bluuebunny/crossref_metadata_2025 huggingface dataset:
VEGF increases permeability of the blood-brain barrier via a nitric oxide synthase/cGMP-dependent pathway
Authors: Mayhan William G.
Published: December 2017

It appears that the expression of vascular endothelial growth factor (VEGF) is increased during brain injury and thus may contribute to disruption of the blood-brain barrier (BBB) during cerebrovascular trauma. The first goal of this study was to determine the effect of VEGF on permeability of the BBB in vivo. The second goal was to determine possible cellular mechanisms by which VEGF increases permeability of the BBB. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the BBB [clearance of FITC-labeled dextran of molecular mass 10,000 Da (FITC-dextran-10K)] and diameter of pial arterioles were measured in absence and presence of VEGF (0.01 and 0.1 nM). During superfusion with vehicle (saline), clearance of FITC-dextran-10K from pial vessels was minimal and diameter of pial arterioles remained constant. Topical application of VEGF (0.01 nM) did not alter permeability of the BBB to FITC-dextran-10K or arteriolar diameter. However, superfusion with VEGF (0.1 nM) produced a marked increase in clearance of FITC-dextran-10K and a modest dilatation of pial arterioles. To determine a potential role for nitric oxide and stimulation of soluble guanylate cyclase in VEGF-induced increases in permeability of the BBB and arteriolar dilatation, we examined the effects of N G-monomethyl-l-arginine (l-NMMA; 10 μM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1.0 μM), respectively.l-NMMA and ODQ inhibited VEGF-induced increases in permeability of the BBB and arteriolar dilatation. The findings of the present study suggest that VEGF, which appears to be increased in brain tissue during cerebrovascular trauma, increases the permeability of the BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase.
Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery
Authors: Raman Narayan , Atkinson Simon J.
Published: December 2017

Actin cytoskeletal disruption is a hallmark of ischemic injury and ATP depletion in a number of cell types, including renal epithelial cells. We manipulated Rho GTPase signaling by transfection and microinjection in LLC-PK proximal tubule epithelial cells and observed actin cytoskeletal organization following ATP depletion or recovery by confocal microscopy and quantitative image analysis. ATP depletion resulted in disruption of stress fibers, cortical F-actin, and apical actin bundles. Constitutively active RhoV14 prevented disruption of stress fibers and cortical F-actin during ATP depletion and enhanced the rate of stress fiber reassembly during recovery. Conversely, the Rho inhibitor C3 or dominant negative RhoN19 prevented recovery of F-actin assemblies upon repletion. Actin bundles in the apical microvilli and cytosolic F-actin were not affected by Rho signaling. Assembly of vinculin and paxillin into focal adhesions was disrupted by ATP depletion, and constitutively active RhoV14, although protecting stress fibers from disassembly, did not prevent dispersion of vinculin and paxillin, resulting in uncoupling of stress fiber and focal adhesion assembly. We propose that ATP depletion causes Rho inactivation during ischemia and that recovery of normal cellular architecture and function requires Rho.
Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels
Authors: Peracchia Camillo , Wang Xiao G. , Peracchia Lillian L.
Published: December 2017

Connexin channels are gated by transjunctional voltage ( V j) or CO2 via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT1) of connexin32 (Cx32) were shown to determine CO2sensitivity, and a gating mechanism involving CL-CT1 association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT1interactions, display atypical V jand CO2 sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH2-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT1 arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to V jpolarity, the existence of a “slow gate” distinct from the conventional V jgate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO2 sensitive than homotypic Cx32 channels, and CO2-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting V jsensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.
Effect of coupling on volume-regulatory response of ciliary epithelial cells suggests mechanism for secretion
Authors: Walker V. E. , Stelling J. W. , Miley H. E. , Jacob T. J. C.
Published: December 2017

The ciliary epithelium of the eye secretes the aqueous humor. It is a double epithelium arranged so that the apical surfaces of the nonpigmented ciliary epithelial (NPCE) and pigmented ciliary epithelial (PCE) cells face each other and the basolateral membranes face the inside of the eye and the blood, respectively. We have investigated the volume responses of both single cells and coupled pairs from this tissue to osmotic challenge. Both NPCE and PCE cells undergo regulatory volume increase (RVI) and decrease (RVD) when exposed to hyper- and hyposmotic solution, respectively. In hyposmotic solution single cells swell and return to their original volumes within ∼3 min. In nonpigmented cells RVD could be inhibited by blockers of volume-activated Cl−channels [tamoxifen (100%) > quinidine (87%) > DIDS (84%) > 5-nitro-2-(3-phenylpropylamino)benzoic acid (80%) > SITS (58%)] and K+ channels [Ba2+(31%)]. However, in PCE cells these inhibitors and additionally tetraethylammonium and Gd3+ were without effect. Only bumetanide, an inhibitor of Na+-K+-2Cl−cotransport, was found to have any effect on RVD in PCE cells. NPCE-PCE cell coupled pairs also underwent RVD, but with altered kinetics. The onset of RVD of the PCE cell in a pair occurred ≈80 s before that of the NPCE cell, and the peak swell was reduced. This is consistent with fluid movement from the PCE to the NPCE cell. The effect of the volume-activated Cl− channel inhibitor tamoxifen was to eliminate this difference in the times of onset of RVD in coupled cell pairs and to inhibit RVD in both the NPCE and PCE cells partially. On the basis of these observations we suggest that fluid is transferred from the PCE to the NPCE cell in coupled pairs during cell swelling and the subsequent RVD. Furthermore, we speculate that reciprocal RVI-RVD could underlie aqueous humor secretion.
Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins
Authors: Gorr Sven-Ulrik , Huang Xue Fen , Cowley Darrin J. , Kuliawat Regina , Arvan Peter
Published: December 2017

For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH4C1cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J.12: 2159–2168, 1993). However, in GH4C1cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH4C1cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.
Human colonic subepithelial myofibroblasts modulate transepithelial resistance and secretory response
Authors: Beltinger J. , McKaig B. C. , Makh S. , Stack W. A. , Hawkey C. J. , Mahida Y. R.
Published: December 2017

The epithelium of the gastrointestinal tract transports ions and water but excludes luminal microorganisms and toxic molecules. The factors regulating these important functions are not fully understood. Intestinal myofibroblasts lie subjacent to the basement membrane, at the basal surface of epithelial cells. We recently showed that primary cultures of adult human colonic subepithelial myofibroblasts express cyclooxygenase (COX)-1 and COX-2 enzymes and release bioactive transforming growth factor-β (TGF-β). In this study we have investigated the role of normal human colonic subepithelial myofibroblasts in the regulation of transepithelial resistance and secretory response in HCA-7 and T84 colonic epithelial cell lines. Cocultures of epithelial cells-myofibroblasts and medium conditioned by myofibroblasts enhanced transepithelial resistance and delayed mannitol flux. A panspecific antibody to TGF-β (but not piroxicam) antagonized this effect. In HCA-7 cells, myofibroblasts downregulated secretagogue-induced change in short-circuit current, and this effect was reversed by pretreatment of myofibroblasts with piroxicam. In contrast to HCA-7 cells, myofibroblasts upregulated the agonist-induced secretory response in T84 cells. This study shows that intestinal subepithelial myofibroblasts enhance barrier function and modulate electrogenic chloride secretion in epithelial cells. The enhancement of barrier function was mediated by TGF-β. In contrast, the modulation of agonist-induced change in short-circuit current was mediated by cyclooxygenase products. These findings suggest that colonic myofibroblasts regulate important functions of epithelial cells via distinct secretory products.
Myosin regulation of NKCC1: effects on cAMP-mediated Cl− secretion in intestinal epithelia
Authors: Hecht Gail , Koutsouris Athanasia
Published: December 2017

The basally located actin cytoskeleton has been demonstrated previously to regulate Cl−secretion from intestinal epithelia via its effects on the Na+-K+-2Cl−cotransporter (NKCC1). In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role of myosin in the regulation of secretagogue-stimulated Cl− secretion in intestinal epithelia. The human intestinal epithelial cell line T84 was used for these studies. Prevention of myosin light chain phosphorylation with the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with butanedione monoxime (BDM) attenuated cAMP but not Ca2+-mediated Cl− secretion. Both ML-9 and BDM diminished cAMP activation of NKCC1. Neither apical Cl− channel activity, basolateral K+ channel activity, nor Na+-K+-ATPase were affected by these agents. Cytochalasin D prevented such attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light chain and subsequent contraction of basal actin-myosin bundles are crucial to the cAMP-driven activation of NKCC1 and subsequent apical Cl− efflux.
Visualization of origins and propagation of excitation in canine gastric smooth muscle
Authors: Stevens Randel J. , Weinert Jeffery S. , Publicover Nelson G.
Published: December 2017

The origin and spread of excitation were visualized with fluo 3 fluorescence in tissues isolated from canine gastric antrum. Sheets of circular muscle (5 × 6 mm) had at least 1 (30%) and up to 3 discrete slow-wave pacing sites located near the longitudinal-circular muscle boundary, whereas similarly sized longitudinal sheets had an average of 5 sites (range 3–12 sites) that initiated Ca2+ waves. Superimposed fluorescent oscillations (circular muscle) and spikes (longitudinal muscle) were seen to initiate and propagate as distinct events, separate from their underlying activities. Average propagation velocities transverse (6–7 mm/s) and parallel (39–45 mm/s) to the long axis of muscle fibers were similar for each type of event in circular and longitudinal tissues; however, distinct regions where velocities of some (but not all) events decreased by up to an order of magnitude were present. The distance propagated by individual events was limited by collisions with concurrent excitable events or recently activated regions. Complex patterns of excitation in gastrointestinal smooth muscle arise as a result of interactions between multiple pacing sites, heterogeneous conduction velocities, and the interplay of adjacent pacemaker domains.
Secretagogue response of goblet cells and columnar cells in human colonic crypts
Authors: Halm Dan R. , Halm Susan Troutman
Published: December 2017

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 μM) or histamine (10 μM) released contents from goblet granules. Stimulation with prostaglandin E2(PGE2, 5 μM) or adenosine (10 μM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine (∼160 fl/cell), but not with PGE2 or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE2 stimulated a prolonged fluid secretion that attained a rate of ∼350 pl/min. Columnar cells lost ∼50% of apical volume during maximal PGE2 stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.
Apical and basolateral CO2- HCO 3 − permeability in cultured bovine corneal endothelial cells
Authors: Bonanno Joseph A. , Guan Yi , Jelamskii Sergey , Kang Xiao Jun
Published: December 2017

Corneal endothelial function is dependent on[Formula: see text] transport. However, the relative[Formula: see text] permeabilities of the apical and basolateral membranes are unknown. Using changes in intracellular pH secondary to removing CO2-[Formula: see text](at constant pH) or removing [Formula: see text]alone (at constant CO2) from apical or basolateral compartments, we determined the relative apical and basolateral [Formula: see text] permeabilities and their dependencies on Na+ and Cl−. Removal of CO2-[Formula: see text]from the apical side caused a steady-state alkalinization (+0.08 pH units), and removal from the basolateral side caused an acidification (−0.05 pH units). Removal of[Formula: see text] at constant CO2 indicated that the basolateral[Formula: see text] fluxes were about three to four times the apical fluxes. Reducing perfusate Na+ concentration to 10 mM had no effect on apical flux but slowed basolateral[Formula: see text] flux by one-half. In the absence of Cl−, there was an apparent increase in apical [Formula: see text] flux under constant-pH conditions; however, no net change could be measured under constant-CO2 conditions. Basolateral flux was slowed ∼30% in the absence of Cl−, but the net flux was unchanged. The steady-state alkalinization after removal of CO2-[Formula: see text]apically suggests that CO2diffusion may contribute to apical[Formula: see text] flux through the action of a membrane-associated carbonic anhydrase. Indeed, apical CO2 fluxes were inhibited by the extracellular carbonic anhydrase inhibitor benzolamide and partially restored by exogenous carbonic anhydrase. The presence of membrane-bound carbonic anhydrase (CAIV) was confirmed by immunoblotting. We conclude that the Na+-dependent basolateral[Formula: see text] permeability is consistent with Na+-[Formula: see text]cotransport. Changes in[Formula: see text] flux in the absence of Cl− are most likely due to Na+-[Formula: see text]cotransport-induced membrane potential changes that cannot be dissipated. Apical [Formula: see text] permeability is relatively low, but may be augmented by CO2 diffusion in conjunction with a CAIV.
Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+signaling
Authors: Blomstrand Fredrik , Giaume Christian , Hansson Elisabeth , Rönnbäck Lars
Published: December 2017

Astrocytes represent a major target for endothelins (ETs), a family of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in the brain. In the present study we have investigated the involvement of different ET receptor subtypes on intercellular dye diffusion, intracellular Ca2+homeostasis, and intercellular Ca2+ signaling in cultured rat astrocytes from hippocampus and striatum. Depending on the ET concentration and the receptor involved, ET-1- and ET-3-induced intracellular Ca2+ increases with different response patterns. Both ET-1 and ET-3 are powerful inhibitors of gap junctional permeability and intercellular Ca2+ signaling. The nonselective ET receptor agonist sarafotoxin S6b and the ETB receptor-selective agonist IRL 1620 mimicked these inhibitions. The ET-3 effects were only marginally affected by an ETA receptor antagonist but completely blocked by an ETB receptor antagonist. However, the ET-1-induced inhibition of gap junctional dye transfer and intercellular Ca2+ signaling was only marginally blocked by ETA or ETB receptor-selective antagonists but fully prevented when these antagonists were applied together. The ET-induced inhibition of gap junction permeability and intercellular Ca2+ signaling indicates that important changes in the function of astroglial communication might occur when the level of ETs in the brain is increased.
Skeletal muscle contractile activity in vitro stimulates mitogen-activated protein kinase signaling
Authors: Hayashi Tatsuya , Hirshman Michael F. , Dufresne Scott D. , Goodyear Laurie J.
Published: December 2017

Physical exercise is a potent stimulator of mitogen-activated protein (MAP) kinase signaling. To determine if this activation is secondary to systemic responses to exercise or due to muscle contractile activity per se, an isolated muscle preparation was developed. Contractile activity in vitro significantly increased p44MAPK and p42MAPK phosphorylation by 2.9- and 2.4-fold, respectively. Contraction-stimulated MAP kinase phosphorylation was not decreased in the presence ofd-tubocurarine or calphostin C, suggesting that neither neurotransmitter release nor diacylglycerol-sensitive protein kinase C mediates the contraction-induced activation of this signaling cascade. However, PD-98059, an inhibitor of MAP kinase kinase (MEK), inhibited the contraction-induced increases in MAP kinase phosphorylation. PD-98059 did not alter contraction-induced increases in glucose uptake or glycogen synthase activity, demonstrating that MAP kinase signaling is not necessary for these important metabolic effects of contractile activity in skeletal muscle. These data suggest that contractile activity of the skeletal muscle fibers per se, and not responses to neurotransmitter release, hormones, or other systemic factors, is responsible for the stimulation of MAP kinase signaling with physical exercise.
Desensitization to ANG II in guinea pig ileum depends on membrane repolarization: role of maxi-K+ channel
Authors: Silva Bagnólia A. , Nouailhetas Viviane L. A. , Aboulafia Jeannine
Published: December 2017

Desensitization of ANG II tonic contractile response of the guinea pig ileum is related to membrane repolarization determined by Ca2+-activated K+(maxi-K+) channel opening. ANG II-stimulated depolarized myocytes presented sustained activation of maxi-K+ channels, characterized by reduction from 415 to 12 ms of the closed time constant. ANG II desensitization was prevented by 100 nM iberiotoxin, being reversible within 30 min. Depolarization by KCl, higher than 4 mM, impaired desensitization, suggesting that the membrane potential must attain a threshold to counteract the repolarization induced by maxi-K+ channel opening. Once this value is attained, there is no time dependency because the desensitization process was shut off by addition of KCl along the time course of the tonic response. In contrast, the sustained ACh tonic component was not altered by these maneuvers. We conclude that desensitization of the ANG II tonic component is foremost due to the opening of maxi-K+ channels, leading to membrane repolarization, thus closing the voltage-dependent Ca2+ channels responsible for the Ca2+ influx that sustains the tonic component in this muscle.
Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts
Authors: Schilling William P. , Sinkins William G. , Estacion Mark
Published: December 2017

Maitotoxin (MTX), a potent cytolytic agent, activates Ca2+ entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca2+ concentration that depended on extracellular Ca2+ and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-d-glucamine (167 Da). Thus MTX initially activates Ca2+-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X7 receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X7 receptor stimulation activate a common cytolytic pore.
Receptor phosphorylation does not mediate cross talk between muscarinic M3 and bradykinin B2 receptors
Authors: Willars Gary B. , Müller-Esterl Werner , Nahorski Stefan R.
Published: December 2017

This study examined cross talk between phospholipase C-coupled muscarinic M3 and bradykinin B2 receptors coexpressed in Chinese hamster ovary (CHO) cells. Agonists of either receptor enhanced phosphoinositide signaling (which rapidly desensitized) and caused protein kinase C (PKC)-independent, homologous receptor phosphorylation. Muscarinic M3 but not bradykinin B2 receptors were also phosphorylated after phorbol ester activation of PKC. Consistent with this, muscarinic M3 receptors were phosphorylated in a PKC-dependent fashion after bradykinin B2 receptor activation, but muscarinic M3 receptor activation did not influence bradykinin B2receptor phosphorylation. Despite heterologous phosphorylation of muscarinic M3 receptors, phosphoinositide and Ca2+signaling were unaffected. In contrast, marked heterologous desensitization of bradykinin-mediated responses occurred despite no receptor phosphorylation. This desensitization was associated with a sustained component of muscarinic receptor-mediated signaling, whereas bradykinin's inability to influence muscarinic receptor-mediated responses was associated with rapid and full desensitization of bradykinin responses. Thus the mechanism of functional cross talk most likely involves depletion of a shared signaling component. These data demonstrate that receptor phosphorylation is not a prerequisite for heterologous desensitization and that such desensitization is not obligatory after heterologous receptor phosphorylation.
Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation
Authors: Su Wanfang , Shmukler Boris E. , Chernova Marina N. , Stuart-Tilley Alan K. , de Franceschi Lucia , Brugnara Carlo , Alper Seth L.
Published: December 2017

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of 86Rb into Xenopus oocytes requires extracellular Cl−, is blocked by the diuretic R(+)-[2- n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1 H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.
Targeting of calsequestrin to sarcoplasmic reticulum after deletions of its acidic carboxy terminus
Authors: Nori Alessandra , Gola Eleonora , Tosato Stefano , Cantini Marcello , Volpe Pompeo
Published: December 2017

Calsequestrin (CS) is the Ca2+ binding protein of the junctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the COOH terminus of CS, was shown to be correctly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe. Am. J. Physiol. 272 ( Cell Physiol. 41): C1420–C1428, 1997]. A putative targeting mechanism of CS to jSR implies electrostatic interactions between negative charges on CS and positive charges on intraluminal domains of jSR integral proteins, such as triadin and junctin. To test this hypothesis, 2 deletion mutants of chimeric CS were engineered: CS-HA1ΔGlu-Asp, in which the 14 acidic residues [-Glu-(Asp)5-Glu-(Asp)7-] of the COOH-terminal tail were removed, and CS-HA1Δ49COOH, in which the last, mostly acidic, 49 residues of the COOH terminus were removed. Both mutant cDNAs were transiently transfected in HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1 mutants were studied by epifluorescence microscopy with use of antibodies against CS or HA1. CS-HA1 mutants were shown to be expressed, sorted, and correctly segregated to jSR. Thus short or long deletions of the COOH-terminal acidic tail do not influence the targeting mechanism of CS.
Epidermal and hepatocyte growth factors stimulate chemotaxis in an intestinal epithelial cell line
Authors: Polk D. Brent , Tong Wei
Published: December 2017

The migration of intestinal cells is important in the development and maintenance of normal epithelium, in a process that may be regulated by growth factors and cytokines. Although a number of growth factor receptors are expressed by intestinal cells, little progress has been made toward assignment of functional roles for these ligand-receptor systems. This study compares several growth factors and cytokines for their chemoattraction of the mouse small intestinal epithelial cell line. Epidermal and hepatocyte growth factors stimulated a rapid 30-fold chemotaxis of cells with delayed threefold migration toward transforming growth factor-β1. Despite stimulating proliferation, keratinocyte, fibroblast, or insulin-like growth factors did not stimulate directed migration. Chemotaxis required tyrosine kinase and phosphatidylinositol phospholipase C activities but not protein kinase C or mitogen-activated protein kinase activity. These findings suggest that the repertoire of growth factors capable of regulating directed intestinal epithelial cell migration is limited and that a divergence exists in the signal transduction pathways for directed vs. nondirected migration.
Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis
Authors: di Wu Qiong , Wang Jiang Huai , Fennessy Fiona , Redmond H. Paul , Bouchier-Hayes David
Published: December 2017

Elevated blood glucose in uncontrolled diabetes is causally correlated with diabetic microangiopathy. Hyperglycemia-triggered accelerated endothelial cell apoptosis is a critical event in the process of diabetes-associated microvascular disease. The conditionally semiessential amino acid taurine has been previously shown to protect against human endothelial cell apoptosis. Therefore, this study was designed to investigate the role of taurine in the prevention of high-glucose-mediated cell apoptosis in human umbilical vein endothelial cells (HUVEC) and the mechanisms involved. Exposure of HUVEC to 30 mM glucose for 48 h (short-term) and 14 days (long-term) resulted in a significant increase in apoptosis, compared with normal glucose (5.5 mM; P < 0.05). High-glucose-induced DNA fragmentation preferentially occurred in the S phase cells. Mannitol (as osmotic control) at 30 mM failed to induce HUVEC apoptosis. Taurine prevented high-glucose-induced HUVEC apoptosis, which correlates with taurine attenuation of high-glucose-mediated increased intracellular reactive oxygen species (ROS) formation and elevated intracellular Ca2+ concentration ([Ca2+]i) level. Antioxidants, DMSO, N-acetyl cysteine, and glutathione, only partly attenuated high-glucose-induced HUVEC apoptosis. Glucose at 30 mM did not cause HUVEC necrosis. However, both glucose and mannitol at 60 mM caused HUVEC necrosis as represented by increased lactate dehydrogenase release and cell lysis. Taurine failed to prevent hyperosmolarity-induced cell necrosis. These results demonstrate that taurine attenuates hyperglycemia-induced HUVEC apoptosis through ROS inhibition and [Ca2+]istabilization and suggest that taurine may exert a beneficial effect in preventing diabetes-associated microangiopathy.
Hypothesis: do voltage-gated H+channels in alveolar epithelial cells contribute to CO2 elimination by the lung?
Authors: DeCoursey Thomas E.
Published: December 2017

Although alveolar epithelial cells were the first mammalian cells in which voltage-gated H+ currents were recorded, no specific function has yet been proposed. Here we consider whether H+ channels contribute to one of the main functions of the lung: CO2 elimination. This idea builds on several observations: 1) some cell membranes have low CO2permeability, 2) carbonic anhydrase is present in alveolar epithelium and contributes to CO2 extrusion by facilitating diffusion, 3) the transepithelial potential difference favors selective activation of H+ channels in apical membranes, and 4) the properties of H+ channels are ideally suited to the proposed role. H+channels open only when the electrochemical gradient for H+ is outward, imparting directionality to the diffusion process. Unlike previous facilitated diffusion models, [Formula: see text] and H+ recombine to form CO2 in the alveolar subphase. Rough quantitative considerations indicate that the proposed mechanism is plausible and indicate a significant capacity for CO2 elimination by the lung by this route. Fully activated alveolar H+ channels extrude acid equivalents at three times the resting rate of CO2 production.
Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells
Authors: Zhuang Shougang , Hirai Syu-Ichi , Ohno Shigeo
Published: December 2017

Protein kinase C (PKC) has been reported to be associated with the activation of extracellular signal-regulated kinase (ERK) by hyperosmolality. However, it is unclear whether hyperosmolality induces PKC activation and which PKC isoforms are involved in ERK activation. In this study, we demonstrate that NaCl increases total PKC activity and induces PKCα, PKCδ, and PKCε translocation from the cytosol to the membrane in NIH/3T3 cells, suggesting that hyperosmotic stress activates conventional PKC (cPKC) and novel PKC (nPKC). Further studies show that NaCl-inducible ERK1 and ERK2 (ERK1/2) activation is a consequence of cPKC and nPKC activation, because either downregulation with 12- O-tetradecanoylphorbol 13-acetate or selective inhibition of cPKC and nPKC by GF-109203X and rottlerin largely inhibited the stimulation of ERK1/2 phosphorylation by NaCl. In addition, we show that NaCl increases diacylglycerol (DAG) levels and that a phospholipase C (PLC) inhibitor, U-73122, inhibits NaCl-induced ERK1/2 phosphorylation. These results, together, suggest that a hyperosmotic NaCl-induced signaling pathway that leads to activation of ERK1/2 may sequentially involve PLC activation, DAG release, and cPKC and nPKC activation.
Temperature-sensitive gating of cation current in guinea pig ileal muscle activated by hyperpolarization
Authors: Yanagida Hiroe , Inoue Ryuji , Tanaka Masao , Ito Yushi
Published: December 2017

The temperature dependence of hyperpolarization-activated current ( I h) was investigated in freshly isolated guinea pig ileal smooth muscle cells, using the nystatin-perforated whole cell recording technique. Hyperpolarizing pulses (−50 to −120 mV) from −40 mV evoked time-dependent inward rectifying currents with a reversal potential of −33 mV and a slow activation time course well approximated by a single exponential. The properties of these currents, such as steady-state variables, dependence on external K, modification by norepinephrine, and blockade by Cs or ZD-7288, coincide well with those of the “classical” I h discovered in the sinoatrial node. Raising the temperature (range: 22–33°C) accelerated the activation time course of this I h and shifted its 50% activation potential positively (12 mV/10 degree) with much less change in the maximum conductance. Based on a simple closed-open model, this can be explained by a high temperature dependence of the opening rate constant (temperature coefficient: 3.4). The activation profile of reconstructed I h at 36°C suggests that a considerable overlap could occur between the ranges of I h activation and physiological membrane potential.
Tyrosine kinase inhibitors reduce bcl-2 expression and induce apoptosis in androgen-dependent cells
Authors: Ohigashi Takashi , Ueno Munehisa , Nonaka Shoichi , Nakanoma Takashi , Furukawa Yusuke , Deguchi Nobuhiro , Murai Masaru
Published: December 2017

The signal transduction pathway showing how androgen withdrawal induces apoptosis in androgen-dependent cells has not been clearly understood. In these studies, we focused on the behavior of tyrosine kinases in androgen-dependent cells and investigated its correlation with apoptosis and bcl-2 expression. We used SC2G, an androgen-dependent mouse mammary carcinoma cell line, which had been cloned from Shionogi Carcinoma 115 (SC115). When SC2G cells were cultured with herbimycin A (HMA), a potent tyrosine kinase inhibitor, the number of viable cells decreased significantly after 24 h. Terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling and flow cytometric analysis of annexin V staining showed that HMA induced apoptosis of SC2G cells. The level of bcl-2 mRNA in SC2G cells was suppressed by HMA in a dose-dependent manner on RT-PCR. Preincubation with caspase inhibitors protected HMA-induced apoptosis of SC2G cells. When a human bcl-2 gene was transfected in SC2G cells and overexpressed, SC2G cells seemed to acquire tolerance for HMA. These data indicate that HMA-sensitive tyrosine kinase(s) can regulate apoptosis and inhibit bcl-2 expression in SC2G mouse androgen-dependent cells. Tyrosine kinase(s) seemed to be a member of signal transduction between androgen receptor activation and bcl-2 expression.
Charged residues in the M2 region of α-hENaC play a role in channel conductance
Authors: Langloh Anne Lynn B. , Berdiev Bakhrom , Ji Hong-Long , Keyser Kent , Stanton Bruce A. , Benos Dale J.
Published: December 2017

The epithelial Na+channel (ENaC) is a low-conductance channel that is highly selective for Na+ and Li+ over K+ and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of α-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in α-subunit human ENaC (α-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na+:K+permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 α-hENaC mutants along with wild-type (wt) β- and γ-hENaC was low (46–93 nA) compared with the wt channel (1–3 μA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant α-hENaC subunits with green fluorescent protein-tagged β- and γ-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of α-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.
Inhibition of ornithine decarboxylase induces STAT3 tyrosine phosphorylation and DNA binding in IEC-6 cells
Authors: Pfeffer Lawrence M. , Yang Chuan He , Pfeffer Susan R. , Murti Aruna , McCormack Shirley A. , Johnson Leonard R.
Published: December 2017

Polyamines are required for the proliferation of the rat intestinal mucosal IEC-6 cell line. Ornithine decarboxylase (ODC) is the enzyme that catalyzes the first step in polyamine synthesis. ODC inhibition not only leads to polyamine depletion but also leads to inhibition of cell proliferation and regulates the expression of the immediate-early genes c- fos, c- myc, and c- jun. Members of the signal transducers and activators of transcription (STAT) transcription factor family bind to the sis-inducible element (SIE) present in the promoters to regulate the expression of a variety of important genes. In the present study, we tested the hypothesis that the STAT3 transcription factor, which is responsible for activation of the acute phase response genes, is activated after inhibition of ODC. We found that inhibition of ODC rapidly induces STAT3 activation as determined by STAT3 tyrosine phosphorylation, translocation of STAT3 from the cytoplasm into the nucleus, and the presence of STAT3 in SIE-dependent DNA-protein complexes. STAT3 activation upon inhibition of ODC was accompanied by the activation of a STAT3-dependent reporter construct. Moreover, prolonged polyamine depletion resulted in downregulation of cellular STAT3 levels.
Ras signaling in the inner medullary cell response to urea and NaCl
Authors: Tian Wei , Boss Gerry R. , Cohen David M.
Published: December 2017

The small guanine nucleotide-binding protein Ras, activated by peptide mitogens and other stimuli, regulates downstream signaling events to influence transcription. The role of Ras in solute signaling to gene regulation was investigated in the murine inner medullary collecting duct (mIMCD3) cell line. Urea treatment (100–200 mM), but not sham treatment, increased Ras activation 124% at 2 min; the effect of NaCl did not achieve statistical significance. To determine the contribution of Ras activation to urea-inducible signal transduction, mIMCD3 cells were stably transfected with an expression plasmid encoding a dominant negative-acting N17Ras mutant driven by a dexamethasone-inducible (murine mammary tumor virus) promoter. After 24 h of induction, selected cell lines exhibited sufficient N17Ras overexpression to abolish epidermal growth factor- and hypotonicity-mediated signaling to extracellular signal-regulated kinase (ERK) phosphorylation, as determined by immunoblotting. Conditional N17Ras overexpression inhibited urea- and NaCl-inducible ERK phosphorylation by 40–50%, but only at 15 min, and not 5 min, of treatment. N17Ras induction, however, almost completely inhibited urea-inducible Egr-1 transcription, as quantitated by luciferase reporter gene assay, but failed to influence tonicity-inducible (TonE-mediated) transcription. N17Ras overexpression also blocked urea-inducible expression of the transcription factor Gadd153 but did not influence osmotic or urea-inducible apoptosis. In addition, urea treatment induced recruitment of the Ras activator Sos to the plasma membrane. Taken together, these observations suggest a role for Ras signaling in the IMCD cell response to urea stress.
Smokeless tobacco potentiates VIP-induced DNA synthesis and inactivates NEP 24.11 in oral keratinocytes
Authors: Rubinstein Israel
Published: December 2017

The purpose of this study was to determine whether exposure of cultured chemically transformed hamster oral keratinocytes (HCPC-1) to an aqueous extract of smokeless tobacco (STE) potentiates DNA synthesis elicited by vasoactive intestinal peptide (VIP), an autocrine neuropeptide, and, if so, whether this response is associated with inactivation of neutral endopeptidase 24.11 (NEP 24.11), an ectoenzyme that cleaves and inactivates VIP very effectively, in these cells. I found that STE and VIP each elicited a modest, albeit significant, increase in DNA synthesis in cultured HCPC-1 cells ( P < 0.05). However, incubation of HCPC-1 cells with STE together with VIP evoked a significant, concentration- dependent increase in DNA synthesis that was mediated by VIP receptors. The effects of STE and VIP were synergistic. Maximal response was observed after a 48-h incubation. STE significantly attenuated NEP 24.11 activity in HCPC-1 cells at a time when VIP-induced DNA synthesis was maximal. Collectively, these data indicate that STE potentiates VIP-induced DNA synthesis in cultured oral keratinocytes, and that this response is temporally related to STE-induced inactivation of NEP 24.11 in these cells. I suggest that NEP 24.11 modulates the mitogenic effects of smokeless tobacco in the oral epithelium, in part, by inactivating VIP.
Folate deficiency reduces the GPI-anchored folate-binding protein in rat renal tubules
Authors: da Costa Maria , Rothenberg Sheldon P. , Sadasivan Easwara , Regec Annette , Qian Lian
Published: December 2017

A folate-binding protein (FBP) anchored to cell membranes by a glycosyl phosphatidylinositol (GPI) adduct is constitutively expressed in some transformed and cultured cell lines. Its expression is upregulated when these cells are grown in medium containing low folate, but whether this occurs in vivo with nutritional folate deficiency is unknown. To address this question, the GPI-FBP in the liver, kidney, and brain of rats on control and folate-deficient (FD) diets was measured. The GPI-FBP in the kidney of FD rats decreased significantly in contrast to the upregulation of this protein in cultured cells. Northern blot analysis and nuclear run-on assays indicated that transcription of the GPI-FBP gene in the kidney was not reduced by folate deficiency. This decrease of the GPI-FBP appears to result from its proteolysis, similar to the enzymatic degradation of the apoprotein that occurs in vitro. Because the GPI-FBP is on the brush borders of the proximal renal tubules and provides for the reabsorption of folate, this function diminishes when the protein decreases in folate deficiency.
Kinase regulation of hENaC mediated through a region in the COOH-terminal portion of the α-subunit
Authors: Volk Kenneth A. , Husted Russell F. , Snyder Peter M. , Stokes John B.
Published: December 2017

In an effort to gain insight into how kinases might regulate epithelial Na+ channel (ENaC) activity, we expressed human ENaC (hENaC) in Xenopus oocytes and examined the effect of agents that modulate the activity of some kinases. Activation of protein kinase C (PKC) by phorbol ester increased the activity of ENaC, but only in oocytes with a baseline current of <2,000 nA. Inhibitors of protein kinases produced varying effects. Chelerythrine, an inhibitor of PKC, produced a significant inhibition of ENaC current, but calphostin C, another PKC inhibitor, had no effect. The PKA/protein kinase G inhibitor H-8 had no effect, whereas the p38 mitogen-activated protein kinase inhibitor, SB-203580 had a significant inhibitory effect. Staurosporine, a nonspecific kinase inhibitor, was the most potent tested. It inhibited ENaC currents in both oocytes and in M-1 cells, a model for the collecting duct. Site-directed mutagenesis revealed that the staurosporine effect did not require an intact COOH terminus of either the β- or γ-hENaC subunit. However, an intact COOH terminus of the α-subunit was required for this effect. These results suggest that an integrated kinase network regulates ENaC activity through an action that requires a portion of the α-subunit.
Development and polarization of cationic amino acid transporters and regulators in the human placenta
Authors: Ayuk Paul T.-Y. , Sibley Colin P. , Donnai Paul , D'Souza Stephen , Glazier Jocelyn D.
Published: December 2017

We have investigatedl-arginine transport systems in the human placental syncytiotrophoblast across gestation using purified microvillous (MVM) and basal (BM) plasma membrane vesicles. In MVM from first-trimester and term placentas, l-arginine transport was by systems y+ and y+L. In BM (term placentas), however, there was evidence for system y+L only. The Michaelis constant of system y+L was significantly lower ( P< 0.05) in first-trimester compared with term MVM and lower in term MVM compared with BM ( P < 0.05). There was no functional evidence for system b0+ in term MVM or BM. Cationic amino acid transporter (CAT) 1, CAT 4, and 4F2hc were detected using RT-PCR in placentas throughout gestation. rBAT was not detected in term placentas. An ∼85-kDa and an ∼135-kDa protein was detected by Western blotting in MVM under reducing and nonreducing conditions, respectively, consistent with the 4F2hc monomer and the 4F2hc-light chain dimer, and their expression was significantly higher ( P< 0.05) in term compared with first-trimester MVM. These proteins were not detected in BM despite functional evidence for system y+L. These data suggest different roles for 4F2hc in the development and polarization of cationic amino acid transporters in the syncytiotrophoblast.