bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells
Authors: Sun Xing Cai , Bonanno Joseph A.
Published: May 2013

HCO[Formula: see text]-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO[Formula: see text]transport have not been reported. Immunoblotting for CFTR showed a single band at ∼170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy- N-ethylquinolinium iodide during Cl−influx in the absence and presence of forskolin (FSK). Apical and basolateral Cl− permeability increased 10- and 3-fold, respectively, in the presence of 50 μM FSK. FSK-activated apical chloride permeability was unaffected by H2DIDs (250 μM); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 μM) and glibenclamide (100 μM) inhibited activated Cl−fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl− permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by H2DIDS. HCO[Formula: see text] permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HCO[Formula: see text]]. FSK (50 μM) increased apical HCO[Formula: see text] permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HCO[Formula: see text] permeability was unaffected by FSK. Genistein (50 μM) significantly increased apical HCO[Formula: see text] and Cl− permeability by 1.8- and 16-fold, respectively. When 50 μM genistein was combined with 50 μM FSK, there was no further increase in Cl− permeability; however, HCO[Formula: see text] permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial Cl− and HCO[Formula: see text] transport. Furthermore, there is a cAMP-activated Cl− pathway on the basolateral membrane that is not CFTR.
Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter
Authors: Brindikova Tatyana A. , Bourcier Nathalie , Torres Brian , Pchejetski Dimitri , Gekle Michel , Maximov Georgy V. , Montminy Valérie , Insel Paul A. , Orlov Sergei N. , Isenring Paul
Published: May 2013

Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones (α-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 μM ATP or 100 μM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.
Transcriptional regulation of the type I myosin heavy chain gene in denervated rat soleus
Authors: Huey K. A. , Haddad F. , Qin A. X. , Baldwin K. M.
Published: May 2013

Denervation (DEN) of rat soleus is associated with a decreased expression of slow type I myosin heavy chain (MHC) and an increased expression of the faster MHC isoforms. The molecular mechanisms behind these shifts remain unclear. We first investigated endogenous transcriptional activity of the type I MHC gene in normal and denervated soleus muscles via pre-mRNA analysis. Our results suggest that the type I MHC gene is regulated via transcriptional processes in the denervated soleus. Deletion and mutational analysis of the rat type I MHC promoter was then used to identify cis elements or regions of the promoter involved in this response. DEN significantly decreased in vivo activity of the −3,500, −2,500, −914, −408, −299, and −215 bp type I MHC promoters, relative to the α-skeletal actin promoter. In contrast, normalized −171 promoter activity was unchanged. Mutation of the βe3 element (−214/−190) in the −215 promoter and deletion of this element (−171 promoter) blunted type I downregulation with DEN. In contrast, βe3 mutation in the −408 promoters was not effective in attenuating the DEN response, suggesting the existence of additional DEN-responsive sites between −408 and −215. Western blotting and gel mobility supershift assays demonstrated decreased expression and DNA binding of transcription enhancer factor 1 (TEF-1) with DEN, suggesting that this decrease may contribute to type I MHC downregulation in denervated muscle.
Characteristics of hyperpolarization-activated cation currents in portal vein smooth muscle cells
Authors: Greenwood I. A. , Prestwich S. A.
Published: May 2013

Voltage-clamp studies of freshly isolated smooth muscle cells from rabbit portal vein revealed the existence of a time-dependent cation current evoked by membrane hyperpolarization (termed I h). Both the rate of activation and the amplitude of I h were enhanced by membrane hyperpolarization. Half-maximal activation of I h was about −105 mV with conventional whole cell and −80 mV when the perforated patch technique was used. In current clamp, injection of hyperpolarizing current produced a marked depolarizing “sag” followed by rebound depolarization. Activation of I h was augmented by an increase in the extracellular K+ concentration and was blocked rapidly by externally applied Cs+ (1–5 mM). The bradycardic agent ZD-7288 (10 μM), a selective inhibitor of I h, produced a characteristically slow inhibition of the portal vein I h. The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs+. Cs+ significantly decreased the frequency of spontaneous contractions in both whole rat portal vein and rabbit portal vein segments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1–4) revealed the existence of cDNA clones corresponding to HCN2, 3, and 4. The present study shows that portal vein myocytes contain genes shown to encode for hyperpolarization-activated channels and exhibit an endogenous current with characteristics similar to I h in other cell types. This conductance appears to determine, in part, the rhythmicity of this vessel.
Serine/threonine phosphorylation regulates HNF-4α-dependent redox-mediated iNOS expression in hepatocytes
Authors: Guo Hongtao , Wei Junping , Inoue Yusuke , Gonzalez Frank J. , Kuo Paul C.
Published: May 2013

Nitric oxide (NO), endogenously synthesized by inducible NO synthase (iNOS), serves antioxidant and antiapoptotic functions in settings characterized by oxidative stress and proinflammatory cytokines such as sepsis and shock. However, the redox-sensitive mechanisms regulating hepatocyte expression of iNOS are largely unknown. In interleukin-1β (IL-1β)-stimulated hepatocytes exposed to superoxide, we demonstrate that hepatocyte nuclear factor-4α (HNF-4α) acts as an activator of redox-associated hepatocyte iNOS expression at the level of protein, mRNA, and promoter activation. In the absence of HNF-4α, this redox-mediated enhancement is ablated. HNF-4α functional activity is associated with a unique serine/threonine kinase-mediated phosphorylation pattern. This suggests that a redox-sensitive kinase pathway targets HNF-4α to augment hepatocyte iNOS expression. Previous studies have not addressed a redox-dependent kinase signaling pathway that targets HNF-4α and enhances hepatocyte iNOS gene transcription. A unique pattern of phosphorylation determines HNF-4α activity as a trans-activator of IL-1β-mediated hepatocyte iNOS expression in the presence of oxidative stress.
Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon
Authors: Ikuma Mutsuhiro , Geibel John , Binder Henry J. , Rajendran Vazhaikkurichi M.
Published: May 2013

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant ( Ki) of ∼26.9 and 30.6 μM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of ∼12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.
Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells
Authors: Alvarez de la Rosa Diego , Canessa Cecilia M.
Published: May 2013

The purpose of this study was to examine the role of the serum- and glucocorticoid-induced kinase (SGK) in the activation of the epithelial sodium channel (ENaC) by aldosterone, arginine vasopressin (AVP), and insulin. We used a tetracycline-inducible system to control the expression of wild-type (SGK[Formula: see text]), constitutively active (S425D mutation; SGK[Formula: see text]), or inactive (K130M mutation; SGK[Formula: see text]) SGK in A6 cells independently of hormonal stimulation. The effect of SGK expression on ENaC activity was monitored by measuring transepithelial amiloride-sensitive short-circuit current ( I sc) of transfected A6 cell lines. Expression of SGK[Formula: see text] or SGK[Formula: see text] and aldosterone stimulation have additive effects on I sc. Although SGK could play some role in the aldosterone response, our results suggest that other mechanisms take place. SGK[Formula: see text] abrogates the responses to AVP and insulin; hence, in the signaling pathways of these hormones there is a shared step that is stimulated by SGK. Because AVP and insulin induce fusion of vesicles to the apical membrane, our results support the notion that SGK promotes incorporation of channels in the apical membrane.
HCO 3 − -dependent soluble adenylyl cyclase activates cystic fibrosis transmembrane conductance regulator in corneal endothelium
Authors: Sun Xing Cai , Zhai Chang-Bin , Cui Miao , Chen Yanqiu , Levin Lonny R. , Buck Jochen , Bonanno Joseph A.
Published: May 2013

cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) regulates fluid transport in many tissues. Secretion by the corneal endothelium is stimulated by cAMP and dependent on HCO[Formula: see text]. We asked whether HCO[Formula: see text] can secondarily increase CFTR permeability in bovine corneal endothelial cells (BCEC) by activating soluble adenylyl cyclase (sAC). Immunofluorescence suggests that sAC is distributed throughout the cytoplasm. HCO[Formula: see text] (40 mM) increased cAMP concentration 42% in the presence of 50 μM rolipram (a phosphodiesterase 4 inhibitor), and a standard HCO[Formula: see text] Ringer solution (28.5 mM) increased apical Cl− permeability by 78% relative to HCO[Formula: see text]-free solution. The HCO[Formula: see text]-dependent increase in Cl−permeability was reduced 60% by 20 mM NaHSO3 (a weak agonist of sAC). NaHSO3 alone increased apical Cl− permeability by only 13%. The HCO[Formula: see text]-dependent increase in Cl−permeability was reduced 57% in the presence of 50 μM Rp-adenosine 3′,5′-cyclic monophosphorothioate, and 86% by 50 μM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid but unaffected by 200 μM apical H2DIDS. CFTR phosphorylation was increased 23, 150, and 32% by 20 mM HSO[Formula: see text], 28.5 mM HCO[Formula: see text], and 28.5 mM HCO[Formula: see text] + 20 mM HSO[Formula: see text], respectively. Activation of apical Cl− permeability by 5 μM genistein was increased synergistically by HCO[Formula: see text] over that due to genistein and HCO[Formula: see text] alone. We conclude that HCO[Formula: see text]-stimulated sAC is a form of autocrine signaling that contributes to baseline cAMP production, thereby affecting baseline CFTR activity in BCEC. This form of autocrine signaling may be important in tissues that express sAC and exhibit robust HCO[Formula: see text] influx (e.g., ocular ciliary epithelium, choroid plexus, and airway epithelium).
CLIC1 inserts from the aqueous phase into phospholipid membranes, where it functions as an anion channel
Authors: Tulk Barry M. , Kapadia Shefalee , Edwards John C.
Published: May 2013

CLIC1 is a member of the CLIC family of proteins, which has been shown to demonstrate chloride channel activity when reconstituted in phospholipid vesicles. CLIC1 exists in cells as an integral membrane protein and as a soluble cytoplasmic protein, implying that CLIC1 might cycle between membrane-inserted and soluble forms. CLIC1 was purified and detergent was removed, yielding an aqueous solution of essentially pure protein. Pure CLIC1 was mixed with vesicles, and chloride permeability was assessed with a chloride efflux assay and with planar lipid bilayer techniques. Soluble CLIC1 confers anion channel activity to preformed membranes that is indistinguishable from the previously reported activity resulting from reconstitution of CLIC1 into membranes by detergent dialysis. The activity is dependent on the amount of CLIC1 added, appears rapidly on mixing of protein and lipid, is inhibited by indanyloxyacetic acid-94, N-ethylmaleimide, and glutathione, is inactivated by heat, and shows sensitivity to pH and to membrane lipid composition. We conclude that CLIC1 in the absence of detergent spontaneously inserts into preformed membranes, where it can function as an anion-selective channel.
Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle
Authors: Walia Mandeep , Samson Sue E. , Schmidt Tracey , Best Kelly , Whittington Melinda , Kwan Chiu Yin , Grover Ashok K.
Published: May 2013

Peroxynitrite generated in arteries from superoxide and nitric oxide (NO) may damage their function. Here, we compare the effects of peroxynitrite and peroxynitrite/NO-generating agents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP (S-nitroso- N-acetyl-penicillamine), SNP (sodium nitroprusside), and NONOate (spermine NONOate) on pig coronary artery. Deendothelialized artery rings were pretreated with these agents and then washed before examining their contractility. Pretreatment with all agents (200 μM) results in a decrease in the force of contraction in response to the sarco(endo)plasmic Ca2+ (SERCA) pump inhibitor cyclopiazonic acid (CPA): SNAP > NONOate ≥ peroxynitrite ≥ SIN-1 > SNP. Pretreatment with SNAP, NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plus superoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor (1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca2+transients produced in response to CPA. Pretreating isolated membrane vesicles with peroxynitrite inhibits the Ca2+ uptake due to the SERCA pump, with all the other agents being less effective. Thus peroxynitrite and NO both inhibit the CPA-induced contractions in deendothelialized artery rings, peroxynitrite by damage to the SERCA pump and NO possibly by a step downstream from the increase in cytosolic Ca2+.
Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter
Authors: Sassani Pejvak , Pushkin Alexander , Gross Eitan , Gomer Alla , Abuladze Natalia , Dukkipati Ramanath , Carpenito Gerardo , Kurtz Ira
Published: May 2013

Sodium-bicarbonate cotransporters are homologous membrane proteins mediating the electrogenic or electroneutral transport of sodium and bicarbonate. Of the functionally characterized sodium-bicarbonate cotransporters (NBC), NBC1proteins are known to be electrogenic. Here we report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein variants and 40% identical to electroneutral NBC3. When expressed in mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate cotransport. The transport of sodium and bicarbonate is chloride independent and is completely inhibited by DIDS. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. The data indicate that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.
Hormonal mechanisms regulating hepatic vitellogenin synthesis in the gilthead sea bream, Sparus aurata
Authors: Mosconi G. , Carnevali O. , Habibi H. R. , Sanyal R. , Polzonetti-Magni A. M.
Published: May 2013

Experiments were carried out to study in vitro the effects of 17β-estradiol (E2), homologous pituitary homogenate (HPH), and recombinant red sea bream growth hormone (sbGH) on vitellogenin (VTG) secretion from cultured sea bream liver fragments. Basal secretion of VTG was found to be significantly higher in the prespawning period, compared with sea bream liver in the spawning and postspawning periods. Similarly, the sea bream liver obtained during the prespawning period responded more significantly to treatments with E2, HPH, or sbGH compared with sea bream liver during spawning. In the postspawning period, treatments with E2, HPH, or sbGH were without significant effect on VTG secretion level in sea bream liver. The level of E2receptors was also analyzed by Western blot analysis. The result demonstrates a significantly higher level of E2 receptors in the sea bream liver at the prespawning stage compared with those at the spawning and postspawning stages. The findings support the hypothesis that homologous upregulation of estrogen receptors plays an important role in the estrogen-sensitive control of VTG synthesis in the sea bream liver.
Rapid and dynamic regulation of TGF-β receptors on blood vessels and fibroblasts during ischemia-reperfusion injury
Authors: Mortazavi-Haghighat Roya , Taghipour-Khiabani Kayvan , David Sam , Kerrigan Carolyn L. , Philip Anie
Published: May 2013

The pathophysiological mechanisms involved in ischemia-reperfusion injury are poorly understood. Although transforming growth factor (TGF)-β has been shown to provide protection against ischemia-reperfusion injury in different organ systems, little is known about the regulation of TGF-β action during this process. Here we analyzed the effect of ischemia and reperfusion on the expression of TGF-β and its receptors in vivo with a pig skin flap model. Analysis of unoperated skin, nonischemic control flap, ischemic flap, and reperfused flap by immunohistochemistry indicates that ischemia and reperfusion result in rapid and dynamic regulation of type I, II, and III TGF-β receptors and TGF-β1 in a cell type-specific manner. Furthermore, hypoxia upregulates type II TGF-β receptor mRNA in skin fibroblasts in culture. Together, our results reveal that TGF-β receptors and TGF-β1 are markedly increased under acute ischemic conditions in the blood vessels and fibroblasts of the skin. We conclude that TGF-β action is enhanced under ischemic conditions and that it may represent an adaptive response to ischemic injury. The augmented TGF-β responsiveness may be a critical determinant of the protective effect of TGF-β during ischemia-reperfusion injury.
Regulation of endothelial barrier function and growth by VE-cadherin, plakoglobin, and β-catenin
Authors: Venkiteswaran Kala , Xiao Kanyan , Summers Susan , Calkins Cathárine C. , Vincent Peter A. , Pumiglia Kevin , Kowalczyk Andrew P.
Published: May 2013

VE-cadherin is an endothelial-specific cadherin that plays a central role in vascular barrier function and angiogenesis. The cytoplasmic domain of VE-cadherin is linked to the cytoskeleton through interactions with the armadillo family proteins β-catenin and plakoglobin. Growing evidence indicates that β-catenin and plakoglobin play important roles in epithelial growth and morphogenesis. To test the role of these proteins in vascular cells, a replication-deficient retroviral system was used to express intercellular junction proteins and mutants in the human dermal microvascular endothelial cell line (HMEC-1). A mutant VE-cadherin lacking an adhesive extracellular domain disrupted endothelial barrier function and inhibited endothelial growth. In contrast, expression of exogenous plakoglobin or metabolically stable mutants of β-catenin stimulated HMEC-1 cell growth, which suggests that the β-catenin signaling pathway was active in HMEC-1 cells. This possibility was supported by the finding that a dominant-negative mutant of the transcription factor TCF-4, designed to inhibit β-catenin signaling, also inhibited HMEC-1 cell growth. These observations suggest that intercellular junction proteins function as components of an adhesion and signaling system that regulates vascular barrier function and growth.
Role of MDR1 and MRP1 in trophoblast cells, elucidated using retroviral gene transfer
Authors: Atkinson Diane E. , Greenwood Susan L. , Sibley Colin P. , Glazier Jocelyn D. , Fairbairn Leslie J.
Published: May 2013

Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [3H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus.
Adenoviral gene transfer of PDGF downregulates gasgene product PDGFαR and prolongs ERK and Akt/PKB activation
Authors: Chen Qi-Ping , Giannobile William V.
Published: May 2013

The delivery of platelet-derived growth factor (PDGF) for tissue engineering of skin and periodontal wounds has become an active area of interest. However, little is known regarding the extended effects of PDGF on cell signaling via gene therapy and how such an approach facilitates the exiting of cells from growth arrest and entry to competence required for cell cycling. We show in vitro expression and secretion of PDGF-AA by recombinant adenovirus encoding the PDGF-A gene (Ad-PDGF-A). The bioactive PDGF-AA protein released induces sustained downregulation of PDGFαR that is encoded by a growth arrest-specific ( gas) gene. Ad-PDGF-A induces sustained phosphorylation of PDGFαR as well as prolonged phosphorylation of downstream extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Furthermore, the phosphorylation of PDGFαR is abolished by cotransducing cells with adenovirus encoding a dominant negative mutant of the PDGF-A gene that disrupts PDGF bioactivity. These findings demonstrate the prolonged effects of adenoviral delivery of PDGF and aid in the better understanding of sustained PDGF signaling.
Progressive decrease of intramyocellular accumulation of H+ and Pi in human skeletal muscle during repeated isotonic exercise
Authors: Rico-Sanz J.
Published: May 2013

The purpose of this study was to evaluate the hypotheses that accumulation of hydrogen ions and/or inorganic phosphate (Pi) in skeletal muscle increases with repeated bouts of isotonic exercise. 31P-Magnetic resonance spectroscopy was used to examine the gastrocnemius muscle of seven highly aerobically trained females during four bouts of isotonic plantar flexion. The exercise bouts ( EX1- 4) of 3 min and 18 s were separated by 3 min and 54 s of complete rest. Muscle ATP did not change during the four bouts. Phosphocreatine (PCr) degradation during EX1 (13.3 ± 2.4 mmol/kg wet weight) was higher ( P < 0.01) compared with EX3- 4(9.7 ± 1.6 and 9.6 ± 1.8 mmol/kg wet weight, respectively). The intramyocellular pH at the end of EX1 (6.87 ± 0.05) was significantly lower ( P < 0.001) than those of EX2 (6.97 ± 0.02), EX3 (7.02 ± 0.01), and EX4 (7.02 ± 0.02). Total Pi and diprotonated Pi were significantly higher ( P < 0.001) at the end of EX1 (17.3 ± 2.7 and 7.8 ± 1.6 mmol/kg wet weight, respectively) compared with the values at the end of EX3 and EX4. The monoprotonated Pi at the end of EX1 (9.5 ± 1.2 mmol/kg wet weight) was also significantly higher ( P < 0.001) than that after EX4 (7.5 ± 1.1 mmol/kg wet weight). Subjects' rating of perceived exertion increased ( P < 0.001) toward exhaustion as the number of exercises progressed (7.1 ± 0.4, EX1; 8.0 ± 0.3, EX2; 8.5 ± 0.3, EX3; and 9.0 ± 0.4, EX4; scale from 0 to 10). The present results indicate that human muscle fatigue during repeated intense isotonic exercise is not due to progressive depletion of high energy phosphates nor to intracellular accumulation of hydrogen ions, total, mono-, or diprotonated Pi.
Ethanol sensitivity of BKCa channels from arterial smooth muscle does not require the presence of the β1-subunit
Authors: Dopico Alejandro M.
Published: May 2013

Ethanol inhibition of large-conductance, Ca2+-activated K+ (BKCa) channels in aortic myocytes may contribute to the direct contraction of aortic smooth muscle produced by acute alcohol exposure. In this tissue, BKCa channels consist of pore-forming ( bslo) and modulatory (β) subunits. Here, modulation of aortic myocyte BKCa channels by acute alcohol was explored by expressing bslo subunits in Xenopus oocytes, in the absence and presence of β1-subunits, and studying channel responses to clinically relevant concentrations of ethanol in excised membrane patches. Overall, average values of bslo channel activity ( NP o, with N = no. of channels present in the patch; P o = probability of a single channel being open) in response to ethanol (3–200 mM) mildly decrease when compared with pre-ethanol, isosmotic controls. However, channel responses show qualitative heterogeneity at all ethanol concentrations. In the majority of patches (42/71 patches, i.e., 59%), a reversible reduction in NP o is observed. In this subset, the maximal effect is obtained with 100 mM ethanol, at which NP o reaches 46.2 ± 9% of control. The presence of β1-subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17β-estradiol, fails to modify ethanol action on bslo channels. Ethanol inhibition of bslo channels results from a marked increase in the mean closed time. Although the voltage dependence of gating remains unaffected, the apparent effectiveness of Ca2+ to gate the channel is decreased by ethanol. These changes occur without modifications of channel conduction. In conclusion, a new molecular mechanism that may contribute to ethanol-induced aortic smooth muscle contraction has been identified and characterized: a functional interaction between ethanol and the bslo subunit and/or its lipid microenvironment, which leads to a decrease in BKCachannel activity.
Oxidative stress inactivates the human DNA mismatch repair system
Authors: Chang Christina L. , Marra Giancarlo , Chauhan Dharam P. , Ha Hannah T. , Chang Dong K. , Ricciardiello Luigi , Randolph Ann , Carethers John M. , Boland C. Richard
Published: May 2013

In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutSα and hMutSβ complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLα heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutSα, hMutSβ, and hMutLα protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.
Maintenance of muscle mass is not dependent on the calcineurin-NFAT pathway
Authors: Dupont-Versteegden Esther E. , Knox Micheal , Gurley Cathy M. , Houlé John D. , Peterson Charlotte A.
Published: May 2013

In this study, the role of the calcineurin pathway in skeletal muscle atrophy and atrophy-reducing interventions was investigated in rat soleus muscles. Because calcineurin has been suggested to be involved in skeletal and cardiac muscle hypertrophy, we hypothesized that blocking calcineurin activity would eliminate beneficial effects of interventions that maintain muscle mass in the face of atrophy-inducing stimuli. Hindlimb suspension and spinal cord transection were used to induce atrophy, and intermittent reloading and exercise were used to reduce atrophy. Cyclosporin (CsA, 25 mg · kg−1 · day−1) was administered to block calcineurin activity. Soleus muscles were studied 14 days after the onset of atrophy. CsA administration did not inhibit the beneficial effects of the two muscle-maintaining interventions, nor did it change muscle mass in control or atrophied muscles, suggesting that calcineurin does not play a role in regulating muscle size during atrophy. However, calcineurin abundance was increased in atrophied soleus muscles, and this was associated with nuclear localization of NFATc1 (a nuclear factor of activated T cells). Therefore, results suggest that calcineurin may be playing opposing roles during skeletal muscle atrophy and under muscle mass-maintaining conditions.
Maturation of neuronal excitability in hippocampal neurons of mice chronically exposed to cyclic hypoxia
Authors: Gu Xiang Q. , Haddad Gabriel G.
Published: May 2013

To examine the effects of chronic cyclic hypoxia on neuronal excitability and function in mice, we exposed mice to cyclic hypoxia for 8 h daily (9 cycles/h) for ∼2 wk (starting at 2–3 days of age) and examined the properties of freshly dissociated hippocampal neurons obtained from slices. Compared with control (Con) hippocampal CA1 neurons, exposed neurons (CYC) had similar resting membrane potentials ( V m) and action potentials (AP). CYC neurons, however, had a lower rheobase than Con neurons. There was also an upregulation of the Na+current density (333 ± 84 pA/pF, n = 18) in CYC compared with that of Con neurons (193 ± 20 pA/pF, n = 27, P < 0.03). Na+channel characteristics were significantly altered by hypoxia. For example, the steady-state inactivation curve was significantly more positive in CYC than in Con (−60 ± 6 mV, n = 8, for CYC and −71 ± 3 mV, n = 14, for Con, P < 0.04). The time constant for deactivation (τd) was much shorter in CYC than in Con (at −100 mV, τd=0.83 ± 0.23 ms in CYC neurons and 2.29 ± 0.38 ms in Con neurons, P = 0.004). We conclude that the increased neuronal excitability in mice neurons treated with cyclic hypoxia is due to alterations in Na+ channel characteristics and/or Na+ channel expression. We hypothesize from these and previous data from our laboratory (Gu XQ and Haddad GG. J Appl Physiol 91: 1245–1250, 2001) that this increased excitability is a reflection of an enhanced central nervous system maturation when exposed to low O2 conditions in early postnatal life.
Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids
Authors: Tucker Torry A. , Varga Karoly , Bebok Zsuzsa , Zsembery Akos , McCarty Nael A. , Collawn James F. , Schwiebert Erik M. , Schwiebert Lisa M.
Published: May 2013

Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase ( LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.
Ca2+-activated Cl− current in cultured myenteric neurons from murine proximal colon
Authors: Kang Sok Han , Vanden Berghe Pieter , Smith Terence K.
Published: May 2013

Whole cell patch-clamp recordings were made from cultured myenteric neurons taken from murine proximal colon. The micropipette contained Cs+ to remove K+ currents. Depolarization elicited a slowly activating time-dependent outward current ( I tdo), whereas repolarization was followed by a slowly deactivating tail current ( I tail). I tdo and I tail were present in ∼70% of neurons. We identified these currents as Cl− currents ( I Cl), because changing the transmembrane Cl− gradient altered the measured reversal potential ( E rev) of both I tdo and I tail with that for I tailshifted close to the calculated Cl− equilibrium potential ( E Cl). I Cl are Ca2+-activated Cl− current [ I Cl(Ca)] because they were Ca2+dependent. E Cl, which was measured from the E rev of I Cl(Ca) using a gramicidin perforated patch, was −33 mV. This value is more positive than the resting membrane potential (−56.3 ± 2.7 mV), suggesting myenteric neurons accumulate intracellular Cl−. ω-Conotoxin GIVA [0.3 μM; N-type Ca2+ channel blocker] and niflumic acid [10 μM; known I Cl(Ca) blocker], decreased the I Cl(Ca). In conclusion, these neurons have I Cl(Ca) that are activated by Ca2+entry through N-type Ca2+ channels. These currents likely regulate postspike frequency adaptation.
Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe
Authors: Virkki Leila V. , Choi Inyeong , Davis Bruce A. , Boron Walter F.
Published: May 2013

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3′ and 5′ directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage ( Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3--induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).
Role of aspartate residues in Ca2+ affinity and permeation of the distal ECaC1
Authors: Jean K. , Bernatchez G. , Klein H. , Garneau L. , Sauvé R. , Parent L.
Published: May 2013

The Ca2+ affinity and permeation of the epithelial Ca2+ channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extracellular Ca2+ and Mg2+ reversibly inhibited ECaC1 whole cell Li+ currents: IC50 = 2.2 ± 0.4 μM ( n = 9) and 235 ± 35 μM ( n= 10), respectively. These values compare well with the Ca2+ affinity of the L-type voltage-gated Ca2+(CaV1.2) channel measured under the same conditions, suggesting that high-affinity Ca2+ binding is a well-conserved feature of epithelial and voltage-gated Ca2+channels. Neutralization of D550 and E535 in the pore region had no significant effect on Ca2+ and Mg2+ affinities. In contrast, neutralization of D542 significantly decreased Ca2+ affinity (IC50 = 1.1 ± 0.2 mM, n = 6) and Mg2+ affinity (IC50 > 25 ± 3 mM, n = 4). Despite a 1,000-fold decrease in Ca2+ affinity in D542N, Ca2+ permeation properties and the Ca2+-to-Ba2+ conductance ratio remained comparable to values for wild-type ECaC1. Together, our observations suggest that D542 plays a critical role in Ca2+ affinity but not in Ca2+ permeation in ECaC1.
Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site
Authors: Giger Julia M. , Haddad Fadia , Qin Anqi X. , Baldwin Kenneth M.
Published: May 2013

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.
Potassium channel expression level is dependent on the proliferation state in the GH3 pituitary cell line
Authors: Czarnecki Antonny , Dufy-Barbe Luce , Huet Sylvie , Odessa Marie-Françoise , Bresson-Bepoldin Laurence
Published: May 2013

Previously, we showed that the peak density of the transient outward K+ current ( I to) expressed in GH3 cells was different in the S phase than in other phases of the cell cycle. Using cell synchronization, we show here that I to drops precisely at the quiescent (G0 phase)/proliferating transition. This change is not due to a modification in the voltage dependence of I to, but rather to a modification in its inactivation kinetics. Molecular determination of K+channel subunits showed that I to required the expression of Kv1.4, Kv4.1, and Kv4.3. We found that the increase in I to density during the quiescent state was accompanied by an increase in Kv1.4 protein expression, whereas Kv4.3 expression remained unchanged. We further demonstrate that the link between I to expression and cell proliferation is not mediated by variations in cell excitability. These results provide new evidence for the cell cycle dependence of I to expression, which could be relevant in understanding the mechanisms leading to pituitary adenomas.
Fast-twitch skeletal muscles of dystrophic mouse pups are resistant to injury from acute mechanical stress
Authors: Grange Robert W. , Gainer Thomas G. , Marschner Krista M. , Talmadge Robert J. , Stull James T.
Published: May 2013

Loss of the dystrophin-glycoprotein complex from muscle sarcolemma in Duchenne's muscular dystrophy (DMD) renders the membrane susceptible to mechanical injury, leaky to Ca2+, and disrupts signaling, but the precise mechanism(s) leading to the onset of DMD remain unclear. To assess the role of mechanical injury in the onset of DMD, extensor digitorum longus (EDL) muscles from C57 (control), mdx, and mdx-utrophin-deficient [ mdx:utrn(−/−); dystrophic] pups aged 9–12 days were subjected to an acute stretch-injury or no-stretch protocol in vitro. Before the stretches, isometric stress was attenuated for mdx:utrn(−/−) compared with control muscles at all stimulation frequencies ( P< 0.05). During the stretches, EDL muscles for each genotype demonstrated similar mean stiffness values. After the stretches, isometric stress during a tetanus was decreased significantly for both mdx and mdx:utrn(−/−) muscles compared with control muscles ( P < 0.05). Membrane injury assessed by uptake of procion orange dye was greater for dystrophic compared with control EDL ( P < 0.05), but, within each genotype, the percentage of total cells taking up dye was not different for the no-stretch vs. stretch condition. These data suggest that the sarcolemma of maturing dystrophic EDL muscles are resistant to acute mechanical injury.
Estrogen pretreatment protects males against hypoxia-induced immune depression
Authors: Knöferl Markus W. , Schwacha Martin G. , Jarrar Doraid , Angele Martin K. , Fragoza Keith , Bland Kirby I. , Chaudry Irshad H.
Published: May 2013

Hypoxemia depresses cell-mediated immune functions in males, whereas proestrous females do not show such a depression. We hypothesized that elevated systemic estradiol levels in proestrous females prevent hypoxemia-induced immune depression. To study this hypothesis, male C3H/HeN mice were pretreated with 17β-estradiol (E2, 40 μg/kg body wt sc) or vehicle for 3 days before induction of hypoxemia and again immediately before induction of hypoxia. The mice were subjected to hypoxemia (95% N2-5% O2) or sham hypoxemia (room air) for 60 min, and plasma and spleen cells were collected 2 h later. In vehicle-treated mice, splenocyte proliferation and interleukin-2 and interleukin-3 production were depressed after hypoxemia. E2-pretreated animals, however, displayed no such depression in splenic T cell parameters after hypoxemia. Splenic macrophage cytokine production was also depressed in vehicle-treated mice subjected to hypoxia, whereas it was normal in E2-pretreated mice. In summary, these findings indicate that administration of E2 before hypoxemia prevented the depression of cell-mediated immune functions. Thus administration of 17β-estradiol in high-risk patients before major surgery might decrease hypoxemia-induced immune depression under those conditions.