bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of Na+-K+-2Cl−cotransporter in neuronal cells by excitatory neurotransmitter glutamate
Authors: Sun Dandan , Murali Sangita G.
Published: December 2017

Na+-K+-2Cl−cotransporters are important in renal salt reabsorption and in salt secretion by epithelia. They are also essential in maintenance and regulation of ion gradients and cell volume in both epithelial and nonepithelial cells. Expression of Na+-K+-2Cl−cotransporters in brain tissues is high; however, little is known about their function and regulation in neurons. In this study, we examined regulation of the Na+-K+-2Cl−cotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitive K+ influx, and protein expression was evaluated by Western blot analysis. Glutamate was found to induce a dose- and time-dependent stimulation of Na+-K+-2Cl−cotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonist N-methyl-d-aspartic acid (NMDA) and the metabotropic receptor agonist (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD) significantly stimulated the cotransport activity in these cells. NMDA-mediated stimulation of the Na+-K+-2Cl−cotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate. trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)-α-methyl-(4-carboxyphenyl)glycine. The results demonstrate that Na+-K+-2Cl−cotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.
Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion
Authors: Gopalakrishnan Shobha , Raman Narayan , Atkinson Simon J. , Marrs James A.
Published: December 2017

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion.
Gating kinetics of ShakerK+ channels are differentially modified by general anesthetics
Authors: Correa Ana M.
Published: December 2017

The ShakerB K+ channel was used as a model voltage-gated channel to probe the interaction of volatile general anesthetics with gating mechanisms. The effects of three anesthetics, chloroform (CHCl3), isoflurane, and halothane, were studied using recombinant native and mutant Shaker channels expressed in Xenopus oocytes. Gating currents and macroscopic ionic currents were recorded with the cut-open oocyte voltage-clamp technique. The effects of CHCl3 and isoflurane on gating kinetics of noninactivating mutants were opposite, whereas halothane had no effect. The effects on ionic currents were also agent dependent: CHCl3 and halothane produced a reduction of the macroscopic conductance, whereas isoflurane increased it. The results indicate that the gating machinery of the channel is mostly insensitive to the anesthetics during activation until near the open state. The effects on the conductance are mainly due to changes in the transitions in and out of the open state. The data give support to direct protein-anesthetic interactions. The magnitude and nature of the effects invite reconsideration of Shaker-like K+ channels as important sites of action of general anesthetics.
Hypotonicity activates transcription through ERK-dependent and -independent pathways in renal cells
Authors: Zhang Zheng , Yang Xiao-Yan , Cohen David M.
Published: December 2017

Acute hypotonic shock (50% dilution of medium with sterile water, but not with isotonic NaCl) activated the extracellular signal response kinase (ERK) mitogen-activated protein (MAP) kinases in renal medullary cells, as measured by Western analysis with a phospho-ERK-specific antibody and by in vitro kinase assay of epitope-tagged ERKs immunoprecipitated from stable HA-ERK transfectants. Hypotonicity also activated the transcription factor and ERK substrate Elk-1 in a partially PD-98059-sensitive fashion, as assessed by chimeric reporter gene assay. Consistent with these data, hypotonic stress activated transcription of the immediate-early gene transcription factor Egr-1 in a partially PD-98059-sensitive fashion. Hypotonicity-inducible Egr-1 transcription was mediated in part through 5′-flanking regions containing serum response elements and in part through the minimal Egr-1 promoter. Elimination of the Ets motifs adjacent to key regulatory serum response elements in the Egr-1 promoter diminished the effect of hypotonicity but failed to abolish it. Interestingly, hypotonicity also transiently activated p38 and c-Jun NH2-terminal kinase 1, as determined by immunoblotting with anti-phospho-MAP kinase antibodies. Taken together, these data strongly suggest that hypotonicity activates immediate-early gene transcription in renal medullary cells via MAP kinase kinase-dependent and -independent mechanisms.
Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl− channel
Authors: Stroffekova Katarina , Kupert Elena Y. , Malinowska Danuta H. , Cuppoletti John
Published: December 2017

Rabbit and human ClC-2G Cl− channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl− channel and to determine which amino acid residues play a role in this acid activation. Channel open probability ( P o) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+concentration (that is, extracellular pH, pH trans ), with an apparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased P o threefold at pH trans 7.4, at which the channel normally exhibits low P o. With extracellular pH reduction (protonation) or amidation, increased P o was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416–419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl− channel by extracellular acid.
Purification of active Na+-K+-ATPase using a new ouabain-affinity column
Authors: Yingst Douglas R. , Yang Shang-You , Schiebinger Rick
Published: December 2017

Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from ∼2 to 15 μmol Pi ⋅ mg−1 ⋅ min−1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the α-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.
In vivo and in vitro induction of c-fos in avian exocrine salt gland cells
Authors: Hildebrandt Jan-Peter , Gerstberger Rüdiger , Schwarz Marion
Published: December 2017

Osmotic stress in ducklings ( Anas platyrhynchos) results in salt secretion and adaptive cell proliferation and differentiation in the nasal glands. We investigated whether osmotic stress in vivo or muscarinic ACh receptor activation in vitro changed the expression levels of the cellular protooncogene products Fos and Jun, which may play a role in the initiation of the adaptive processes. Using Fos- and Jun-specific polyclonal antisera in Western blot experiments, we demonstrated that Jun is constitutively expressed in nasal gland tissue, whereas Fos is not detectable in tissue from unstressed (naive) animals. Under conditions of osmotic stress imposed by replacing the drinking water of the animals with a 1% NaCl solution, Jun protein remains constant in nasal gland tissue, whereas Fos protein is transiently upregulated. Treatment of cultured nasal gland tissue with muscarinic agonists results in a transcriptionally regulated expression of Fos in an atropine-sensitive manner. Immunohistochemical experiments show that Fos accumulation occurs in the nuclei of the secretory cells. These results indicate that the activation of the c- fos gene induced by muscarinic ACh receptor-mediated signaling pathways may play an important role in the initiation of adaptive growth and differentiation processes in nasal glands of osmotically stressed ducklings.
Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms
Authors: He Zhaoping , Raman Sasikala , Guo Yi , Reenstra William W.
Published: December 2017

Recent studies have demonstrated that several compounds with diverse structures can activate wild-type cystic fibrosis transmembrane conductance regulator (CFTR) by non-receptor-mediated mechanisms. Some of these compounds have been shown to enhance cAMP-dependent activation of ΔF508-CFTR. This study was undertaken to compare the mechanisms by which genistein, IBMX, milrinone, 8-cyclopentyl-1,3-dipropylxanthine (CPX), the benzimidazolone NS004, and calyculin A increase CFTR activity. Our studies demonstrate that, in transfected NIH-3T3 cells, maximal enhancements of forskolin-dependent ΔF508-CFTR activity are greatest with genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, and calyculin A do not increase cellular cAMP. Because forskolin and calyculin A increase in vivo phosphorylation of cAMP binding response element (CREB), the inability of milrinone, genistein, CPX, and NS004 to increase CREB phosphorylation suggests that they do not stimulate protein kinase A or inhibit phosphatase activity. Our data suggest that the mechanisms by which genistein and NS004 activate CFTR differ. We also demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement of cAMP-dependent CFTR activity is not due to an increase in cellular cAMP and may involve a mechanism like that of genistein.
Role of the α-subunit326GRV sequence in the surface expression of fibrinogen and vitronectin receptors
Authors: Ferrer Milagros , Ayuso Matilde S. , Butta Nora , Parrilla Roberto , González-Manchón Consuelo
Published: December 2017

The platelet GPIIb-GPIIIa heterodimer (integrin αIIbβ3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The 326GRV motif in GPIIb is highly conserved in the α-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327→His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327 or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327 or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327 in the α-subunit of the vitronectin receptor, to His did not prevent the surface expression of αv-GPIIIa complexes. Thus the conformational constraint imposed by His327 seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.
Analysis of a Ca2+-activated K+ channel that mediates hyperpolarization via the thrombin receptor pathway
Authors: Sullivan Richard , Koliwad Suneil K. , Kunze Diana L.
Published: December 2017

Dami human leukemia cells express G protein-coupled thrombin receptors that operate through the phospholipase C pathway. When these receptors are activated by α-thrombin or by thrombin receptor-activating peptide, an elevation in cytosolic Ca2+ concentration develops that is accompanied by hyperpolarization of the plasma membrane. This transitory phase of hyperpolarization is primarily mediated by inwardly rectifying, Ca2+-activated K+ channels that have an inward conductance of ∼24 pS. In cell-attached patches the channels open within seconds after superfusion of the cell with thrombin receptor-activating peptide. In inside-out patches, perfusion of submicromolar Ca2+ onto the cytosolic surface of the membrane is sufficient to activate the channels. In outside-out patches, channel opening can be blocked by nanomolar concentrations of charybdotoxin. The function of these intermediate-sized inwardly rectifying, Ca2+-activated K+ channels has not been established; however, by analogy with other cell systems, they may serve to regulate cell volume during cellular activation or to increase the electromotive drive that sustains Na+ and/or Ca2+ influx through ligand-gated cation channels.
A water channel of the nematode C. elegans and its implications for channel selectivity of MIP proteins
Authors: Kuwahara Michio , Ishibashi Kenichi , Gu Yong , Terada Yoshio , Kohara Yuji , Marumo Fumiaki , Sasaki Sei
Published: December 2017

A genome project focusing on the nematode Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein superfamily. We functionally characterized one of these cDNAs named C01G6.1. Injection of C01G6.1 cRNA increased the osmotic water permeability ( P f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed to increase the glycerol permeability. It has been speculated that the MIP family may be separated into two large subfamilies based on the presence or absence of two segments of extra amino acid residues (∼15 amino acids) at the second and third extracellular loops. Because C01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with those of AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that of wild-type AQP-CE1, although the values of P f and urea permeability were decreased by 39–74% and 28–65%, respectively. These results suggest that the two segments of extra amino acid residues may not contribute to channel selectivity or formation of the route for small solutes.
Cell-cell contact between adult rat cardiac myocytes regulates Kv1.5 and Kv4.2 K+ channel mRNA expression
Authors: Hershman Kenneth M. , Levitan Edwin S.
Published: December 2017

Regulation of voltage-gated K+ channel genes represents an important mechanism for modulating cardiac excitability. Here we demonstrate that expression of two K+ channel mRNAs is reciprocally controlled by cell-cell interactions between adult cardiac myocytes. It is shown that culturing acutely dissociated rat ventricular myocytes for 3 h results in a dramatic downregulation of Kv1.5 mRNA and a modest upregulation of Kv4.2 mRNA. These effects are specific, because similar changes are not detected with other channel mRNAs. Increasing myocyte density promotes maintenance of Kv1.5 gene expression, whereas Kv4.2 mRNA expression was found to be inversely proportional to cell density. Conditioned culture medium did not mimic the effects of high cell density. However, paraformaldehyde-fixed myocytes were comparable to live cells in their ability to influence K+ channel message levels. Thus the reciprocal effects of cell density on the expression of Kv1.5 and Kv4.2 genes are mediated by direct contact between adult cardiac myocytes. These findings reveal for the first time that cardiac myocyte gene expression is influenced by signaling induced by cell-cell contact.
Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC
Authors: Khayat Zayna A. , Tsakiridis Theodoros , Ueyama Atsunori , Somwar Romel , Ebina Yousuke , Klip Amira
Published: December 2017

2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative chain from ATP production, preventing oxidative metabolism. The consequent increase in energy demand is, however, contested by cells increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal muscle cells, DNP rapidly doubles glucose transport, reminiscent of the effect of insulin. However, glucose transport stimulation by DNP does not require insulin receptor substrate-1 phosphorylation and is wortmannin insensitive. We report here that, unlike insulin, DNP does not activate phosphatidylinositol 3-kinase, protein kinase B/Akt, or p70 S6 kinase. However, chelation of intra- and extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose uptake by 78.9 ± 3.5%. Because Ca2+-sensitive, conventional protein kinase C (cPKC) can activate glucose transport in L6 muscle cells, we examined whether cPKC may be translocated and activated in response to DNP in L6 myotubes. Acute DNP treatment led to translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from plasma membranes exhibited a twofold increase in kinase activity in response to DNP. Overnight treatment with 4-phorbol 12-myristate 13-acetate downregulated cPKC isoforms α, β, and γ and partially inhibited (45.0 ± 3.6%) DNP- but not insulin-stimulated glucose uptake. Consistent with this, the PKC inhibitor bisindolylmaleimide I blocked PKC enzyme activity at the plasma membrane (100%) and inhibited DNP-stimulated 2-[3H]deoxyglucose uptake (61.2 ± 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC-β inhibitor LY-379196 partially inhibited DNP effects on glucose uptake (66.7 ± 1.6%). The results suggest interfering with mitochondrial ATP production acts on a signal transduction pathway independent from that of insulin and partly mediated by Ca2+ and cPKCs, of which PKC-β likely plays a significant role.
Activation of the JAK-STAT pathway by reactive oxygen species
Authors: Simon Amy R. , Rai Usha , Fanburg Barry L. , Cochran Brent H.
Published: December 2017

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson’s disease, pulmonary fibrosis, and Alzheimer’s disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c- fos and c- myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-l-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.
Phosphorylation of p70S6kcorrelates with increased skeletal muscle mass following resistance exercise
Authors: Baar Keith , Esser Karyn
Published: December 2017

High-resistance exercise training results in an increase in muscle wet mass and protein content. To begin to address the acute changes following a single bout of high-resistance exercise, a new model has been developed. Training rats twice a week for 6 wk resulted in 13.9 and 14.4% hypertrophy in the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, respectively. Polysome profiles after high-resistance lengthening contractions suggest that the rate of initiation is increased. The activity of the 70-kDa S6 protein kinase (p70S6k), a regulator of translation initiation, is also increased following high-resistance lengthening contractions (TA, 363 ± 29%; EDL, 353 ± 39%). Furthermore, the increase in p70S6k activity 6 h after exercise correlates with the percent change in muscle mass after 6 wk of training ( r = 0.998). The tight correlation between the activation of p70S6k and the long-term increase in muscle mass suggests that p70S6k phosphorylation may be a good marker for the phenotypic changes that characterize muscle hypertrophy and may play a role in load-induced skeletal muscle growth.
CFTR drives Na+- n HCO 3 − cotransport in pancreatic duct cells: a basis for defective HCO 3 − secretion in CF
Authors: Shumaker Holli , Amlal Hassane , Frizzell Raymond , Ulrich Charles D. , Soleimani Manoocher
Published: December 2017

Pancreatic dysfunction in patients with cystic fibrosis (CF) is felt to result primarily from impairment of ductal[Formula: see text] secretion. We provide molecular evidence for the expression of NBC-1, an electrogenic Na+-[Formula: see text]cotransporter (NBC) in cultured human pancreatic duct cells exhibiting physiological features prototypical of CF duct fragments (CFPAC-1 cells) or normal duct fragments [CAPAN-1 cells and CFPAC-1 cells transfected with wild-type CF transmembrane conductance regulator (CFTR)]. We further demonstrate that 1)[Formula: see text] uptake across the basolateral membranes of pancreatic duct cells is mediated via NBC and 2) cAMP potentiates NBC activity through activation of CFTR-mediated Cl− secretion. We propose that the defect in agonist-stimulated ductal[Formula: see text] secretion in patients with CF is predominantly due to decreased NBC-driven[Formula: see text] entry at the basolateral membrane, secondary to the lack of sufficient electrogenic driving force in the absence of functional CFTR.
Intracellular signaling mechanisms of interleukin-1β in synovial fibroblasts
Authors: Kolomytkin Oleg V. , Marino Andrew A. , Sadasivan Kalia K. , Wolf Robert E. , Albright James A.
Published: December 2017

The possibility that membrane depolarization of synovial fibroblasts caused by interleukin-1β (IL-1β) was mediated by protein kinase C (PKC) and Ca2+influx was studied using inhibitor and activator analysis. The effect of IL-1β was blocked by bisindolylmaleimide I, an inhibitor of PKC, and by the Ca2+ channel blockers nifedipine and verapamil. In other experiments, PKC was activated using phorbol 12-myristate 13-acetate, and Ca2+ influx was increased by means of a Ca2+ ionophore. Simultaneous application of phorbol ester and Ca2+ ionophore in the absence of IL-1β mimicked the depolarization caused by IL-1β. The results were consistent with the hypothesis that, under the conditions studied, activation of PKC and Ca2+ influx are necessary and sufficient processes in the transduction of IL-1β by synovial cells leading to membrane depolarization. The essential role of protein phosphorylation and Ca2+ influx in the early electrophysiological response of synovial fibroblasts to IL-1β was therefore established. The role of IL-1β-induced depolarization in regulating protein expression by the cells remains to be determined, but the results reported here, taken together with observations that protein phosphorylation and Ca2+influx also mediate the effect of IL-1β on protease production (1, 2), suggest that electrophysiological changes are actually part of the pathway for expression of proteases in response to IL-1β.
Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium
Authors: Amsler Kurt , Kuwada Scott K.
Published: December 2017

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-γ (PLC-γ) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-γ protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.
Hypoxemia in the absence of blood loss upregulates iNOS expression and activity in macrophages
Authors: Angele Martin K. , Schwacha Martin G. , Smail Nadia , Catania Robert A. , Ayala Alfred , Cioffi William G. , Chaudry Irshad H.
Published: December 2017

Regional hypoxia, associated with hemorrhage, is thought to induce a variety of alterations in immune cell function, including upregulation of macrophage-inducible nitric oxide synthase (iNOS) expression and activity (NO production). Furthermore, NO may cause immune cell dysfunction similar to that associated with hemorrhagic shock. However, it remains unknown whether hypoxia per se in the absence of any blood loss is a sufficient stimulus to cause iNOS expression and NO production by macrophages. To study this, male Sprague-Dawley rats (275–325 g) were placed in a plastic box flushed with a gas mixture containing 5% O2-95% N2 for 60 min. Peritoneal and splenic macrophages were isolated 0–5.5 h thereafter, and blood samples were obtained. Nitrite and nitrate (stable degradation products of NO) production by splenic and peritoneal macrophages cultured for 48 h was significantly increased 3 and 5.5 h after hypoxemia. The increase in NO production by macrophages was preceded by elevated expression of iNOS mRNA at 1.5 h after hypoxia. Additionally, interferon-γ (IFN-γ) levels in plasma from rats subjected to hypoxemia were significantly elevated soon after the insult (0–1.5 h posthypoxemia), suggesting a causal relationship between IFN-γ production and upregulation of iNOS activity. We propose that a hypoxemia-induced increase in macrophage iNOS activity following hemorrhage may in part be responsible for the observed immune dysfunction. Thus attempts to suppress macrophage iNOS activity after this form of trauma may be helpful in improving immune function under those conditions.
Preservation of murine embryos in a state of dormancy at 4°C
Authors: Wiggins Philippa M. , Rowlandson Jamie , Ferguson Alexander B.
Published: December 2017

With the aim of improving preservation of blood products and organs for transplantation, we designed solutions to induce a state of dormancy in cells and tissues at 4°C. The solutions were devoid of combinations of ions (e.g., K+, Rb+, Cs+, and[Formula: see text] with[Formula: see text],[Formula: see text], and Cl−) that are believed to break down low-density water in the entrance compartments of ion channels, resulting in cyclical open states (normal water) and closed states (low-density water). The total osmolality was always 0.29–0.3 osmol/kgH2O, made up of combinations of a di- or trisaccharide, a compatible solute, sodium sulfate, citrate, or chloride, and 1.75 mM CaCl2. The end point was the ability of murine embryos to progress to hatching in culture after preservation in such a solution at 4°C. Embryos hatched after 5 or 6 days in some preservative solutions compared with 1–3 days in most saline solutions; survival was improved by pretreatment with sodium butyrate.
Colonic H-K-ATPase β-subunit: identification in apical membranes and regulation by dietary K depletion
Authors: Sangan Pitchai , Kolla Sarah S. , Rajendran Vazhaikkurichi M. , Kashgarian Michael , Binder Henry J.
Published: December 2017

P-type ATPases require both α- and β-subunits for functional activity. Although an α-subunit for colonic apical membrane H-K-ATPase (HKcα) has been identified and studied, its β-subunit has not been identified. We cloned putative β-subunit rat colonic H-K-ATPase (HKcβ) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat β-subunits. Northern blot analysis demonstrates that this HKcβ is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcβ mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase β1 mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKcβ mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcβ fusion protein established expression of a 45-kDa HKcβ protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcβ protein expression only in apical membranes. Physical association between HKcβ and HKcα proteins was demonstrated by Western blot analysis performed with HKcβ antibody on immunoprecipitate of apical membranes of rat distal colon and HKcα antibody. Tissue-specific upregulation of this β-subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKcα protein provide compelling evidence that HKcβ is the putative β-subunit of colonic H-K-ATPase.
Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes
Authors: Ibrahimi Azeddine , Abumrad Nada , Maghareie Hengameh , Golia Michael , Shoshani Ilana , Désaubry Laurent , Johnson Roger A.
Published: December 2017

Differentiation of Ob1771 preadipocytes to adipocytes was characterized by morphological changes and elevated expression of the specific marker enzyme, glycerol-3-phosphate dehydrogenase. A differentiation response substantially more complete and rapid than that obtained with insulin and 3,5,3′-triiodothyronine was observed with established inhibitors of adenylyl cyclases: 2′,5′-dideoxyadenosine (2′,5′-dd-Ado), 9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or by N 6-(2-phenylisopropyl)adenosine or 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively, for adenosine A1 receptors. Also ineffective were IBMX or forskolin, agents that can raise intracellular cAMP levels. Potency of the differentiation response followed the order 2′,5′-dd-Ado (1–20 μM) > 9-CP-Ade (10–100 μM) = 9-Ara-Ade (10–100 μM) >> inosine, consistent with their potencies to inhibit adenylyl cyclases. The data suggest that inhibition of adenylyl cyclase via the P-site and the consequent reduction in cell cAMP levels facilitate the induction of differentiation in Ob1771 cells. The findings raise the question whether the known endogenous P-site ligands participate in the differentiation response induced by hormones.
Ca2+ sensitization of smooth muscle contractility induced by ruthenium red
Authors: Yamada Aki , Ohya Susumu , Hirano Masaru , Watanabe Minoru , Walsh Michael P. , Imaizumi Yuji
Published: December 2017

The effects of ruthenium red (RuR) on contractility were examined in skinned fibers of guinea pig smooth muscles, where sarcoplasmic reticulum function was destroyed by treatment with A-23187. Contractions of skinned fibers of the urinary bladder were enhanced by RuR in a concentration-dependent manner (EC50 = 60 μM at pCa 6.0). The magnitude of contraction at pCa 6.0 was increased to 320% of control by 100 μM RuR. Qualitatively, the same results were obtained in skinned fibers prepared from the ileal longitudinal smooth muscle layer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuR was not reversed by the addition of guanosine 5′- O-(2-thiodiphosphate) or a peptide inhibitor of protein kinase C [PKC-(19—31)]. The application of microcystin, a potent protein phosphatase inhibitor, induced a tonic contraction of skinned smooth muscle at low Ca2+ concentration ([Ca2+]; pCa > 8.0). RuR had a dual effect on the microcystin-induced contraction-to- enhancement ratio at low concentrations and suppression at high concentrations. The relaxation following the decrease in [Ca2+] from pCa 5.0 to >8.0 was significantly slowed down by an addition of RuR. Phosphorylation of the myosin light chain at pCa 6.3 was significantly increased by RuR in skinned fibers of the guinea pig ileum. These results indicate that RuR markedly increases the Ca2+ sensitivity of the contractile system, at least in part via inhibition of myosin light chain phosphatase.
Polyamine depletion arrests cell cycle and induces inhibitors p21Waf1/Cip1, p27Kip1, and p53 in IEC-6 cells
Authors: Ray Ramesh M. , Zimmerman Barbara J. , McCormack Shirley A. , Patel Tarun B. , Johnson Leonard R.
Published: December 2017

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence ofdl-α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21Waf1/Cip1 and p27Kip1. Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.
Chemokine expression in CF epithelia: implications for the role of CFTR in RANTES expression
Authors: Schwiebert Lisa M. , Estell Kim , Propst Stacie M.
Published: December 2017

To delineate the mechanisms that facilitate leukocyte migration into the cystic fibrosis (CF) lung, expression of chemokines, including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES, was compared between CF and non-CF airway epithelia. The findings presented herein demonstrate that, under either basal conditions or tumor necrosis factor-α (TNF-α)- and/or interferon-γ (IFN-γ)-stimulated conditions, a consistent pattern of differences in the secretion of IL-8 and MCP-1 between CF and non-CF epithelial cells was not observed. In contrast, CF epithelial cells expressed no detectable RANTES protein or mRNA under basal conditions or when stimulated with TNF-α and/or IFN-γ ( P ≤ 0.05), unlike their non-CF counterparts. Correction of the CF transmembrane conductance regulator (CFTR) defect in CF airway epithelial cells restored the induction of RANTES protein and mRNA by TNF-α in combination with IFN-γ ( P ≤ 0.05) but had little effect on IL-8 or MCP-1 production compared with mock controls. Transfection studies utilizing RANTES promoter constructs suggested that CFTR activates the RANTES promoter via a nuclear factor-κB-mediated pathway. Together, these results suggest that 1) RANTES expression is altered in CF epithelia and 2) epithelial expression of RANTES, but not IL-8 or MCP-1, is dependent on CFTR.
EGF precursor mRNA and membrane-associated EGF precursor protein in rat exorbital lacrimal gland
Authors: Maréchal Hervé , Jammes Hélene , Rossignol Bernard , Mauduit Philippe
Published: December 2017

This study was designed to demonstrate the presence of epidermal growth factor (EGF) in the rat exorbital lacrimal gland. EGF precursor gene transcription was demonstrated first by RT-PCR analysis of lacrimal gland RNA using a set of specific primers and second by Northern blot analysis of rat lacrimal gland mRNA. A rabbit polyclonal antibody (rEGF2) directed against rat submaxillary gland EGF was used to detect EGF-containing proteins by RIA. Results indicate that the rat lacrimal gland does not contain detectable soluble and mature EGF but that the EGF immunoreactivity is associated with the membrane-enriched fraction. Analysis of the detergent-solubilized membrane proteins by gel filtration shows that membrane-associated EGF immunoreactivity was present as a high-molecular-mass protein. Moreover, as shown by Western blot analysis, a specific anti-rat EGF precursor antibody (ppEGF1) can immunoprecipitate a 152-kDa EGF-containing protein. Taken together, these results demonstrate for the first time both EGF precursor gene transcription and EGF precursor protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. The demonstration that EGF appears to be stored only as its full-length membrane precursor may provide important information to study the regulation of its secretory process.
Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging
Authors: Jayaraman Sujatha , Biwersi Joachim , Verkman A. S.
Published: December 2017

The fluorescence of quinolinium-based Cl− indicators such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl− by a collisional mechanism without change in spectral shape. A series of “chimeric” dual-wavelength Cl− indicators were synthesized by conjugating Cl−-sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl−-sensitive moiety [excitation wavelength (λex) 350 nm, emission wavelength (λem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl−-insensitive moiety because of its different spectral characteristics (λex 380 nm, λem 546 nm), insensitivity to Cl−, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl−-sensitive indicators were α-MQ-α′-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-α′-MQ-1′-α′-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl− sensitive; Stern-Volmer constants 82 and 98 M−1) and 565 nm (Cl−insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 μM, 10 min). Changes in Cl− concentration in response to Cl−/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl− indicators for ratiometric measurement of Cl− concentration.
Hormonal regulation of oligopeptide transporter Pept-1 in a human intestinal cell line
Authors: Thamotharan Manikkavasagar , Bawani Shahab Zare , Zhou Xiaodong , Adibi Siamak A.
Published: December 2017

The intestinal oligopeptide transporter (cloned as Pept-1) has major roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. In this experiment, we investigated whether insulin has such a role. We used a human intestinal cell monolayer (Caco-2) as the in vitro model of human small intestine and glycylglutamine (Gly-Gln) as the model substrate for Pept-1. Results showed that addition of insulin at a physiological concentration (5 nM) to incubation medium greatly stimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blocked when genistein, an inhibitor of tyrosine kinase, was added to incubation medium. Studies of the mechanism of insulin stimulation showed the following. 1) Stimulation occurred promptly (30–60 min) after exposure to insulin. 2) There was no significant change in the Michaelis-Menten constant of Gly-Gln transport, but there was a nearly twofold increase in its maximal velocity. 3) Insulin effect persisted even when Golgi apparatus, which is involved in trafficking of newly synthesized Pept-1, was dismantled. 4) However, there was complete elimination of insulin effect by disruption of microtubules involved in trafficking of preformed Pept-1. 5) Finally, with insulin treatment, there was no change in Pept-1 gene expression, but the amount of Pept-1 protein in the apical membrane was increased. In conclusion, the results show that insulin, when it binds to its receptor, stimulates Gly-Gln uptake by Caco-2 cells by increasing the membrane population of Pept-1. The mechanism appears to be increased translocation of this transporter from a preformed cytoplasmic pool.
UTP inhibits Na+ absorption in wild-type and ΔF508 CFTR-expressing human bronchial epithelia
Authors: Devor Daniel C. , Pilewski Joseph M.
Published: December 2017

Ca2+-mediated agonists, including UTP, are being developed for therapeutic use in cystic fibrosis (CF) based on their ability to modulate alternative Cl− conductances. As CF is also characterized by hyperabsorption of Na+, we determined the effect of mucosal UTP on transepithelial Na+transport in primary cultures of human bronchial epithelia (HBE). In symmetrical NaCl, UTP induced an initial increase in short-circuit current ( I sc) followed by a sustained inhibition. To differentiate between effects on Na+ absorption and Cl− secretion, I sc was measured in the absence of mucosal and serosal Cl−( I Na). Again, mucosal UTP induced an initial increase and then a sustained decrease that reduced amiloride-sensitive I Na by 73%. The Ca2+-dependent agonists histamine, bradykinin, serosal UTP, and thapsigargin similarly induced sustained inhibition (62–84%) of I Na. Mucosal UTP induced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) of I Na in primary cultures of human CF airway homozygous for the ΔF508 mutation. BAPTA-AM blunted UTP-dependent inhibition of I Na, but inhibitors of protein kinase C (PKC) and phospholipase A2 had no effect. Indeed, direct activation of PKC by phorbol 12-myristate 13-acetate failed to inhibit Na+ absorption. Apyrase, a tri- and diphosphatase, did not reverse inhibitory effects of UTP on I Na, suggesting a long-term inhibitory effect of UTP that is independent of receptor occupancy. After establishment of a mucosa-to-serosa K+ concentration gradient and permeabilization of the mucosal membrane with nystatin, mucosal UTP induced an initial increase in K+current followed by a sustained inhibition. We conclude that increasing cellular Ca2+ induces a long-term inhibition of transepithelial Na+transport across normal and CF HBE at least partly due to downregulation of a basolateral membrane K+ conductance. Thus UTP may have a dual therapeutic effect in CF airway: 1) stimulation of a Cl− secretory response and 2) inhibition of Na+ transport.
TGF-α reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells
Authors: Beltinger J. , Hawkey C. J. , Stack W. A.
Published: December 2017

The effect of chronic exposure to transforming growth factor-α (TGF-α) on bradykinin-stimulated acute prostanoid production and ion secretion in monolayers of HCA-7 colony 29 colonic epithelial cells has been studied. Monolayers synthesized prostaglandin E2(PGE2) at a basal rate of 2.10 ± 0.31 pg ⋅ monolayer−1 ⋅ min−1over 24 h. Bradykinin (10−8–10−5M) dose dependently increased acute PGE2 release by three orders of magnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer ( P < 0.02) and a dose-dependent increase in short-circuit current (SCC). When monolayers were primed by a 24-h exposure to TGF-α, basal PGE2 release rose to 6.31 ± 0.38 pg ⋅ monolayer−1 ⋅ min−1(TGF-α concn 10 ng/ml; P = 0.001). However, the stimulation of acute prostaglandin release, intracellular cAMP, and increased SCC by bradykinin was significantly reduced by preincubation with TGF-α. Priming with PGE2(10−8–10−6M) over 24 h mimicked the effect of TGF-α on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF-α may downregulate acute responses to bradykinin. In vivo, TGF-α could have an important modulatory function in regulating secretion under inflammatory conditions.