bluuebunny/crossref_metadata_2025 huggingface dataset:
An electrogenic Na+- HCO 3 − cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain
Authors: Bevensee Mark O. , Schmitt Bernhard M. , Choi Inyeong , Romero Michael F. , Boron Walter F.
Published: December 2017

We screened rat brain cDNA libraries and used 5′ rapid amplification of cDNA ends to clone two electrogenic Na+-[Formula: see text] cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3′ end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (αrb1NBC) and rb2NBC (αrb2NBC). αrb1NBC labels an ∼130-kDa protein predominantly from kidney, and αrb2NBC labels an ∼130-kDa protein predominantly from brain. αrb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; αrb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO2/10 mM [Formula: see text] to Xenopus oocytes injected with rb2NBC cRNA causes 1) pHi to recover from the initial CO2-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na+ reverses the pHi increase and elicits a rapid depolarization. In the presence of 450 μM DIDS, removing external Na+ has no effect on pHi and elicits a small hyperpolarization. The rate of the pHidecrease elicited by removing Na+ is insensitive to removing external Cl−. Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.
Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells
Authors: Bello-Reuss Elsa , Ernest Sylvain , Holland O. Bryan , Hellmich Mark R.
Published: December 2017

We determined the role of the multidrug resistance (MDR1) gene product, P-glycoprotein (PGP), in the secretion of aldosterone by the adrenal cell line NCI-H295. Aldosterone secretion is significantly decreased by the PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, and vinblastine. Aldosterone inhibits the efflux of the PGP substrate rhodamine 123 from NCI-H295 cells and from human mesangial cells (expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2 significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells, expressing multidrug resistance-associated protein (MRP) but not PGP, and in the parental cell line MCF7 (expressing no MRP and no PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3 cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosterone was rapid and it was inhibited by verapamil, indicating that transfection with MDR1 cDNA confers the ability to transport FL-aldosterone. These results strongly indicate that PGP plays a role in the secretion of aldosterone by NCI-H295 cells and in other cells expressing MDR1, including normal adrenal cells.
Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts
Authors: Ko Kevin , Arora Pamela , Lee Wilson , McCulloch Christopher
Published: December 2017

Despite their significance in wound healing, little is known about the molecular determinants of cell-to-cell adhesion and gap junctional communication in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required Ca2+ and was >30-fold stronger than cell-to-fibronectin adhesion at 15 min. Electron micrographs showed rapid formation of adherens junction-like structures at ∼15 min that matured by ∼2–3 h; distinct gap junctional complexes were evident by ∼3 h. Immunoblotting showed that HGF expressed β-catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye transfer in a cohort of Texas red dextran-labeled cells, but this was almost completely abolished by the gap junction inhibitor β-glycyrrhetinic acid and the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model allows large numbers (>105) of cells to form synchronous cell-to-cell contacts, thereby enabling the simultaneous functional and molecular studies of adherens junctions and gap junctions.
Contribution of intermediate filaments to cell stiffness, stiffening, and growth
Authors: Wang Ning , Stamenović Dimitrije
Published: December 2017

It has been shown previously that intermediate filament (IF) gels in vitro exhibit stiffening at high-applied stress, and it was suggested that this stiffening property of IFs might be important for maintaining cell integrity at large deformations (Janmey PA, Evtenever V, Traub P, and Schliwa M, J Cell Biol 113: 155–160, 1991). In this study, the contribution of IFs to cell mechanical behavior was investigated by measuring cell stiffness in response to applied stress in adherent wild-type and vimentin-deficient fibroblasts using magnetic twisting cytometry. It was found that vimentin-deficient cells were less stiff and exhibited less stiffening than wild-type cells, except at the lowest applied stress (10 dyn/cm2) where the difference in the stiffness was not significant. Similar results were obtained from measurements on wild-type fibroblasts and endothelial cells after vimentin IFs were disrupted by acrylamide. If, however, cells were plated over an extended period of time (16 h), they exhibited a significantly greater stiffness before than after acrylamide, even at the lowest applied stress. A possible reason could be that the initially slack IFs became fully extended due to a high degree of cell spreading and thus contributed to the transmission of mechanical stress across the cell. Taken together, these findings were consistent with the notion that IFs play important roles in the mechanical properties of the cell during large deformation. The experimental data also showed that depleting or disrupting IFs reduced, but did not entirely abolish, cell stiffening. This residual stiffening might be attributed to the effect of geometrical realignment of cytoskeletal filaments in the direction of applied load. It was also found that vimentin-deficient cells exhibited a slower rate of proliferation and DNA synthesis than wild-type cells. This could be a direct consequence of the absence of the intracellular IFs that may be necessary for efficient mediation of mechanical signals within the cell. Taken together, results of this study suggest that IFs play important roles in the mechanical properties of cells and in cell growth.
ROCK mediates thrombin's endothelial barrier dysfunction
Authors: Carbajal José M. , Gratrix Max L. , Yu Chung-Ho , Schaeffer Richard C.
Published: December 2017

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to β-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125FAK, it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.
H2O2-mediated permeability: role of MAPK and occludin
Authors: Kevil Christopher G. , Oshima Tadayuki , Alexander Brett , Coe Laura L. , Alexander J. Steven
Published: December 2017

H2O2-mediated elevation in endothelial solute permeability is associated with pathological events such as ischemia-reperfusion and inflammation. To understand how H2O2 mediates increased permeability, we investigated the effects of H2O2 administration on vascular endothelial barrier properties and tight junction organization and function. We report that H2O2exposure caused an increase in endothelial solute permeability in a time-dependent manner through extracellularly regulated kinase 1 and 2 (ERK1/ERK2) signal pathways. H2O2 exposure caused the tight junctional protein occludin to be rearranged from endothelial cell-cell junctions. Occludin rearrangement involved redistribution of occludin on the cell surface and dissociation of occludin from ZO-1. Occludin also was heavily phosphorylated on serine residues upon H2O2 administration. H2O2 mediates changes in ERK1/ERK2 phosphorylation, increases endothelial solute permeability, and alters occludin localization and phosphorylation were all blocked by PD-98059, a specific mitogen-activated protein (MAP) or ERK kinase 1 inhibitor. These data strongly suggest that H2O2-mediated increased endothelial solute permeability involves the loss of endothelial tight junction integrity through increased ERK1/ERK2 activation.
IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells
Authors: Wei Liu Hua , Jacobs Aaron T. , Morris Sidney M. , Ignarro Louis J.
Published: December 2017

The objectives of this study were to determine whether rat aortic smooth muscle cells (RASMC) express arginase and to elucidate the possible mechanisms involved in the regulation of arginase expression. The results show that RASMC contain basal arginase I (AI) activity, which is significantly enhanced by stimulating the cells with either interleukin (IL)-4 or IL-13, but arginase II (AII) expression was not detected under any condition studied here. We further investigated the signal transduction pathways responsible for AI induction. AI mRNA and protein levels were enhanced by addition of forskolin (1 μM) and inhibited by H-89 (30 μM), suggesting positive regulation of AI by a protein kinase A pathway. Genistein (10 μg/ml) and sodium orthovanadate (Na3VO4; 10 μM) were used to investigate the role of tyrosine phosphorylation in the control of AI expression. Genistein inhibited, whereas Na3VO4enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate the JAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon (IFN)-γ were investigated for their effects on AI induction. Dex (1 μM) and IFN-γ (100 U/ml) alone had no effect on basal AI expression in RASMC, but both reduced AI induction by IL-4 and IL-13. In combination, Dex and IFN-γ abolished AI induction by IL-4 and IL-13. Finally, both IL-4 and IL-13 significantly increased RASMC DNA synthesis as monitored by [3H]thymidine incorporation, demonstrating that upregulation of AI is correlated with an increase in cell proliferation. Blockade of AI induction by IFN-γ, H-89, or genistein also blocked the increase in cell proliferation. These observations are consistent with the possibility that upregulation of AI might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.
Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts
Authors: Bell Andrea , Gagnon Annemarie , Grunder Laura , Parikh Sonia J. , Smith Terry J. , Sorisky Alexander
Published: December 2017

Controversy continues about whether, and to what levels of abundance, thyroid-stimulating hormone receptors (TSHR) are found in human tissues other than the thyroid gland. Restricted expression to the thyroid and orbit would suggest that TSHR represents the target autoantigen in thyroid-associated ophthalmopathy. A more generalized pattern of tissue expression would be inconsistent with TSHR acting as the autoantigen that is solely responsible for selectively targeting the immune system to the orbit. We have detected TSHR mRNA in human abdominal adipose tissue by Northern blot analysis. TSHR protein was also detected, by immunoblotting with two different antibodies, in preadipocytes isolated from human abdominal subcutaneous and omental adipose tissue and in derivative adipocytes differentiated in primary culture. Preadipocytes treated with thyroid-stimulating hormone (TSH) exhibited a sevenfold increase in the activity of p70 S6 kinase, a serine/threonine kinase recently recognized as a downstream target of TSHR in thyroid cells. Activation of p70 S6 kinase by TSH was also observed in orbital fibroblasts. Thus TSHR protein expression is found in fibroblasts from several anatomic locations, suggesting that factors other than site-limited TSHR expression must be involved in restricting the distribution of Graves' disease manifestations. Furthermore, the presence of functional TSHR in preadipocytes raises the possibility of a novel role for TSHR signaling in adipose tissue development.
Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle
Authors: Yamboliev Ilia A. , Wiesmann Kevin M. , Singer Cherie A. , Hedges Jason C. , Gerthoffer William T.
Published: December 2017

In canine colon, M2/M3 muscarinic receptors are coupled to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. We tested the hypothesis that this coupling is mediated by enzymes of the phosphatidylinositol (PI) 3-kinase family. RT-PCR and Western blotting demonstrated expression of two isoforms, PI 3-kinase-α and PI 3-kinase-γ. Muscarinic stimulation of intact muscle strips (10 μM ACh) activated PI 3-kinase-γ, ERK and p38 MAP kinases, and MAP kinase-activated protein kinase-2, whereas PI 3-kinase-α activation was not detected. Wortmannin (25 μM) abolished the activation of PI 3-kinase-γ, ERK, and p38 MAP kinases. MAP kinase inhibition was a PI 3-kinase-γ-specific effect, since wortmannin did not inhibit recombinant activated murine ERK2 MAP kinase, protein kinase C, Raf-1, or MAP kinase kinase. In cultured muscle cells, newborn calf serum (3%) activated PI 3-kinase-α and PI 3-kinase-γ isoforms, ERK and p38 MAP kinases, and stimulated chemotactic cell migration. Using wortmannin and LY-294002 to inhibit PI 3-kinase activity and PD-098059 and SB-203580 to inhibit ERK and p38 MAP kinases, we established that these enzymes are functionally important for regulation of chemotactic migration of colonic myocytes.
Genistein activates CFTR-mediated Cl−secretion in the murine trachea and colon
Authors: Goddard Catharine A. , Evans Martin J. , Colledge William H.
Published: December 2017

The action of the isoflavone genistein on the cystic fibrosis transmembrane conductance regulator (CFTR) has been studied in many cell systems but not in intact murine tissues. We have investigated the action of genistein on murine tissues from normal and cystic fibrosis (CF) mice. Genistein increased the short-circuit current ( I sc) in tracheal (16.4 ± 2.8 μA/cm2) and colonic (40.0 ± 4.4 μA/cm2) epithelia of wild-type mice. This increase was inhibited by furosemide, diphenylamine-2-carboxylate, and glibenclamide, but not by DIDS. In contrast, genistein produced no significant change in the I sc of the tracheal epithelium (0.9 ± 1.1 μA/cm2) and decreased the I sc of colons from CF null (−13.1 ± 2.3 μA/cm2) and ΔF508 mice (−10.3 ± 1.3 μA/cm2). Delivery of a human CFTRcDNA-liposome complex to the airways of CF null mice restored the genistein response in the tracheas to wild-type levels. Tracheas from ΔF508 mice were also studied: 46% of trachea showed no response to genistein, whereas 54% gave an increase in I scsimilar to that in wild type. We conclude that genistein activates CFTR-mediated Cl− secretion in the murine trachea and distal colon.
Hypertrophic effect of selective β1-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat
Authors: Schäfer Matthias , Frischkopf Karen , Taimor Gerhild , Piper Hans Michael , Schlüter Klaus-Dieter
Published: December 2017

We investigated whether selective β1-adrenoceptor stimulation causes hypertrophic growth on isolated ventricular cardiomyocytes from adult rat. As parameters for the induction of hypertrophic growth, the increases of [14C]phenylalanine incorporation, protein and RNA mass, and cell size were determined. Isoproterenol (Iso, 10 μM) alone had no growth effect. In the presence of the β2-adrenoceptor antagonist ICI-118551 (ICI, 10 μM), Iso caused an increase in [14C]phenylalanine incorporation, protein and RNA mass, cell volume, and cross-sectional area. We showed for phenylalanine incorporation that the growth effect of Iso+ICI could be antagonized by β1-adrenoceptor blockade with atenolol (10 μM) or metoprolol (10 μM), indicating that it was caused by selective β1-adrenoceptor stimulation. The growth response to Iso+ICI was accompanied by an increase in ornithine decarboxylase (ODC) activity and expression. Inhibition of ODC by the ODC antagonist difluoromethylornithine (1 mM) attenuated this hypertrophic response, indicating that ODC induction is causally involved. The growth response to Iso+ICI was found to be cAMP independent but was sensitive to genistein (100 μM) or rapamycin (0.1 μM). The reaction was enhanced in the presence of pertussis toxin (10 μM). We conclude that selective β1-adrenoceptor stimulation causes hypertrophic growth of ventricular cardiomyocytes by a mechanism that is independent of cAMP but dependent on a tyrosine kinase and ODC.
Skeletal muscle fiber quality in older men and women
Authors: Frontera Walter R. , Suh Dongwon , Krivickas Lisa S. , Hughes Virginia A. , Goldstein Richard , Roubenoff Ronenn
Published: December 2017

Whole muscle strength and cross-sectional area (WMCSA), and contractile properties of chemically skinned segments from single fibers of the quadriceps were studied in 7 young men (YM, 36.5 ± 3.0 yr), 12 older men (OM, 74.4 ± 5.9 yr), and 12 older women (OW, 72.1 ± 4.3 yr). WMCSA was smaller in OM compared with YM (56.1 ± 10.1 vs. 79.7 ± 13.1 cm2; P = 0.031) and in OW (44.9 ± 7.5; P < 0.003) compared with OM. Age-related, but not sex-related, differences in strength were eliminated after adjusting for WMCSA. Maximal force was measured in 552 type I and 230 type IIA fibers. Fibers from YM (type I = 725 ± 221; type IIA = 792 ± 271 μN) were stronger ( P < 0.001) than fibers from OM (I = 505 ± 179; IIA = 577 ± 262 μN) even after correcting for size. Type IIA fibers were stronger ( P < 0.005) than type I fibers in YM and OM but not in OW (I = 472 ± 154; IIA = 422 ± 97 μN). Sex-related differences in type I and IIA fibers were dependent on fiber size. In conclusion, differences in WMCSA explain age-related differences in strength. An intrinsic defect in contractile proteins could explain weakness in single fibers from OM. Sex-related differences exist at the whole muscle and single fiber levels.
Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes
Authors: Himms-Hagen J. , Melnyk A. , Zingaretti M. C. , Ceresi E. , Barbatelli G. , Cinti S.
Published: December 2017

Multilocular, mitochondria-rich adipocytes appear in white adipose tissue (WAT) of rats treated with the β3-adrenoceptor agonist, CL-316243 (CL). Objectives were to determine whether these multilocular adipocytes derived from cells that already existed in the WAT or from proliferation of precursor cells and whether new mitochondria contained in them were typical brown adipocyte mitochondria. Use of 5-bromodeoxyuridine to identify cells that had undergone mitosis during the CL treatment showed that most multilocular cells derived from cells already present in the WAT. Morphological techniques showed that at least a subpopulation of unilocular adipocytes underwent conversion to multilocular mitochondria-rich adipocytes. A small proportion of multilocular adipocytes (∼8%) was positive for UCP1 by immunohistochemistry. Biochemical techniques showed that mitochondrial protein recovered from WAT increased 10-fold and protein isolated from brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels showed a different protein composition of new mitochondria isolated from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much lower concentration than in BAT mitochondria, and UCP3, at a higher concentration than that in BAT mitochondria. We hypothesize that multilocular adipocytes present at 7 days of CL treatment have two origins. First, most come from convertible unilocular adipocytes that become multilocular and make many mitochondria that contain UCP3. Second, some come from a cell that gives rise to more typical brown adipocytes that express UCP1.
Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat
Authors: Putman Charles T. , Düsterhöft Sabine , Pette Dirk
Published: December 2017

Satellite cell proliferation was assessed in low-frequency-stimulated hypothyroid rat fast-twitch muscle by 5-bromo-2′-deoxyuridine (BrdU) labeling and subsequent staining of labeled muscle nuclei, and by staining for proliferating cell nuclear antigen (PCNA). BrdU labeling and PCNA staining were highly correlated and increased approximately fourfold at 5 days of stimulation, decayed thereafter, but remained elevated over control in 10- and 20-day stimulated muscles. Myogenin mRNA was ∼4-fold elevated at 5 days and 1.5-fold at 10 days. Staining for myogenin protein yielded results similar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequency stimulation. By 20 days, myogenin staining was observed in transforming fast fibers that coexpressed embryonic and adult myosin heavy chain isoforms. In the slower fiber populations (i.e., IIA and I), myogenin-positive transforming fibers that coexpressed embryonic myosin heavy chain, appeared already at 5 days. Thus the satellite cell progeny on slower fibers seemed to proliferate less and to fuse earlier to their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.
A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle
Authors: Jeneson J. A. L. , Westerhoff H. V. , Kushmerick M. J.
Published: December 2017

A system analysis of ATP free energy metabolism in skeletal muscle was made using the principles of metabolic control theory. We developed a network model of ATP free energy metabolism in muscle consisting of actomyosin ATPase, sarcoplasmic reticulum (SR) Ca2+-ATPase, and mitochondria. These components were sufficient to capture the major aspects of the regulation of the cytosolic ATP-to-ADP concentration ratio (ATP/ADP) in muscle contraction and had inherent homeostatic properties regulating this free energy potential. As input for the analysis, we used ATP metabolic flux and the cytosolic ATP/ADP at steady state at six contraction frequencies between 0 and 2 Hz measured in human forearm flexor muscle by 31P-NMR spectroscopy. We used the mathematical formalism of metabolic control theory to analyze the distribution of fractional kinetic control of ATPase flux and the ATP/ADP in the network at steady state among the components over this experimental range and an extrapolated range of stimulation frequencies (up to 10 Hz). The control analysis showed that the contractile actomyosin ATPase has dominant kinetic control of ATP flux in forearm flexor muscle over the 0- to 1.6-Hz range of contraction frequencies that resulted in steady states, as determined by 31P-NMR. However, flux control begins to shift toward mitochondria at >1 Hz. This inversion of flux control from ATP demand to ATP supply control hierarchy progressed as the contraction frequency increased past 2 Hz and was nearly complete at 10 Hz. The functional significance of this result is that, at steady state, ATP free energy consumption cannot outstrip the ATP free energy supply. Therefore, this reduced, three-component muscle ATPase system is inherently homeostatic.
Elevation of resting mitochondrial membrane potential of neural cells by cyclosporin A, BAPTA-AM, and Bcl-2
Authors: Kowaltowski Alicia J. , Smaili Soraya S. , Russell James T. , Fiskum Gary
Published: December 2017

This study tested the hypothesis that the activity of the mitochondrial membrane permeability transition pore (PTP) affects the resting mitochondrial membrane potential (ΔΨ) of normal, healthy cells and that the anti-apoptotic gene product Bcl-2 inhibits the basal activity of the PTP. ΔΨ was measured by both fluorometric and nonfluorometric methods with SY5Y human neuroblastoma cells and with GT1–7 hypothalamic cells and PC12 pheochromocytoma cells in the absence and presence of Bcl-2 gene overexpression. The resting ΔΨ of Bcl-2 nonexpressing PC12 and wild-type SY5Y cells was increased significantly by the presence of the PTP inhibitor cyclosporin A (CsA) or by intracellular Ca2+ chelation through exposure to the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA-AM). The ΔΨ of Bcl-2-overexpressing PC12 cells was larger than that of Bcl-2-negative cells and not significantly increased by CsA or by Ca2+ chelation. CsA did not present a significant effect on the ΔΨ monitored in unstressed GT1–7 cells but did inhibit the decrease in ΔΨ elicited by the addition of t-butyl hydroperoxide, an oxidative inducer of the mitochondrial permeability transition. These results support the hypothesis that an endogenous PTP activity can contribute to lowering the basal ΔΨ of some cells and that Bcl-2 can regulate the endogenous activity of the mitochondrial PTP.
Alterations in expression of myosin and myosin light chain kinases in response to vascular injury
Authors: Gallagher Patricia J. , Jin Yijun , Killough Gayla , Blue Emily K. , Lindner Volkhard
Published: December 2017

Histochemical analysis of balloon-injured rat carotid arteries revealed a coordinated expression of nonmuscle myosin heavy chain-A and -B (NM-A and NM-B) in response to injury. Expression of these nonmuscle myosin forms shifts from the media to the adventitia and intima. In contrast, expression of smooth muscle myosin heavy chain-1 (SM-1) within the media is not altered, whereas smooth muscle myosin heavy chain-2 (SM-2) expression declines. Western blotting shows a statistically significant increase in expression of NM-A that occurs within 6 h in response to carotid injury, suggesting this myosin form may be an appropriate experimental marker for proliferating, migrating cells in injured vessels. No overall change in the relative expression level of NM-B was detected, suggesting that compensatory declines in media expression are balanced by increases in the intima and adventitia. Expression of SM-1 did not change in response to injury, whereas the expression of SM-2 significantly declined between 24 h and 7 days. Expression of myosin light chain kinase is also negatively regulated, and the decline in its expression parallels downregulation of SM-2.
Evidence against the acidification hypothesis in cystic fibrosis
Authors: Gibson Gregory A. , Hill Warren G. , Weisz Ora A.
Published: December 2017

The pleiotropic effects of cystic fibrosis (CF) result from the mislocalization or inactivity of an apical membrane chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR may also modulate intracellular chloride conductances and thus affect organelle pH. To test the role of CFTR in organelle pH regulation, we developed a model system to selectively perturb the pH of a subset of acidified compartments in polarized cells and determined the effects on various protein trafficking steps. We then tested whether these effects were observed in cells lacking wild-type CFTR and whether reintroduction of CFTR affected trafficking in these cells. Our model system involves adenovirus-mediated expression of the influenza virus M2 protein, an acid-activated ion channel. M2 expression selectively slows traffic through the trans-Golgi network (TGN) and apical endocytic compartments in polarized Madin-Darby canine kidney (MDCK) cells. Expression of M2 or treatment with other pH perturbants also slowed protein traffic in the CF cell line CFPAC, suggesting that the TGN in this cell line is normally acidified. Expression of functional CFTR had no effect on traffic and failed to rescue the effect of M2. Our results argue against a role for CFTR in the regulation of organelle pH and protein trafficking in epithelial cells.
Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-α
Authors: Merial Christelle , Bouloumie Anne , Trocheris Véronique , Lafontan Max , Galitzky Jean
Published: December 2017

Uncoupling protein-2 (UCP-2) is a mitochondrial protein expressed in adipocytes and has recently been involved in the control of energy dissipation. Because obesity is characterized by an imbalance between energy intake and expenditure and by an enhanced adipocyte-derived secretion of tumor necrosis factor-α (TNF-α), we asked whether TNF-α could directly influence UCP-2 expression in adipocytes. Experiments performed in differentiated 3T3F442A preadipocytes showed that TNF-α (10 ng/ml) induced a reduction of UCP-2 trancripts, assessed by Northern blot analysis. A significant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF-α stimulation of the cells. The characterization of the mechanisms responsible for the TNF-α effect on UCP-2 expression demonstrates an involvement of the TNF-α-induced inducible (i) nitric oxide synthase (NOS) expression. Cell treatment with the NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME; 1 mmol/l) significantly diminished the TNF-α-mediated sustained downregulation of UCP-2 expression, whereas cell treatment with a nitric oxide (NO) donor (10−3 mol/l S-nitroso-l-glutathione) mimicked the TNF-α effect on UCP-2 expression. Moreover, Western blot analysis clearly showed that TNF-α alone induces the expression of iNOS after 12–24 h treatment of differentiated 3T3F442A cells. These experiments demonstrate that TNF-α directly downregulates UCP-2 expression via NO-dependent pathways that involve the induction of iNOS expression.
Upregulation of Kv1.3 K+ channels in microglia deactivated by TGF-β
Authors: Schilling Tom , Quandt Fred N. , Cherny Vladimir V. , Zhou Wei , Heinemann Uwe , Decoursey Thomas E. , Eder Claudia
Published: December 2017

Microglial activation is accompanied by changes in K+ channel expression. Here we demonstrate that a deactivating cytokine changes the electrophysiological properties of microglial cells. Upregulation of delayed rectifier (DR) K+channels was observed in microglia after exposure to transforming growth factor-β (TGF-β) for 24 h. In contrast, inward rectifier K+ channel expression was unchanged by TGF-β. DR current density was more than sixfold larger in TGF-β-treated microglia than in untreated microglia. DR currents of TGF-β-treated cells exhibited the following properties: activation at potentials more positive than −40 mV, half-maximal activation at −27 mV, half-maximal inactivation at −38 mV, time dependent and strongly use-dependent inactivation, and a single channel conductance of 13 pS in Ringer solution. DR channels were highly sensitive to charybdotoxin (CTX) and kaliotoxin (KTX), whereas α-dendrotoxin had little effect. With RT-PCR, mRNA for Kv1.3 and Kir2.1 was detected in microglia. In accordance with the observed changes in DR current density, the mRNA level for Kv1.3 (assessed by competitive RT-PCR) increased fivefold after treatment of microglia with TGF-β.
Sterol carrier protein-2 localization in endoplasmic reticulum and role in phospholipid formation
Authors: Starodub O. , Jolly C. A. , Atshaves B. P. , Roths J. B. , Murphy E. J. , Kier A. B. , Schroeder F.
Published: December 2017

Although sterol carrier protein-2 (SCP-2; also called nonspecific lipid transfer protein) binds fatty acids and fatty acyl-CoAs, its role in fatty acid metabolism is not fully understood. L-cell fibroblasts stably expressing SCP-2 were used to resolve the relationship between SCP-2 intracellular location and fatty acid transacylation in the endoplasmic reticulum. Indirect immunofluorescence double labeling and laser scanning confocal microscopy detected SCP-2 in peroxisomes > endoplasmic reticulum > mitochondria > lysosomes. SCP-2 enhanced incorporation of exogenous [3H]oleic acid into phospholipids and triacylglycerols of overexpressing cells 1.6- and 2.5-fold, respectively, stimulated microsomal incorporation of [1-14C]oleoyl-CoA into phosphatidic acid in vitro 13-fold, and exhibited higher specificity for unsaturated versus saturated fatty acyl-CoA. SCP-2 enhanced the rate-limiting step in microsomal phosphatidic acid biosynthesis mediated by glycerol-3-phosphate acyltransferase. SCP-2 also enhanced microsomal acyl-chain remodeling of phosphatidylethanolamine up to fivefold and phosphatidylserine twofold, depending on the specific fatty acyl-CoA, but had no effect on other phospholipid classes. In summary, these results were consistent with a role for SCP-2 in phospholipid synthesis in the endoplasmic reticulum.
Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools
Authors: Dagher Pierre C.
Published: December 2017

Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.
Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability
Authors: Tinsley John H. , De Lanerolle Primal , Wilson Emily , Ma Weiya , Yuan Sarah Y.
Published: December 2017

The actomyosin complex is the major cytoskeletal component that controls cell contraction. In this study, we investigated the effects of actomyosin interaction on endothelial barrier function and gap formation. Activated myosin light chain kinase (MLCK) protein was transferred into coronary venular endothelial cell (CVEC) monolayers. Uptake of the activated protein resulted in a significant shift in myosin light chain (MLC) from an unphosphorylated to a diphosphorylated form. In addition, MLCK induced a hyperpermeability response of the monolayer as measured by albumin transendothelial flux. Microscopic examination of MLCK-treated CVECs revealed widespread gap formation in the monolayer, loss of peripheral β-catenin, and increases in actin stress fibers. Inhibition of all of the above responses by a specific MLCK inhibitor suggests they are the direct result of exogenously added MLCK. These data suggest that activation of MLCK in CVECs causes phosphorylation of MLC and contraction of CVECs, resulting in gap formation and concomitant increases in permeability. This study uses a novel technique to measure the effects of an activated kinase on both its substrate and cellular morphology and function through direct transference into endothelial cells.
Role of thyroglobulin endocytic pathways in the control of thyroid hormone release
Authors: Marinò Michele , McCluskey Robert T.
Published: December 2017

Thyroglobulin (Tg), the thyroid hormone precursor, is synthesized by thyrocytes and secreted into the colloid. Hormone release requires uptake of Tg by thyrocytes and degradation in lysosomes. This process must be precisely regulated. Tg uptake occurs mainly by micropinocytosis, which can result from both fluid-phase pinocytosis and receptor-mediated endocytosis. Because Tg is highly concentrated in the colloid, fluid-phase pinocytosis or low-affinity receptors should provide sufficient Tg uptake for hormone release; high-affinity receptors may serve to target Tg away from lysosomes, through recycling into the colloid or by transcytosis into the bloodstream. Several apical receptors have been suggested to play roles in Tg uptake and intracellular trafficking. A thyroid asialoglycoprotein receptor may internalize and recycle immature forms of Tg back to the colloid, a function also attributed to an as yet unidentified N-acetylglucosamine receptor. Megalin mediates Tg uptake by thyrocytes, especially under intense thyroid-stimulating hormone stimulation, resulting in transcytosis of Tg from the colloid to the bloodstream, a function that prevents excessive hormone release.
Ca2+-activated Cl− current in sheep lymphatic smooth muscle
Authors: Toland H. M. , McCloskey K. D. , Thornbury K. D. , McHale N. G. , Hollywood M. A.
Published: December 2017

Freshly dispersed sheep mesenteric lymphatic smooth muscle cells were studied at 37°C using the perforated patch-clamp technique with Cs+- and K+-filled pipettes. Depolarizing steps evoked currents that consisted ofl-type Ca2+ [ I Ca(L)] current and a slowly developing current. The slow current reversed at 1 ± 1.5 mV with symmetrical Cl− concentrations compared with 23.2 ± 1.2 mV ( n = 5) and −34.3 ± 3.5 mV ( n = 4) when external Cl− was substituted with either glutamate (86 mM) or I− (125 mM). Nifedipine (1 μM) blocked and BAY K 8644 enhanced I Ca(L), the slow-developing sustained current, and the tail current. The Cl− channel blocker anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing inward and tail currents. Application of caffeine (10 mM) to voltage-clamped cells evoked currents that reversed close to the Cl− equilibrium potential and were sensitive to 9-AC. Small spontaneous transient depolarizations and larger action potentials were observed in current clamp, and these were blocked by 9-AC. Evoked action potentials were triphasic and had a prominent plateau phase that was selectively blocked by 9-AC. Similarly, fluid output was reduced by 9-AC in doubly cannulated segments of spontaneously pumping sheep lymphatics, suggesting that the Ca2+-activated Cl− current plays an important role in the electrical activity underlying spontaneous activity in this tissue.
The Egr-1 gene is induced by epidermal growth factor in ECV304 cells and primary endothelial cells
Authors: Tsai Jo C. , Liu Lixin , Guan Jiazhen , Aird William C.
Published: December 2017

The early growth response (Egr)-1 transcription factor serves to couple changes in the extracellular environment to alterations in gene expression. An understanding of the mechanisms that underlie Egr-1 gene regulation should provide important insights into how environmental signals are transduced by endothelial cells. The aim of the present study was to determine whether epidermal growth factor (EGF) induces Egr-1 expression in endothelial cells. In ECV304 cells, Egr-1 mRNA and protein levels were increased in response to EGF. In stable transfection assays, the 1,200-bp promoter of the mouse Egr-1 gene contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent pathway. The endogenous Egr-1 gene was similarly responsive to EGF in primary human umbilical vein endothelial cells, human coronary artery endothelial cells, and rat fat pad endothelial cells, but not in bovine aortic endothelial cells, calf pulmonary artery endothelial cells, or PY-4-1 endothelial cells. Together, these results suggest that the Egr-1 gene is responsive to EGF in a subset of endothelial cells.
Impaired Gsα and adenylyl cyclase cause β-adrenoceptor desensitization in chronically hypoxic rat hearts
Authors: Pei Jian-Ming , Yu Xiao-Chun , Fung Man-Lung , Zhou Jing-Jun , Cheung Chi-Sing , Wong Nai-Sum , Leung Maurrice-Ping , Wong Tak-Ming
Published: December 2017

The effects of β-adrenoceptor stimulation with isoproterenol on electrically induced contraction and intracellular calcium ([Ca2+]i) transient, and cAMP in myocytes from both hypertrophied right and nonhypertrophied left ventricles of rats exposed to 10% oxygen for 4 wk, were significantly attenuated. The increased [Ca2+]i transient in response to cholera toxin was abolished, whereas increased cAMP after NaF significantly attenuated. The biologically active isoform, Gsα-small (45 kDa), was reduced while the biologically inactive isoform, Gsα-large (52 kDa), increased. The increased electrically induced [Ca2+]i transient and cAMP with 10–100 μM forskolin were significantly attenuated in chronically hypoxic rats. The content of Giα2, the predominant isoform of Gi protein in the heart, was unchanged. Results indicate that impaired functions of Gs protein and adenylyl cyclase cause β-adrenoceptor desensitization. The impaired function of the Gs protein may be due to reduced Gsα-small and/or increased Gsα-large, which does not result from changes in Gi protein. Responses to all treatments were the same for right and left ventricles, indicating that the impaired cardiac functions are not secondary to cardiac hypertrophy.
Regulation of Na+-K+-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein
Authors: Kurihara Kinji , Nakanishi Nobuo , Ueha Takao
Published: December 2017

Na+-K+- ATPase α-subunits in basolateral membrane vesicles (BLMVs) purified from rat parotid glands were 32P-labeled within 5 s by incubation with [γ-32P]ATP at 37°C in the presence of cAMP, but no labeling occurred without cAMP. Phosphorylation of Na+-K+-ATPase was associated with a decrease in its activity. This α-subunit phosphorylation disappeared when BLMVs were briefly incubated with cAMP and subsequent washing before the incubation with [γ-32P]ATP, indicating that catalytic subunit of protein kinase A (PKA) associated to BLMVs via binding with its RII regulatory subunit anchored on the membrane. In the absence of cAMP, a PKA catalytic subunit readily reassociated with the membrane-bound RII subunit. HT-31 peptide inhibited the Na+-K+-ATPase phosphorylation by membrane-bound endogenous PKA, indicating an involvement of A-kinase anchoring protein (AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and an RII overlay assay and was coimmunoprecipitated by anti-RII antibody. These results show that Na+-K+-ATPase of rat parotid gland acinar cells is regulated in vivo by membrane-anchored PKA via AKAP rather than by free cytosolic PKA.
Amino acid deprivation induces translation of branched-chain α-ketoacid dehydrogenase kinase
Authors: Doering Christopher B. , Danner Dean J.
Published: December 2017

Leucine, isoleucine, and valine are used by cells for protein synthesis or are catabolized into sources for glucose and lipid production. These branched-chain amino acids influence proteolysis, hormone release, and cell cycle progression along with their other metabolic roles. The branched-chain amino acids play a central role in regulating cellular protein turnover by reducing autophagy. These essential amino acids are committed to their catabolic fate by the activity of the branched-chain α-ketoacid dehydrogenase complex. Activity of the branched-chain α-ketoacid dehydrogenase complex is regulated by phosphorylation/inactivation of the α-subunit performed by a complex specific kinase. Here we show that elimination of the branched-chain amino acids from the medium of cultured cells results in a two- to threefold increased production of the branched-chain α-ketoacid dehydrogenase kinase with a decrease in the activity state of the branched-chain α-ketoacid dehydrogenase complex. The mechanism cells use to increase kinase production under these conditions involves recruitment of the kinase mRNA into polyribosomes. Promoter activity and the steady-state concentration of the mRNA are unchanged by these conditions.
Endogenous nitric oxide is implicated in the regulation of lipolysis through antioxidant-related effect
Authors: Gaudiot Nicolas , Ribière Catherine , Jaubert Anne-Marie , Giudicelli Yves
Published: December 2017

We studied the influence of nitric oxide (NO) endogenously produced by adipocytes in lipolysis regulation. Diphenyliodonium (DPI), a nitric oxide synthase (NOS) inhibitor, was found to completely suppress NO synthesis in intact adipocytes and was thus used in lipolysis experiments. DPI was found to decrease both basal and dibutyryl cAMP (DBcAMP)-stimulated lipolysis. Inhibition of DBcAMP-stimulated lipolysis by DPI was prevented by S-nitroso- N-acetyl-penicillamine (SNAP), a NO donor. This antilipolytic effect of DPI was also prevented by two antioxidants, ascorbate or diethyldithiocarbamic acid (DDC). Preincubation of isolated adipocytes with DPI (30 min) before exposure to DBcAMP almost completely abolished the stimulated lipolysis. Addition of SNAP or antioxidant during DPI preincubation restored the lipolytic response to DBcAMP, whereas no preventive effects were observed when these compounds were added simultaneously to DBcAMP. Exposure of isolated adipocytes to an extracellular generating system of oxygen species (xanthine/xanthine oxidase) or to H2O2 also resulted in an inhibition of the lipolytic response to DBcAMP. H2O2 or DPI decreased cAMP-dependent protein kinase (PKA) activation. The DPI effect on PKA activity was prevented by SNAP, ascorbate, or DDC. These results provide clear evidence that 1) the DPI antilipolytic effect is related to adipocyte NOS inhibition leading to PKA alterations, and 2) endogenous NO is required for the cAMP lipolytic process through antioxidant-related effect.