bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride
Authors: Lytle Christian , McManus Thomas
Published: May 2013

Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl− transport. Reductions in either cell volume or cytosolic Cl− concentration ([Cl−]i) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl−]i was evaluated in model duck red blood cells. Replacement of [Cl−]i with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl−]i at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl−]iis associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl−]i accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl−] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl−]. The major effect of Cl− on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein.
Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction
Authors: Liu Tom S. , Musch Mark W. , Sugi Kazunori , Walsh-Reitz Margaret M. , Ropeleski Mark J. , Hendrickson Barbara A. , Pothoulakis Charalabos , Lamont J. Thomas , Chang Eugene B.
Published: May 2013

We determined whether the cytoprotective heat shock protein HSP72 protects against the injurious effects of Clostridium difficile toxin A (TxA) on intestinal epithelial cells. Colonic epithelial Caco-2/bbe (C2) cells were stably transfected with HSP72 antisense (C2AS) or vector only (C2VC), resulting in low and high HSP72 expression, respectively. Measurements of epithelial barrier integrity, mitochondrial function, and apoptosis activation were assessed after TxA exposure. HSP72 and RhoA interactions were evaluated with immunoprecipitations. In C2AS cells, TxA was associated with a greater decrease in transepithelial resistance (TER), an increase in [3H]mannitol flux, and increased dissociation of perijunctional actin. Although HSP72 binds RhoA, it failed to prevent RhoA glucosylation. TxA caused a more rapid decrease in ATP, release of cytochrome c, and activation of caspase-9 in C2AS cells. To determine whether ATP depletion decreases TER, we treated cells with antimycin A, which caused a decline in TER. We conclude that HSP72 may protect intestinal epithelial cells from TxA-mediated damage through several mechanisms, including actin stabilization, mitochondrial protection, and inhibition of apoptosis activation, but not by prevention of RhoA glucosylation.
External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly
Authors: Putnam Andrew J. , Cunningham James J. , Pillemer Brendan B. L. , Mooney David J.
Published: May 2013

Transmission of externally applied mechanical forces to the interior of a cell requires coordination of biochemical signaling pathways with changes in cytoskeletal assembly and organization. In this study, we addressed one potential mechanism for this signal integration by applying uniform single external mechanical strains to aortic smooth muscle cells (SMCs) via their adhesion substrate. A tensile strain applied to the substrate for 15 min significantly increased microtubule (MT) assembly by 32 ± 7%, with no apparent effect on the cells' focal adhesions as revealed by immunofluorescence and quantitative analysis of Triton X-100-insoluble vinculin levels. A compressive strain decreased MT mass by 24 ± 9% but did not influence the level of vinculin in focal adhesions. To understand the decoupling of these two cell responses to mechanical strain, we examined a redistribution of the small GTPases RhoA and Rac. Tensile strain was found to decrease the amount of membrane-associated RhoA and Rac by 70 ± 9% and 45 ± 11%, respectively, compared with static controls. In contrast, compressive strain increased membrane-associated RhoA and Rac levels by 74 ± 17% and 36 ± 13%, respectively. Disruption of the MT network by prolonged treatments with low doses of either nocodazole or paclitaxel before the application of strain abolished the redistribution of RhoA and Rac in response to the applied forces. Combined, these results indicate that the effects of externally applied mechanical strain on the distribution and activation of the Rho family GTPases require changes in the state of MT polymerization.
Adenoviral gene transfer of eNOS: high-level expression in ex vivo expanded marrow stromal cells
Authors: Deng Weiwen , Bivalacqua Trinity J. , Chattergoon Natasha N. , Hyman Albert L. , Jeter James R. , Kadowitz Philip J.
Published: May 2013

Endothelial nitric oxide synthase (eNOS) is an attractive target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold great promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated eNOS gene transfer into ex vivo expanded MSCs, rat MSCs (rMSCs) were isolated, expanded ex vivo, and transduced with Ad5RSVeNOS, an adenoviral vector containing the eNOS gene under the control of the Rous sarcoma virus promoter. The presence of eNOS protein in Ad5RSVeNOS-transduced rMSCs was confirmed by immunohistochemical and Western blot analysis. Transduction efficiency was dose dependent, and eNOS transgene expression in rMSCs persisted for ≥21 days in culture. The rMSCs retained multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Furthermore, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum, and stem cells were identified within corporal sinusoids. These findings demonstrate that replication-deficient recombinant adenovirus can be used to engineer ex vivo expanded rMSCs and that high-level eNOS transgene expression can be achieved, pointing out the clinical potential of using this novel adult stem cell-based gene therapy method for the treatment of cardiovascular diseases.
Some sweet and bitter tastants stimulate inhibitory pathway of adenylyl cyclase via melatonin and α2-adrenergic receptors in Xenopus laevis melanophores
Authors: Zubare-Samuelov Meirav , Peri Irena , Tal Michael , Tarshish Mark , Spielman Andrew I. , Naim Michael
Published: May 2013

The sweeteners saccharin, d-tryptophan, and neohesperidin dihydrochalcone (NHD) and the bitter tastant cyclo(Leu-Trp) stimulated concentration-dependent pigment aggregation in a Xenopus laevis melanophore cell line similar to melatonin. Like melatonin, these tastants inhibited (by 45-92%) cAMP formation in melanophores; pertussis toxin pretreatment almost completely abolished the tastant-induced cAMP inhibition, suggesting the involvement of the inhibitory pathway (Gi) of adenylyl cyclase. The presence of luzindole (melatonin receptor antagonist) almost completely abolished the inhibition of cAMP formation induced by saccharin, d-tryptophan, and cyclo(Leu-Trp) but only slightly affected the inhibitory effect of NHD. In contrast, the presence of an α2-adrenergic receptor antagonist, yohimbine, almost completely abolished the inhibition of cAMP formation induced by NHD but had only a minor effect on that induced by the other tastants. Thus saccharin, d-tryptophan, and cyclo(Leu-Trp) are melatonin receptor agonists whereas NHD is an α2-adrenergic receptor agonist, but both pathways lead to the same transduction output and cellular response. Formation of d- myo-inositol 1,4,5-trisphosphate (IP3) in melanophores was reduced (15-58%, no concentration dependence) by saccharin, d-tryptophan, and cyclo(Leu-Trp) stimulation but increased by NHD stimulation. Tastant stimulation did not affect cGMP. Although some of the above tastants were found to be membrane permeant, their direct activation of downstream transduction components in this experimental system is questionable. MT1 and MT2 melatonin receptor mRNAs were identified in rat circumvallate papilla taste buds and nonsensory epithelium, suggesting the occurrence of MT1 and MT2 receptors in these tissues. Melatonin stimulation reduced the cellular content of cAMP in taste cells, which may or may not be related to taste sensation.
Laminins and TGF-β maintain cell polarity and functionality of human gastric glandular epithelium
Authors: Basque Jean-René , Chailler Pierre , Ménard Daniel
Published: May 2013

The human gastric glandular epithelium produces a gastric lipase enzyme (HGL) that plays an important role in digestion of dietary triglycerides. To assess the involvement of extracellular matrix components and transforming growth factor-β1 (TGF-β1) in the regulation of this enzymic function, normal gastric epithelial cells were cultured on collagen type I, Matrigel, and laminins (LN)-1 and -2 with or without TGF-β1. Epithelial morphology and HGL expression were evaluated using microscopy techniques, enzymic assays, Western blot, Northern hybridization, and RT-PCR. A correlation was observed between the cell polarity status and the level of HGL expression. TGF-β1 alone or individual matrix components stimulated cell spreading and caused a downfall of HGL activity and mRNA. By contrast, Matrigel preserved the morphological features of differentiated epithelial cells and maintained HGL expression. The combination of LNs with TGF-β1 (two constituents of Matrigel) exerted similar beneficial effects on epithelial cell polarity and evoked a 10-fold increase of HGL levels that was blunted by a neutralizing antibody against the α2-integrin subunit and by mitogen-activated protein kinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). This investigation demonstrates for the first time that a powerful synergism between a growth factor and basement membrane LNs positively influences cell polarity and functionality of the human gastric glandular epithelium through an activation of the α2β1-integrin and effectors of two MAPK pathways.
α1-Adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland
Authors: Ota Isao , Zoukhri Driss , Hodges Robin R. , Rios José D. , Tepavcevic Vanja , Raddassi Isabelle , Chen Li Li , Dartt Darlene A.
Published: May 2013

The purpose of this study was to determine the role of p42/p44 mitogen-activated protein kinase (MAPK) in α1-adrenergically and cholinergically stimulated protein secretion in rat lacrimal gland acinar cells and the pathways used by these agonists to activate MAPK. Acini were isolated by collagenase digestion and incubated with the α1-adrenergic agonist phenylephrine or the cholinergic agonist carbachol, and activation of MAPK and protein secretion were then measured. Phenylephrine and carbachol activated MAPK in a time- and concentration-dependent manner. Inhibition of MAPK significantly increased phenylephrine- and carbachol-induced protein secretion. Inhibition of EGF receptor (EGFR) with AG1478, an inhibitor of the EGFR tyrosine kinase activity, significantly increased phenylephrine- but not carbachol-induced protein secretion. Whereas phenylephrine-induced activation of MAPK was completely inhibited by AG1478, activation of MAPK by carbachol was not. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60Src, and possibly Pyk2, to activate MAPK. We conclude that, in the lacrimal gland, activation of MAPK plays an inhibitory role in α1-adrenergically and cholinergically stimulated protein secretion and that these agonists use different signaling mechanisms to activate MAPK.
Effects of organ culture on arterial gene expression and hypoxic relaxation: role of the ryanodine receptor
Authors: Thorne George D. , Paul Richard J.
Published: May 2013

Organ culture specifically inhibits vasorelaxation to acute hypoxia and preferentially decreases specific voltage-dependent K+channel expression over other K+ and Ca2+channel subtypes. To isolate further potential oxygen-sensing mechanisms correlated with altered gene expression, we performed differential display analysis on RNA isolated from control and cultured coronary arterial rings. We hypothesize that organ culture results in altered gene expression important for vascular smooth muscle contractility important to the mechanism of hypoxia-induced relaxation. Our results indicate a milieu of changes suggesting both up- and downregulation of several genes. The altered expression pattern of two positive clones was verified by Northern analysis. Subsequent screening of a porcine cDNA library indicated homology to the ryanodine receptor (RyR). RT-PCR using specific primers to the three subtypes of RyR shows an upregulation of RyR2 and RyR3 after organ culture. Additionally, the caffeine- and/or ryanodine-sensitive intracellular Ca2+store was significantly more responsive to caffeine activation after organ culture. Our data indicate that organ culture increases expression of specific RyR subtypes and inhibits hypoxic vasorelaxation. Importantly, ryanodine blunted hypoxic relaxation in control coronary arteries, suggesting that upregulated RyR might play a novel role in altered intracellular Ca2+ handling during hypoxia.
Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells
Authors: Hu Shaohua , Chen Jianxin , Fabry Ben , Numaguchi Yasushi , Gouldstone Andrew , Ingber Donald E. , Fredberg Jeffrey J. , Butler James P. , Wang Ning
Published: May 2013

We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-l-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.
Immunocompetence of macrophages in rats exposed toCandida albicans infection and stress
Authors: Rodriguez-Galán Maria Cecilia , Sotomayor Claudia , Costamagna Maria Eugenia , Cabanillas Ana Maria , Renterı́a Beatriz Salido , Masini-Repiso Ana Maria , Correa Silvia
Published: May 2013

The integration of innate and adaptive immune responses is required for efficient control of Candida albicans. The present work aimed to assess, at the local site of the infection, the immunocompetence of macrophages in rats infected intraperitoneally with C. albicans and exposed simultaneously to stress during 3 days (CaS group). We studied the 1) ability to remove and kill C. albicans, 2) tumor necrosis factor-α (TNF-α) release, 3) balance of the inducible enzymes NO synthase (iNOS) and arginase, and 4) expression of interleukin (IL)-1β and IL-1 receptor antagonist (ra) mRNA. Compared with only infected animals (Ca group), the number of colony-forming units was significantly higher in CaS rats ( P < 0.01), and the macrophage candidicidal activity was ∼2.5-fold lower ( P < 0.01). Release of TNF-α was diminished in both unstimulated and heat-killed C. albicans restimulated macrophages of the CaS group (Ca vs. CaS, P < 0.03 and P < 0.05, respectively). In Ca- and CaS-group rats, the rates for both the arginase activity and the NO synthesis were significantly enhanced. However, the stress exposure downregulated the activity of both enzymes (CaS vs. Ca, P < 0.05). After in vitro restimulation, the IL-1ra/IL-1β ratio was significantly diminished in CaS-group rats ( P < 0.05). Our results indicate that a correlation exists between early impairment of macrophage function and stress exposure.
Virtual cloning, functional expression, and gating analysis of human connexin31.9
Authors: White Thomas W. , Srinivas Miduturu , Ripps Harris , Trovato-Salinaro Angela , Condorelli Daniele F. , Bruzzone Roberto
Published: May 2013

We have identified a novel gap junction gene by searching the human genome sequence database that encodes a protein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologous to human Cx32.4 and did not cluster with either the purported α- or β-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCR in human mRNA from several tissues including cerebral cortex, heart, liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9 formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by an apparent low unitary conductance (12–15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channels were gated by cytoplasmic acidification or exposure to halothane like other connexins. Cx31.9 was able to form heterotypic channels with the highly voltage-sensitive Xenopus Cx38 (XenCx38), which provides an opportunity to study gating in heterotypic channels formed by hemichannels (connexons) composed of connexins with widely divergent properties. Thus Cx31.9 is a novel human connexin that forms channels with unique functional properties.
Methemoglobin is a potent activator of endothelial cells by stimulating IL-6 and IL-8 production and E-selectin membrane expression
Authors: Liu Xueying , Spolarics Zoltán
Published: May 2013

Infection and injury are frequently accompanied by hemolysis. Endothelial cells are direct targets of free Hb or its oxidative derivatives, including methemoglobin (MHb) and hemin. This study tested whether Hb or its derivatives alter chemokine (IL-8) and cytokine (IL-6) production and the membrane expression of cell adhesion molecule (E-selectin) in human umbilical vein endothelial cells ( passages 2-4, HUVECs). E-selectin membrane content and IL-6 and IL-8 release were quantified by ELISA; cellular mRNA levels were determined by RT-PCR. MHb in vitro resulted in a dose (1-50 μM)- and time (2-16 h)-dependent increase in E-selectin membrane content and IL-6 and IL-8 release in HUVECs. The stimulatory effect of MHb (12 μM) on E-selectin membrane expression and IL-6 and IL-8 release was similar to that produced after treatment with TNF-α (5 ng/ml) and IL-1β (0.25 ng/ml). In contrast, Hb or hemin had no effects. As expected, MHb, Hb, and hemin markedly induced heme oxygenase-1 expression in HUVECs. Haptoglobin, cytochalasin D, and actinomycin inhibited the MHb-induced responses, whereas zinc protoporphyrin IX (a heme oxygenase inhibitor) or desferroxamine (an iron chelator) did not inhibit MHb-induced responses. MHb also increased cellular mRNA levels of E-selectin, IL-6, and IL-8. MHb treatment activated cellular NF-κB and NF-κB inhibitors; N-acetyl cysteine, SN50, and caffeic acid phenylethyl ester inhibited the MHb-induced responses. These data indicate that MHb is a potent activator of endothelial cells through NF-κB-mediated upregulation of cell adhesion molecule expression and chemokine and cytokine production. MHb-induced endothelial cell activation may have clinical significance after infections, hemolysis, or methemoglobinemia.
Localization of Na+-HCO 3 − cotransporter (NBC-1) variants in rat and human pancreas
Authors: Satoh Hiroaki , Moriyama Nobuo , Hara Chiaki , Yamada Hideomi , Horita Shoko , Kunimi Motoei , Tsukamoto Kazuhisa , Iso-o Naoyuki , Inatomi Jun , Kawakami Hayato , Kudo Akihiko , Endou Hitoshi , Igarashi Takashi , Goto Atsuo , Fujita Toshiro , Seki George
Published: May 2013

Mutations in Na+-HCO[Formula: see text] cotransporter (NBC-1) cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities. One pRTA patient had increased serum amylase, suggesting possible evidence of pancreatitis. To further delineate a link between NBC-1 inactivation and pancreatic dysfunction, immunohistochemical analysis was performed on rat and human pancreas using antibodies against kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters. In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and both apical and basolateral membranes of medium and large duct cells. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label acinar cells, although it did label the basolateral membranes of the entire duct system. The labeling by anti-kNBC-1 antibody was detected in only a limited number of rat pancreatic duct cells. To examine the effects of pRTA-related mutations, R342S and R554H, on pNBC-1 function, we performed functional analysis and found that both mutants had reduced transport activities compared with the wild-type pNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO[Formula: see text] uptake into duct cells in both rat and human pancreas. The loss of pNBC-1 function is predicted to have significant impact on overall ductal HCO[Formula: see text] secretion, which could potentially lead to pancreatic dysfunction.
Uptake of modified low-density lipoproteins alters actin distribution and locomotor forces in macrophages
Authors: Zerbinatti Celina V. , Gore Robert W.
Published: May 2013

It is postulated that macrophage-derived foam cells accumulate in the arterial wall because they lose the ability to migrate after excessive ingestion of modified forms of low-density lipoproteins (LDL). To assess changes in locomotor force generating capacity of foam cells, we measured isometric forces in J774A.1 macrophages after cholesterol loading with oxidized (Ox-LDL) or aggregated (Agg-LDL) LDL using a novel magnetic force transducer. Ox-LDL loading reduced the ability of J774A.1 macrophages to generate isometric forces by 50% relative to control cells. Changes in force frequency consistent with reduced motility were detected as well. Agg-LDL loading was also detrimental to J774A.1 motility but to a lesser extent than Ox-LDL. Ox-LDL loading significantly reduced total actin levels and induced changes in the F-actin to G-actin distribution, whereas Agg-LDL loaded cells had significantly increased levels of total actin. These data provide evidence that cholesterol loading and subsequent accumulation decreases macrophage motility by reducing the cells' force generating capacity and that Ox-LDL appears to be more effective than Agg-LDL in disrupting the locomotor machinery.
H+-linked transport of salicylic acid, an NSAID, in the human trophoblast cell line BeWo
Authors: Emoto Akiko , Ushigome Fumihiko , Koyabu Noriko , Kajiya Hiroshi , Okabe Koji , Satoh Shoji , Tsukimori Kiyomi , Nakano Hitoo , Ohtani Hisakazu , Sawada Yasufumi
Published: May 2013

We investigated the transport of salicylic acid and l-lactic acid across the placenta using the human trophoblast cell line BeWo. We performed uptake experiments and measured the change in intracellular pH (pHi). The uptakes of [14C]salicylic acid andl-[14C]lactic acid were temperature- and extracellular pH-dependent and saturable at higher concentrations. Both uptakes were also reduced by FCCP, nigericin, and NaN3. Various nonsteroidal anti-inflammatory drugs (NSAIDs) strongly inhibited the uptake of l-[14C]lactic acid. Salicylic acid and ibuprofen noncompetitively inhibited the uptake ofl-[14C]lactic acid. α-Cyano-4-hydroxycinnamate (CHC), a monocarboxylate transporter inhibitor, suppressed the uptake ofl-[14C]lactic acid but not that of [14C]salicylic acid. CHC also suppressed the decrease of pHi induced by l-lactic acid but had little effect on that induced by salicylic acid or diclofenac. These results suggest that NSAIDs are potent inhibitors of lactate transporters, although they are transported mainly by a transport system distinct from that for l-lactic acid.
Circadian rhythm in intracellular Cl− activity of acutely dissociated neurons of suprachiasmatic nucleus
Authors: Shimura Masahiko , Akaike Norio , Harata Nobutoshi
Published: May 2013

A link between the circadian rhythm and the function of Cl−-permeable γ-aminobutyric acid (GABA) type A (GABAA) receptors on suprachiasmatic nucleus (SCN) neurons was studied by measuring intracellular activity of Cl−( aCl[Formula: see text]) at different times during a circadian cycle in SCN neurons acutely dissociated from rat brains. To measure aCl[Formula: see text], the voltage-clamp mode of the gramicidin-perforated patch-clamp technique was used, and reversal potential of GABA-induced currents ( E GABA) was converted to aCl[Formula: see text]. Measured aCl[Formula: see text] was significantly higher at around noon (20.1 ± 1.4 mM) than at three other time zones of a circadian cycle (means ranging from 11.6 to 14.3 mM). Chord conductance of GABA-induced currents showed no circadian changes, indicating a lack of circadian changes in the number or single-channel conductance of GABAA receptors. These results suggest that aCl[Formula: see text] participates in modulating GABAA receptor functions on SCN neurons during the circadian rhythm.
Sustained norepinephrine contraction in the rat portal vein is lost when Ca2+ is replaced with Sr2+
Authors: Bonnevier Johan , Malmqvist Ulf , Sonntag Dagmar , Schroeter Mechthild , Nilsson Holger , Pfitzer Gabriele , Arner Anders
Published: May 2013

Agonist-induced activation of smooth muscle involves a rise in intracellular Ca2+ concentration and sensitization of myosin light chain phosphorylation to Ca2+. Sr2+ can enter through Ca2+channels, be sequestered and released from sarcoplasmic reticulum, and replace Ca2+ in activation of myosin light chain phosphorylation. Sr2+ cannot replace Ca2+ in facilitation of agonist-activated Ca2+-dependent nonselective cation channels. It is not known whether Sr2+can replace Ca2+ in small G protein-mediated sensitization of phosphorylation. To explore mechanisms involved in α-receptor-activated contractions in smooth muscle, effects of replacing Ca2+ with Sr2+ were examined in rat portal vein. Norepinephrine (NE) at >3.0 × 10−7 M in the presence of Ca2+ resulted in a strong sustained contraction, whereas this sustained component was absent in the presence of Sr2+; only the amplitude of phasic contractions increased. Pretreatment with low (∼0.05 mM) free Ca2+followed by 2.5 mM Sr2+ resulted in a sustained component of the NE response. In β-escin-permeabilized preparations, phenylephrine in the presence of GTP or guanosine 5′- O-(3-thiotriphosphate) alone induced sensitization to Sr2+. In conclusion, a Ca2+-regulated membrane/channel process is required for the sustained component of NE responses in rat portal vein. Sensitization alone is not responsible for the sustained phase of the NE contraction.
High glucose-induced, endothelin-dependent fibronectin synthesis is mediated via NF-κB and AP-1
Authors: Chen Shali , Mukherjee Suranjana , Chakraborty Chandan , Chakrabarti Subrata
Published: May 2013

Human endothelial cells cultured under high glucose (HG) conditions were shown before to upregulate several basement membrane proteins, including fibronectin (FN), thus mimicking effects of diabetes. Using human macrovascular (HUVEC) and microvascular (HMEC) endothelial cell lines, we evaluated in the present study some of the key molecular signaling events involved in HG-induced FN overexpression. This expression was shown to be dependent on endogenous endothelin (ET) receptor-mediated signaling. We also examined the roles played by protein kinase C (PKC) and the transcription factors nuclear factor κB (NF-κB) and activating protein (AP)-1 with respect to such changes. HG, PKC activators, and ETs (ET-1 and ET-3) that increased FN expression also caused activation of NF-κB and AP-1. Inhibitors of both NF-κB and AP-1 prevented HG- and ET-induced FN production. ET receptor blockade also prevented these HG- and ET-mediated changes. The results of this study indicate that glucose-induced increased FN production in diabetes may be mediated via ET-dependent NF-κB and AP-1 activation.
Fibroblast growth factor 2 promotes microvessel formation from mouse embryonic aorta
Authors: Akimoto Tetsu , Hammerman Marc R.
Published: May 2013

To delineate the roles that oxygen and fibroblast growth factors (FGFs) play in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O2, but not room air, the addition of FGF2 to explants stimulated the formation of Gs-IB4-positive, CD31-positive, and Flk-1-positive microvessels in a concentration-dependent manner. FGF2-stimulated microvessel formation was inhibited by sequestration of FGF2 via addition of soluble FGF receptor (FGFR) chimera protein or anti-FGF2 antibodies. FGFR1 and FGFR2 were present on explants. Levels of FGFR1, but not FGFR2, were increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low oxygen upregulates FGFR1 expression in embryonic aorta in vitro and renders it more responsive to FGF2.
A2B receptor activation promotes glycogen synthesis in astrocytes through modulation of gene expression
Authors: Allaman Igor , Lengacher Sylvain , Magistretti Pierre J. , Pellerin Luc
Published: May 2013

Adenosine has been proposed as a key factor regulating the metabolic balance between energy supply and demand in the central nervous system. Because astrocytes represent an important cellular element in the control of brain energy metabolism, we investigated whether adenosine could induce long-term changes of glycogen levels in primary cultures of mouse cortical astrocytes. We observed that adenosine increased glycogen content, up to 300%, in a time- (maximum at 8 h) and concentration-dependent manner with an EC50 of 9.69 μM. Pharmacological experiments using the broad-spectrum agonist 5′-( N-ethylcarboxamido)adenosine (NECA) and specific agonists for the A1, A2A, and A3receptors [ N 6-cyclopentyladenosine (CPA), CGS-21680, and IB-MECA, respectively] suggest that the effect of adenosine is mediated through activation of the low-affinity A2B adenosine receptor subtype. Interestingly, adenosine induces in parallel the expression of the protein targeting to glycogen (PTG), one of the protein phosphatase-1 glycogen-targeting subunits that has been implicated in the control of glycogen levels in various tissues. These results indicate that adenosine can exert long-term control over glycogen levels in astrocytes and might therefore play a significant role in physiological and/or pathological processes involving long-term modulation of brain energy metabolism.
Excess plasma membrane and effects of ionic amphipaths on mechanics of outer hair cell lateral wall
Authors: Morimoto Noriko , Raphael Robert M. , Nygren Anders , Brownell William E.
Published: May 2013

The interaction between the outer hair cell (OHC) lateral wall plasma membrane and the underlying cortical lattice was examined by a morphometric analysis of cell images during cell deformation. Vesiculation of the plasma membrane was produced by micropipette aspiration in control cells and cells exposed to ionic amphipaths that alter membrane mechanics. An increase of total cell and vesicle surface area suggests that the plasma membrane possesses a membrane reservoir. Chlorpromazine (CPZ) decreased the pressure required for vesiculation, whereas salicylate (Sal) had no effect. The time required for vesiculation was decreased by CPZ, indicating that CPZ decreases the energy barrier required for vesiculation. An increase in total volume is observed during micropipette aspiration. A deformation-induced increase in hydraulic conductivity is also seen in response to micropipette-applied fluid jet deformation of the lateral wall. Application of CPZ and/or Sal decreased this strain-induced hydraulic conductivity. The impact of ionic amphipaths on OHC plasma membrane and lateral wall mechanics may contribute to their effects on OHC electromotility and hearing.
Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice
Authors: Roman Brian B. , Meyer Ronald A. , Wiseman Robert W.
Published: May 2013

Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by31P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 ± 0.6% of initial PCr in MMKO muscle vs. 9.7 ± 1.6% in WT muscles (mean ± SE, n = 7, P < 0.001). Initial PCr/ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 ± 3.5 vs. WT 35.5 ± 4.4% of initial PCr; n = 5–6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.
CLCA protein and chloride transport in canine retinal pigment epithelium
Authors: Loewen Matthew E. , Smith Nicola K. , Hamilton Don L. , Grahn Bruce H. , Forsyth George W.
Published: May 2013

Problems in ion and fluid transfer across the retinal pigment epithelium (RPE) are a probable cause of inappropriate accumulations of fluid between the photoreceptors of the retina and the RPE. The activities of Cl- transporters involved in basal fluid transfer across the RPE have been compared to determine whether Ca2+- or cAMP-dependent channels may be responsible for basal housekeeping levels of secretory activity in this tissue. The role of a candidate Ca2+-dependent CLCA protein in the basal RPE transport of Cl- has been investigated. Low concentrations of the Cl- conductance inhibitors glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoate reduced the short-circuit current in dog RPE preparations mounted in Ussing chambers and decreased the Ca2+-dependent Cl- efflux from fibroblasts expressing the pCLCA1 Cl- conductance regulator. However, these same agents did not inhibit the rate of Cl- release from cultured fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive Cl- channel. Addition of ionomycin to primary cultures of canine RPE cells or to fibroblasts expressing the pCLCA1 channel regulator increased the rate of release of Cl- from both types of cultured cells. However, the presence of pCLCA1 also increased cAMP-dependent Cl- release from fibroblasts expressing CFTR. We conclude that Ca2+-dependent Cl- transport may be more important than cAMP-dependent Cl- transport for normal fluid secretion across the RPE. Furthermore, CLCA proteins expressed in the RPE appear to regulate the activity of other Cl- transporters, rather than functioning as primary ion transport proteins.
Overexpression of p73 causes apoptosis in vascular smooth muscle cells
Authors: Davis Benjamin B. , Dong Yao , Weiss Robert H.
Published: May 2013

Abnormal vascular smooth muscle (VSM) cell proliferation contributes to the development of atherosclerosis and its associated disorders, including angioplasty restenosis. The tumor-suppressor protein p53 has been linked to the development of atherosclerotic lesions, and its homolog, p73, is proving to have contrasting functions in a variety of tissues. As an outgrowth of our previous finding that p73 is increased in serum-stimulated VSM cells and human atherosclerotic tissue, we examined p73 overexpression in VSM cells to elucidate causality of p73 expression with growth response. Overexpression of p73 results in decreased cell cycle transit and is accompanied by apoptosis. The apoptotic changes in p73 overexpressing VSM cells are independent of p53 and are associated with a decrease in levels of p21waf1/cip1. In conjunction with our previous data finding that p73 is increased in serum-stimulated VSM cells, this work suggests a role for p73 in vascular proliferative diseases.
Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity
Authors: Neuhofer Wolfgang , Woo Seung Kyoon , Na Ki Young , Grünbein Rita , Park Won Kun , Nahm Ohnn , Beck Franz-X. , Kwon H. Moo
Published: May 2013

In response to ambient hypertonicity, TonEBP (tonicity-responsive enhancer binding protein) stimulates certain genes including those encoding cytokines, transporters for organic solutes, and a molecular chaperone. TonEBP is regulated in a bidirectional manner, upregulated by an increase in ambient tonicity while downregulated by a decrease. To investigate the role of intracellular ionic strength in the activity of TonEBP, we subjected Madin-Darby canine kidney cells to a variety of conditions. Electron microprobe analysis was performed to measure intracellular electrolytes. Under conditions in which changes in cell volume were similar, TonEBP activity correlated with the intracellular ionic strength regardless of the external tonicity. On the other hand, inhibition of the Na+/K+-ATPase and high external K+ concentration led to a decreased activity of TonEBP despite a marked increase in the intracellular ionic strength. Because isotonic swelling is known to occur under these conditions, these data suggest that dilution of the cytoplasmic constituents inhibits the activity of TonEBP. We conclude that intracellular ionic strength and water content are major factors that determine the activity of TonEBP.
Termination of immediate-early gene expression after stimulation by parathyroid hormone or isoproterenol
Authors: Chen Xin , Dai Jia-Chun , Greenfield Edward M.
Published: May 2013

cAMP/PKA signaling transiently stimulates mRNA expression of immediate-early genes, including IL-6 and c- fos. We confirmed that these mRNAs are transiently stimulated by parathyroid hormone (PTH) in ROS 17/2.8 osteoblastic cells. Consistent with the role for cAMP/PKA signaling in this response, PTH induces transient cAMP elevation, PKA activation, and cAMP-responsive element-binding protein (CREB) phosphorylation. Our goal was to determine whether termination of immediate-early gene expression is due to receptor desensitization or cAMP degradation. The approaches used were 1) inhibition of PTH receptor desensitization with G protein-coupled receptor kinase 2 (GRK2) antisense oligonucleotides or antisense plasmids, 2) sustained activation of adenyl cyclase with forskolin, and 3) inhibition of cAMP degradation with 3-isobutyl-1-methylxanthine. These experiments show that mechanisms downstream of receptor desensitization and cAMP degradation are primarily responsible for termination of PKA activity, CREB phosphorylation, and immediate-early gene expression. Similar conclusions were also obtained in response to PTH in a second osteoblastic cell line (MC3T3-E1) and in response to isoproterenol in NIH3T3 fibroblasts. This conclusion may therefore reflect a general mechanism for termination of immediate-early gene expression after induction by cAMP/PKA.
Src-dependent, neutrophil-mediated vascular hyperpermeability and β-catenin modification
Authors: Tinsley John H. , Ustinova Elena E. , Xu Wenjuan , Yuan Sarah Y.
Published: May 2013

The hyperpermeability response of microvessels in inflammation involves complex signaling reactions and structural modifications in the endothelium. Our goal was to determine the role of Src-family kinases (Src) in neutrophil-mediated venular hyperpermeability and possible interactions between Src and endothelial barrier components. We found that inhibition of Src abolished the increases in albumin permeability caused by C5a-activated neutrophils in intact, perfused coronary venules, as well as in cultured endothelial monolayers. Activated neutrophils increased Src phosphorylation at Tyr416, which is located in the catalytic domain, and decreased phosphorylation at Tyr527 near the carboxyl terminus, events consistent with reports that phosphorylating and transforming activities of Src are upregulated by Tyr416 phosphorylation and negatively regulated by Tyr527 phosphorylation. Furthermore, neutrophil stimulation resulted in association of Src with the endothelial junction protein β-catenin and β-catenin tyrosine phosphorylation. These phenomena were abolished by blockage of Src activity. Taken together, our studies link for the first time neutrophil-induced hyperpermeability to a pathway involving Src kinase activation, Src/β-catenin association, and β-catenin tyrosine phosphorylation in the microvascular endothelium.
P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia
Authors: Blaug Sasha , Rymer Jodi , Jalickee Stephen , Miller Sheldon S.
Published: May 2013

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were −4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4 Ω · cm2, respectively. The apical membrane potential was −43.9 ± 1.7 mV, and the ratio of apical to basolateral membrane resistance ( R A/ R B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC50 of ∼24 μM (apical) and ∼30 μM (basal). Apical ATP/UTP (100 μM) depolarized apical membrane potential by 17.6 ± 0.8 mV ( n = 7) and decreased R A/ R B by a factor of ≈3. The addition of adenosine to either side (100 μM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca2+ concentration (427 ± 206 nM; 15–25 μM ATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 ± 3 μl · cm−2 · h−1( n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y2 purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca2+-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.
Recovery time affects immunoendocrine responses to a second bout of endurance exercise
Authors: Ronsen Ola , Kjeldsen-Kragh Jens , Haug Egil , Bahr Roald , Pedersen Bente Klarlund
Published: May 2013

The purpose of this study was to examine the effect of different durations of rest between two bouts of exercise on immunoendocrine responses during and after the second bout of exercise. Nine endurance athletes participated in three 25-h trials: 1) complete bed rest (REST), 2) two bouts of exercise separated by 3 h of rest (SHORT), and 3) two bouts of exercise separated by 6 h of rest (LONG). Each cycle ergometer exercise bout lasted 75 min at 75% of maximal O2uptake. We observed a more pronounced increase in epinephrine, norepinephrine, adrenocorticotropic hormone, and cortisol, but not in growth hormone, and a larger neutrophilia and lymphocytopenia in connection with the second bout of exercise in trial SHORT compared with trial LONG. Lymphocyte activation was unaltered by the difference in rest protocol. In conclusion, a second bout of exercise elicited more pronounced change in neuroendocrine factors and leukocyte counts when preceded by 3 h of rest as opposed to 6 h of rest after the first bout of exercise.
p53 protein and p21 mRNA levels and caspase-3 activity are altered by zinc status in aortic endothelial cells
Authors: Fanzo Jessica C. , Reaves Scott K. , Cui Libin , Zhu Lei , Lei Kai Y.
Published: May 2013

The influence of zinc status on the levels of p53, as well as downstream targets of p53 in cell repair and survival, was examined in human aortic endothelial cells (HAECs). A serum-reduced low-zinc medium (ZD) was used to deplete zinc over one passage. Other treatments included zinc-normal control (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) treatment with 3.0, 16.0, and 32.0 μM zinc, respectively. Cellular zinc levels in the ZD cells were 64% of ZN controls; levels in the ZA cells were not different, but levels in ZS cells were significantly higher (40%) than in ZN cells. No difference in p53 mRNA abundance was detected among all treatments; however, p53 nuclear protein levels were >100% higher in the ZD and ZS cells and almost 200% higher in the ZA cells than in ZN controls. In addition, p21 mRNA abundance, a downstream target of p53 protein, was increased in the ZS cells compared with both the ZN control and ZD cells. In the ZS cells, bax and mcl-1 were also ∼50% higher compared with ZN controls, whereas bcl-2 mRNA was increased compared with ZA cells. Moreover, caspase-3 activity of ZD cells was not different from that of ZN controls but was reduced to 83 and 69% of ZN controls in ZA and ZS cells, respectively. Thus p53 protein and p53 downstream target genes appeared to be modulated by intracellular zinc status in HAECs.
Basolateral membrane expression of the Kir 2.3 channel is coordinated by PDZ interaction with Lin-7/CASK complex
Authors: Olsen Olav , Liu Hui , Wade James B. , Merot Jean , Welling Paul A.
Published: May 2013

The basolateral membrane sorting determinant of an inwardly rectifying potassium channel, Kir 2.3, is comprised of a unique arrangement of trafficking motifs containing tandem, conceivably overlapping, biosynthetic targeting and PDZ-based signals. In the present study, we elucidate a mechanism by which a PDZ interaction coordinates one step in a basolateral membrane sorting program. In contrast to apical missorting of channels lacking the entire sorting domain, deletion of the PDZ binding motif caused channels to accumulate into an endosomal compartment. Here, we identify a new human ortholog of a Caenorhabditis elegans PDZ protein, hLin-7b, that interacts with the COOH-terminal tail of Kir 2.3 in renal epithelia. hLin-7b associates with the channel as a part of a multimeric complex on the basolateral membrane similar to a basolateral membrane complex in C. elegans vulva progenitor cells. Coexpression of hLin-7b with Kir 2.3 dramatically increases channel activity by stabilizing plasma membrane expression. The discovery identifies one component of the sorting machinery and provides evidence for a retention mechanism in a hierarchical basolateral trafficking program.