bluuebunny/crossref_metadata_2025 huggingface dataset:
Contractile properties of diaphragm muscle segments from old mdx and old transgenic mdx mice
Authors: Lynch G. S. , Rafael J. A. , Hinkle R. T. , Cole N. M. , Chamberlain J. S. , Faulkner J. A.
Published: December 2017

Diaphragm muscles of young (4- to 6-mo-old) mdx mice show severe fiber necrosis and have normalized forces and powers 60 and 46% of the values for control C57BL/10 mice. In contrast, microinjection of mdx mouse embryos with a truncated dystrophin minigene has produced young transgenic mdx (tg-mdx) mice with a level of dystrophin expression and structural and functional properties of diaphragm muscle strips measured in vitro not different from those of control mice. Whether dystrophin expression and functional corrections persist for the life span of these animals is not know. We tested the null hypothesis that, in old (24 mo) tg-mdx mice, dystrophin expression is adequate and diaphragm muscle strips have forces and powers not different from values for diaphragm muscle strips from young tg-mdx mice or control mice. Compared with control values, diaphragm muscle strips from old mdx mice had normalized forces and powers of 48 and 31%, respectively. Expression of dystrophin persisted in diaphragm muscles of old tg-mdx mice, and functional properties were not different from diaphragm muscles of young tg-mdx or young or old control mice. These results suggest that, with a transgenic animal approach, dystrophin expression and functional corrections persist for the life span of the animals.
Colocalization of gold-labeled LDL and fibrinogen on platelets: enhanced fibrinogen binding induced by LDL
Authors: Volf I. , Koller E. , Bielek E. , Koller F.
Published: December 2017

Low-density lipoproteins (LDL) specifically bind to the human platelet integrin-alpha IIb and -beta 3 (Koller et al., J. Biol. Chem. 264: 12412-12418, 1989). We show by electron microscopy (EM) that gold (Au)-labeled LDL bind to sites randomly distributed on the surface of platelets in suspension. Within a few minutes, mobile ligand-receptor complexes are translocated from the surface to the open canalicular system (OCS), which finally centralizes as a broad belt. Binding and translocation of Au-LDL are independent of stimulation of platelets by ADP and are completely reversible. Au-fibrinogen shows a strikingly similar, though agonist-dependent, redistribution behavior. Platelets are markedly activated by LDL. This activation is initiated by the binding of LDL to the plasma membrane receptor, and receptor internalization is probably not required for the activation but may instead be one of its consequences. Coincubation with Au-LDL and Au-fibrinogen results in more pronounced activation. The amount of OCS-localized ligands is significantly increased, most likely reflecting enhanced receptor recycling. The two ligands show a tendency to segregate in separate clusters, indicating differences in their postbinding pathways.
Volume transport across tracheal and bronchial airway epithelia in a tubular culture system
Authors: Grubb B. R. , Schiretz F. R. , Boucher R. C.
Published: December 2017

Airway epithelia are thought to play an important role in maintaining the depth (volume) and composition of airway surface liquid (ASL). However, due to the difficulty in measuring airway epithelial volume flow (Jv) and ASL composition, our knowledge of ASL homeostasis is limited. We have developed a permeable tubular culture system (biofiber) suitable for growing airway epithelia on the biofiber luminal surface, which allows measurements of bioelectric properties and Jv. Canine tracheal and bronchial epithelia readily attach, grow to confluence, and develop an electrical potential difference (-10 to -40 mV) across the biofiber. Using a six-hormone-supplemented medium, we detected a significant basal absorptive Jv across both the tracheal cells (0.65 +/- 0.08 microliter.cm-2.h-1) and bronchial cells (2.21 +/- 0.42 microliters.cm-2.h-1), which was significantly reduced by amiloride. Forskolin stimulated a net secretory Jv in tracheal biofibers (-0.56 +/- 0.19 microliter.h-1.cm-2) only. When the culture medium was supplemented with cholera toxin (CT), the basal absorptive Jv was significantly reduced in the bronchial biofibers and the tracheal biofibers exhibited net secretion. The forskolin-stimulated secretory Jv in the tracheal biofibers was significantly greater in the presence of CT than in its absence (-1.30 +/- 0.29 microliters.h-1.cm-2), whereas bronchial biofibers exhibited no significant Jv response to forskolin. We conclude that the Jv measured in tubular culture is highly dependent on the region from which the cells originated as well as the composition of the culture medium. Use of the biofiber culture system to study airway epithelia should give further insight into factors regulating Jv and composition of ASL.
A volume-sensitive, IP3-insensitive Ca2+ store in vascular endothelial cells
Authors: Jena M. , Minore J. F. , O'Neill W. C.
Published: December 2017

The mechanism by which cell swelling and other physical forces increase the intracellular Ca2+ concentration ([Ca2+]i) is poorly defined. In vascular endothelial cells, the increase in [Ca2+]i after hypotonic swelling was independent of external Ca2+ and membrane potential, was not blocked by La3+ or Gd3+, and was prevented by thapsigargin, indicative of intracellular release. This release also occurred after depletion of agonist-sensitive Ca2+ stores. In cells in which the plasma membrane was permeabilized with saponin, hypotonic medium stimulated release of 45Ca2+ from a thapsigargin-sensitive site. Isosmotic substitutions with sucrose or urea revealed that this release was due specifically to swelling and not to changes in osmolarity or ion concentrations. This volume-sensitive release was inhibited by high concentrations of La3+ and Gd3+ in a time-dependent manner, suggesting inhibition from within the storage compartment. Release was not inhibited by ruthenium red or by prior stimulation with inositol 1,4,5-trisphosphate (IP3), indicating that the volume-sensitive storage site is distinct from mitochondria and from stores sensitive to ryanodine or IP3. The results suggest the presence of a novel, stretch-activated Ca2+ store in endothelial cells that could contribute to their mechanosensitivity.
Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-
Authors: Taguchi Y. , Ono Y. , Lin L. , Storey B. T. , Dodgson S. J. , Forster R. E.
Published: December 2017

To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.
Unopposed phosphatase action initiates ezrin dysfunction: a potential mechanism for anoxic injury
Authors: Chen J. , Mandel L. J.
Published: December 2017

Because extensive kinase inhibition during anoxia has previously been reported, we investigated the role of kinase inhibition in anoxic cell injury by studying the effects of kinase inhibitors on a membrane-microvillar cytoskeleton linker protein, ezrin, in rabbit renal proximal tubules. Like anoxia, kinase inhibitors caused ezrin dephosphorylation in a dose-dependent manner under normoxia. The kinase inhibitor chelerythrine also induced ezrin dissociation from the cytoskeleton, i.e., causing it to lose its membrane-cytoskeleton linker function. Blockage of kinase inhibitor-induced ezrin dephosphorylation by a phosphatase inhibitor, calyculin A, ameliorated ezrin dissociation. Stimulation of the kinase during anoxia did not improve ezrin phosphorylation, suggesting that anoxia-induced kinase inhibition might be due to the lack of the substrate ATP. Finally, in vitro study of ezrin phosphatase revealed no increase in its activity during anoxia, suggesting the principal role of kinase inhibition in the loss of the linker function of ezrin during anoxia. Our results provide, for the first time at the molecular level, a mechanistic insight into anoxic cell injury caused by unopposed phosphatase action.
ARF-independent inhibition of the carbachol-induced contractions by brefeldin A in intestinal smooth muscle
Authors: Loirand G. , Cario-Toumaniantz C. , Chardin P. , Pacaud P.
Published: December 2017

The aim of this study was to determine whether an ADP ribosylation factor (ARF)-regulated pathway is involved in the carbachol-induced contraction in rat intestinal smooth muscle. Brefeldin A, a known inhibitor of the guanine nucleotide exchange activity on ARF, reversibly inhibited the carbachol-induced contraction in intact ileal muscle strips, whereas the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced increases in the Ca2+ sensitivity of myofilaments in beta-escin-permeabilized strips were not affected. The high-K(+)-induced contraction in intact strips was also inhibited by brefeldin A. In isolated ileal myocytes, brefeldin A inhibited the Ca2+ channel current, indicating that the inhibitory effect of brefeldin A in intact cells is related to an inhibition of voltage-dependent Ca2+ channels. Furthermore, the loading of permeabilized strips with the combination of the recombinant fully myristoylated ARF1, the guanine nucleotide exchange factor ARNO, and guanosine 5'-triphosphate did not change the tone at constant pCa (6.45) and did not modify the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca2+ sensitization. Taken together, these findings suggest that an ARF-dependent pathway is not involved in the carbachol-induced contraction.
CGP-42112 partially activates human monocytes and reduces their stimulation by lipopolysaccharides
Authors: Egidy G. , Friedman J. , Viswanathan M. , Wahl L. M. , Saavedra J. M.
Published: December 2017

CGP 42112, a high-affinity ligand for angiotensin II AT2 receptors, binds to rat macrophage/microglia lacking AT2 receptors. Here we report that CGP-42112 binds to human monocytes and exerts specific effects. Binding studies revealed a single site, highly specific for CGP-42112, not displaceable by angiotensin II, angiotensin fragments, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4, IL-10, transforming growth factor-beta, or lipopolysaccharide (LPS). Incubation of purified human monocytes in serum-free medium with CGP-42112 enhanced, in a dose-dependent manner, cell attachment to fibronectin and collagen-coated dishes as well as matrix metalloproteinase-9 secretion. CGP-42112 did not promote cytokine secretion. In contrast, when added in the presence of low doses of LPS, CGP-42112 reduced the LPS-stimulated secretion of TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6 without affecting IL-10 and decreased the LPS-stimulated matrix metalloproteinase-9 activity. Additionally, CGP-42112 inhibited the increase in protein kinase A activity produced by LPS. Our results indicate that CGP-42112 may modulate monocyte activation through binding to a novel receptor.
Inhibition of oxidative phosphorylation, vascular tone, and [Ca2+]i in the perfused rat tail artery
Authors: Tran N. N. , Robert A. , Atkinson J. , Capdeville-Atkinson C.
Published: December 2017

We investigated the possibility that the inhibition of oxidative phosphorylation in vascular smooth muscle attenuates norepinephrine- or KCl-evoked vasoconstriction with no change in mobilization of intracellular calcium concentration ([Ca2+]i). Experiments were performed in perfused segments of the rat tail artery loaded with the intracellular calcium dye fura 2, in the absence and presence of dinitrophenol or sodium cyanide; inhibition of oxidative phosphorylation was evaluated from the fall in intracellular ATP levels. The metabolic inhibitors reduced vasoconstriction with no change in [Ca2+]i handling, suggesting that 1) inhibition of oxidative phosphorylation attenuates vasoconstriction via a mechanism downstream of [Ca2+]i, and 2) [Ca2+]i homeostasis (both increases and decreases in [Ca2+]i) can be maintained in the presence of inhibitors of oxidative phosphorylation.
Modulation of high- and low-voltage-activated calcium currents in smooth muscle by calcium
Authors: McCarron J. G. , McGeown J. G. , Walsh J. V. , Fay F. S.
Published: December 2017

Ca2+ currents (ICa) and cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in isolated gastric myocytes from Bufo marinus using whole cell voltage clamp and fura 2, respectively. After a conditioning train of depolarizing pulses, high-voltage-activated ICa (test potential of +10 mV) was increased, returning to control values after approximately 85 s. This enhancement was [Ca2+]c dependent, with a maximal increase at approximately 600 nM [Ca2+]c. During the conditioning train, ICa measured at 70 ms, which provides a measure of high-voltage-activated current, initially decreased with each successive pulse to a minimum of 56 +/- 5% of the first pulse in the train. Thereafter, the 70-ms current showed considerable recovery. Blockade of calmodulin activity with a peptide (RS20) or calmidazolium did not affect the early inhibition but did abolish current recovery. A peptide inhibitor of calmodulin-dependent protein kinase II (CK3AA) had similar effects. Substraction of currents measured in the presence and absence of RS20 revealed a 2-s delay between the start of the train and the onset of current enhancement. It was also observed that low-voltage-activated current (test potential of -17 mV) was reduced to 76 +/- 7% of control 5 s after the conditioning train; this inhibition recovered to 92 +/- 4% after 35 s and was not dependent on [Ca2+]c elevation.
Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29
Authors: Sand P. , Svenberg T. , Rydqvist B.
Published: December 2017

The patch-clamp technique was used to study the effects of carbachol (CCh) on HT-29 cells. During CCh exposure, the cells ( n = 23) depolarized close to the equilibrium potential for Cl−([Formula: see text]; −48 mV) and the membrane potential then started to oscillate (16/23 cells). In voltage-clamp experiments, similar oscillations in whole cell currents could be demonstrated. The whole cell conductance increased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). In substitution experiments (22 mM Cl− in bath solution,[Formula: see text]= 0 mV), the reversal potential changed from −41.6 ± 2.2 mV (means ± SE, n = 9) to −3.2 ± 2.0 mV (means ± SE, n = 7). When the cells were loaded with the calcium-sensitive fluorescent dye, fluo 3, and simultaneously patch clamped, CCh caused a synchronous oscillating pattern of fluorescence and membrane potential. In cell-attached patches, the CCh-activated currents reversed at a relative membrane potential of 1.9 ± 3.7 mV (means ± SE, n = 11) with control solution in the pipette and at 46.2 ± 5.3 mV (means ± SE, n = 10) with a 15 mM Cl− solution in the pipette. High K+ (144 mM) did not change the reversal potential significantly ( P ≤ 0.05, n = 8). In inside-out patches, calcium-dependent Cl−channels could be demonstrated with a conductance of 19 pS ( n = 7). It is concluded that CCh causes oscillations in membrane potential that involve calcium-dependent Cl−channels and a K+ permeability.
Effect of osmolarity on LDL binding and internalization in hepatocytes
Authors: Krämer-Guth Annette , Busch Gillian L. , Kaba Nubia Kristen , Schwedler Susanne , Wanner Christoph , Lang Florian
Published: December 2017

The present study has been performed to elucidate a possible role of cell volume in low-density lipoprotein (LDL) binding and internalization (LDLb+i). As shown previously, increase of extracellular osmolarity (OSMe) and K+ depletion, both known to shrink cells, interfere with the formation of clathrin-coated pits and thus with LDLb+i. On the other hand, alterations of cell volume have been shown to modify lysosomal pH, which is a determinant of LDLb+i. LDLb+i have been estimated from heparin-releasable (binding) or heparin-insensitive (internalization) uptake of 125I-labeled LDL. OSMe was modified by alterations of extracellular concentrations of ions, glucose, urea, or raffinose. When OSMe was altered by varying NaCl concentrations, LDLb+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe and LDLb+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to altered affinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 μg/ml at 219, 293, and 435 mosM, respectively. A 25% increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreased lysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMe decreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by 0.17 ± 0.02) pH. Partial replacement of extracellular Na+ with K+ had little effect on LDLb+i, although it swelled hepatocytes and increased lysosomal and cytosolic pH. Hypertonic glucose, urea, or raffinose did not exert similar effects despite a shrinking effect of hypertonic raffinose. Monensin, which completely dissipates lysosomal acidity, virtually abolished LDLb+i. In conclusion, the observations reveal a significant effect of ionic strength on LDLb+i. The effect is, however, not likely to be mediated by alterations of cell volume or alterations of lysosomal pH.
Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells
Authors: Korenaga Risa , Ando Joji , Kosaki Keisuke , Isshiki Masashi , Takada Yoshio , Kamiya Akira
Published: December 2017

To explore the mechanism of shear stress-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1) expression in murine endothelial cells (ECs), we examined the effect of shear stress on VCAM-1 gene transcription and assessed the cis-acting elements involved in this phenomenon. VCAM-1 mRNA expression was downregulated at the transcriptional level as defined by nuclear run-on assay and transient transfection of VCAM-1 promoter-luciferase gene constructs. The luciferase assay on the VCAM-1 deletion mutants revealed that the cis-acting element is contained between −694 and −329 bp upstream from the transcription initiation site. Gel shift assay using overlapping oligonucleotide probes of this region showed that oligonucleotides containing a double AP-1 consensus sequence (TGACTCA) formed distinct complexes with nuclear proteins extracted from shear-stressed cells. Mutation of either one or both of two AP-1 consensus sequences completely abolished the ability of the promoter to respond to shear stress. These results suggest that fluid shear stress downregulates the transcription of the VCAM-1 gene via an upstream cis-element, a double AP-1 consensus sequence, in murine lymph node venule ECs.
Aqp1 expression in erythroleukemia cells: genetic regulation of glucocorticoid and chemical induction
Authors: Moon Chulso , King Landon S. , Agre Peter
Published: December 2017

The aquaporin-1 (AQP1) water channel protein is expressed in multiple mammalian tissues by several different developmental programs; however, the genetic regulation is undefined. The proximal promoter of mouse Aqp1 contains multiple putative cis-acting regulatory elements, and mouse erythroleukemia (MEL) cells are a well-characterized model for erythroid differentiation. Corticosteroid or dimethyl sulfoxide (DMSO) exposure induces AQP1 protein expression in MEL cells, and transcriptional regulation was investigated by transient transfections with Aqp1 promoter-reporter constructs. Dexamethasone induction is abrogated by deletion of two glucocorticoid response elements −0.5 kilobases (kb) from the transcription initiation site. Mutation of the GATA element at −0.62 kb has no effect, whereas mutation of the CACCC site at −37 bp significantly reduces DMSO-induced promoter activity. Hydroxyurea induces expression of AQP1 protein without acting through the proximal promoter. The MEL cell line is a reproducible erythroid model system for studying transcriptional regulation of the Aqp1 gene while determining the consequences on AQP1 protein biosynthesis.
Hypoxia enhances induction of endothelial ICAM-1: role for metabolic acidosis and proteasomes
Authors: Zünd Gregor , Uezono Shoichi , Stahl Gregory L. , Dzus Andrea L. , McGowan Francis X. , Hickey Paul R. , Colgan Sean P.
Published: December 2017

Intercellular adhesion molecule 1 (ICAM-1) is an important molecule in promotion of polymorphonuclear neutrophil transendothelial migration during inflammation. Coincident with many inflammatory diseases is tissue hypoxia. Thus we hypothesized that combinations of hypoxia and inflammatory stimuli may differentially regulate expression of endothelial ICAM-1. Human endothelial cells were exposed to hypoxia in the presence or absence of added lipopolysaccharide (LPS) and examined for expression of functional ICAM-1. Although hypoxia alone did not induce ICAM-1, the combination of LPS and hypoxia enhanced (3 ± 0.4-fold over normoxia) ICAM-1 expression. Combinations of hypoxia and LPS significantly increased lymphocyte binding, and such increases were inhibited by addition of anti-ICAM-1 antibodies or antisense oligonucleotides. Hypoxic endothelia showed a >10-fold increase in sensitivity to inhibitors of proteasome activation, and combinations of hypoxia and LPS enhanced proteasome-dependent cytoplasmic-to-nuclear localization of the nuclear transcription factor-κB p65 ( Rel A) subunit. Such proteasome activation correlated with hypoxia-evoked decreases in both extracellular and intracellular pH. We conclude from these studies that endothelial hypoxia provides a novel, proteasome-dependent stimulus for ICAM-1 induction.
Nitric oxide enhances hydrogen peroxide-mediated endothelial permeability in vitro
Authors: Okayama Naotsuka , Kevil Christopher G. , Correia Loisann , Jourd’Heuil David , Itoh Makoto , Grisham Matthew B. , Alexander J. Steven
Published: December 2017

The objective of this study was to evaluate the effects of nitric oxide (NO) on H2O2-mediated endothelial permeability. H2O2(0.1 mM) increased permeability at 90 min to 298% of baseline. Spermine NONOate (SNO), an NO donor, at 0.1 or 1 mM did not alter permeability. However, 0.1 mM H2O2+ 1 mM SNO increased permeability to 764%, twice that of 0.1 mM H2O2alone. These treatments were not directly toxic to endothelial cells. This NO effect was concentration dependent, inasmuch as 0.1 mM SNO did not significantly change H2O2-mediated permeability. The NO-enhanced, H2O2-dependent permeability required the simultaneous presence of NO and H2O2, inasmuch as preincubation with SNO for 30 min followed by 0.1 mM H2O2did not alter permeability. Staining of endothelial junctions showed widening of the intercellular space only in junctions of cells exposed to H2O2(0.1 mM) + SNO (1 mM). Furthermore, NO did not affect H2O2metabolism by endothelial cells but significantly depleted intracellular glutathione. This reduction of cell glutathione produced by NO exposure recovered 15–30 min after removal of the NO donor. NO-enhanced permeability was completely blocked by methionine (1 mM), a scavenger of reactive oxygen species, and by the iron chelator desferrioxamine (0.1 mM). These results suggest that NO may exacerbate the effects of H2O2-dependent increase in endothelial monolayer permeability via the iron-catalyzed formation of reactive oxygen metabolites.
Influence of bafilomycin A1on pHi responses in cultured rabbit nonpigmented ciliary epithelium
Authors: Wu Qiang , Delamere Nicholas A.
Published: December 2017

Aqueous humor secretion is in part linked to [Formula: see text]transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H+-ATPase, the present study was conducted to examine whether vacuolar H+-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H+-ATPase expression as judged by H+-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K+-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 μM; an inhibitor of vacuolar H+-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), omeprazole, low-Cl−medium, [Formula: see text]-free medium, or Na+-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H+-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.
Functional coupling in bovine ciliary epithelial cells is modulated by carbachol
Authors: Stelling J. W. , Jacob T. J. C.
Published: December 2017

The functional coupling of the ciliary epithelium was studied in isolated pairs (couplets) of pigmented ciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE) cells using the whole cell patch clamp and the fluorescent dye lucifer yellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2–5 MΩ electrode, containing 1–2 mM lucifer yellow, in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells were coupled. The electrical coupling of the cells was also studied by calculating the capacitance (using the time-domain technique), assuming a “supercell” model for coupled cells. The mean capacitance of coupled pairs was 79.8 ± 4.3 (SE) pF ( n = 47) compared with single cell capacitances of 36.8 ± 3.4 pF ( n = 10) for PCE cells and 38.1 ± 3.1 pF ( n = 15) for NPCE cells. Octanol, carbachol (CCh), and raised extracellular Ca2+ concentration ([Ca2+]o) all caused uncoupling in pairs (couplets) of coupled NPCE and PCE cells. At room temperature (22–24°C), the capacitance of the couplets decreased from 70.5 ± 8.0 to 48.0 ± 5.2 pF ( n = 5) when exposed to octanol (1 mM), from 73.8 ± 9.2 to 43.2 ± 9.5 pF ( n = 4) when exposed to CCh (100 μM), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF ( n = 4) when exposed to 10 mM [Ca2+]o. The response to CCh was dose dependent; at higher temperatures of 34–37°C, 10 μM CCh caused a 38% reduction in capacitance, from 53.7 ± 9.7 to 33.5 ± 3.3 pF ( n = 7) with a half-time of 249 s, and 100 μM CCh caused a 49% reduction in capacitance, from 51.3 ± 5.6 to 26.0 ± 2.4 pF ( n = 7) with a half-time of 124 s. After pairs uncoupled and the uncoupling agent was washed out, the cell pairs often exhibited an increase in capacitance that we interpreted as “recoupling” or a reopening of the gap junctional communication pathway; the half-time for this process was 729 s after uncoupling with 100 μM CCh and 211 s after uncoupling with 10 μM CCh. This interpretation was confirmed optically by the spread of lucifer yellow into both cells of an uncoupled pair with a time course corresponding to the increase in electrical coupling. The controllable coupling of ciliary epithelial cells extends the idea of a functional syncytium involved in active transport. PCE cells take up solute and water from the blood, which then cross to NPCE cells via gap junctions and from there are secreted into the posterior chamber of the eye. Modulation of the coupling between NPCE and PCE cells may provide a mechanism to control secretion.
Mechanical stimulation increases expression of acetylcholinesterase in cultured myotubes
Authors: Hubatsch Douglas A. , Jasmin Bernard J.
Published: December 2017

We tested the hypothesis that acetylcholinesterase (AChE) expression in skeletal muscle cells is increased by passive mechanical stimulation. To this end, primary cultures of myotubes were subjected to repeated cycles of stretch-relaxation for 5 min, 30 min, 3 h, and 24 h, using the Flexercell FX-2000 strain unit. Although mechanical stimulation did not affect AChE expression at early time points, it led to a significant increase (42%; P < 0.05) in total AChE activity at 24 h. This increase reflected a general elevation in the activity of all AChE molecular forms as opposed to a preferential increase in a specific form. Tetrodotoxin (TTX) treatment did not prevent the increase in AChE expression, whereas nifedipine partially blocked it. These changes in enzyme expression were accompanied by increases in the levels of AChE mRNA, suggesting the involvement of pretranslational regulatory mechanisms. Together, these results illustrate that, in addition to neural activation and trophic factors, passive mechanical forces modulate expression of AChE in skeletal muscle cells. Because TTX did not prevent the increase in AChE expression, it appears that the effects of mechanical stimulation are independent of electrical activity, which further indicates the use of an alternate signaling pathway.
Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart
Authors: Poderoso Juan J. , Peralta Jorge G. , Lisdero Constanza L. , Carreras Maria Cecilia , Radisic Marcelo , Schöpfer Francisco , Cadenas Enrique , Boveris Alberto
Published: December 2017

Isolated rat heart perfused with 1.5–7.5 μM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 μM NO, n = 18, P < 0.05) and to 1.2 ± 0.1 μM O2 ⋅ min−1 ⋅ g tissue−1 (10 μM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent ( r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 μM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1–0.3 μM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.
Role of H2O2and heme-containing O2 sensors in hypoxic regulation of tyrosine hydroxylase gene expression
Authors: Kroll Sandra L. , Czyzyk-Krzeska Maria F.
Published: December 2017

In the current study, we investigated links between O2-regulated H2O2formation and the hypoxic induction of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in O2-sensitive PC-12 cells. During exposure of PC-12 cells to 5% O2, H2O2concentration decreased by 40% as measured with 2′,7′-dichlorofluorescein (DCF). Treatment with H2O2reduced TH mRNA during normoxia and prevented the induction of TH mRNA during hypoxia. Treatment with catalase or N-(2-mercaptopropionyl)-glycine, a reducing antioxidant agent that decreases H2O2concentration, also induced TH mRNA. Deferoxamine (DF), an iron chelator, failed to affect H2O2formation but induced TH mRNA in normoxia and hypoxia. CoCl2 led to a decrease in H2O2at 20 h of treatment but induced TH mRNA during normoxia and hypoxia before it affected H2O2. In conclusion, TH gene expression correlates inversely with H2O2formation. DF and Co2+ seem to affect TH gene expression in the mechanism downstream from the H2O2formation rather than by interfering with the H2O2-generating activity of the O2 sensor.
Polyamine deficiency alters EGF receptor distribution and signaling effectiveness in IEC-6 cells
Authors: McCormack Shirley A. , Blanner Patrick M. , Zimmerman Barbara J. , Ray Ramesh , Poppleton Helen M. , Patel Tarun B. , Johnson L. R.
Published: December 2017

Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipatated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.
Amino acids stimulate phosphorylation of p70 S6k and organization of rat adipocytes into multicellular clusters
Authors: Fox Heather L. , Kimball Scot R. , Jefferson Leonard S. , Lynch Christopher J.
Published: December 2017

In previous studies we have shown that rat adipocytes suspended in Matrigel and placed in primary culture migrate through the gel to form multicellular clusters over a 5- to 6-day period. In the present study, phosphorylation of the insulin-regulated 70-kDa ribosomal protein S6 kinase (p70 S6k ) was observed within 30 min of establishment of adipocytes in primary culture. Two inhibitors of the p70 S6k signaling pathway, rapamycin and LY-294002, greatly reduced phosphorylation of p70 S6k and organization of adipocytes into multicellular clusters. Of all the components of the cell culture medium, amino acids, and in particular a subset of neutral amino acids, were found to promote both phosphorylation of p70 S6k and cluster formation. Lowering the concentrations of amino acids in the medium to levels approximating those in plasma of fasted rats decreased both phosphorylation of p70 S6k and cluster formation. Furthermore, stimulation of p70 S6k phosphorylation by amino acids was prevented by either rapamycin or LY-294002. These findings demonstrate that amino acids stimulate the p70 S6k signaling pathway in adipocytes and imply a role for this pathway in multicellular clustering.
A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry
Authors: Lytle Christian , McManus Thomas J. , Haas Mark
Published: December 2017

In the duck red blood cell, Na-K-2Cl cotransport exhibits two modes of ion movement: net cotransport and obligate cation exchange. In high-K cells, the predominant exchange is K/K (or K/Rb). In high-Na cells, it becomes Na/Na (or Na/Li). Both represent partial reactions in which a fully loaded carrier releases part of its cargo, rebinds fresh ions, and returns back across the membrane fully loaded. Net cotransport occurs when the carrier unloads completely and returns empty. This mode has a fixed stoichiometry of 1Na:1K:2Cl under all conditions tested. The ion requirements of the two exchanges differ: K/K exchange requires only K and Cl outside but all three ions inside. Na/Na exchange requires all three ions outside but only Na inside. We propose a simple model in which the carrier can only move when either fully loaded or completely empty and in which the ions bind in a strictly ordered sequence. For example, externally, a Na binds first and then a Cl, followed by a K and a second Cl. Internally, the first on is the first off (glide symmetry), so the Na is released first and then the first Cl, followed by the K and finally by the second Cl. Only then can the empty form return to the outside to start a new cycle.
[Ca2+]i-reducing action of cAMP in rat pancreatic β-cells: involvement of thapsigargin-sensitive stores
Authors: Yaekura Kazuro , Yada Toshihiko
Published: December 2017

In the present study, we examined the ability of adenosine 3′,5′-cyclic monophosphate (cAMP) to reduce elevated levels of cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic β-cells. [Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single β-cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 μM), and an incretin glucagon-like peptide-1-(7–36) amide (10−9 M), as well as by glucose (16.7 mM). The [Ca2+]i-reducing effects of cAMP were greater at elevated glucose (8.3–16.7 mM) than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca2+]i-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10–100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in β-cells. [Ca2+]i-reducing effects of cAMP were inhibited by 0.3 μM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. In contrast, [Ca2+]i-reducing effects of cAMP were not altered by ryanodine, an ER Ca2+-release inhibitor, Na+-free conditions, or diazoxide, an ATP-sensitive K+ channel opener. In conclusion, the cAMP-PKA pathway reduces [Ca2+]ielevation by sequestering Ca2+ in thapsigargin-sensitive stores. This process does not involve, but is potentiated by, activation of β-cell metabolism. Together with the known [Ca2+]i-increasing action of cAMP, our results reveal dual regulation of β-cell [Ca2+]iby the cAMP-signaling pathway and by a physiological incretin.
Basic fibroblast growth factor destabilizes osteonectin mRNA in osteoblasts
Authors: Delany Anne M. , Canalis Ernesto
Published: December 2017

Osteonectin (secreted protein acidic and rich in cysteine, 40-kDa basement membrane) is a glycoprotein abundantly expressed in bone and in other tissues undergoing active remodeling. Fibroblast growth factors (FGFs) are important in skeletal development and fracture repair, events associated with extracellular matrix remodeling. We used the murine osteoblastic cell line MC3T3 to determine whether basic FGF (bFGF) regulates osteonectin expression in bone. Northern blot analysis showed that bFGF decreased osteonectin transcripts in a dose- and time-dependent manner. This regulation was independent of the mitogenic effect of bFGF but was dependent on new protein synthesis. Immunoprecipitation of [35S]methionine-cysteine osteoblast-conditioned medium and cell layer proteins showed that bFGF decreased osteonectin synthesis. Nuclear runoff assays failed to reveal regulation of osteonectin gene transcription by bFGF. However, bFGF dramatically decreased the stability of osteonectin mRNA in transcriptionally arrested osteoblasts. This destabilization of osteonectin mRNA may be one means by which bFGF regulates extracellular matrix remodeling.
Oxidant-induced arachidonic acid release and impairment of fatty acid acylation in vascular smooth muscle cells
Authors: Cane Agnès , Breton Michelyne , Koumanov Kamen , Béréziat Gilbert , Colard Odile
Published: December 2017

Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with A[Formula: see text], a process not involving phospholipase A2(PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327–337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2or A[Formula: see text] as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.
ANG II controls Na+-K+( NH 4 + )-2Cl−cotransport via 20-HETE and PKC in medullary thick ascending limb
Authors: Amlal Hassane , LeGoff Christian , Vernimmen Catherine , Soleimani Manoocher , Paillard Michel , Bichara Maurice
Published: December 2017

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na+-K+([Formula: see text])-2Cl−cotransport. ANG II at 10−16to 10−12 M inhibited 30–50% ( P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10−12 M ANG II level cotransport activity; eventually, 10−6 M ANG II stimulated 34% cotransport activity ( P < 0.003). Inhibition by 10−12M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10−12 M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10−6 M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus ≤10−12 M ANG II inhibits via 20-HETE, whereas ≥5 × 10−11 M ANG II stimulates via PKC Na+-K+([Formula: see text])-2Cl−cotransport; all ANG II effects involve AT1 receptors and PLC activation.
Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal tracheal xenografts in SCID mice
Authors: Tirouvanziam Rabindra , Desternes Mama , Saari Anouar , Puchelle Edith , Péault Bruno , Chinet Thierry
Published: December 2017

We measured the bioelectric properties of 14 cystic fibrosis (CF) and 33 non-CF human fetal tracheal xenografts in severe combined immunodeficiency (SCID) mice. All xenografts exhibited a mature airway-type epithelium irrespective of their gestational age, duration of engraftment, and genotype. The in vivo potential difference and the in vitro baseline short-circuit current ( I sc) were significantly higher in non-CF than in CF xenografts. In non-CF xenografts, sequential addition of amiloride, forskolin, and ATP resulted in a 39.4% decrease, a 24.1% increase, and a 43.6% increase in I sc, respectively. In CF xenografts, forskolin had no significant effect on I sc, whereas amiloride- and ATP-induced changes in I sc were proportionally higher than in non-CF xenografts (−60.0 and +68.8%, respectively). These results indicate that the bioelectric properties of non-CF xenografts are similar to those of postnatal airways and that CF xenografts exhibit lower baseline electrogenic activity than non-CF xenografts but similar regulation of ion transport processes to postnatal CF airways. This model of mature human fetal tracheal mucosa may help gain insight into early CF airway pathogenesis.
Effect of cell swelling on membrane and cytoplasmic distribution of pICln
Authors: Emma Francesco , Breton Sylvie , Morrison Rebecca , Wright Stephen , Strange Kevin
Published: December 2017

pICln is found ubiquitously in mammalian cells and is postulated to play a critical role in cell volume regulation. Mutagenesis studies led to the proposal that pICln is a swelling-activated anion channel. However, recent studies in Madin-Darby canine kidney cells and endothelial cells have shown that the protein is localized primarily to the cytoplasm. It has therefore been postulated that activation involves reversible translocation of pICln from the cytoplasm and insertion into the plasma membrane. We tested this hypothesis using several different approaches. Fractionation of C6 glioma cells into plasma membrane- and cytoplasm-containing fractions demonstrated that ∼90% of the recovered pICln was confined to the cytosol. Swelling had no effect on the relative amount of protein present in the plasma membrane fraction. Immunofluorescence microscopy revealed that pICln is localized primarily, if not exclusively, to the cytoplasm of swollen and nonswollen cells. Similarly, transfection of cells with a green fluorescent protein-labeled pIClnconstruct failed to reveal any membrane localization of the protein. These findings do not support the hypothesis that pICln is a volume regulatory anion channel activated by swelling-induced membrane insertion.