bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon-γ induces C/EBPβ expression and activity through MEK/ERK and p38 in T84 colon epithelial cells
Authors: Salmenperä Pertteli , Hämäläinen Sammy , Hukkanen Mika , Kankuri Esko
Published: May 2013

We investigated the role of IFN-γ and MAPKs on the expression and activity of the transcription factor CCAAT/enhancer-binding protein-beta (C/EBPβ) in the T84 colon epithelial cell line. IFN-γ induced the expression and activity of C/EBPβ and subsequently increased the secretion of IL-6 from these cells. Treatment with the p38 inhibitor SB-203580, the MEK1 and MEK2 inhibitor U-0126, or the translational inhibitor cycloheximide inhibited the induction of C/EBPβ and IL-6 by IFN-γ, whereas the MEK1 inhibitor PD-98059 or the tyrosine kinase inhibitor genistein had no effect. These results suggest a role for MEK2 and p38 in IFN-γ-mediated signal transduction and induction of C/EBPβ expression and activity associated with interleukin-6 (IL-6) secretion in colon epithelial cells.
NO and TNF-α released from activated macrophages stabilize HIF-1α in resting tubular LLC-PK1 cells
Authors: Zhou Jie , Fandrey Joachim , Schümann Jens , Tiegs Gisa , Brüne Bernhard
Published: May 2013

Hypoxic/ischemic conditions provoke activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1α (subjected to protein stability regulation) and constitutively expressed HIF-1β. Besides hypoxia, diverse agonists are identified that stabilize HIF-1α during normoxia. Here we used a coculture system of RAW 264.7 macrophage cells and tubular LLC-PK1 cells to establish that lipopolysaccharide- and interferon-γ-stimulated but not resting macrophages elicited HIF-1α accumulation in LLC-PK1cells. Via pharmacological interventions such as blockade of nitric oxide (NO) production in macrophages, scavenging of NO with the use of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or application of tumor necrosis factor-α (TNF-α)-neutralizing antibodies, we identified NO and TNF-α as signaling molecules. Working in concert, NO and TNF-α have a stronger response when allowed direct cell-to-cell contact instead of contact with only the cell supernatant of activated macrophages. We show that signal transmission by NO with TNF-α in LLC-PK1 cells is mediated via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, because it is blocked by wortmannin or dominant-negative forms of PI3-K as well as protein kinase B. We conclude that NO and TNF-α, derived from activated macrophages, provoke HIF-1α stabilization in LLC-PK1 cells under normoxic conditions, which underscores HIF-1α stabilization due to intercellular regulation.
Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a
Authors: Grover Ashok K. , Kwan Chiu-Yin , Samson Sue E.
Published: May 2013

Sarco(endo)plasmic reticulum Ca2+ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca2+ uptake, Ca2+-Mg2+-ATPase, Ca2+-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca2+ uptake, Ca2+-Mg2+-ATPase, and Ca2+-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress.
ET-1- and NO-mediated signal transduction pathway in human brain capillary endothelial cells
Authors: Chen Y. , McCarron R. M. , Golech S. , Bembry J. , Ford B. , Lenz F. A. , Azzam N. , Spatz M.
Published: May 2013

Previous studies have demonstrated that functional interaction between endothelin (ET)-1 and nitric oxide (NO) involves changes in Ca2+ mobilization and cytoskeleton in human brain microvascular endothelial cells. The focus of this investigation was to examine the possible existence of analogous interplay between these vasoactive substances and elucidate their signal transduction pathways in human brain capillary endothelial cells. The results indicate that ET-1-stimulated Ca2+mobilization in these cells is dose-dependently inhibited by NOR-1 (an NO donor). This inhibition was prevented by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of protein kinase G). Treatment of endothelial cells with 8-bromo-cGMP reduced ET-1-induced Ca2+ mobilization in a manner similar to that observed with NOR-1 treatment. In addition, NOR-1 or cGMP reduced Ca2+ mobilization induced by mastoparan (an activator of G protein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitor of Ca2+-ATPase). Interestingly, alterations in endothelial cytoskeleton (actin and vimentin) were associated with these effects. The data indicate for the first time that the cGMP-dependent protein kinase colocalizes with actin. These changes were accompanied by altered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 and significantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functional interrelationship between ET-1 and NO plays a role in regulating capillary tone, microcirculation, and blood-brain barrier function.
Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication
Authors: Villars F. , Guillotin B. , Amédée T. , Dutoya S. , Bordenave L. , Bareille R. , Amédée J.
Published: May 2013

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This “juxtacrine signaling” could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18α-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.
KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential
Authors: Marcus Daniel C. , Wu Tao , Wangemann Philine , Kofuji Paulo
Published: May 2013

Stria vascularis of the cochlea generates the endocochlear potential and secretes K+. K+ is the main charge carrier and the endocochlear potential the main driving force for the sensory transduction that leads to hearing. Stria vascularis consists of two barriers, marginal cells that secrete potassium and basal cells that are coupled via gap junctions to intermediate cells. Mice lacking the KCNJ10 (Kir4.1) K+ channel in strial intermediate cells did not generate an endocochlear potential. Endolymph volume and K+ concentration ([K+]) were reduced. These studies establish that the KCNJ10 K+ channel provides the molecular mechanism for generation of the endocochlear potential in concert with other transport pathways that establish the [K+] difference across the channel. KCNJ10 is also a limiting pathway for K+ secretion.
Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells
Authors: Zhang Jing , Lauf Peter K. , Adragna Norma C.
Published: May 2013

Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.
Somatostatin stimulates ductal bile absorption and inhibits ductal bile secretion in mice via SSTR2 on cholangiocytes
Authors: Gong Ai-Yu , Tietz Pamela S. , Muff Melissa A. , Splinter Patrick L. , Huebert Robert C. , Strowski Mathias Z. , Chen Xian-Ming , LaRusso Nicholas F.
Published: May 2013

With an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and somatostatin receptor (SSTR) subtype 2 knockout mice, we tested the effects of somatostatin, secretin, and a selective SSTR2 agonist (L-779976) on fluid movement across the bile duct epithelial cell layer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected in cholangiocytes in wild-type mice. In contrast, SSTR2A/2B were completely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 and SSTR4 were expressed in these cholangiocytes. Somatostatin induced a decrease of luminal area of IBDUs isolated from wild-type mice, reflecting net fluid absorption; L-779976 also induced a comparable decrease of luminal area. No significant decrease of luminal area by either somatostatin or L-779976 was observed in IBDUs from SSTR2 knockout mice. Secretin, a choleretic hormone, induced a significant increase of luminal area of IBDUs of wild-type mice, reflecting net fluid secretion; somatostatin and L-779976 inhibited ( P< 0.01) secretin-induced fluid secretion. The inhibitory effect of both somatostatin and L-779976 on secretin-induced IBDU secretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibited secretin-stimulated cAMP synthesis in cholangiocytes; depletion of SSTR2 blocked these effects of somatostatin. These data suggest that somatostatin regulates ductal bile formation in mice not only by inhibition of ductal fluid secretion but also by stimulation of ductal fluid absorption via interacting with SSTR2 on cholangiocytes, a process involving the intracellular cAMP/cGMP second messengers.
Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes
Authors: Kimball Scot R. , Horetsky Rick L. , Ron David , Jefferson Leonard S. , Harding Heather P.
Published: May 2013

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNAi(Met-tRNAi) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2 · GTP · Met-tRNAi complex is thought to bind to the 40S ribosomal subunit before the eIF4G · eIF4E · mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the α-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes.
Cell swelling-induced ATP release and gadolinium-sensitive channels
Authors: Boudreault Francis , Grygorczyk Ryszard
Published: May 2013

ATP release induced by hypotonic swelling is an ubiquitous phenomenon in eukaryotic cells, but its underlying mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has been implicated because gadolinium (Gd3+), an inhibitor of stretch-activated channels, suppressed ATP efflux monitored by luciferase bioluminescence. We examined the effect of Gd3+on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells. We found that luciferase was inhibited by ≤10 μM Gd3+, and this may have contributed to the previously reported inhibition of ATP release. In ATP efflux experiments, luciferase inhibition could be prevented by chelating Gd3+with EGTA before luminometric ATP determinations. Using this approach, we found that 10–100 μM Gd3+, i.e., concentrations typically used to block MS channels, actually stimulated hypotonically induced ATP release from fibroblasts. Inhibition of ATP release required at least 500, 200, or 100 μM Gd3+ for fibroblasts, A549 cells, and 16HBE14o− cells, respectively. Such biphasic and cell-specific effects of Gd3+ are most consistent with its action on membrane lipids and membrane-dependent processes such as exocytosis.
ECM is required for skeletal muscle differentiation independently of muscle regulatory factor expression
Authors: Osses Nelson , Brandan Enrique
Published: May 2013

Transcription of specific skeletal muscle genes requires the expression of the muscle regulatory factor myogenin. To assess the role of the extracellular matrix (ECM) in skeletal muscle differentiation, the specific inhibitors of proteoglycan synthesis, sodium chlorate and β-d-xyloside, were used. Treatment of cultured skeletal muscle cells with each inhibitor substantially abolished the expression of creatine kinase and α-dystroglycan. This inhibition was totally reversed by the addition of exogenous ECM. Myoblast treatment with each inhibitor affected the deposition and assembly of the ECM constituents glypican, fibronectin, and laminin. These treatments did not affect MyoD, MEF2A, and myogenin expression and nuclear localization. Differentiated myoblast treatment with RGDS peptides completely inhibited myogenesis without affecting the expression or nuclear localization of myogenin. Integrin-mediated signaling of focal adhesion kinase was partially inhibited by chlorate and β-d-xyloside, an effect reversed by the addition of exogenous ECM gel. These results suggested that the expression of myogenin is not sufficient to successfully drive skeletal muscle formation and that ECM is required to complete the skeletal muscle differentiation process.
Injury-elicited differential transcriptional regulation of phospholipid growth factor receptors in the cornea
Authors: Wang De-An , Du Haiming , Jaggar Jonathan H. , Brindley David N. , Tigyi Gabor J. , Watsky Mitchell A.
Published: May 2013

The phospholipid growth factors (PLGFs), including lysophosphatidic acid (LPA), have been implicated in corneal wound healing. PLGF concentrations and activities are elevated after corneal injury. Using real-time PCR, we quantified receptor mRNA levels in the healing rabbit cornea. In intact corneas, transcripts for S1P1, LPA1, and LPA3 receptor subtypes were detected, as was lipid phosphate phosphatase 1 (LPP1). After wounding, the trend for endothelium and keratocytes was for significant decreases in transcript numbers for the three receptor subtypes, whereas epithelial cells showed increased transcript numbers, except for an S1P1 decrease in healing cells. LPP1 transcript numbers were decreased in keratocytes and endothelium, although LPP-specific activity was unchanged. LPA-elicited Ca2+ transients were significantly reduced in the healing endothelium. Consistent with reduced LPA3 receptor numbers, dioctylglycerol pyrophosphate, a selective antagonist, reduced LPA-induced Ca2+ transients 2.7-fold in nonwounded epithelium but only 1.5-fold in wound-healing endothelium. These data for the first time establish physiologically relevant differential changes in the expression of PLGF receptor subtypes and provide evidence for the changing role of LPA3 receptors in endothelial cells.
Altered muscle force and stiffness of skeletal muscles in α-sarcoglycan-deficient mice
Authors: Patel Nisha D. , Jannapureddy Suneal R. , Hwang Willy , Chaudhry Imran , Boriek Aladin M.
Published: May 2013

α-Sarcoglycan (ASG) is a transmembrane protein of the dystrophin-associated complex, and absence of ASG causes limb-girdle muscular dystrophy. We hypothesize that disruption of the sarcoglycan complex may alter muscle extensibility and disrupt the coupling between passive transverse and axial contractile elements in the diaphragm. We determined the length-tension relationships of the diaphragm of young ASG-deficient mice and their controls during uniaxial and biaxial loading. We also determined the isometric contractile properties of the diaphragm muscles from mutant and normal mice in the absence and presence of passive transverse stress. We found that the diaphragm muscles of the null mutants for the protein ASG show 1) significant decrease in muscle extensibility in the directions of the muscle fibers and transverse to fibers, 2) significant reductions in force-generating capacity, and 3) significant reductions in coupling between longitudinal and transverse properties. Thus these findings suggest that the sarcoglycan complex serves a mechanical function in the diaphragm by contributing to muscle passive stiffness and to the modulation of the contractile properties of the muscle.
Topographic requirements and dynamics of signaling via L-selectin on neutrophils
Authors: Green Chad E. , Pearson David N. , Christensen Nadine B. , Simon Scott I.
Published: May 2013

Cross-linking of L-selectin on leukocytes signals phosphorylation of mitogen-activated protein kinases (MAPKs) leading to activation of CD18 function and enhanced transmigration on inflamed endothelium. We examined how alterations in the topography of L-selectin correlate with the dynamics of CD18 activation and phosphorylation of MAPK. Simultaneous ligation of humanized antibodies DREG55 and DREG200 provided a strategy for regulating the extent of cross-linking. Triggering of CD11b/CD18 upregulation and adhesion required clustering of L-selectin to microvillus-sized patches of ∼0.2 μm2. Immunofluorescence revealed that L-selectin was colocalized with high-affinity CD18. Anti-L-selectin-coated protein A microspheres indicated that a single site of contact to a 5.5-μm bead, or multiple contacts to 0.94- or 0.3-μm beads, elicited maximum neutrophil activation. Adhesion signaled via L-selectin coincided with the kinetics of MAPK phosphorylation and was inhibited by blocking p38 or p42/44 activity. These data demonstrate the capacity of L-selectin to transduce signals effecting rapid (∼1 s) neutrophil adhesion that is regulated by the size and frequency of receptor clustering.
220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns
Authors: Blue Emily K. , Goeckeler Zoe M. , Jin Yijun , Hou Ling , Dixon Shelley A. , Herring B. Paul , Wysolmerski Robert B. , Gallagher Patricia J.
Published: May 2013

To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.
High-temporal-resolution analysis demonstrates that ICAM-1 stabilizes WEHI 274.1 monocytic cell rolling on endothelium
Authors: Kevil Christopher G. , Chidlow John H. , Bullard Daniel C. , Kucik Dennis F.
Published: May 2013

Leukocyte rolling, adhesion, and migration on vascular endothelium involve several sets of adhesion molecules that interact simultaneously. Each of these receptor-ligand pairs may play multiple roles. We examined the role of ICAM-1 in adhesive interactions with mouse aortic endothelial cells (MAECs) in an in vitro flow system. Average rolling velocity of the monocytic cell line WEHI 274.1 was increased on ICAM-1-deficient MAECs compared with wild-type MAECs, both with and without TNF-α stimulation. High-temporal-resolution analysis provided insights into the underlying basis for these differences. Without TNF-α stimulation, average rolling velocity was slower on wild-type than on ICAM-1-deficient endothelium because of brief (<1 s) pauses. On TNF-α-stimulated ICAM-1-deficient endothelium, cells rolled faster because of transient accelerations, producing “jerky” rolling. Firm adhesion to ICAM-1-deficient MAECs was significantly reduced compared with wild-type MAECs, although the number of rolling cells was similar. These results demonstrate directly that ICAM-1 affects rolling velocity by stabilizing leukocyte rolling.
Measurement of stretch-induced loss of alveolar epithelial barrier integrity with a novel in vitro method
Authors: Cavanaugh Kenneth J. , Margulies Susan S.
Published: May 2013

Mechanical ventilation with high tidal volumes has been shown to contribute to the formation or worsening of interstitial and alveolar edema. Previously we showed that application of large biaxial deformations in vitro perturbs the concentration and distribution of functional tight junction proteins in alveolar epithelial cells. Using a novel method, we determined that applied epithelial strain increases paracellular permeability in a dose- and rate-dependent manner. Primary rat alveolar epithelial cells were subjected to 12%, 25%, or 37% change in surface area (ΔSA) cyclic equibiaxial stretch for 1 h. Cells were also stretched noncyclically at 25% ΔSA for 1 h. During the experimental period, a fluorescently tagged ouabain derivative was added to the apical fluid. Evidence of binding indicated functional failure of the paracellular transport barrier. The percentage of field area stained was quantified from microscopic images. There was no significant evidence of basolateral fluorescent staining at 12% ΔSA or at 25% ΔSA applied cyclically or statically. However, cyclic stretch at 37% ΔSA resulted in significantly more staining than in unstretched cells ( P < 0.0001) or those stretched at either 12% ( P < 0.0001) or 25% cyclic ( P < 0.0005) or static ( P < 0.05) ΔSA. These results suggest that large cyclic tidal volumes may increase paracellular permeability, potentially resulting in alveolar flooding.
Hyperglycemia increases endothelial superoxide that impairs smooth muscle cell Na+-K+-ATPase activity
Authors: Gupta Sandeep , Chough Eugene , Daley Jennifer , Oates Peter , Tornheim Keith , Ruderman Neil B. , Keaney John F.
Published: May 2013

Nitric oxide (NO) plays an important role in the control of numerous vascular functions including basal Na+-K+-ATPase activity in arterial tissue. Hyperglycemia inhibits Na+-K+-ATPase activity in rabbit aorta, in part, through diminished bioactivity of NO. The precise mechanism(s) for such observations, however, are not yet clear. The purpose of this study was to examine the role of superoxide in modulating NO-mediated control of Na+-K+-ATPase in response to hyperglycemia. Rabbit aorta incubated with hyperglycemic glucose concentrations (44 mM) demonstrated a 50% reduction in Na+-K+-ATPase activity that was abrogated by superoxide dismutase. Hyperglycemia also produced a 50% increase in steady-state vascular superoxide measured by lucigenin-enhanced chemiluminescence that was closely associated with reduced Na+-K+-ATPase activity. Specifically, the hyperglycemia-induced increase in vascular superoxide was endothelium dependent, inhibited by l-arginine, and stimulated by N ω-nitro-l-arginine. Aldose reductase inhibition with zopolrestat also inhibited the hyperglycemia-induced increase in vascular superoxide. In each manipulation of vascular superoxide, a reciprocal change in Na+-K+-ATPase activity was observed. Finally, a commercially available preparation of Na+-K+-ATPase was inhibited by pyrogallol, a superoxide generator. These data suggest that hyperglycemia induces an increase in endothelial superoxide that inhibits the stimulatory effect of NO on vascular Na+-K+-ATPase activity.
Epidermal growth factor upregulates β-adrenergic receptor signaling in a human salivary cell line
Authors: Yeh Chih-Ko , Hymer Tazuko K. , Sousa April L. , Zhang Bin-Xian , Lifschitz Meyer D. , Katz Michael S.
Published: May 2013

The effects of epidermal growth factor (EGF) on the β-adrenergic receptor-coupled adenylyl cyclase system were studied in a human salivary cell line (HSY). The β-adrenergic agonist isoproterenol (10−5 M) stimulated adenylyl cyclase activity by ∼2-fold, and the isoproterenol response was increased 1.8-fold after prolonged (48 h) exposure to EGF (5 × 10−10 M). In contrast, enzyme activation via stimulatory prostaglandin receptors and by agents acting on nonreceptor components of the adenylyl cyclase system was not enhanced by EGF. β-Adrenergic receptor density, assessed by binding of the β-adrenergic receptor antagonist (−)-[125I]iodopindolol, was increased threefold after EGF treatment. Competition binding studies with unlabeled antagonists selective for β1- and β2-adrenergic receptor subtypes indicated that the increase in (−)-[125I]iodopindolol binding sites induced by EGF reflected an increased number of β2-adrenergic receptors. Likewise, Northern blot analysis of RNA from EGF-treated cells revealed selective induction of β2-adrenergic receptor mRNA, which was blocked by the RNA synthesis inhibitor actinomycin D. The increase in β-adrenergic receptor density produced by EGF was unaltered after phorbol ester-induced downregulation of protein kinase C (PKC). Enhancement of isoproterenol-responsive adenylyl cyclase activity and phosphorylation of mitogen-activated protein kinase (MAPK) by EGF were both blocked by the MAPK pathway inhibitor PD-98059. The results suggest that in HSY cells EGF enhances β-adrenergic responsiveness by upregulating β2-adrenergic receptor expression at the transcriptional level. Moreover, the stimulatory effect of EGF on β2-adrenergic receptor signaling appears to be mediated by the MAPK pathway and independent of PKC activation.
Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K+ and Ca2+channels
Authors: Filosa Jessica A. , Putnam Robert W.
Published: May 2013

We studied chemosensitive signaling in locus coeruleus (LC) neurons using both perforated and whole cell patch techniques. Upon inhibition of fast Na+ spikes by tetrodotoxin (TTX), hypercapnic acidosis [HA; 15% CO2, extracellular pH (pHo) 6.8] induced small, slow spikes. These spikes were inhibited by Co2+ or nifedipine and were attributed to activation of L-type Ca2+ channels by HA. Upon inhibition of both Na+ and Ca2+ spikes, HA resulted in a membrane depolarization of 3.52 ± 0.61 mV ( n = 17) that was reduced by tetraethylammonium (TEA) (1.49 ± 0.70 mV, n = 7; P < 0.05) and absent (−0.97 ± 0.73 mV, n = 7; P < 0.001) upon exposure to isohydric hypercapnia (IH; 15% CO2, 77 mM HCO[Formula: see text], pHo 7.45). Either HA or IH, but not 50 mM Na-propionate, activated Ca2+ channels. Inhibition of L-type Ca2+channels by nifedipine reduced HA-induced increased firing rate and eliminated IH-induced increased firing rate. We conclude that chemosensitive signals (e.g., HA or IH) have multiple targets in LC neurons, including TEA-sensitive K+ channels and TWIK-related acid-sensitive K+ (TASK) channels. Furthermore, HA and IH activate L-type Ca2+ channels, and this activation is part of chemosensitive signaling in LC neurons.
Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1
Authors: Sacchi V. Franca , Castagna Michela , Mari Stefania A. , Perego Carla , Bossi Elena , Peres Antonio
Published: May 2013

KAAT1 is a neutral amino acid transporter activated by K+ or by Na+ ( 9 ). The protein shows significant homology with members of the Na+/Cl–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of Vmax and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na+ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1.
Phosphorylation of the salivary Na+-K+-2Cl− cotransporter
Authors: Kurihara Kinji , Nakanishi Nobuo , Moore-Hoon Marilyn L. , Turner R. James
Published: May 2013

We studied the phosphorylation of the secretory Na+-K+-2Cl− cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to β-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by β-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.
A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis
Authors: Kelly K. J. , Sandoval Ruben M. , Dunn Kenneth W. , Molitoris Bruce A. , Dagher Pierre C.
Published: May 2013

Apoptosis is an important mode of cell death under both physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.
Robust induction of PGHS-2 by IL-1 in orbital fibroblasts results from low levels of IL-1 receptor antagonist expression
Authors: Cao H. James , Han Rui , Smith Terry J.
Published: May 2013

Human orbital fibroblasts are more susceptible to some actions of proinflammatory cytokines than are fibroblasts from other anatomic regions. These cells produce high levels of PGE2 when activated by cytokines. Here we report that they express high levels of prostaglandin-endoperoxide H synthase (PGHS)-2, the inflammatory cyclooxygenase, when treated with IL-1β. This induction results from enhanced PGHS-2 mRNA stability and small increases in gene promoter activity. The enhanced transcript stability is a result of actions of the cytokine on the 3′-untranslated region. Orbital fibroblasts, unlike those from skin, fail to express high levels of IL-1 receptor antagonist (IL-1ra) when treated with IL-1β, leading to loss of modulation of IL-1 action. This can be overcome by transiently transfecting cells with IL-1ra. Thus a decreased level of IL-1ra expression in orbital fibroblasts may underlie the exaggerated responses to IL-1 observed in those cells and, therefore, the susceptibility of the orbit to inflammation.
Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells
Authors: Rajasekaran Sigrid A. , Hu Jane , Gopal Jegan , Gallemore Ron , Ryazantsev Sergey , Bok Dean , Rajasekaran Ayyappan K.
Published: May 2013

Na,K-ATPase regulates a variety of transport functions in epithelial cells. In cultures of human retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPase by ouabain and K+ depletion decreased transepithelial electrical resistance (TER) and increased permeability of tight junctions to mannitol and inulin. Electrophysiological studies demonstrated that the decrease in TER was due to an increase in paracellular shunt conductance. At the light microscopy level, this increased permeability was not accompanied by changes in the localization of the tight junction proteins ZO-1, occludin, and claudin-3. At the ultrastructural level, increased tight junction permeability correlated with a decrease in tight junction membrane contact points. Decreased tight junction membrane contact points and increased tight junction permeability were reversible in K+-repletion experiments. Confocal microscopy revealed that in control cells, Na,K-ATPase was localized at both apical and basolateral plasma membranes. K+ depletion resulted in a large reduction of apical Na,K-ATPase, and after K+repletion the apical Na,K-ATPase recovered to control levels. These results suggest a functional link exists between Na,K-ATPase and tight junction function in human RPE cells.
PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo
Authors: Kuo Shiuhyang , Chokas Ann L. , Rogers Richard J. , Nick Harry S.
Published: May 2013

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.
Cyclooxygenase 2-mediated suppression of macrophage interleukin-12 production after thermal injury
Authors: Schwacha Martin G. , Chung Chun-Shiang , Ayala Alfred , Bland Kirby I. , Chaudry Irshad H.
Published: May 2013

Macrophage (Mφ) prostaglandin (PG)E2 production has been implicated in immunosuppression and increased susceptibility to sepsis after thermal injury. Deficient interleukin (IL)-12 production has also been implicated in these postburn complications. The present study examined the relationship between Mφ cyclooxygenase (COX)-2 activity and IL-12 production after thermal injury. C57BL/6 female mice were subjected to a 25% total body surface area full-thickness burn. Mφ were isolated 7 days later, or the mice were subjected to sepsis by cecal ligation and puncture (CLP). IL-12 production by Mφ from injured mice was suppressed by >50%, whereas COX-2 expression and PGE2production were increased twofold. The COX-2 inhibitor NS-398 suppressed PGE2 production and normalized IL-12 production in the injury group, whereas it had no effect on IL-10 production. Injured mice subjected to CLP had lower IL-12 plasma levels compared with sham-treated mice subjected to CLP. NS-398 treatment prevented the suppression in plasma IL-12 levels in the injury group. Thus elevated Mφ COX-2 activity, independent of IL-10, suppresses Mφ IL-12 production after thermal injury and may play an important role in the observed immunosuppression under such conditions.
Stability of actin cytoskeleton and PKC-δ binding to actin regulate NKCC1 function in airway epithelial cells
Authors: Liedtke Carole M. , Hubbard Melinda , Wang Xiangyun
Published: May 2013

Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-δ, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-δ were verified by Western blot analysis of immunoprecipitated proteins. PKC-δ was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-δ. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-δ. Binding of recombinant PKC-δ to NKCC1 was not detected in overlay assays. Rather, activated PKC-δ bound to actin, and this interaction was prevented by a peptide encoding δC2, a C2-like domain based on the amino acid sequence of PKC-δ. δC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-δ in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-δ indicated colocalization of PKC-δ and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-δ.
Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule
Authors: Blumenthal Edward M.
Published: May 2013

The Malpighian (renal) tubule of Drosophila melanogaster is a useful model for studying epithelial transport. The purpose of this study was to identify factors responsible for modulating transepithelial chloride conductance in isolated tubules. I have found that tyrosine and several of its metabolites cause an increase in chloride conductance. The most potent of these agonists is tyramine, which is active at low nanomolar concentrations; the pharmacology of this response matches that of the previously published cloned insect tyramine receptor. In addition, the tubule appears capable of synthesizing tyramine from applied tyrosine, as shown by direct measurement of tyrosine decarboxylase activity. Immunohistochemical staining of tubules with an antibody against tyramine indicates that the principal cells are the sites of tyramine production, whereas previous characterization of the regulation of chloride conductance suggests that tyramine acts on the stellate cells. This is the first demonstration of a physiological role for an insect tyramine receptor.
Regional distribution of the Na+ and K+currents around the crystalline lens of rabbit
Authors: Candia Oscar A. , Zamudio Aldo C.
Published: May 2013

Early studies described asymmetrical electrical properties across the ocular lens in the anterior-to-posterior direction. More recent results obtained with a vibrating probe indicated that currents around the lens surface are not uniform by showing an outwardly directed K+ efflux at the lens equator and Na+ influx at the poles. The latter studies have been used to support theoretical models for fluid recirculation within the avascular lens. However, the existence of a nonuniform current distribution in the lens epithelium from the anterior pole to the equator has never been confirmed. The present work developed a modified short-circuiting technique to examine the net flows of Na+ and K+ across arbitrarily defined lens surface regions. Results indicate that passive inflows of Na+ occur at both the anterior polar region and posterior lens surface, consistent with suggestions derived from the vibrating probe data, whereas K+ efflux plus the Na+-K+ pump-generated current comprise the currents at the equatorial surface and an area anterior to it. Furthermore, Na+-K+ pump activity was absent at the posterior surface and its polar region in all lenses examined, as well as from the anterior polar region in most lenses. The latter unexpected observation suggests that the monolayered epithelium, which is confined to the anterior surface of the lens, does not express an active Na+-K+ pump at its anterior-most aspect. Nevertheless, this report represents the first independent confirmation that positive currents leave the lens around the equator and reenter across the polar and posterior surfaces.
Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells
Authors: Clarke Alison L. , Petrou Steven , Walsh John V. , Singer Joshua J.
Published: May 2013

Fatty acids and other negatively charged single-chain lipids increase large-conductance Ca2+-activated K+(BKCa) channel activity, whereas sphingosine and other positively charged single-chain lipids suppress activity. Because these molecules are effective on both inside-out and outside-out patches and because they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids on this channel, we used two compounds that are unlikely to flip across the lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify the site of action of negatively charged lipids, and a positively charged myristoylated pentapeptide (myr-KPRPK) was used to investigate the site of action of positively charged lipids. The effect of these compounds on channel activity was studied in excised patches using patch-clamp techniques. In “normal” ionic strength solutions and in experiments where high-ionic strength solutions were used to shield membrane surface charge, PCoA increased channel activity only when applied to outside-out patches, suggesting that the site of action of negatively charged lipids is located on the outer surface of the membrane. A decrease in activity, similar to that of other positively charged lipids, was observed only when myr-KPRPK was applied to outside-out patches, suggesting that positively charged lipids suppress activity by also acting on the outer membrane surface. Some channel blockade effects of myr-KPRPK and KPRPK are also described. The sidedness of action suggests that modulation of channel activity by single-chain lipids can occur by their interaction with the channel protein.