bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation
Authors: D'Souza Fiona M. , Sparks Rodney L. , Chen Huiying , Kadowitz Philip J. , Jeter James R.
Published: May 2013

Endothelial nitric oxide synthase (eNOS) is responsible for the production of nitric oxide (NO) in blood vessels. NO has been shown to be involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation. In the present study, the eNOS gene was transferred into rat aortic smooth muscle cells by using an adenoviral vector, and the effect of endogenously produced NO on VSMC proliferation was investigated. The presence of eNOS in eNOS-transfected cells was confirmed by immunocytochemistry and Western blot analysis. eNOS transfection resulted in inhibition of VSMC proliferation. This effect was accompanied by increased levels of p53 and p21. This effect was abrogated in the presence of the protein kinase A (PKA) inhibitor Rp-8-bromoadenosine 3′,5′-cyclic monophosphothioate. The increased levels of p53 and p21 observed in eNOS-transfected cells were reduced in the presence of the PKA inhibitor. These data suggest that p21 and p53 play a role in the inhibition of proliferation in eNOS-transfected cells and that levels of these two proteins are regulated by PKA.
Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells
Authors: Chen Lixin , Wang Liwei , Zhu Linyan , Nie Sihai , Zhang Jin , Zhong Ping , Cai Bo , Luo Haibing , Jacob Tim J. C.
Published: May 2013

Patch-clamping and cell image analysis techniques were used to study the expression of the volume-activated Cl− current, I Cl(vol), and regulatory volume decrease (RVD) capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a Cl− current with a linear conductance, negligible time-dependent inactivation, and a reversal potential close to the Cl− equilibrium potential. The sequence of anion permeability was I− > Br− > Cl− > gluconate. The Cl− channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and ATP inhibited I Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a double chemical-block (thymidine and hydroxyurea) technique. The expression of I Cl(vol) was cell cycle dependent, being high in G1 phase, downregulated in S phase, but increasing again in M phase. Hypotonic solution activated RVD, which was cell cycle dependent and inhibited by the Cl− channel blockers NPPB, tamoxifen, and ATP. The expression of I Cl(vol) was closely correlated with the RVD capacity in the cell cycle, suggesting a functional relationship. Inhibition of I Cl(vol) by NPPB (100 μM) arrested cells in G0/G1. The data also suggest that expression of I Cl(vol) and RVD capacity are actively modulated during the cell cycle. The volume-activated Cl− current associated with RVD may therefore play an important role during the cell cycle progress.
Role of Ca2+-activated K+ channels in human erythrocyte apoptosis
Authors: Lang Philipp A. , Kaiser Stefanie , Myssina Swetlana , Wieder Thomas , Lang Florian , Huber Stephan M.
Published: May 2013

Exposure of erythrocytes to the Ca2+ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca2+-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca2+-sensitive Gardos K+ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 μM) induces activation of inwardly rectifying K+-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K+ concentration to 25 mM and exposure to the Gardos K+ channel blockers charybdotoxin (230 nM) and clotrimazole (5 μM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K+ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 μM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K+ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca2+-activated K+ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca2+-sensitive K+ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis.
Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation
Authors: Nobe Hiromi , Nobe Koji , Fazal Fabeha , de Lanerolle Primal , Paul Richard J.
Published: May 2013

Fibroblasts form fibers when grown in culture medium containing native type 1 collagen. The contractile forces generated can be precisely quantified and used to analyze the signal transduction pathways regulating fibroblast contraction. Calf serum (30%) induces a sustained contraction that is accompanied by a transient increase in intracellular calcium ([Ca2+]i). W-7, a calmodulin inhibitor, KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, and ML-7, a myosin light-chain kinase inhibitor, had no effects on either the contraction or the [Ca2+]i responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca2+]i. In contrast, the Rho kinase inhibitors (R)-(+)- trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca2+]iresponse. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did not alter mono- or diphosphorylation of the myosin regulatory light chain (MRLC) compared with control untreated fibers. These results suggest that the sustained contraction of NIH 3T3 fibroblast fibers induced by calf serum is mediated by Rho kinase but is independent of a sustained increase in [Ca2+]i, calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.
Glucose transporter in bovine mammary epithelial cells is an asymmetric carrier that exhibits cooperativity and trans-stimulation
Authors: Xiao Changting , Cant John P.
Published: May 2013

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3- O-methyl-d-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37°C under zero- trans, equilibrium-exchange, high- cis, and high- trans concentrations of 3- O-methyl-d-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2 was 0.97. Forward Vmax was estimated at 18.2 nmol·min-1·mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.
Ultraviolet A radiation transiently disrupts gap junctional communication in human keratinocytes
Authors: Provost Nicolas , Moreau Marielle , Leturque Armelle , Nizard Carine
Published: May 2013

Ultraviolet A (UVA) (320–400 nm) radiation is known to cause cutaneous aging and skin cancer. We studied the effect of UVA (365 nm) radiation on the human epidermis by focusing on keratinocyte gap junction-mediated intercellular communication (GJIC). We observed a dose-dependent 10-fold decrease in GJIC induced by UVA in normal human keratinocytes. This decrease in GJIC was associated with time-dependent internalization of connexin43 (Cx43). UVA radiation also damaged the actin cytoskeleton, as shown by microfilament disappearance. Importantly, the decrease in GJIC was transient when keratinocytes were irradiated with 10 J/cm2UVA, with a return to baseline values after 8 h. Concomitantly, Cx43 was relocalized and the actin cytoskeleton was restored. UVA irradiation and 12- O-tetradecanoylphorbol 13-acetate (TPA) treatment activated protein kinase C and reduced GJIC. However, Cx43 localization and phosphorylation were differently regulated by the two treatments. This suggests that at least two different pathways may mediate the observed fall in GJIC. These findings identify keratinocyte GJIC as a new UVA target that might sensitize human skin to photoaging and cancer formation.
Aging-related satellite cell differentiation defect occurs prematurely after Ski-induced muscle hypertrophy
Authors: Chargé Sophie B. P. , Brack Andrew S. , Hughes Simon M.
Published: May 2013

To investigate the cause of skeletal muscle weakening during aging we examined the sequence of cellular changes in murine muscles. Satellite cells isolated from single muscle fibers terminally differentiate progressively less well with increasing age of donor. This change is detected before decline in satellite cell numbers and all histological changes examined here. In MSVski transgenic mice, which show type IIb fiber hypertrophy, initial muscle weakness is followed by muscle degeneration in the first year of life. This degeneration is accompanied by a spectrum of changes typical of normal muscle aging and a more marked decline in satellite cell differentiation efficiency. On a myoD-null genetic background, in which satellite cell differentiation is defective, the MSVski muscle phenotype is aggravated. This suggests that, on a wild-type genetic background, satellite cells are capable of repairing MSVski fibers and preserving muscle integrity in early life. We propose that decline in myogenic cell differentiation efficiency is an early event in aging-related loss of muscle function, both in normal aging and in some late-onset muscle degenerative conditions.
Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro
Authors: Allen David L. , Teitelbaum Daniel H. , Kurachi Kotoku
Published: May 2013

We investigated the role of growth factors and fibronectin on matrix metalloproteinase (MMP) expression and on migration and invasion of mouse skeletal myoblasts in vitro. None of the growth factors tested significantly affected MMP-1 or MMP-2 activity as revealed by gelatin zymography, but both basic FGF (bFGF) and tumor necrosis factor (TNF)-α significantly increased MMP-9 activity (10- and 30-fold, respectively). The increase in secreted MMP-9 activity with TNF-α stimulation was due at least in part to an increase in MMP-9 gene transcription, because an MMP-9 promoter construct was approximately fivefold more active in TNF-α-treated myoblasts than in control myoblasts, as well as an increase in MMP-9 proteolytic activation. However, whereas fibronectin, bFGF, hepatocyte growth factor, and TGF-β1 significantly augmented migration of mouse myoblasts, TNF-α did not, nor did PDGF-BB or IGF-I. Fibronectin and bFGF also significantly augmented invasion of myoblasts across a Matrigel barrier, and plasmin cotreatment potentiated whereas N-acetyl cysteine suppressed the effects of bFGF and fibronectin on myoblast migration and invasion. Finally, transient transfection with an MMP-9 overexpression construct had only minimal effects on myoblast migration/invasion, whereas overexpression of either MMP-2 or MMP-1 significantly augmented myoblast migration and invasion. These observations support the hypothesis that MMP activity is a necessary component of growth factor-mediated myoblast migration but suggest that other consequences of growth factor signaling are also necessary for migration to occur.
Glutamine delays spontaneous apoptosis in neutrophils
Authors: Pithon-Curi Tania C. , Schumacher Robert Ivan , Freitas Jofre J. S. , Lagranha Cláudia , Newsholme Philip , Palanch Adrianne C. , Doi Sonia Q. , Curi Rui
Published: May 2013

Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or presence of glutamine (0.5, 1.0, and 2.0 mM). Condensation of chromatin was reduced after 24 or 48 h of culture in the presence of glutamine compared with its absence as assessed by Hoechst 33342 staining. The level of Escherichia coli phagocytosis in the presence of glutamine was markedly increased compared with the level achieved by cells cultured in the absence of glutamine. Annexin V binding to externalized phosphatidylserine was reduced in the presence of glutamine. Sensitive fluorochrome rhodamine 123, as determined by fluorescence-activated cell sorting and confocal microscopy, was used to monitor loss of the mitochondrial transmembrane potential. In the absence of glutamine, neutrophils exhibited a marked reduction in the uptake of rhodamine 123. In the presence of 1.0 or 2.0 mM glutamine, the uptake of rhodamine was 20 or 38% higher, respectively. Similar effect was found in human neutrophils by measuring DNA fragmentation and mitochondrial transmembrane potential. Therefore, glutamine protects from events associated with triggering and executing apoptosis in both rat and human neutrophils.
The COOH termini of NBC3 and the 56-kDa H+-ATPase subunit are PDZ motifs involved in their interaction
Authors: Pushkin Alexander , Abuladze Natalia , Newman Debra , Muronets Vladimir , Sassani Pejvak , Tatishchev Sergei , Kurtz Ira
Published: May 2013

The electroneutral sodium bicarbonate cotransporter 3 (NBC3) coimmunoprecipitates from renal lysates with the vacuolar H+-ATPase. In renal type A and B intercalated cells, NBC3 colocalizes with the vacuolar H+-ATPase. The involvement of the COOH termini of NBC3 and the 56-kDa subunit of the proton pump in the interaction of these proteins was investigated. The intact and modified COOH termini of NBC3 and the 56-kDa subunit of the proton pump were synthesized, coupled to Sepharose beads, and used to pull down kidney membrane proteins. Both the 56- and the 70-kDa subunits of the proton pump, as well as a PDZ domain containing protein Na+/H+ exchanger regulatory factor 1 (NHERF-1), were bound to the intact 18 amino acid NBC3 COOH terminus. A peptide truncated by five COOH-terminal amino acids did not bind these proteins. Replacement of the COOH-terminal leucine with glycine blocked binding of both the proton pump subunits but did not affect binding of NHERF-1. The 18 amino acid COOH terminus of the 56-kDa subunit of the proton pump bound NHERF-1 and NBC3, but the truncated and modified peptide did not. A complex of NBC3, the 56-kDa subunit of the proton pump, and NHERF-1 was identified in rat kidney. The data indicate that the COOH termini of NBC3 and the 56-kDa subunit of the vacuolar proton pump are PDZ-interacting motifs that are necessary for the interaction of these proteins. NHERF-1 is involved in the interaction of NBC3 and the vacuolar proton pump.
Ca2+ channels involved in endothelin-induced mitogenic response in carotid artery vascular smooth muscle cells
Authors: Kawanabe Yoshifumi , Hashimoto Nobuo , Masaki Tomoh
Published: May 2013

Endothelin (ET)-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in rabbit internal carotid artery (ICA) vascular smooth muscle cells (VSMCs) in addition to the voltage-operated Ca2+channel (VOCC). These channels can be discriminated using the Ca2+ channel blockers SK&F-96365 and LOE-908. SK&F-96365 is sensitive to NSCC-2 and SOCC, and LOE-908 is sensitive to NSCC-1 and NSCC-2. On the basis of sensitivity to nifedipine, a specific blocker of the L-type VOCC, VOCCs have a minor role in ET-1-induced mitogenesis. Both LOE-908 and SK&F-96365 inhibited ET-1-induced mitogenesis in a concentration-dependent manner, and the combination of LOE-908 and SK&F-96365 abolished it. The IC50 values of these blockers for ET-1-induced mitogenesis correlated well with those of the ET-1-induced intracellular free Ca2+concentration responses. These results indicate that the inhibitory action of these blockers on ET-1-induced mitogenesis may be mediated by blockade of NSCC-1, NSCC-2, and SOCC. Collectively, extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for ET-1-induced mitogenesis in ICA VSMCs.
Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells
Authors: Hallows Kenneth R. , Kobinger Gary P. , Wilson James M. , Witters Lee A. , Foskett J. Kevin
Published: May 2013

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-gated Cl− channel and cellular conductance regulator, but the detailed mechanisms of CFTR regulation and its regulation of other transport proteins remain obscure. We previously identified the metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and inhibited CFTR-dependent whole cell conductances when coexpressed with CFTR in Xenopus oocytes. To address the physiological relevance of the CFTR-AMPK interaction, we have now studied polarized epithelia and have evaluated the localization of endogenous AMPK and CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical distribution in several rat epithelial tissues, including nasopharynx, submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit currents ( Isc ) were measured in polarized T84 cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog 5-amino-4-imidazolecarboxamide-1-β-d-ribofuranoside (AICAR) inhibited forskolin-stimulated CFTR-dependent I sc in nonpermeabilized monolayers and monolayers with nystatin permeabilization of the basolateral membrane. Raising intracellular AMP concentration in monolayers with basolateral membranes permeabilized with α-toxin also inhibited CFTR, an effect that was unrelated to adenosine receptors. Finally, overexpression of a kinase-dead mutant AMPK-α1 subunit (α1-K45R) enhanced forskolin-stimulated I sc in polarized T84 monolayers, consistent with a dominant-negative reduction in the inhibition of CFTR by endogenous AMPK. These results indicate that AMPK plays a physiological role in modulating CFTR activity in polarized epithelia and suggest a novel paradigm for the coupling of ion transport to cellular metabolism.
Mutations throughout the S6 region of the hKv1.5 channel alter the stability of the activation gate
Authors: Rich Thomas C. , Yeola Sarita W. , Tamkun Michael M. , Snyders Dirk J.
Published: May 2013

First published September 21, 2001; 10.1152/ ajpcell.00232.2001.—The S6 segment of voltage-gated K+ channels is thought to contribute to the gate that opens the central permeation pathway. Here we present evidence that mutations throughout the cytoplasmic end of S6 strongly influence hKv1.5 channel gating characteristics. Modification of hKv1.5 at positions T505, V512, and S515 resulted in large negative shifts in the voltage dependence of activation, whereas modifications at position Y519 resulted in negative (Y519N) and positive (Y519F) shifts. When adjusted for the altered voltage sensitivity, activation kinetics of mutated channels were similar to those of the wild-type (WT) channel; however, deactivation kinetics of mutations T505I, T505V, V512A, and V512M [time constant (τ) = 35, 250, 170, and 420 ms, respectively] were still slower than WT (τ = 8.3 ms). In addition, deactivation of WT channels was highly temperature sensitive. However, deactivation of T505I and V512A channels was largely temperature insensitive. Together, these data suggest that mutations in S6 decouple activation from deactivation by altering the open-state stability and that residues on both sides of the highly conserved Pro-X-Pro sequence influence the movement of S6 during channel gating.
Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells
Authors: Naro Fabio , De Arcangelis Vania , Coletti Dario , Molinaro Mario , Zani Bianca , Vassanelli Stefano , Reggiani Carlo , Teti Anna , Adamo Sergio
Published: May 2013

Cytoplasmic Ca2+concentration ([Ca2+]i) variation is a key event in myoblast differentiation, but the mechanism by which it occurs is still debated. Here we show that increases of extracellular Ca2+ concentration ([Ca2+]o) produced membrane hyperpolarization and a concentration-dependent increase of [Ca2+]i due to Ca2+influx across the plasma membrane. Responses were not related to inositol phosphate turnover and Ca2+-sensing receptor. [Ca2+]o-induced [Ca2+]i increase was inhibited by Ca2+ channel inhibitors and appeared to be modulated by several kinase activities. [Ca2+]i increase was potentiated by depletion of intracellular Ca2+ stores and depressed by inactivation of the Na+/Ca2+exchanger. The response to arginine vasopressin (AVP), which induces inositol 1,4,5-trisphosphate-dependent [Ca2+]i increase in L6-C5 cells, was not modified by high [Ca2+]o. On the contrary, AVP potentiated the [Ca2+]i increase in the presence of elevated [Ca2+]o. Other clones of the L6 line as well as the rhabdomyosarcoma RD cell line and the satellite cell-derived C2-C12 line expressed similar responses to high [Ca2+]o, and the amplitude of the responses was correlated with the myogenic potential of the cells.
Cellular distribution of parchorin, a chloride intracellular channel-related protein, in various tissues
Authors: Mizukawa Yumiko , Nishizawa Tomohiro , Nagao Taku , Kitamura Ken , Urushidani Tetsuro
Published: May 2013

The cellular distribution of parchorin, a new chloride intracellular channel family member, was investigated in rabbit tissues by immunohistochemistry using an antibody recognizing the sequence containing a parchorin-specific repeat. Parchorin preferentially resides in the epithelium of the ducts of the lacrymal, parotid, submandibular, and mammary glands and the pancreas, prostate, and testis. In the trachea and lung, parchorin was found in the airway epithelium and type II alveolar cells. In the kidney, parchorin was distributed mainly from the thick ascending limb to the distal convoluted tubule. In the eye, both pigment and nonpigment epithelia of the ciliary body were positive, whereas only the pigment epithelium was positive in the retina. Parchorin was also present in the cochlea and semicircular canal. The amount of parchorin in the gastric mucosa, but not in the submandibular glands, increased after weaning. In the mammary gland, parchorin expression was greater in a lactating rabbit (1 wk after delivery) compared with a pregnant (3 wk) rabbit. The cellular distribution and changes in expression indicate that parchorin plays an important role, possibly in chloride transport, in the cells that create an ion gradient for water movement.
Effect of interleukin-1β and tumor necrosis factor-α on gene expression in human endothelial cells
Authors: Zhao Baiteng , Stavchansky Salomon A. , Bowden Robert A. , Bowman Phillip D.
Published: May 2013

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are two major cytokines that rise to relatively high levels during systemic inflammation, and the endothelial cell (EC) response to these cytokines may explain some of the dysfunction that occurs. To better understand the cytokine-induced responses of EC at the gene expression level, human umbilical vein EC were exposed to IL-1β or TNF-α for various times and subjected to cDNA microarray analyses to study alterations in their mRNA expression. Of ∼4,000 genes on the microarray, expression levels of 33 and 58 genes appeared to be affected by treatment with IL-1β and TNF-α, respectively; 25 of these genes responded to both treatments. These results suggest that the effects of IL-1β and TNF-α on EC are redundant and that it may be necessary to suppress both cytokines simultaneously to ameliorate the systemic response.
Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line
Authors: Fernández-Fernández José M. , Nobles Muriel , Currid Aoife , Vázquez Esther , Valverde Miguel A.
Published: May 2013

The cell regulatory volume decrease (RVD) response triggered by hypotonic solutions is mainly achieved by the coordinated activity of Cl− and K+ channels. We now describe the molecular nature of the K+ channels involved in the RVD response of the human bronchial epithelial (HBE) cell line 16HBE14o−. These cells, under isotonic conditions, present a K+current consistent with the activity of maxi K+ channels, confirmed by RT-PCR and Western blot. Single-channel and whole cell maxi K+ currents were readily and reversibly activated following the exposure of HBE cells to a 28% hypotonic solution. Both maxi K+ current activation and RVD response showed calcium dependency, inhibition by TEA, Ba2+, iberiotoxin, and the cationic channel blocker Gd3+ but were insensitive to clofilium, clotrimazole, and apamin. The presence of the recently cloned swelling-activated, Gd3+-sensitive cation channels (TRPV4, also known as OTRPC4, TRP12, or VR-OAC) was detected by RT-PCR in HBE cells. This channel, TRPV4, which senses changes in volume, might provide the pathway for Ca2+ influx under hypotonic solutions and, consequently, for the activation of maxi K+channels.
Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells
Authors: Romanenko Victor G. , Davies Peter F. , Levitan Irena
Published: May 2013

The key mechanism responsible for maintaining cell volume homeostasis is activation of volume-regulated anion current (VRAC). The role of hemodynamic shear stress in the regulation of VRAC in bovine aortic endothelial cells was investigated. We showed that acute changes in shear stress have a biphasic effect on the development of VRAC. A shear stress step from a background flow (0.1 dyn/cm2) to 1 dyn/cm2 enhanced VRAC activation induced by an osmotic challenge. Flow alone, in the absence of osmotic stress, did not induce VRAC activation. Increasing the shear stress to 3 dyn/cm2, however, resulted in only a transient increase of VRAC activity followed by an inhibitory phase during which VRAC was gradually suppressed. When shear stress was increased further (5–10 dyn/cm2), the current was immediately strongly suppressed. Suppression of VRAC was observed both in cells challenged osmotically and in cells that developed spontaneous VRAC under isotonic conditions. Our findings suggest that shear stress is an important factor in regulating the ability of vascular endothelial cells to maintain volume homeostasis.
SK4/IK1-like channels mediate TEA-insensitive, Ca2+-activated K+ currents in bovine parotid acinar cells
Authors: Takahata T. , Hayashi M. , Ishikawa T.
Published: May 2013

Although Ca2+-activated K+ (KCa) channels distinct from maxi-K+ channels have been suggested to contribute to muscarinically stimulated K+ currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native KCacurrents in bovine parotid acinar (BPA) cells. Ca2+-dependent K+ efflux from perfused bovine parotid tissues was not inhibited by a maxi-K+ channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive KCa conductance, which was highly permeable to Rb+. In inside-out macropatches, TEA-insensitive Rb+ currents were activated by Ca2+ with half-maximal values of 0.4 μM. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca2+sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the Ca2+-activated Rb+ currents. In outside-out macropatches, Ca2+-activated Rb+ currents were inhibited by Ba2+, quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native KCa currents in BPA cells.
Activation of NMDA receptors linked to modulation of voltage-gated ion channels and functional implications
Authors: Davis S. F. , Linn C. L.
Published: May 2013

Catfish ( Ictalurus punctatus) cone horizontal cells contain N-methyl-d-aspartate (NMDA) receptors, the function of which has yet to be determined. In the present study, we have examined the effect of NMDA receptor activation on voltage-gated ion channel activity. NMDA receptor activation produced a long-term downregulation of voltage-gated sodium and calcium currents but had no effect on the delayed rectifying potassium current. NMDA's effect was eliminated in the presence of AP-7. To determine whether NMDA receptor activation had functional implications, isolated catfish cone horizontal cells were current clamped to mimic the cell's physiological response. When horizontal cells were depolarized, they elicited a single depolarizing overshoot and maintained a depolarized steady state membrane potential. NMDA reduced the amplitude of the depolarizing overshoot and increased the depolarized steady-state membrane potential. Both effects of NMDA were eliminated in the presence of AP-7. These results support the hypothesis that activation of NMDA receptors in catfish horizontal cells may affect the type of visual information conveyed through the distal retina.
Phosphatidic acid stimulates cardiac KATPchannels like phosphatidylinositols, but with novel gating kinetics
Authors: Fan Zheng , Gao Lizhi , Wang Wenxia
Published: May 2013

Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (KATP) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native KATP channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2–1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg2+-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 ± 2.0 to 3.3 ± 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on KATP channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.
Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles
Authors: Szymanski Pawel T. , Szymanska Grazyna , Goyal Raj K.
Published: May 2013

To determine whether densities of calmodulin (CaM) and CaM-binding proteins are related to phasic and tonic behavior of smooth muscles, we quantified these proteins in the opossum esophageal body (EB) and lower esophageal sphincter (LES), which represent phasic and tonic smooth muscles, respectively. Gel electrophoresis, immunoprecipitation, Western blot, and hemagglutinin epitope-tagged CaM (HA-CaM) overlay assay with quantitative scanning densitometry and phosphorylation measurements were used. Total protein content in the two smooth muscles was similar (∼30 mg protein/g frozen tissue). Total tissue concentration of CaM was significantly (25%) higher in EB than in LES ( P < 0.05). HA-CaM-binding proteins were qualitatively similar in LES and EB extracts. Myosin, myristoylated alanine-rich C kinase substrate protein, Ca2+/CaM kinase II, and calponin contents were also similar in the two muscles. However, content and total activity of myosin light chain kinase (MLCK) and content of caldesmon (CaD) were three- to fourfold higher in EB than in LES. Increased CaM and MLCK content may allow for a wide range of contractile force varying from complete relaxation in the basal state to a large-amplitude, high-velocity contraction in EB phasic muscle. Increased content of CaD, which provides a braking mechanism on contraction, may further contribute to the phasic contractile behavior. In contrast, low CaM, MLCK, and CaD content may be responsible for a small range of contractile force seen in tonic muscle of LES.
Nongastric H-K-ATPase in rodent prostate: lobe-specific expression and apical localization
Authors: Pestov Nikolay B. , Korneenko Tatyana V. , Adams Gail , Tillekeratne Manoranjani , Shakhparonov Mikhail I. , Modyanov Nikolai N.
Published: May 2013

The molecular basis of active ion transport in secretory glands such as the prostate is not well characterized. Rat nongastric H-K-ATPase is expressed at high levels in distal colon surface cell apical membranes and thus is referred to as “colonic.” Here we show that the ATPase is expressed in rodent prostate complex in a lobe-specific manner. RT-PCR and Western blot analyses indicate that rat nongastric H-K-ATPase α-subunit (αng) mRNA and protein are present in coagulating gland (anterior prostate) and lateral and dorsal prostate and absent from ventral lobe, whereas Na-K-ATPase α-subunit is present in all lobes. RT-PCR analysis shows that Na-K-ATPase α4 and α3 and gastric H-K-ATPase α-subunit are not present in significant amounts in all prostate lobes. Relatively low levels of Na-K-ATPase α2were found in lateral, dorsal, and anterior lobes. αngprotein expression is anteriodorsolateral: highest in coagulating gland, somewhat lower in dorsal lobe, and even lower in lateral lobe. Na-K-ATPase protein abundance has the reverse order: expression in ventral lobe is higher than in coagulating gland. αngprotein abundance is higher in coagulating gland than distal colon membranes. Immunohistochemistry shows that in rat and mouse coagulating gland epithelium αng protein has an apical polarization and Na-K-ATPase α1 is localized in basolateral membranes. The presence of nongastric H-K-ATPase in rodent prostate apical membranes may indicate its involvement in potassium concentration regulation in secretions of these glands.
Blockade of maitotoxin-induced endothelial cell lysis by glycine and l-alanine
Authors: Estacion Mark , Weinberg Justin S. , Sinkins William G. , Schilling William P.
Published: May 2013

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca2+ concentration ([Ca2+]i), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; the second phase reflects cell lysis. In the present study, the effect of the cytoprotective amino acid glycine was examined. Glycine had no effect on MTX-induced change in [Ca2+]i or on the first phase of vital dye uptake but produced a concentration-dependent (EC50 ∼1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic “pore,” the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 162 kDa was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine. The data suggest that the second “lytic” phase of MTX-induced endothelial cell death reflects formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents glycine andl-alanine.
Autocrine loops with positive feedback enable context-dependent cell signaling
Authors: Shvartsman S. Y. , Hagan M. P. , Yacoub A. , Dent P. , Wiley H. S. , Lauffenburger D. A.
Published: May 2013

We describe a mechanism for context-dependent cell signaling mediated by autocrine loops with positive feedback. We demonstrate that the composition of the extracellular medium can critically influence the intracellular signaling dynamics induced by extracellular stimuli. Specifically, in the epidermal growth factor receptor (EGFR) system, amplitude and duration of mitogen-activated protein kinase (MAPK) activation are modulated by the positive-feedback loop formed by the EGFR, the Ras-MAPK signaling pathway, and a ligand-releasing protease. The signaling response to a transient input is short-lived when most of the released ligand is lost to the cellular microenvironment by diffusion and/or interaction with an extracellular ligand-binding component. In contrast, the response is prolonged or persistent in a cell that is efficient in recapturing the endogenous ligand. To study functional capabilities of autocrine loops, we have developed a mathematical model that accounts for ligand release, transport, binding, and intracellular signaling. We find that context-dependent signaling arises as a result of dynamic interaction between the parts of an autocrine loop. Using the model, we can directly interpret experimental observations on context-dependent responses of autocrine cells to ionizing radiation. In human carcinoma cells, MAPK signaling patterns induced by a short pulse of ionizing radiation can be transient or sustained, depending on cell type and composition of the extracellular medium. On the basis of our model, we propose that autocrine loops in this, and potentially other, growth factor and cytokine systems may serve as modules for context-dependent cell signaling.
Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity
Authors: Nahm Ohnn , Woo Seung Kyoon , Handler Joseph S. , Kwon H. Moo
Published: May 2013

Osmolality of the mammalian renal medulla is high because of the operation of the urinary concentrating mechanism. To understand molecular events during the early phase of cellular adaptation to hypertonicity, we performed comprehensive searches for genes induced in response to hypertonicity using a cell line (mIMCD3) derived from the inner medullary collecting duct of mouse kidney. PCR-based subtractive hybridization of cDNA pools and cDNA microarray analysis were used. We report 12 genes whose mRNA expression is significantly increased within 4 h after exposure to hypertonicity. The increase in mRNA expression was the result of increased transcription. Many are either stress response genes or growth regulatory genes, supporting the notion that hypertonicity evokes the stress response and growth regulation in cells. Experiments using inhibitors revealed that mitogen-activated protein kinases were commonly involved in signaling for the induction of genes by hypertonicity. Tyrosine kinases and phosphatidylinositol 3-kinase also play a significant role. Signaling pathways for stimulation of transcription appeared quite diverse in that each gene was sensitive to different combinations of inhibitors.
Traction fields, moments, and strain energy that cells exert on their surroundings
Authors: Butler James P. , Tolić-Nørrelykke Iva Marija , Fabry Ben , Fredberg Jeffrey J.
Published: May 2013

Adherent cells exert tractions on their surroundings. These tractions can be measured by observing the displacements of beads embedded on a flexible gel substrate on which the cells are cultured. This paper presents an exact solution to the problem of computing the traction field from the observed displacement field. The solution rests on recasting the relationship between displacements and tractions into Fourier space, where the recovery of the traction field is especially simple. We present two subcases of the solution, depending on whether or not tractions outside the observed cell boundaries are set to be zero. The implementation is computationally efficient. We also give the solution for the traction field in a representative human airway smooth muscle cell contracted by treatment with histamine. Finally, we give explicit formulas for reducing the traction and displacement fields to contraction moments, the orientation of the principal axes of traction, and the strain energy imparted by the cell to the substrate.
Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains
Authors: Lohi Hannes , Lamprecht Georg , Markovich Daniel , Heil Anders , Kujala Minna , Seidler Ursula , Kere Juha
Published: May 2013

The solute carrier gene family SLC26 consists of tissue-specific anion exchanger genes, three of them associated with distinct human recessive disorders. By a genome-driven approach, several new SLC26 family members have been identified, including a kidney- and pancreas-specific gene, SLC26A6. We report the functional characterization of SLC26A6 and two new alternatively spliced variants, named SLC26A6c and SLC26A6d. Immunofluorescence studies on transiently transfected cells indicated membrane localization and indicated that both NH2- and COOH-terminal tails of the SLC26A6 variants are located intracellularly, suggesting a topology with an even number of transmembrane domains. Functional expression of the three proteins in Xenopus oocytes demonstrated Cl− and SO[Formula: see text] transport activity. In addition, the transport of SO[Formula: see text] and Cl− was inhibited by DIDS and HCO[Formula: see text]. We demonstrated also that the COOH terminus of SLC26A6 binds to the first and second PDZ domains of the Na+/H+ exchanger (NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor (NHERF) proteins in vitro. Truncation of the last three amino acids (TRL) of SLC26A6 abrogated the interaction but did not affect transport function. These results demonstrate that SLC26A6 and its two splice variants can function as anion transporters linked to PDZ-interaction pathways. Our results support the general concept of microdomain organization for ion transport and suggest a mechanism for cystic fibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulation in pancreatic duct cells.
TNF-α-induced endothelial cell adhesion molecule expression is cytochrome P-450 monooxygenase dependent
Authors: Sasaki Makoto , Ostanin D. , Elrod J. W. , Oshima T. , Jordan P. , Itoh M. , Joh T. , Minagar A. , Alexander J. S.
Published: May 2013

It is strongly suspected that cytokine-induced gene expression in inflammation is oxidant mediated; however, the intracellular sources of signaling oxidants remain controversial. In inflammatory bowel disease (IBD) proinflammatory cytokines, such as TNF-α, trigger gene expression of endothelial adhesion molecules including mucosal addressin cell adhesion molecule-1 (MAdCAM-1). MAdCAM-1 plays an essential role in gut inflammation by governing the infiltration of leukocytes into the intestine. Several groups suggest that endothelial-derived reduced NADP (NADPH) oxidase produces signaling oxidants that control the expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1). In addition to NADPH oxidase, cytochrome P-450 (CYP450) monooxygenases have also been shown to trigger cytokine responses. We found that in high endothelial venular cells (SVEC4-10), multiple inhibitors of CYP450 monooxygenases (SKF-525a, ketoconazole, troleandomycin, itraconazole) attenuated TNF-α induction of MAdCAM-1, whereas NADPH oxidase inhibition (PR-39) did not. Conversely, E-selectin, ICAM-1, and VCAM-1 induction requires both NADPH oxidase and CYP450-derived oxidants. We show here that MAdCAM-1 induction may depend exclusively on CYP450-derived oxidants, suggesting that CYP450 blockers might represent a possible novel therapeutic treatment for human IBD.
Divergence in species and regulatory role of β-myosin heavy chain proximal promoter muscle-CAT elements
Authors: Tsika Richard W. , McCarthy John , Karasseva Natalia , Ou Yangsi , Tsika Gretchen L.
Published: May 2013

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair β-myosin heavy chain (βMyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized βMyHC proximal promoter elements contribute to NWB regulation.
Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents
Authors: Ekhterae Daryoush , Platoshyn Oleksandr , Zhang Shen , Remillard Carmelle V. , Yuan Jason X.-J.
Published: May 2013

Cell shrinkage is an early prerequisite in programmed cell death, and cytoplasmic K+ is a dominant cation that controls intracellular ion homeostasis and cell volume. Blockade of K+ channels inhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K+ efflux through voltage-gated K+ (Kv) channels. In heart-derived H9c2 cells, whole cell Kv currents ( I K(V)) were isolated by using Ca2+-free extracellular (bath) solution and including 5 mM ATP and 10 mM EGTA in the intracellular (pipette) solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), a blocker of Kv channels, reversibly reduced I K(V)by 50–60% in H9c2 cells. The remaining currents during 4-AP treatment may be generated by K+ efflux through 4-AP-insensitive K+ channels. Overexpression of ARC in heart-derived H9c2 cells significantly decreased I K(V), whereas treatment with staurosporine, a potent apoptosis inducer, enhanced I K(V)in wild-type cells. The staurosporine-induced increase in I K(V) was significantly suppressed and the staurosporine-mediated apoptosis was markedly inhibited in cells overexpressing ARC compared with cells transfected with the control neomycin vector. These results suggest that the antiapoptotic effect of ARC is, in part, due to inhibition of Kv channels in cardiomyocytes.