bluuebunny/crossref_metadata_2025 huggingface dataset:
Shear stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells
Authors: Chatterjee Shampa , Al-Mehdi Abu-Bakr , Levitan Irena , Stevens Troy , Fisher Aron B.
Published: May 2013

We have shown previously that acute ischemia leads to depolarization of pulmonary microvascular endothelial cells that is prevented with cromakalim, suggesting the presence of ATP-sensitive K+ (KATP) channels in these cells. Thus KATP channel expression and activity were evaluated in rat pulmonary microvascular endothelial cells (RPMVEC) by whole cell current measurements, dot blot (mRNA), and immunoblot (protein) for the inwardly rectifying K+ channel (KIR) 6.2 subunit and fluorescent ligand binding for the sulfonylurea receptor (SUR). Low-level expression of a KATP channel was detected in endothelial cells in routine (static) culture and led us to examine whether its expression is inducible when endothelial cells are adapted to flow. Channel expression (mRNA and both KIR6.2 and SUR proteins) and inwardly rectified membrane current by patch clamp increased significantly when RPMVEC were adapted to flow at 10 dyn/cm2 for 24 h in either a parallel plate flow chamber or an artificial capillary system. Induction of the KATP channel with flow adaptation was also observed in bovine pulmonary artery endothelial cells. Flow-adapted but not static RPMVEC showed cellular plasma membrane depolarization upon stop of flow that was inhibited by a KATP channel opener and prevented by addition of cycloheximide to the medium during the flow adaptation period. These studies indicate the induction of KATP channels by flow adaptation in pulmonary endothelium and that the expression and activity of this channel are essential for the endothelial cell membrane depolarization response with acute decrease in shear stress.
Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction
Authors: DiSanto Michael E. , Stein Raimund , Chang Shaohua , Hypolite Joseph A. , Zheng Yongmu , Zderic Stephen , Wein Alan J. , Chacko Samuel
Published: May 2013

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders.
Mitochondrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism
Authors: Samavati Lobelia , Monick Martha M. , Sanlioglu Salih , Buettner Garry R. , Oberley Larry W. , Hunninghake Gary W.
Published: May 2013

Extracellular signal-regulated kinases (ERKs) are key regulatory proteins that mediate cell survival, proliferation, and differentiation. Reactive oxygen species (ROS) may play a role in activation of the ERK pathway. Because mitochondria are a major source of ROS, we investigated whether mitochondria-derived ROS play a role in ERK activation. Diazoxide, a potent mitochondrial ATP-sensitive K+ (KATP) channel opener, is known to depolarize the mitochondrial membrane potential and cause a reversible oxidation of respiratory chain flavoproteins, thus increasing mitochondrial ROS production. Using THP-1 cells as a model, we postulated that opening mitochondrial KATP channels would increase production of ROS and, thereby, regulate the activity of the ERK kinase. We found that opening mitochondrial KATP channels by diazoxide induced production of ROS as determined by an increased rate of dihydroethidium and dichlorofluorescein fluorescence. This increased production of ROS was associated with increased phosphorylation of ERK kinase in a time-dependent fashion. The MEK inhibitors PD-98059 and U-0126 blocked ERK activation mediated by diazoxide. N-acetylcysteine, but not diphenyleneiodonium, attenuated ERK activation mediated by diazoxide. Adenovirus-mediated overexpression of manganese superoxide dismutase, which is expressed in mitochondria, decreased the rate of dihydroethidium oxidation as well as ERK activation. We conclude that mitochondrial KATP channel openers trigger ERK activation via mitochondria-derived ROS.
Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells
Authors: Li F. , Mao H. P. , Ruchalski K. L. , Wang Y. H. , Choy W. , Schwartz J. H. , Borkan S. C.
Published: May 2013

The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-d-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.
Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle
Authors: Afroze Talat , Yang Li L. , Wang Changsen , Gros Robert , Kalair Waseem , Hoque Abu N. , Mungrue Imran N. , Zhu Ziping , Husain Mansoor
Published: May 2013

Calcineurin mediates repression of plasma membrane Ca2+-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca2+ concentrations ([Ca2+]i) increased twofold, and real-time RT-PCR and Western blot revealed a ∼40% decrease in PMCA4 expression levels from G0 to G1/S in the cell cycle, where PMCA4 constituted ∼20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca2+-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca2+]i through alternate targets.
The nonclassic secretion of thioredoxin is not sensitive to redox state
Authors: Tanudji Marcel , Hevi Sarah , Chuck Steven L.
Published: May 2013

Thioredoxin (Trx) is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular Trx are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of Trx could be altered by the redox status of the cell or the protein. To study Trx mutants, we characterized the secretion of human Trx from Chinese hamster ovary cells. Secretion of human Trx is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of Trx but not the proportion secreted; thus this chemical does not block the nonclassic pathway for Trx secretion. Furthermore, we find that mutations in either the active site or the dimerization site of Trx do not alter its secretion. Thus the nonclassic secretion of Trx is not dependent on the redox status of either the cell or the protein.
NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells
Authors: Brar Sukhdev S. , Corbin Zachary , Kennedy Thomas P. , Hemendinger Richelle , Thornton Lisa , Bommarius Bettina , Arnold Rebecca S. , Whorton A. Richard , Sturrock Anne B. , Huecksteadt Thomas P. , Quinn Mark T. , Krenitsky Kevin , Ardie Kristia G. , Lambeth J. David , Hoidal John R.
Published: May 2013

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22 phox and gp91 phox, as well as the Ca2+ concentration-responsive gp91 phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22 phox was not detectable, both gp91 phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2′,7′-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22 phox or gp91 phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.
Sarcoplasmic reticulum Ca2+ pump mRNA stability in cardiac and smooth muscle: role of the 3′-untranslated region
Authors: Misquitta Christine M. , Mwanjewe James , Nie Lin , Grover Ashok K.
Published: May 2013

Stomach smooth muscle (SSM) and left ventricular muscle (LVM) express the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump gene SERCA2. Alternative splicing yields two major isoforms, SERCA2a in LVM and slow twitch muscle and SERCA2b in SSM and most other tissues. The splices have different 3′-untranslated regions (UTR) and also encode proteins that differ slightly in their COOH-terminal domains. SERCA2 transcription rates are similar in the two tissues, yet LVM has a much higher level of SERCA2 mRNA than SSM. To understand the control of SERCA2 RNA expression, we inhibited transcription and showed that the half-life of SERCA2 mRNA is significantly longer ( P < 0.05) in primary cultures of LVM cells than in SSM cells. Nuclear SERCA2 mRNA levels were also higher in LVM than in SSM. In vitro decay assays using synthetic RNA corresponding to the 3′-UTR of SERCA2a and -2b showed that nuclear extracts produced a faster decay of SERCA2 RNA than cytoplasmic extracts and that nuclear extracts produced a faster decay of SERCA2b than -2a. This was also true when the full-length native mRNA was used instead of the 3′-UTR RNA, and SERCA2b decay by cytoplasmic extracts was faster for LVM than for SSM. We propose that nuclear decay is an initial step in the control of SERCA2 RNA abundance and that this control is maintained or modulated in the cytoplasm. We discuss how these control mechanisms may be part of a control switch in cardiac development and pathophysiology.
Evidence that signal transduction for osmoreception is mediated by stretch-activated ion channels in tilapia
Authors: Seale A. P. , Richman N. H. , Hirano T. , Cooke I. , Grau E. G.
Published: May 2013

Prolactin (PRL) plays a central role in the freshwater osmoregulation of teleost fish, including the tilapia ( Oreochromis mossambicus). Consistent with this action, PRL release from the tilapia pituitary increases as extracellular osmolality is reduced both in vitro and in vivo. Dispersed tilapia PRL cells were incubated in a perfusion chamber that allowed simultaneous measurements of cell volume and PRL release. Intracellular Ca2+ concentrations were measured from fura 2-loaded PRL cells treated in a similar way. Gadolinium (Gd3+), known to block stretch-activated cation channels, inhibited hyposmotically induced PRL release in a dose-related manner without preventing cell swelling. Nifedipine, an L-type Ca2+ channel blocker, did not prevent the increase in PRL release during hyposmotic stimulation. A high, depolarizing concentration of KCl induced a transient and marked increase of intracellular Ca2+ and release of PRL but did not prevent the rise in intracellular Ca2+ and PRL release evoked by exposure to hyposmotic medium. These findings suggest that a decrease in extracellular osmolality stimulates PRL release through the opening of stretch-activated ion channels, which allow extracellular Ca2+ to enter the cell when it swells.
Role of nitric oxide synthase isoforms in glucose-stimulated insulin release
Authors: Henningsson Ragnar , Salehi Albert , Lundquist Ingmar
Published: May 2013

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N G-monomethyl-l-arginine (l-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of l-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.
Two pore residues mediate acidosis-induced enhancement of C-type inactivation of the Kv1.4 K+ channel
Authors: Claydon T. W. , Boyett M. R. , Sivaprasadarao A. , Orchard C. H.
Published: May 2013

Acidosis inhibits current through the Kv1.4 K+ channel, perhaps as a result of enhancement of C-type inactivation. The mechanism of action of acidosis on C-type inactivation has been studied. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Δ2–146) was expressed in Xenopus oocytes, and currents were recorded using two-microelectrode voltage clamp. Acidosis increased fKv1.4 Δ2–146 C-type inactivation. Replacement of a pore histidine with cysteine (H508C) abolished the increase. Application of positively charged thiol-specific methanethiosulfonate to fKv1.4 Δ2–146 H508C increased C-type inactivation, mimicking the effect of acidosis. Replacement of a pore lysine with cysteine (K532C) abolished the acidosis-induced increase of C-type inactivation. A model of the Kv1.4 pore, based on the crystal structure of KcsA, shows that H508 and K532 lie close together. It is suggested that the acidosis-induced increase of C-type inactivation involves the charge on H508 and K532.
Human trabecular meshwork cell volume regulation
Authors: Mitchell Claire H. , Fleischhauer Johannes C. , Stamer W. Daniel , Peterson-Yantorno K. , Civan Mortimer M.
Published: May 2013

The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl−/HCO[Formula: see text]exchange, and K+-Cl− efflux mechanisms have on the volume of TM cells. Volume, Cl− currents, and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+channel blockers Ba2+ and tetraethylammonium, and the K+-Cl− symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+ exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl− symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl− channel displaying the permeability ranking Cl− > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl− channels, Na+/H+ antiports, and possibly K+-Cl− symports in addition to Na+-K+-2Cl− symports.
Gα12 regulates epithelial cell junctions through Src tyrosine kinases
Authors: Meyer Tobias N. , Hunt Jennifer , Schwesinger Catherine , Denker Bradley M.
Published: May 2013

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Gα12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Gα12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Gα12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Gα12 (QLα12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLα12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLα12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLα12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLα12-expressing MDCK cells as assessed by Src autophosphorylation, and β-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Gα12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.
The role of regulated CFTR trafficking in epithelial secretion
Authors: Bertrand Carol A. , Frizzell Raymond A.
Published: May 2013

The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.
Novel regulation of adrenomedullin receptor by PDGF: role of receptor activity modifying protein-3
Authors: Nowak Wojciech , Parameswaran Narayanan , Hall Carolyn S. , Aiyar Nambi , Sparks Harvey V. , Spielman William S.
Published: May 2013

Receptor activity modifying protein-3 (RAMP-3) has been shown to complex with the calcitonin receptor-like receptor, establishing a functional receptor for adrenomedullin (AM). AM exhibits potent antiproliferative and antimigratory effects on rat mesangial cells (RMCs). In this study we investigated the effect of platelet-derived growth factor (PDGF) on RAMP-3 expression in RMCs. We show here that PDGF-BB stimulates RAMP-3 mRNA expression in a concentration-dependent manner. Pretreatment with actinomycin-D and α-amanitin demonstrates that this effect is independent of new RNA synthesis. Furthermore, PDGF increased the half-life of RAMP-3 mRNA from 66.5 to 331.6 min. Using selective inhibitors, our results also indicate that the increase in RAMP-3 mRNA is mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK and p38 MAPK dependent. PDGF also caused a corresponding elevation in membrane-associated RAMP-3 protein. Associated with this increase, PDGF pretreatment led to a significantly higher AM-mediated adenylate cyclase activity, suggesting a functional consequence for the PDGF-induced increase in RAMP-3 expression. Taken together, these data identify PDGF-dependent regulation of RAMP-3 expression as a possible mechanism for modulating the responsiveness of the mesangial cell to AM.
Posttranslational inactivation of human xanthine oxidoreductase by oxygen under standard cell culture conditions
Authors: Linder Nina , Martelin Eeva , Lapatto Risto , Raivio Kari O.
Published: May 2013

Xanthine oxidoreductase (XOR) catalyzes the final reactions of purine catabolism and may account for cell damage by producing reactive oxygen metabolites in cells reoxygenated after hypoxia. We found a three- to eightfold higher XOR activity in cultured human bronchial epithelial cells exposed to hypoxia (0.5–3% O2) compared with cells grown in normoxia (21% O2) but no difference in XOR protein or mRNA. XOR promoter constructs failed to respond to hypoxia. The cellular XOR activity at 3% O2 returned to basal levels when the cells were returned to 21% O2, and hyperoxia (95% O2) abolished enzyme activity with no change in XOR protein. Our data suggest reversible enzyme inactivation by oxygen or its metabolites. NADH was normally oxidized by the oxygen-inactivated enzyme, which rules out damage to the flavin adenine dinucleotide cofactor. Hydrogen peroxide partially inactivated the molybdenum center of XOR, as shown by a parallel decrease in XOR-catalyzed xanthine oxidation and dichlorophenolindophenol reduction. We conclude that the transcription or translation of XOR is not influenced by hypoxia or hyperoxia. Instead, the molybdenum center of XOR is posttranslationally inactivated by oxygen metabolites in “normal” (21% O2) cell culture atmosphere. This inactivation is reversed in hypoxia and accounts for the apparent induction.
Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A
Authors: Pedersen Stine F. , King Scott A. , Rigor Robert R. , Zhuang Zhenpeng , Warren Jaimie M. , Cala Peter M.
Published: May 2013

In this report, we describe the cloning, cellular localization, and functional characteristics of Na+/H+ exchanger 1 (NHE1) from red blood cells of the winter flounder Pseudopleuronectes americanus (paNHE1). The paNHE1 protein localizes primarily to the marginal band and exhibits a 74% similarity to the trout β-NHE, and 65% to the human NHE1 (hNHE1). Functionally, paNHE1 shares characteristics of both β-NHE and hNHE1 in that it is activated both by manipulations that increase cAMP and by cell shrinkage, respectively. In accordance, the paNHE1 protein exhibits both protein kinase A consensus sites as in β-NHE and a region of high homology to that required for shrinkage-dependent activation of hNHE1. After shrinkage-dependent activation of paNHE1 and resulting activation of a Cl−/HCO[Formula: see text] exchanger, their parallel operation results in net uptake of NaCl and osmotically obliged water. Activation of paNHE1 by cAMP is at least additive to that elicited by osmotic shrinkage, suggesting that these stimuli regulate paNHE1 by distinct mechanisms. Finally, exposure to the serine/threonine phosphatase inhibitor calyculin A potently activates paNHE1, and this activation is also additive to that induced by shrinkage or cAMP.
A novel form of cellular communication among thymic epithelial cells: intercellular calcium wave propagation
Authors: Nihei O. K. , Campos de Carvalho A. C. , Spray D. C. , Savino W. , Alves L. A.
Published: May 2013

We here describe intercellular calcium waves as a novel form of cellular communication among thymic epithelial cells. We first characterized the mechanical induction of intercellular calcium waves in different thymic epithelial cell preparations: cortical 1-4C18 and medullary 3-10 thymic epithelial cell lines and primary cultures of thymic “nurse” cells. All thymic epithelial preparations responded with intercellular calcium wave propagation after mechanical stimulation. In general, the propagation efficacy of intercellular calcium waves in these cells was high, reaching 80-100% of the cells within a given confocal microscopic field, with a mean velocity of 6-10 μm/s and mean amplitude of 1.4- to 1.7-fold the basal calcium level. As evaluated by heptanol and suramin treatment, our results suggest the participation of both gap junctions and P2 receptors in the propagation of intercellular calcium waves in thymic nurse cells and the more prominent participation of gap junctions in thymic epithelial cell lines. Finally, in cocultures, the transmission of intercellular calcium wave was not observed between the mechanically stimulated thymic epithelial cell and adherent thymocytes, suggesting that intercellular calcium wave propagation is limited to thymic epithelial cells and does not affect the neighboring thymocytes. In conclusion, these data describe for the first time intercellular calcium waves in thymic epithelial cells and the participation of both gap junctions and P2 receptors in their propagation.
Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation
Authors: Spangenburg Espen E. , Booth Frank W.
Published: May 2013

There are many known growth factors/cytokines that induce skeletal muscle satellite cell proliferation. Currently, the signaling mechanisms in which these growth factors/cytokines activate satellite cell proliferation are not completely understood. Here, we sought to determine signaling mechanisms by which leukemia inhibitory factor (LIF) induces satellite cell proliferation in culture. First, we confirmed that LIF induces proliferation of C2C12 immortalized myoblasts and cultured primary rat satellite cells. In addition, we also found that this increase in proliferation can be inhibited by incubation of the cells in tyrphostin AG 490, a specific inhibitor of Janus-activated kinase (JAK) 2 activity. Furthermore, we also found that incubation of the cells at various time points with LIF (10 ng/ml) induces a significant, transient increase in JAK2 phosphorylation, signal transducers and activators of transcription (STAT3) phosphorylation, and STAT3 transcriptional activity. Increases in the STAT3-sensitive endogenous SOC3 protein followed these transient increases in STAT3 activation. In addition, AG 490 inhibited the increase in STAT3 phosphorylation. Finally, LIF did not change the phosphorylation status of extracellular signal-regulated protein kinase (ERK)1/2 or affect the phosphorylation status of Akt/protein kinase B. However, LY-294002, an inhibitor of phosphoinositide 3-kinase, blocked LIF-induced proliferation of satellite cells. These data suggest that LIF induces satellite cell proliferation by activation of the JAK2-STAT3 signaling pathway, suggesting that this may be an important pathway in muscle growth and/or hypertrophy.
Substance P inhibits bicarbonate secretion from guinea pig pancreatic ducts by modulating an anion exchanger
Authors: Hegyi Peter , Gray Michael A. , Argent Barry E.
Published: May 2013

The stimulatory pathways controlling HCO3- secretion by the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, which may play an important role in the physiological control of the pancreas. The aim of this study was to investigate the cellular mechanism by which substance P (SP) inhibits pancreatic ductal HCO3- secretion. Small intra/interlobular ducts were isolated from the pancreas of guinea pigs. During overnight culture the ducts seal to form a closed sac. Transmembrane HCO3- fluxes were calculated from changes in intracellular pH (measured using the pH-sensitive dye BCECF) and the buffering capacity of the cells. We found that secretin can stimulate HCO3- secretion in guinea pig pancreatic ducts about fivefold and that this effect could be totally blocked by SP. The inhibitory effect of SP was relieved by spantide, an SP receptor antagonist. SP had no effect on the activity of basolateral Na+-HCO3- cotransporters and Na+/H+ exchangers. However, the peptide did inhibit a Cl--dependent HCO3- efflux (secretory) mechanism, most probably the Cl-/HCO3 exchanger on the apical membrane of the duct cell.
High glucose inhibits apoptosis in human coronary artery smooth muscle cells by increasing bcl-xL and bfl-1/A1
Authors: Sakuma Hiroya , Yamamoto Mayumi , Okumura Mie , Kojima Toshihiro , Maruyama Takako , Yasuda Keigo
Published: May 2013

Cardiovascular disease is a serious complication in diabetic patients. To elucidate the precise mechanisms of atherosclerosis in diabetic patients, the effects of high glucose concentration (25 mM) on apoptosis regulation and bcl-2 family protein expression in human coronary artery smooth muscle cells (CASMC) were examined. Treatment with a high level of glucose (25 mM) caused a significant decrease in apoptosis in CASMC compared with the same cells treated with a physiologically normal glucose concentration (5.5 mM) (23.9 ± 2.4% vs. 16.5 ± 1.8%; P < 0.01). With respect to apoptosis regulation, treatment of CASMC with high glucose concentration markedly increased mRNA expressions of bcl-xL and bfl-1/A1 compared with cells treated with normal glucose. High glucose induced phosphorylation of phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase (ERK)1/2 along with bcl-xL and bfl-1/A1 upregulation. These results suggest that high glucose suppresses apoptosis via upregulation of bcl-xL and bfl-1/A1 levels through PI 3-K and ERK1/2 pathways in CASMC. High glucose-induced increase in the expression of antiapoptotic proteins may be important in the development of atherosclerosis in diabetic patients.
Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions
Authors: Khadeer Mohammed A. , Tang Zhihui , Tenenhouse Harriet S. , Eiden Maribeth V. , Murer Heini , Hernando Natividad , Weinman Edward J. , Chellaiah Meenakshi A. , Gupta Anandarup
Published: May 2013

We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Pi cotransporters in the osteoclast plasma membrane provide Pi for ATP synthesis and that the osteoclast may utilize part of the Pi released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Pi cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Pi cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Pi cotransporters in the osteoclast.
Cross signaling, cell specificity, and physiology
Authors: Dumont J. E. , Dremier S. , Pirson I. , Maenhaut C.
Published: May 2013

The literature on intracellular signal transduction presents a confusing picture: every regulatory factor appears to be regulated by all signal transduction cascades and to regulate all cell processes. This contrasts with the known exquisite specificity of action of extracellular signals in different cell types in vivo. The confusion of the in vitro literature is shown to arise from several causes: the inevitable artifacts inherent in reductionism, the arguments used to establish causal effect relationships, the use of less than adequate models (cell lines, transfections, acellular systems, etc.), and the implicit assumption that networks of regulations are universal whereas they are in fact cell and stage specific. Cell specificity results from the existence in any cell type of a unique set of proteins and their isoforms at each level of signal transduction cascades, from the space structure of their components, from their combinatorial logic at each level, from the presence of modulators of signal transduction proteins and of modulators of modulators, from the time structure of extracellular signals and of their transduction, and from quantitative differences of expression of similar sets of factors.
Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion
Authors: Kanno Harumi , Horikawa Yoshitaka , Hodges Robin R. , Zoukhri Driss , Shatos Marie A. , Rios Jose D. , Dartt Darlene A.
Published: May 2013

Conjunctival goblet cells are the primary source of mucins in the mucous layer, the innermost layer of the tear film. Conjunctival goblet cell mucin secretion is under neural control because exogenous addition of parasympathetic agonists stimulates goblet cell secretion. To elucidate the intracellular signal pathways used by cholinergic agonists to stimulate goblet cell mucin secretion, we determined whether p42/p44 mitogen-activated protein kinase (MAPK) is activated during cholinergic agonist-stimulated mucin secretion. Rat conjunctiva was removed, preincubated with or without antagonists, and stimulated with the cholinergic agonist carbachol (10−4M). Carbachol statistically significantly stimulated the phosphorylation of MAPK in a time- and concentration-dependent manner. U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M1 muscarinic antagonist pirenzepine, the M2 muscarinic antagonist gallamine, and the M1/M3 muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP) also inhibited carbachol-stimulated MAPK activation. Increasing the intracellular Ca2+ concentration with a Ca2+ionophore increased MAPK activation, and chelation of extracellular Ca2+ inhibited carbachol-stimulated activation. Carbachol also increased tyrosine phosphorylation of Pyk2, p60Src, and the epidermal growth factor receptor (EGFR). The Src inhibitor PP1 and the EGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPK activation. AG-1478 also inhibited goblet cell secretion. We conclude that carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.
Cytokine response to eccentric exercise in young and elderly humans
Authors: Toft Anders Dyhr , Jensen Lars Bjørn , Bruunsgaard Helle , Ibfelt Tobias , Halkjær-Kristensen Jens , Febbraio Mark , Pedersen Bente Klarlund
Published: May 2013

To examine the plasma interleukin (IL)-6 response in elderly (E) and young (Y) humans, 10 E and 10 Y subjects completed 60 min of eccentric lower limb exercise at the same relative oxygen uptake. Plasma IL-6 was measured before, immediately after, and 5 days into recovery from exercise, as were the biochemical markers of muscle damage, creatine kinase (CK), and myoglobin. In both groups, IL-6 increased ( P < 0.05) immediately after exercise and peaked 4 h after exercise at 4.35 ± 1.7 vs. 5.05 ± 3.17 pg/ml for E and Y subjects, respectively. However, the increase in IL-6 in both groups was modest relative to the increases in CK peaking at 539 ± 413 vs. 10,301 ± 5,863 U/l for E and Y subjects, respectively. In addition, the increase in IL-6 was less pronounced ( P < 0.05) in E subjects compared with Y subjects. These results suggest that IL-6 increases progressively after eccentric exercise, suggesting that this increase is related to muscle damage. However, the modest increase in IL-6, despite large increases in CK, suggests that the IL-6 response to muscle damage does not make an important contribution to the large increase in IL-6 observed during concentric exercise of long duration. Our data also suggest that aging may be associated with impaired repair mechanisms for exercise-induced muscle damage.
Differential regulation of junctional complex assembly in renal epithelial cell lines
Authors: Gopalakrishnan Shobha , Hallett Mark A. , Atkinson Simon J. , Marrs James. A.
Published: May 2013

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and β-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.
Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-Iα activation by NO/cGMP
Authors: Rivero-Vilches F. J. , De Frutos S , Saura M , Rodriguez-Puyol D , Rodriguez-Puyol M
Published: May 2013

cGMP is generated in endothelial cells after stimulation of soluble guanylyl cyclase (sGC) by nitric oxide (NO) or of particulate guanylyl cyclase (pGC) by natriuretic peptides (NP). We examined whether localized increases in cytosolic cGMP have distinct regulatory roles on the contraction induced by H2O2 treatment in human umbilical vein endothelial cells. cGMP concentrations and temporal dynamics were different upon NO stimulation of sGC or C-type NP (CNP) activation of pGC and did not correlate with their relaxing effects measured as planar cell surface area after H2O2 challenge. cGMP production due to sGC stimulation was always smaller and more brief than that induced by pGC stimulation with CNP, which was greater and remained elevated longer. The NO effects on cell relaxation were cGMP dependent because they were blocked by sGC inhibition with 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxaline-1-one and mimicked by 8-Br-cGMP. An antagonist of the cGMP-dependent protein kinase type-I (PKG-I) also inhibited the NO-induced effects. The cell contraction induced by H2O2 produces myosin light chain (MLC) phosphorylation and NO prevented it completely, whereas CNP only produced a partial inhibition. Transfection with a dominant negative form of PKG type-Iα completely reversed the NO-induced effects on MLC phosphorylation, whereas it only partially inhibited the effects due to CNP. Taken together, these results demonstrate that the NO/sGC/cGMP pathway induces endothelial cell relaxation in a more efficient manner than does CNP/pGC/cGMP pathway, an effect that might be related to a selective stimulation of PKG-1α by NO-derived cGMP. Consequently, stimulated PKG-Iα may phosphorylate important protein targets that are necessary to inhibit the endothelial contractile machinery activated by oxidative stress.
Resistance of isolated mammalian spinal cord white matter to oxygen-glucose deprivation
Authors: Peasley Melissa A. , Shi Riyi
Published: May 2013

We found that isolated guinea pig spinal cord white matter is resistant to acute oxygen-glucose deprivation. Sixty minutes of oxygen-glucose deprivation resulted in a 60% reduction of compound action potential (CAP) conductance, and there was a near complete recovery after 60 min reperfusion. Corresponding horseradish peroxidase-exclusion assay showed little axonal membrane damage. To further deprive the axons of metabolic substrate, we added 2 mM sodium cyanide or 2 mM sodium azide, both mitochondrial suppressors, to the ischemic medium, which completely abolished CAP and resulted in a 15 to ∼30% recovery postreperfusion. Both compounds preferentially reduced the conductance of large diameter axons. We suggest the residual ATP in our ischemic model can protect anatomic integrity and physiological functioning of spinal axons following ischemic insult. This further suggests that oxygen-glucose deprivation alone cannot be solely responsible for short-term functional and anatomic damage. The damaging effects of ischemia in vivo may be mediated by factors originating from the gray matter of the cord or other systemic factors; both were largely eliminated in our in vitro white matter preparation.
Expression of SERCA2a is independent of innervation in regenerating soleus muscle
Authors: Zádor Ernő , Wuytack Frank
Published: May 2013

The speed of contraction of a skeletal muscle largely depends on the myosin heavy chain isoforms (MyHC), whereas the relaxation is initiated and maintained by the sarcoplasmic reticulum Ca2+-ATPases (SERCA). The expression of the slow muscle-type myosin heavy chain I (MyHCI) is entirely dependent on innervation, but, as we show here, innervation is not required for the expression of the slow-type sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in regenerating soleus muscles of the rat, although it can play a modulator role. Remarkably, the SERCA2a level is even higher in denervated than in innervated regenerating soleus muscles on day 7 when innervation is expected to resume. Later, the level of SERCA2a protein declines in denervated regenerated muscles but it remains expressed, whereas the corresponding mRNA level is still increasing. SERCA1 (i.e., the fast muscle-type isoform) expression shows only minor changes in denervated regenerating soleus muscles compared with innervated regenerating controls. When the soleus nerve was transected instead of the sciatic nerve, SERCA2a and MyHCI expressions were found to be even more uncoupled because the MyHCI nearly completely disappeared, whereas the SERCA2a mRNA and protein levels decreased much less. The transfection of regenerating muscles with constitutively active mutants of the Ras oncogene, known to mimic the effect of innervation on the expression of MyHCI, did not affect SERCA2a expression. These results demonstrate that the regulation of SERCA2a expression is clearly distinct from that of the slow myosin in the regenerating soleus muscle and that SERCA2a expression is modulated by neuronal activity but is not entirely dependent on it.