bluuebunny/crossref_metadata_2025 huggingface dataset:
Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia
Authors: Evans D. J. , Matsumoto P. S. , Widdicombe J. H. , Li-Yun C. , Maminishkis A. A. , Miller S. S.
Published: December 2017

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption ( J v) across bovine cells of 3.2 μl ⋅ cm−2 ⋅ h−1was inhibited by ∼78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance ( R t). Effects of P. aeruginosa were blocked by amiloride, which decreased J v by 112% from baseline of 2.35 ± 1.25 μl ⋅ cm−2 ⋅ h−1, increased R t by 101% from baseline of 610 ± 257 Ω ⋅ cm2, and decreased TEP by 91% from baseline of −55 ± 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of ∼2.5 ± 0.4 μl ⋅ cm−2 ⋅ h−1and decreased R twithout changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on J v, TEP, or R t. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a ς factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.
Hydrogen peroxide inhibits cAMP-induced Cl− secretion across colonic epithelial cells
Authors: DuVall Michael D. , Guo Yi , Matalon Sadis
Published: December 2017

We examined the effects of H2O2on Cl− secretion across human colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, a measure of Cl− secretion, was inhibited in a concentration-dependent fashion when monolayers were pretreated with H2O2for 30 min (30–100% inhibition between 500 μM and 5 mM). Moreover, H2O2inhibited 76% of the Cl−current across monolayers when the basolateral membranes were permeabilized with nystatin (200 μg/ml). When the apical membrane was permeabilized with amphotericin B, H2O2inhibited the Na+ current (a measure of Na+-K+-ATPase activity) by 68% but increased the K+ current more than threefold. In addition to its effects on ion transport pathways, H2O2also decreased intracellular ATP levels by 43%. We conclude that the principal effect of H2O2on colonic Cl− secretion is inhibitory. This may be due to a decrease in ATP levels following H2O2treatment, which subsequently results in an inhibition of the apical membrane Cl− conductance and basolateral membrane Na+-K+-ATPase activity. Alternatively, H2O2may alter Cl− secretion by direct action on the transporters or alterations in signal transduction pathways.
Na+-K+-Cl−cotransport in human fibroblasts is inhibited by cytomegalovirus infection
Authors: Maglova Lilia M. , Crowe William E. , Smith Peter R. , Altamirano Aníbal A. , Russell John M.
Published: December 2017

We examined the effects of human cytomegalovirus (HCMV) infection on the Na+-K+-Cl−cotransporter (NKCC) in a human fibroblast cell line. Using the Cl−-sensitive dye MQAE, we showed that the mock-infected MRC-5 cells express a functional NKCC. 1) Intracellular Cl− concentration ([Cl−]i) was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mM following bumetanide treatment. 2) Net Cl− efflux caused by replacement of external Cl−with gluconate was bumetanide sensitive. 3) In Cl−-depleted mock-infected cells, the Cl− reuptake rate (in [Formula: see text]-free media) was reduced in the absence of external Na+ and by treatment with bumetanide. After HCMV infection, we found that although [Cl−]iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide and Na+ sensitivities of [Cl−]iand net Cl− uptake and loss were reduced by 24 h PE and abolished by 72 h PE. Western blots using the NKCC-specific monoclonal antibody T4 showed an approximately ninefold decrease in the amount of NKCC protein after 72 h of infection. Thus HCMV infection resulted in the abolition of NKCC function coincident with the severe reduction in the amount of NKCC protein expressed.
Antisense oligonucleotide to PKC-ε alters cAMP-dependent stimulation of CFTR in Calu-3 cells
Authors: Liedtke Carole M. , Cole Thomas S.
Published: December 2017

Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (−)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3′,5′- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-α, -βII, -δ, -ε, and -ζ were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-ε for 48 h prevented stimulation of CFTR with (−)-epinephrine, reduced PKC-ε activity in unstimulated cells by 52.1%, and decreased PKC-ε mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-ε and antisense oligonucleotide to PKC-δ and -ζ did not alter (−)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-ε.
Gap junction permeability is diminished in proliferating vascular smooth muscle cells
Authors: Kurjiaka David T. , Steele Timothy D. , Olsen Mary V. , Burt Janis M.
Published: December 2017

In atherosclerosis and hypertension, vascular smooth muscle cells (SMCs) are stimulated to proliferate and exhibit enhanced gap junction protein expression. Our goal was to determine whether gap junction function differs in proliferating vs. growth-arrested SMCs. A7r5 cells (embryonic rat aortic SMCs) did not proliferate in media with reduced serum (∼90% of cells in G0/G1phase after 48–96 h in 1% fetal bovine serum). Dye coupling was less but electrical coupling was comparable in proliferating vs. growth-arrested A7r5 cells, suggesting differences in junctional permselectivity. In growth-arrested cells, junctional conductances measured with potassium glutamate, tetraethylammonium chloride, and KCl were well predicted by the conductivities of these solutions. In contrast, junctional conductances measured with potassium glutamate and tetraethylammonium chloride in proliferating cells were significantly greater than predicted by the conductivities of these solutions. These results suggest that junctions between growth-arrested cells are permeated equally well and simultaneously by anions and cations, whereas junctions between proliferating cells are poorly permeated by large molecules of either charge and equally well but not simultaneously by small anions and cations. The data indicate that A7r5 cells regulate chemical coupling independent of electrical coupling, a capacity that could facilitate growth control while protecting vasomotor responsiveness of vessels.
Association of intrinsic pICln with volume-activated Cl− current and volume regulation in a native epithelial cell
Authors: Chen Lixin , Wang Liwei , Jacob Tim J. C.
Published: December 2017

We investigated the relationship between pICln, the volume-activated Cl−current, and volume regulation in native bovine nonpigmented ciliary epithelial (NPCE) cells. Immunofluorescence studies demonstrated the presence of pICln protein in the NPCE cells. Exposure to hypotonic solution activated a Cl− current and induced regulatory volume decrease (RVD) in freshly isolated bovine NPCE cells. Three antisense oligonucleotides complementary to human pICln mRNA were used in the experiments. The antisense oligonucleotides were taken up by the cells in a dose-dependent manner. The antisense oligonucleotides, designed to be complementary to the initiation codon region of the human pICln mRNA, “knocked down” the pICln protein immunofluorescence, delayed the activation of volume-activated Cl− current, diminished the value of the current, and reduced the ability of the cells to volume regulate. We conclude that pIClnis involved in the activation pathway of the volume-activated Cl− current and RVD following hypotonic swelling.
Measurements of mitochondrial K+ fluxes in whole rat hearts using 87Rb-NMR
Authors: Gruwel Marco L. H. , Kuzio Bozena , Deslauriers Roxanne , Kupriyanov Valerie V.
Published: December 2017

The rubidium efflux from hypothermic rat hearts perfused by the Langendorff method at 20°C was studied. At this temperature 87Rb-NMR efflux experiments showed the existence of two 87Rb pools: cytoplasmic and mitochondrial. Rat heart mitochondria showed a very slow exchange of mitochondrial Rb+ for cytoplasmic K+. After washout of cytosolic Rb+, mitochondria kept a stable Rb+level for >30 min. Rb+ efflux from mitochondria was stimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmal permeabilization and concomitant cellular energy depletion by saponin (0.01 mg/ml for 4 min) in the presence of a perfusate mimicking intracellular conditions, or by ATP-sensitive K (KATP) channel openers. DNP, a mitochondrial uncoupler, caused the onset of mitochondrial Rb+ exchange; however, the washout was not complete (80 vs. 56% in control). Energy deprivation by saponin, which permeabilizes the sarcolemma, resulted in a rapid and complete Rb+ efflux. The mitochondrial Rb+ efflux rate constant ( k) decreased in the presence of glibenclamide, a KATP channel inhibitor (5 μM; k = 0.204 ± 0.065 min−1; n = 8), or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM, respectively; k = 0.134 ± 0.021 min−1; n = 4) in the saponin experiments (saponin only; k = 0.321 ± 0.079 min−1; n = 3), indicating the inhibition of mitochondrial KATP channels. Thus hypothermia in combination with 87Rb-NMR allowed the probing of the mitochondrial K+ pool in whole hearts without mitochondrial isolation.
Thrombin-, bradykinin-, and arachidonic acid-induced Ca2+ signaling in Ehrlich ascites tumor cells
Authors: Jørgensen Nanna Koschmieder , Petersen Stine Falsig , Hoffmann Else Kay
Published: December 2017

Stimulation of single Ehrlich ascites tumor cells with agonists (bradykinin, thrombin) and with arachidonic acid (AA) induces increases in the free intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of extracellular Ca2+, measured using the Ca2+-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increases in [Ca2+]i, unlike addition of the same agonist twice. Bradykinin and thrombin have additive effects on [Ca2+]iin Ca2+-free medium. The phosphoinositidase C inhibitor U-73122 inhibits the agonist-induced increases in [Ca2+]i, whereas ryanodine has no effect. Pretreatment of cells in Ca2+-free medium with thapsigargin abolishes the bradykinin-induced increase in [Ca2+]ibut not the response to thrombin. The AA-induced response is not inhibited by U-73122 and cannot be mimicked by the inactive structural analog trifluoromethylarachidonyl ketone. Pretreatment of the cells with 50 μM AA (but not with 10 μM AA) abolishes the agonist-induced increase in [Ca2+]i. Thus bradykinin, thrombin, and AA induce increases in [Ca2+]iin Ehrlich cells due to Ca2+ entry and release from intracellular stores. Thrombin causes release of Ca2+ from an intracellular store that is insensitive to bradykinin and is not depleted by thapsigargin but is depleted by AA.
Isolated epithelial cells from amphibian urinary bladder express functional gap junctional hemichannels
Authors: Vanoye Carlos G. , Vergara Leoncio A. , Reuss Luis
Published: December 2017

Exposure of the urinary bladder epithelium of Necturus maculosus (NUB) to protease and collagenase yields ∼50% isolated polarized cells. These cells express a membrane current slowly activated by depolarization or by removal of external divalent cations. The biophysical and pharmacological properties of the current are largely consistent with those of gap junctional hemichannels. After removal of divalent cations, the cells can also be loaded with 5(6)-carboxyfluorescein, a hydrophilic fluorescent anionic dye, and exposure to dye reduces the current in a manner dependent on membrane voltage and side of application. In contrast, Necturus gallbladder (NGB) cells exhibit no membrane conductance attributable to gap junctional hemichannels, although previous studies reveal the persistence of gap junction plaques on the plasma membrane. We conclude that functional gap junctional hemichannels can be expressed on the surface of certain isolated epithelial cells and that this is not a necessary consequence of the isolation procedure. These structures may contribute to cell damage under pathological conditions involving cell detachment.
Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA
Authors: Gueorguiev Volodia D. , Zeman Richard J. , Hiremagalur Bhargava , Menezes Ana , Sabban Esther L.
Published: December 2017

The involvement of cAMP- and Ca2+-mediated pathways in the activation of tyrosine hydroxylase (TH) gene expression by nicotine was examined in PC-12 cells. Extracellular Ca2+ and elevations in intracellular Ca2+ concentration ([Ca2+]i) were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in [Ca2+]iwas inhibited by blockers of either L-type or N-type, and to a lesser extent P/Q-, but not T-type, voltage-gated Ca2+ channels. With continual nicotine treatment, [Ca2+]ireturned to basal levels within 3–4 min. After a lag of ∼5–10 min, there was a smaller elevation in [Ca2+]ithat persisted for 6 h and displayed different responsiveness to Ca2+ channel blockers. This second phase of elevated [Ca2+]iwas blocked by an inhibitor of store-operated Ca2+ channels, consistent with the observed generation of inositol trisphosphate. 1,2-Bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine, prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine (DDA) prevented the nicotine-elicited phosphorylation of cAMP response element binding protein. DDA also blocked the elevation of TH mRNA only when added after the initial transient rise in [Ca2+]iand not after 1 h. This study reveals that several temporal phases are involved in the induction of TH gene expression by nicotine, each of them with differing requirements for Ca2+ and cAMP.
Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl− currents
Authors: Vanoye Carlos G. , Castro Ariel F. , Pourcher Thierry , Reuss Luis , Altenberg Guillermo A.
Published: December 2017

Several proteins belonging to the ATP-binding cassette superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1). We measured whole cell swelling-activated Cl− currents ( I Cl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol ester reduced the rate of increase in I Cl,swell only in cells that express MDR1. PKC stimulation had no effect on steady-state I Cl,swell. Stimulation of protein kinase A (PKA) with 8-bromoadenosine 3′,5′-cyclic monophosphate reduced steady-state I Cl,swell only in MDR1-expressing cells. PKA stimulation had no effect on the rate of I Cl,swellactivation. The effects of stimulation of PKA and PKC on I Cl,swell were additive (i.e., decrease in the rate of activation and reduction in steady-state I Cl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the phosphorylation-defective mutant. In summary, it is likely that phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl− channels by independent mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the responses of I Cl,swell to stimulation of PKA and PKC. These results support the notion that MDR1 phosphorylation affects I Cl,swell.
Imaging of intracellular calcium stores in single permeabilized lens cells
Authors: Churchill Grant C. , Louis Charles F.
Published: December 2017

Intracellular Ca2+ stores in permeabilized sheep lens cells were imaged with mag-fura 2 to characterize their distribution and sensitivity to Ca2+-releasing agents. Inositol 1,4,5-trisphosphate (IP3) or cyclic ADP-ribose (cADPR) released Ca2+ from intracellular Ca2+ stores that were maintained by an ATP-dependent Ca2+ pump. The IP3 antagonist heparin inhibited IP3- but not cADPR-mediated Ca2+ release, whereas the cADPR antagonist 8-amino-cADPR inhibited cADPR- but not IP3-mediated Ca2+ release, indicating that IP3 and cADPR were operating through separate mechanisms. A Ca2+ store sensitive to IP3, cADPR, and thapsigargin appeared to be distributed throughout all intracellular regions. In some cells a Ca2+ store insensitive to IP3, cADPR, thapsigargin, and 2,4-dinitrophenol, but not ionomycin, was present in a juxtanuclear region. We conclude that lens cells contain intracellular Ca2+ stores that are sensitive to IP3, cADPR, and thapsigargin, as well as a Ca2+store that appears insensitive to all these agents.
Glucose induces an acute increase of superoxide dismutase activity in incubated rat pancreatic islets
Authors: Oliveira Henriette R. , Curi Rui , Carpinelli Angelo R.
Published: December 2017

The activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSP) in isolated rat pancreatic islets exposed to high glucose concentration for a short period of time (60 min) was determined. High glucose concentration (16.7 mM) did not significantly alter catalase activity. GSP activity was increased by glucose at 5.6 mM, remaining elevated at higher concentrations up to 16.7 mM. However, the activity of SOD increased with glucose concentration, and this increment was closely correlated with the rate of insulin secretion ( r = 0.96). High potassium (30 mM) did not increase SOD activity, suggesting that the increase in intracellular ionic calcium concentration does not stimulate this enzyme activity. α-Ketoisocaproic acid and pyruvate, which are metabolized through the TCA cycle, did not increase SOD activity, indicating that the stimulation of SOD activity might be triggered by a factor produced through glycolysis or the pentose phosphate pathway.
Ca2+-ATPases and their expression in the mammary gland of pregnant and lactating rats
Authors: Reinhardt Timothy A. , Horst Ronald L.
Published: December 2017

The transcellular Ca2+fluxes required for milk production must be rigorously regulated to maintain the low cytosolic Ca2+concentrations critical to cell function. Ca2+-ATPases play a critical role in the maintenance of this cellular Ca2+ homeostasis. Using RT-PCR and sequencing, we identified six Ca2+pumps in lactating mammary tissue. Three plasma membrane Ca2+-ATPases (PMCAs) were found (PMCA1b, PMCA2b, and PMCA4b). Two sarco (endo)plasmic reticulum Ca2+-ATPases (SERCAs) were identified (SERCA2 and SERCA3), and the rat homologue to the yeast Golgi Ca2+-ATPase RS-10 was also found. The pattern of mRNA expression of each of these pumps was examined in rat mammary tissue from the 7th day of pregnancy to the 21st day of lactation. Northern blots revealed increased mRNA expression for all Ca2+ pumps by the 14th day of lactation, and transcripts continued to increase through the 18th day of lactation. PMCA1b, PMCA4b, SERCA2, and SERCA3 showed the lowest levels of expression. RS-10 transcripts were more abundant than SERCA2, SERCA3, PMCA1b, and PMCA4b. RS-10 was the only pump to increase in expression before parturition. PMCA2b was the most abundant transcript found in lactating mammary tissue. At peak lactation, expression of PMCA2b approached that of actin. The high expression, high affinity for Ca2+, and high activity at low calmodulin concentrations exhibited by PMCA2b suggest that it is uniquely suited for maintenance of Ca2+ homeostasis in the lactating mammary gland. The pattern of expression and abundance of RS-10 suggest that it is a candidate for the Golgi Ca2+-ATPase shown to be important in maintaining the Golgi Ca2+concentration required for casein synthesis and micelle formation.
Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO
Authors: Fischer Silvia , Clauss Matthias , Wiesnet Marion , Renz Dieter , Schaper Wolfgang , Karliczek Gerhard F.
Published: December 2017

In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor N G-monomethyl-l-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.
Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS
Authors: Yeh Li-Hong , Park Young J. , Hansalia Riple J. , Ahmed Imraan S. , Deshpande Shailesh S. , Goldschmidt-Clermont Pascal J. , Irani Kaikobad , Alevriadou B. Rita
Published: December 2017

The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2 for 5–30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.
Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis
Authors: Li Li , Li Ji , Rao Jaladanki N. , Li Minglin , Bass Barbara L. , Wang Jian-Ying
Published: December 2017

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.
Mitogen-induced proliferation increases biotin uptake into human peripheral blood mononuclear cells
Authors: Zempleni Janos , Mock Donald M.
Published: December 2017

We sought to determine whether the proliferation of immune cells affects the cellular uptake of the vitamin biotin. Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults. The proliferation of PBMC was induced by either pokeweed lectin, concanavalin A, or phytohemagglutinin. When the medium contained a physiological concentration of [3H]biotin, nonproliferating PBMC accumulated 406 ± 201 amol [3H]biotin ⋅ 106cells−1 ⋅ 30 min−1. For proliferating PBMC, [3H]biotin uptake increased to between 330 and 722% of nonproliferating values. Maximal transport rates of [3H]biotin in proliferating PBMC were also about four times greater than those in nonproliferating PBMC, suggesting that proliferation was associated with an increase in the number of biotin transporters on the PBMC membrane. The biotin affinities and specificities of the transporter for proliferating and nonproliferating PBMC were similar, providing evidence that the same transporter mediates biotin uptake in both states. [14C]urea uptake values for proliferating and nonproliferating PBMC were similar, suggesting that the increased [3H]biotin uptake was not caused by a global upregulation of transporters during proliferation. We conclude that PBMC proliferation increases the cellular accumulation of biotin.
Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue
Authors: Dreja Karl , Hellstrand Per
Published: December 2017

To investigate the Ca2+-dependent plasticity of sarcoplasmic reticulum (SR) function in vascular smooth muscle, transient responses to agents releasing intracellular Ca2+ by either ryanodine (caffeine) ord- myo-inositol 1,4,5-trisphosphate [IP3; produced in response to norepinephrine (NE), 5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors in rat tail arterial rings were evaluated after 4 days of organ culture. Force transients induced by all agents were increased compared with those induced in fresh rings. Stimulation by 10% FCS during culture further potentiated the force and Ca2+ responses to caffeine (20 mM) but not to NE (10 μM), 5-HT (10 μM), or AVP (0.1 μM). The effect was persistent, and SR capacity was not altered after reversible depletion of stores with cyclopiazonic acid. The effects of serum could be mimicked by culture in depolarizing medium (30 mM K+) and blocked by the addition of verapamil (1 μM) or EGTA (1 mM) to the medium, lowering intracellular Ca2+ concentration ([Ca2+]i) during culture. These results show that modulation of SR function can occur in vitro by a mechanism dependent on long-term levels of basal [Ca2+]iand involving ryanodine- but not IP3 receptor-mediated Ca2+release.
Ni2+ transport by the human Na+/Ca2+ exchanger expressed in Sf 9 cells
Authors: Egger M. , Ruknudin A. , Niggli E. , Lederer W. J. , Schulze D. H.
Published: December 2017

The mechanism of Ni2+ block of the Na+/Ca2+exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured63Ni2+entry. The63Ni2+entry was observed in Sf 9 cells expressing the Na+/Ca2+exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+ and the other permits Ni2+ translocation. We conclude that, because Ni2+ entry was inhibited by extracellular Ca2+and enhanced by extracellular Na+, the Ca2+ translocation step moved Ni2+, whereas the Na+ translocation step was inhibited by Ni2+. A model is presented to discuss these findings.
Heterogeneity of mitochondrial matrix free Ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ
Authors: Monteith Gregory R. , Blaustein Mordecai P.
Published: December 2017

The role of mitochondria in Ca2+ homeostasis is controversial. We employed the Ca2+-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca2+ concentration of individual mitochondria ([Ca2+]m) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 μM serotonin (5-HT) evoked modest cytosolic Ca2+ transients [cytosolic free Ca2+ concentration ([Ca2+]cyt) <500 nM; measured with fura 2] and triggered contractions in short-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca2+]m) in only 4% of cells. This revealed heterogeneity in the responses of individual mitochondria, all of which stained with MitoTracker Green FM. In contrast, stimulation with 100 μM ATP evoked large cytosolic Ca2+ transients (>1,000 nM) and induced pronounced, reversible elevation of [Ca2+]m(measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behind the cytosolic Ca2+ transient peak by 3–5 s, and [Ca2+]mdeclined more slowly than did bulk [Ca2+]cyt. The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca2+]cyt. The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca2+]m.
Isoproterenol potentiates α-adrenergic and muscarinic receptor-mediated Ca2+ response in rat parotid cells
Authors: Tanimura Akihiko , Nezu Akihiro , Tojyo Yosuke , Matsumoto Yoshito
Published: December 2017

The effects of the cAMP pathway on the Ca2+ response elicited by phospholipase C-coupled receptor stimulations were studied in rat parotid cells. Although 1 μM isoproterenol (Iso) itself had no effect on the cytosolic Ca2+ concentration, the pretreatment with Iso potentiated Ca2+ responses evoked by phenylephrine. The potentiating effect of Iso was attributed to a shifting of the concentration-response curves of phenylephrine to the left and an increase in the maximal response. Half-maximal potentiation occurred at 3 nM Iso. Iso also potentiated the Ca2+ response elicited by carbachol. The potentiating effect of Iso was mimicked by forskolin (10 μM) and dibutyryl adenosine 3′,5′-cyclic monophosphate (2 mM) and was blocked by 10 μM H-89. Iso potentiated the phenylephrine-induced Ca2+response in the absence of extracellular Ca2+, but Iso did not increase the inositol trisphosphate (IP3) production induced by phenylephrine. These results suggest that the potentiation of the Ca2+ response can be attributed to a sensitization of IP3 receptors by cAMP-dependent protein kinase.
Long-term effects of Ca2+ on structure and contractility of vascular smooth muscle
Authors: Lindqvist Anders , Nordström Ina , Malmqvist Ulf , Nordenfelt Patrik , Hellstrand Per
Published: December 2017

Culture of dispersed smooth muscle cells is known to cause rapid modulation from the contractile to the synthetic cellular phenotype. However, organ culture of smooth muscle tissue, with maintained extracellular matrix and cell-cell contacts, may facilitate maintenance of the contractile phenotype. To test the influence of culture conditions, structural, functional, and biochemical properties of rat tail arterial rings were investigated after culture. Rings were cultured for 4 days in the absence and presence of 10% FCS and then mounted for physiological experiments. Intracellular Ca2+concentration ([Ca2+]i) after stimulation with norepinephrine was similar in rings cultured with and without FCS, whereas force development after FCS was decreased by >50%. The difference persisted after permeabilization with β-escin. These effects were associated with the presence of vasoconstrictors in FCS and were dissociated from its growth-stimulatory action. FCS treatment increased lactate production but did not affect ATP, ADP, or AMP contents. The contents of actin and myosin were decreased by culture but similar for all culture conditions. There was no effect of FCS on calponin contents or myosin SM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration not found after culture without FCS or with FCS + verapamil (1 μM) to lower [Ca2+]i. The decreased force-generating ability after culture with FCS is thus associated with increased [Ca2+]iduring culture and not primarily caused by growth-associated modulation of cells from the contractile to the synthetic phenotype.
Voltage-dependent outward K+current in intermediate cell of stria vascularis of gerbil cochlea
Authors: Takeuchi Shunji , Ando Motonori
Published: December 2017

A voltage-dependent outward K+(KV) current in the intermediate cell (melanocyte) of the cochlear stria vascularis was studied using the whole cell patch-clamp technique. The KV current had an activation threshold voltage of approximately −80 mV, and 50% activation was observed at −42.6 mV. The time courses of activation and inactivation were well fitted by two exponential functions: the time constants at 0 mV were 7.9 and 58.8 ms for activation and 0.6 and 4.3 s for inactivation. The half-maximal activation time was 13.8 ms at 0 mV. Inactivation of the current was incomplete even after a prolonged depolarization of 10 s. This current was independent of intracellular Ca2+. Quinine, verapamil, Ba2+, and tetraethylammonium inhibited the current in a dose-dependent manner, but 4-aminopyridine was ineffective at 50 mM. We conclude that the KV conductance in the intermediate cell may stabilize the membrane potential, which is thought to be closely related to the endocochlear potential, and may provide an additional route for K+ secretion into the intercellular space.
AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells
Authors: Park John M. , Adam Rosalyn M. , Peters Craig A. , Guthrie Paul D. , Sun Zijie , Klagsbrun Michael , Freeman Michael R.
Published: December 2017

Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5′ deletion mutants of a promoter-reporter construct containing 1.7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions −1301 to −881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides −993 to −973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces.
Intracellular elasticity and viscosity in the body, leading, and trailing regions of locomoting neutrophils
Authors: Yanai Masaru , Butler James P. , Suzuki Tomoko , Kanda Akio , Kurachi Masashi , Tashiro Hideo , Sasaki Hidetada
Published: December 2017

To investigate the mechanisms underlying pseudopod protrusion in locomoting neutrophils, we measured the intracellular stiffness and viscosity in the leading region, main body, and trailing region from displacements of oscillating intracellular granules driven with an optical trap. Experiments were done in control conditions and after treatment with cytochalasin D or nocodazole. We found 1) in the body and trailing region, the granules divided into a “fixed” population (too stiff to measure) and a “free” population (easily oscillated; fixed fraction 65%, free fraction 35%). By contrast, the fixed fraction in the leading region was <5%. 2) In the body and trailing region, there was no difference in stiffness or viscosity, but both were sharply lower in the leading region (respectively, 20-fold and 5-fold). 3) Neither cytochalasin D nor nocodazole caused a decrease in stiffness, but both treatments markedly reduced the fixed fraction in the body and trailing region to <20% and <40%, respectively. These observations suggest a discrete lattice structure in the body and trailing region and suggest that the developing pseudopod has a core that is more fluidlike, in the sense of a much lower viscosity and an almost total loss of stiffness. This is consistent with the contraction/solation hypothesis of pseudopodial formation.
Inhibition of Na+-K+-2Cl−cotransport by mercury
Authors: Jacoby Steven C. , Gagnon Edith , Caron Luc , Chang John , Isenring Paul
Published: December 2017

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH−) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl− channels. In this study, we determined whether the Na+-K+-Cl−cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+inhibits Na+-K+-Cl−cotransport, with inhibitor constant ( K i) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additional SH− groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.
Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3
Authors: Bahar Sonya , Gunter Christopher T. , Wu Cheryl , Kennedy Scott D. , Knauf Philip A.
Published: December 2017

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward’s reagent K ( N-ethyl-5-phenylisoxazolium-3′-sulfonate; WRK). Measurements of Cl− binding by35Cl-NMR show that external Cl− binds to band 3 even when Cl− transport is inhibited ∼95% by WRK modification of Glu-681. This indicates that the external Cl− binding site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl− binding even when WRK is bound to Glu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facing Cl−transport site are located nearer to the external surface of the membrane than Glu-681.
Differential regulation of renal sodium-phosphate transporter by glucocorticoids during rat ontogeny
Authors: Guner Yigit S. , Kiela Pawel R. , Xu Hua , Collins James F. , Ghishan Fayez K.
Published: December 2017

The effects of chronic administration of methylprednisolone (MP) were studied on the ontogeny of the renal type II Na-Pi transporter (NaPi-2). Immunoblot analysis showed that MP did not alter the expression of NaPi-2 protein levels in suckling and weanling rats; however, there was an ∼50% decrease in adolescent and adult rats. There was no change in Na-dependent Pi uptake in brush-border membrane vesicles in suckling rats, but there was an almost twofold decrease in adolescent rats induced by MP treatment. MP administration did not alter mRNA levels in suckling or adolescent rats. Dual injections with the glucocorticoid receptor blocker RU-486 (mifepristone) and MP did not reverse the downregulation of NaPi-2 immunoreactive protein levels in adolescent rats. To control for RU-486 antagonism efficiency, Na/H exchanger isoform 3 (NHE3) protein levels were also assayed after injection with RU-486 and MP. As expected, NHE3 protein levels increased after MP injection; however, the increase was blocked in adolescent rats by RU-486. We conclude that there is an age-dependent responsiveness to glucocorticoids and that the marked decrease in NaPi-2 immunoreactive protein levels and activity in adolescent rats is due to posttranscriptional mechanisms.
Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton
Authors: Park J. H. , Okayama N. , Gute D. , Krsmanovic A. , Battarbee H. , Alexander J. S.
Published: December 2017

The effects of hypoxia/aglycemia on microvascular endothelial permeability were evaluated, and the second messenger systems and the cytoskeletal-junctional protein alterations in this response were also examined. Monolayers of human dermal microvascular endothelial cells on microcarrier beads were exposed to either thioglycolic acid (5 mM, an O2 chelator), glucose-free medium, or both stresses together. Permeability measurements were performed over a 90-min time course. Although neither hypoxia alone nor aglycemia alone increased endothelial permeability significantly, the combination of both increased significantly as early as 15 min. Intracellular Ca2+ measurements with fura 2-AM showed that hypoxia/aglycemia treatment increased Ca2+ influx. To determine the second messengers involved in increased permeability, monolayers were incubated for 30 min with the cytosolic Ca2+ scavenger 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mM), a classical protein kinase C (PKC) blocker, Gö-6976 (10 nM), a cGMP-dependent protein kinase (PKG) antagonist, KT-5823 (0.5 μM), or the mitogen-activated protein (MAP) kinase inhibitor PD-98059 (20 μM). Hypoxia/aglycemia-mediated permeability changes were blocked by chelating cell Ca2+, PKC blockade, PKG blockade, and by inhibiting p38 MAP kinase-1. Finally, changes in the binding of junctional proteins to the cytoskeleton under the same conditions were assessed. The concentrations of occludin and pan-reactive cadherin binding to the cytoskeleton were significantly decreased by only both stresses together. However, these effects were also blocked by pretreatment with TMB-8, Gö-6976, KT-5823 (not in occludin), and PD-98059. These data suggest that hypoxia/aglycemia-mediated endothelial permeability may occur through PKC, PKG, MAP kinase, and Ca2+related to dissociation of cadherin-actin and occludin-actin junctional bonds.
Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity
Authors: Baudouin-Legros Maryvonne , Brouillard Franck , Cougnon Marc , Tondelier Danielle , Leclerc Tony , Edelman Aleksander
Published: December 2017

Hypertonicity has pleiotropic effects on cell function, including activation of transporters and regulation of gene expression. It is important to investigate the action of hypertonicity on cystic fibrosis gene expression because cystic fibrosis transmembrane conductance regulator (CFTR), the cAMP-regulated Cl− channel, regulates ion transport across the secretory epithelia, which are often in a hypertonic environment. We found that adding >150 mosmol/l NaCl, urea, or mannitol to the culture medium reduced the amount of CFTR mRNA in colon-derived HT-29 cells in a time-dependent manner. Studies with inhibitors of various kinases [H-89 (protein kinase A inhibitor), bisindolylmaleimide (protein kinase C inhibitor), staurosporine (serine/threonine kinase inhibitor) and herbimycin A (tyrosine kinase inhibitor), SB-203580 and PD-098059 (mitogen-activated protein kinase inhibitors)] showed that CFTR gene expression and its decrease by added NaCl required p38 kinase cascade activity. The CFTR gene activity is regulated at the transcriptional level, since adding NaCl diminished the luciferase activity of HeLa cells transiently transfected with the CFTR promoter. This regulation requires protein synthesis. The complexity of the reactions involved in blocking CFTR gene transcription by NaCl strongly suggests that the decrease in CFTR mRNA is part of a general cell response to hyperosmolar stress.