others. This review focuses on the physiology of the ASL and submucosal glands with regard to their proposed role in CF lung disease. Experimental evidence for defective ASL properties and gland function in CF is reviewed, and deficiencies in understanding ASL/gland physiology are identified as areas for further investigation. New model systems and measurement technologies are being developed to make progress in establishing lung disease mechanisms in CF, which should facilitate mechanism-based design of therapies for CF.
Human Na+/H+ exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria
Authors: Brett Christopher L. , Wei Ying , Donowitz Mark , Rao Rajini
Published: May 2013

Since the discovery of the first intracellular Na+/H+exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na+ into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na+/H+ exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na+/H+ exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis.
Cytokines synergistically induce osteoclast differentiation: support by immortalized or normal calvarial cells
Authors: Ragab Ashraf A. , Nalepka Jennifer L. , Bi Yanming , Greenfield Edward M.
Published: May 2013

Conditionally immortalized murine calvarial (CIMC) cells that support differentiation of precursors into mature osteoclasts were isolated. All six CIMC cell lines supported osteoclast differentiation in response to 1,25-dihydroxyvitamin D3 or interleukin (IL)-11. CIMC-4 cells also supported osteoclast differentiation in response to tumor necrosis factor (TNF)-α, IL-1β, or IL-6. The resultant multinucleated cells expressed tartrate-resistant acid phosphatase and formed resorption lacunae on mineralized surfaces. CIMC-4 cells, therefore, establish an osteoclast differentiation assay that is responsive to many cytokines and does not rely on isolation of primary stromal support cells. Low concentrations of the cytokines synergistically stimulated differentiation when osteoclast precursors were cocultured with either CIMC-4 cells or primary calvarial cells. Osteoclast differentiation induced by all stimuli other than TNF-α was completely blocked by osteoprotegerin, whether the stimulators were examined alone or in combination. Moreover, study of precursors that lack TNF-α receptors showed that TNF-α induces osteoclast differentiation primarily through direct actions on osteoclast precursors, which is a distinct mechanism from that used by the other bone-resorptive agents examined in this study.
Colon carcinoma cell glycolipids, integrins, and other glycoproteins mediate adhesion to HUVECs under flow
Authors: Burdick Monica M. , McCaffery J. Michael , Kim Young S. , Bochner Bruce S. , Konstantopoulos Konstantinos
Published: May 2013

This study was undertaken to investigate the molecular constituents mediating LS174T colon adenocarcinoma cell adhesion to 4-h TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) under flow. At 1 dyn/cm2, ∼57% of cells rolled and then became firmly adherent, whereas others continuously rolled on endothelium. Initial cell binding was primarily mediated by endothelial E-selectin. By using neuraminidase, glycolipid biosynthesis inhibitor d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol · HCl, trypsin, and flow cytometry, LS174T cells were shown to express sialyl Lewisx (sLex)- and di-sLex-decorated, but not sLea-decorated, glycolipid and glycoprotein ligands for E-selectin. The cells preferentially employed sialylated glycoproteins over glycolipids in adhesion as measured by conversion of rolling to firm adhesion, resistance to detachment by increased shear stress, and rolling velocity. However, a nonsialylated E-selectin counterreceptor also exists. Furthermore, LS174T α2, α6, and β1 integrins support a minor pathway in adhesion to HUVECs. Finally, tumor cell attachment specifically increases HUVEC endocytosis of E-selectin. Altogether, the data indicate the complexity of carcinoma cell-endothelium adhesion via sialylated glycoconjugates, integrins, and their respective counterreceptors.
Hyperoxia induces retinal vascular endothelial cell apoptosis through formation of peroxynitrite
Authors: Gu Xiaolin , El-Remessy Azza B. , Brooks Steven E. , Al-Shabrawey Mohamed , Tsai Nai-Tsi , Caldwell Ruth B.
Published: May 2013

Hyperoxia exposure induces capillary endothelial cell apoptosis in the developing retina, leading to vaso-obliteration followed by proliferative retinopathy. Previous in vivo studies have shown that endothelial nitric oxide synthase (NOS3) and peroxynitrite are important mediators of the vaso-obliteration. Now we have investigated the relationship between hyperoxia, NOS3, peroxynitrite, and endothelial cell apoptosis by in vitro experiments using bovine retinal endothelial cells (BREC). We found that BREC exposed to 40% oxygen (hyperoxia) for 48 h underwent apoptosis associated with activation of caspase-3 and cleavage of the caspase substrate poly(ADP-ribose) polymerase. Hyperoxia-induced apoptosis was associated with increased formation of nitric oxide, peroxynitrite, and superoxide anion and was blocked by treatment with uric acid, nitro-l-arginine methyl ester, or superoxide dismutase. Analyses of the phosphatidylinositol 3-kinase/Akt kinase survival pathway in cells directly treated with peroxynitrite revealed inhibition of VEGF- and basic FGF-induced activation of Akt kinase. These results suggest that hyperoxia-induced formation of peroxynitrite induces BREC apoptosis by crippling key survival pathways and that blocking peroxynitrite formation prevents apoptosis. These data may have important clinical implications for infants at risk of retinopathy of prematurity.
Essential role of Ca2+-dependent phospholipase A2 in estradiol-induced lysosome activation
Authors: Burlando Bruno , Marchi Barbara , Panfoli Isabella , Viarengo Aldo
Published: May 2013

The mechanism of lysosome activation by 17β-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was completely prevented by preincubating the cells with the Ca2+ chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+-dependent PLA2(cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+-independent PLA2, ( E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2 H-pyran-2-one (BEL). The Ca2+ ionophore A-23187 yielded similar effects on [Ca2+]i and lysosomes. Exposure to estradiol also resulted in cPLA2 translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+-dependent mechanism involving activation of Ca2+-dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.
Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane
Authors: Lee So Yeong , Maniak Peter J. , Ingbar David H. , O'Grady Scott M.
Published: May 2013

Whole cell perforated patch-clamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was −60 mV, and depolarizing voltage steps activated voltage-gated K+ (Kv) channels. The voltage-activated currents exhibited a mean reversal potential of −32 mV. Complete activation was achieved at −10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in K+ selectivity, suggesting contributions of multiple Kv α-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between −30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv α-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three β-subunits (Kvβ1.1, Kvβ2.1, and Kvβ3.1), and two K+ channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv α-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv α-, β-, and KChIP subunits explains the variability in inactivation gating and K+ selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal K+ secretion across the alveolar epithelium.