bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional significance of human trp1 and trp3 in store-operated Ca2+ entry in HEK-293 cells
Authors: Wu Xiaoyan , Babnigg György , Villereal Mitchel L.
Published: December 2017

The Drosophila trp (transient receptor potential) gene appears to encode the Drosophilastore-operated channel (SOC), and some mammalian trp homologues have been proposed to encode mammalian SOCs. This study provides evidence for the expression of three trp homologues (Mtrp2, Mtrp3, and Mtrp4) in fibroblasts from wild-type and src knockout mice, and four trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) in human embryonic kidney (HEK-293) cells based on RT-PCR techniques. In HEK-293 cells stably transfected with a 323-bp Htrp3 antisense construct (Htrp3AS), Northern blot analysis revealed that the expression of a 4-kb transcript was dramatically suppressed in comparison to that observed in cells stably transfected with a short Htrp3 sense construct (Htrp3S). Activity of SOCs, monitored as Ba2+ influx following Ca2+ store depletion with thapsigargin, was reduced by 32% in Htrp3AS cells in comparison with Htrp3S cells. Transient transfection of a 369-bp Htrp1 antisense construct in cells stably expressing Htrp3AS induced a higher level of inhibition (55%) of store-operated Ca2+ entry. These data suggest that Htrp1 and Htrp3 may be functional subunits of SOCs.
Voltage-dependent stimulation of the Na+-K+ pump by insulin in rabbit cardiac myocytes
Authors: Hansen Peter S. , Buhagiar Kerrie A. , Gray David F. , Rasmussen Helge H.
Published: December 2017

Insulin enhances Na+-K+ pump activity in various noncardiac tissues. We examined whether insulin exposure in vitro regulates Na+-K+ pump function in rabbit ventricular myocytes. Pump current ( I p) was measured using the whole-cell patch-clamp technique at test potentials ( V ms) from −100 to +60 mV. When the Na+ concentration in the patch pipette ([Na]pip) was 10 mM, insulin caused a V m-dependent increase in I p. The increase was ∼70% when V m was at near physiological diastolic potentials. This effect persisted after elimination of extracellular voltage-dependent steps and when K+ and K+-congeners were excluded from the patch pipettes. When [Na]pip was 80 mM, causing near-maximal pump stimulation, insulin had no effect, suggesting that it did not cause an increase in membrane pump density. Effects of tyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I in pipette solutions suggested that the insulin-induced increase in I p involved activation of tyrosine kinase, phosphatidylinositol 3-kinase, and protein phosphatase 1, whereas protein phosphatase 2A and protein kinase C were not involved.
EGF stimulates gastrin promoter through activation of Sp1 kinase activity
Authors: Chupreta Sergey , Du Ming , Todisco Andrea , Merchant Juanita L.
Published: December 2017

Epidermal growth factor (EGF) receptor activation stimulates gastrin gene expression through a GC-rich element called gastrin EGF response element (gERE). This element is bound by Sp1 family members and is a target of the ras-extracellular signal-regulated kinase (Erk) signal transduction cascade. This raised the possibility that Sp1 may be phosphorylated by kinases of this signaling pathway. Erk is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk and Sap, suggesting that Erk may also mediate EGF-dependent phosphorylation of Sp1. This possibility was tested by studying Sp1-dependent kinase activity in extracts prepared from EGF-activated AGS cells by use of solid-phase kinase assays and immunoprecipitation of metabolically labeled Sp1. The results revealed that Sp1 kinase activity (like gastrin promoter activation) is inhibited by PD-98059 and, therefore, is dependent on mitogen-activated protein kinase kinase 1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not account for most of the Sp1 kinase activity, since Erk and additional Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from an ion-exchange column in different fractions. Phosphoamino acid analysis of in vivo radiolabeled Sp1 demonstrated that the kinase phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk.
LabPatch, an acquisition and analysis program for patch-clamp electrophysiology
Authors: Robinson Tim , Thomsen Lars , Huizinga Jan D.
Published: December 2017

An acquisition and analysis program, “LabPatch,” has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/ .
Hypertonic perfusion inhibits intracellular Na and Ca accumulation in hypoxic myocardium
Authors: Ho Hung S. , Liu Hong , Cala Peter M. , Anderson Steven E.
Published: December 2017

Much evidence supports the view that hypoxic/ischemic injury is largely due to increased intracellular Ca concentration ([Ca]i) resulting from 1) decreased intracellular pH (pHi), 2) stimulated Na/H exchange that increases Na uptake and thus intracellular Na (Nai), and 3) decreased Na gradient that decreases or reverses net Ca transport via Na/Ca exchange. The Na/H exchanger (NHE) is also stimulated by hypertonic solutions; however, hypertonic media may inhibit NHE's response to changes in pHi (Cala PM and Maldonado HM. J Gen Physiol 103: 1035–1054, 1994). Thus we tested the hypothesis that hypertonic perfusion attenuates acid-induced increases in Nai in myocardium and, thereby, decreases Cai accumulation during hypoxia. Rabbit hearts were Langendorff perfused with HEPES-buffered Krebs-Henseleit solution equilibrated with 100% O2 or 100% N2. Hypertonic perfusion began 5 min before hypoxia or normoxic acidification (NH4Cl washout). Nai, [Ca]i, pHi, and high-energy phosphates were measured by NMR. Control solutions were 295 mosM, and hypertonic solutions were adjusted to 305, 325, or 345 mosM by addition of NaCl or sucrose. During 60 min of hypoxia (295 mosM), Nai rose from 22 ± 1 to 100 ± 10 meq/kg dry wt while [Ca]i rose from 347 ± 11 to 1,306 ± 89 nM. During hypertonic hypoxic perfusion (325 mosM), increases in Nai and [Ca]i were reduced by 65 and 60%, respectively ( P < 0.05). Hypertonic perfusion also diminished Na uptake after normoxic acidification by 87% ( P < 0.05). The data are consistent with the hypothesis that mild hypertonic perfusion diminishes acid-induced Na accumulation and, thereby, decreases Na/Ca exchange-mediated Caiaccumulation during hypoxia.
Viscosity and solute dependence of F-actin translocation by rabbit skeletal heavy meromyosin
Authors: Chase P. Bryant , Chen Ying , Kulin Kristi L. , Daniel Thomas L.
Published: December 2017

We tested the hypothesis that solvent viscosity affects translocation of rhodamine phalloidin-labeled F-actin by rabbit skeletal heavy meromyosin (HMM). When viscosity was increased using either glycerol, fructose, sucrose, or dextran (1.5, 6.0, or 15–20 kDa mol mass), there was little or no effect on the fraction of moving filaments, whereas sliding speed decreased in inverse proportion to viscosity. The results could be explained neither by an effect of osmotic pressure at high solute concentrations nor by altered solvent drag on the actin filament. Elevated viscosity inhibited HMM ATPase activity in solution, but only at much higher viscosities than were needed to reduce sliding speed. Polyethylene glycols (300, 1,000, or 3,000 mol wt) also inhibited speed via elevated viscosity but secondarily inhibited by enhancing electrostatic interactions. These results demonstrate that a diffusion-controlled process intrinsic to cross-bridge cycling can be limiting to actomyosin function.
The cGMP-dependent protein kinase stimulates the basolateral 18-pS K channel of the rat CCD
Authors: Wang Wen Hui
Published: December 2017

We used the patch-clamp technique to study the effect of cGMP on the 18-pS K channel in the basolateral membrane of the rat cortical collecting duct. Addition of 100 μM 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased the activity of the 18-pS K channel, defined by NP o, by 95%. In contrast, applying 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) has no effect on channel activity. The effect of 8-Br-cGMP was observed only in cell-attached but not in inside-out patches. Application of 1 μM KT-5823, an inhibitor of the cGMP-dependent protein kinase (PKG), not only reduced the channel activity, but also completely abolished the stimulatory effect of 8-Br-cGMP, suggesting that the 18-pS K channel is not a cGMP-gated K channel. Addition of H-89, an agent that also blocks the PKG, mimicked the effect of KT-5823. To examine the possibility that the effect of 8-Br-cGMP is the result of inhibiting cGMP-dependent phosphodiesterase (PDE) and, accordingly, increasing cAMP or cGMP levels, we explored the effect on the 18-pS K channel of IBMX, an agent that inhibits the PDE. The addition of 100 μM IBMX had no significant effect on channel activity in cell-attached patches. Moreover, in the presence of IBMX, 8-Br-cGMP increased the channel activity to the same extent as that observed in the absence of IBMX, suggesting that the effect of cGMP is not mediated by inhibiting the cGMP-dependent PDE. That the effect of cGMP is mediated by stimulating PKG was further indicated by experiments in which application of exogenous PKG restored the channel activity when it decreased after the excision of the patches. In contrast, adding exogenous cAMP-dependent protein kinase catalytic subunit failed to reactivate the run-down channels. We conclude that cGMP stimulates the 18-pS channel, and the effect of cGMP is mediated by PKG.
Sucrose-stimulated subsecond transient increase in cGMP level in rat intact circumvallate taste bud cells
Authors: Krizhanovsky Valery , Agamy Orly , Naim Michael
Published: December 2017

Initial sweet taste transduction is expected to occur in the subsecond time range. We demonstrate a rapid and transient (75–250 ms) increase of cGMP (but not cAMP) level in rat intact circumvallate taste cells after stimulation by sucrose. This rapid increase does not occur in nonsensory epithelial cells. Pretreatment with a nonspecific phosphodiesterase (PDE) inhibitor (IBMX), a specific cAMP-PDE4 inhibitor (denbufylline), or an adenylyl cyclase activator (forskolin) all increased basal cAMP and abolished the sucrose-stimulated cGMP increase at 150 ms. Pretreatment with a soluble guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) reduced, whereas a specific cGMP-PDE inhibitor (zaprinast) abolished, the sucrose-stimulated cGMP increase. It is proposed that cGMP is involved in the initial stage of sugar taste transduction and that cGMP is more significant than cAMP at this stage. Activation of soluble guanylyl cyclase and inhibition of cGMP-PDE may be involved in the transient elevation of cGMP in response to sucrose stimulation. Moreover, it appears that cAMP level must remain low for sucrose to stimulate an increase in cGMP.
Ca2+-dependent activation of c-jun NH2-terminal kinase in primary rabbit proximal tubule epithelial cells
Authors: Cui Xiao-Lan , Jin Wen-Wu , Ding Ya-Xian , Alexander Larry D. , Hopfer Ulrich , Douglas Janice G.
Published: December 2017

Previous work from this laboratory demonstrated that arachidonic acid activates c- junNH2-terminal kinase (JNK) through oxidative intermediates in a Ca2+-independent manner (Cui X and Douglas JG. Arachidonic acid activates c- jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci USA 94: 3771–3776, 1997.). We now report that JNK can also be activated via a Ca2+-dependent mechanism by agents that increase the cytosolic Ca2+ concentration (Ca2+ ionophore A23187, Ca2+-ATPase inhibitor thapsigargin) or deplete intracellular Ca2+stores [intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite a decrease in cytosolic Ca2+ concentration as detected by the indicator dye fura 2, but appears to be related to Ca2+metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca2+, but also the potency for JNK activation. BAPTA-AM stimulates Ca2+ influx across the plasma membrane, and the resulting local Ca2+ increases are probably involved in activation of JNK because Ca2+ influx inhibitors (SKF-96365, nifedipine) and lowering of the free extracellular Ca2+ concentration with EGTA reduce the BAPTA-induced JNK activation.
Characterization of outward K+currents in isolated smooth muscle cells from sheep urethra
Authors: Hollywood M. A. , McCloskey K. D. , McHale N. G. , Thornbury K. D.
Published: December 2017

The perforated-patch technique was used to measure membrane currents in smooth muscle cells from sheep urethra. Depolarizing pulses evoked large transient outward currents and several components of sustained current. The transient current and a component of sustained current were blocked by iberiotoxin, penitrem A, and nifedipine but were unaffected by apamin or 4-aminopyridine, suggesting that they were mediated by large-conductance Ca2+-activated K+ (BK) channels. When the BK current was blocked by exposure to penitrem A (100 nM) and Ca2+-free bath solution, there remained a voltage-sensitive K+ current that was moderately sensitive to blockade with tetraethylammonium (TEA; half-maximal effective dose = 3.0 ± 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increased the spike amplitude and plateau potential in slow waves evoked in single cells, whereas addition of TEA (10 mM) further increased the plateau potential and duration. In conclusion, both Ca2+-activated and voltage-dependent K+currents were found in urethral myocytes. Both of these currents are capable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.
Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes
Authors: Adrie Christophe , Richter Christoph , Bachelet Maria , Banzet Nathalie , François Dominique , Dinh-Xuan A. Tuan , Dhainaut Jean François , Polla Barbara S. , Richard Marie-Jeanne
Published: December 2017

The free radicals nitric oxide (·NO) and superoxide (O2 −·) react to form peroxynitrite (ONOO−), a highly toxic oxidant species. In this study we investigated the respective effects of NO and ONOO− in monocytes from healthy human donors. Purified monocytes were incubated for 6 or 16 h with a pure NO donor ( S-nitroso- N-acetyl-dl-penicillamine, 0–2 mM), an ·NO/ONOO− donor (3-morpholinosydnonimine chlorhydrate, 0–2 mM) with and without superoxide dismutase (200 IU/ml), or pure ONOO−. We provide evidence that 3-morpholinosydnonimine chlorhydrate alone represents a strong stress to human monocytes leading to a dose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis and necrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO−, but not ·NO, was responsible for the observed effects. This observation was further strengthened by the absence of a stress response in cells exposed to S-nitroso- N-acetyl-dl-penicillamine. Conversely, exposure of cells to ONOO− alone also induced mitochondrial membrane depolarization and cell death by apoptosis and necrosis. Thus ONOO− formation may well explain the toxic effect generally attributed to ·NO.
Negative feedback regulation of activated macrophages via Fas-mediated apoptosis
Authors: Niinobu Tadaaki , Fukuo Keisuke , Yasuda Osamu , Tsubakimoto Maki , Mogi Masaki , Nishimaki Hiroyuki , Morimoto Shigeto , Ogihara Toshio
Published: December 2017

Apoptosis is a critical event for eliminating activated macrophages. Here we show that Fas-mediated apoptosis may participate in the mechanism of negative feedback regulation of activated macrophages. Cytokine-activated macrophages released high levels of nitric oxide (NO) that induced apoptosis in macrophages themselves. This NO-induced macrophage apoptosis was inhibited by a Fas-Fc chimeric molecule that binds to Fas ligand (FasL) and prevents its interaction with endogenous cell surface Fas. High levels of NO stimulated the release of the soluble form of FasL that was inhibited by a matrix metalloproteinase inhibitor KB-8301. High levels of NO also upregulated the expression of Fas mRNA in macrophages. In addition, macrophages isolated from Fas-lacking mice were resistant to NO-induced apoptosis. Finally, inhibition of apoptosis by a caspase inhibitor augmented peroxide production from activated macrophages. These findings suggest that high levels of NO released from activated macrophages may promote the Fas-mediated macrophage apoptosis that may be a negative feedback mechanism for elimination and the downregulation of activated macrophages in the vessel wall.
EGF inhibits muscarinic receptor-mediated calcium signaling in a human salivary cell line
Authors: Zhang Bin-Xian , Yeh Chih-Ko , Hymer Tazuko K. , Lifschitz Meyer D. , Katz Michael S.
Published: December 2017

The effects of epidermal growth factor (EGF) on intracellular calcium ([Ca2+]i) responses to the muscarinic agonist carbachol were studied in a human salivary cell line (HSY). Carbachol (10−4 M)-stimulated [Ca2+]i mobilization was inhibited by 40% after 48-h treatment with 5 × 10−10 M EGF. EGF also reduced carbachol-induced [Ca2+]i in Ca2+-free medium and Ca2+ influx following repletion of extracellular Ca2+. Under Ca2+-free conditions, thapsigargin, an inhibitor of Ca2+ uptake to internal stores, induced similar [Ca2+]i signals in control and EGF-treated cells, indicating that internal Ca2+ stores were unaffected by EGF; however, in cells exposed to thapsigargin, Ca2+influx following Ca2+ repletion was reduced by EGF. Muscarinic receptor density, assessed by binding of the muscarinic receptor antagonistl-[benzilic-4,4′-3HCN]quinuclidinyl benzilate ([3H]QNB), was decreased by 20% after EGF treatment. Inhibition of the carbachol response by EGF was not altered by phorbol ester-induced downregulation of protein kinase C (PKC) but was enhanced upon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the carbachol response by EGF were both blocked by the MAP kinase pathway inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca2+ release from internal stores and also exerts a direct inhibitory action on Ca2+ influx. A decline in muscarinic receptor density may contribute to EGF inhibition of carbachol responsiveness. The inhibitory effect of EGF is mediated by the MAP kinase pathway and is potentiated by a distinct modulatory cascade involving activation of PKC. EGF may play a physiological role in regulating muscarinic receptor-stimulated salivary secretion.
Expression and regulation of parathyroid hormone-related peptide in normal and malignant melanocytes
Authors: El Abdaimi Khadija , Papavasiliou Vasilios , Goltzman David , Kremer Richard
Published: December 2017

We examined parathyroid hormone-related peptide (PTHrP) production and regulation in both normal human melanocytes and in a human amelanotic melanoma cell line (A375). Northern blot and immunocytochemical analysis demonstrated that both cultured A375 cells and normal human melanocytes express PTHrP, but A375 cells expressed much higher levels of the peptide. PTHrP secretory rate increased at least 10-fold after treatment with 10% fetal bovine serum (100.2 ± 2.8 pmol/106 cells vs. basal <15 pmol/106 cells) in proliferating A375 cells but only twofold in confluent cells. Treatment of A375 cells with increasing concentrations of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] or its low-calcemic analog EB-1089 revealed that EB-1089 was 10-fold more potent than 1,25-(OH)2D3 on inhibition of both cell proliferation and PTHrP expression. Furthermore, inoculation of A375 cells into the mammary fat pad of female severe combined immunodeficiency mice resulted in the development of hypercalcemia and elevated concentrations of plasma immunoreactive PTHrP in the absence of detectable skeletal metastases. Our study, therefore, demonstrates a stepwise increase in PTHrP expression when cells progress from normal to malignant phenotype and suggests that EB-1089 should be further evaluated as a therapeutic agent in human melanoma.
Elevated ceramide is downstream of altered calcium homeostasis in low serum-induced apoptosis
Authors: Jayadev Supriya , Barrett J. Carl , Murphy Elizabeth
Published: December 2017

Two immortalized cell lines, sup (+) and sup (−), derived from mutagenized Syrian hamster embryo cells, were used to study the relationship and temporal order between calcium and ceramide signals during apoptosis. The early preneoplastic cells, termed sup (+), suppress tumorigenicity when hybridized with tumor cells, whereas later-stage sup (−) cells do not. In reduced serum conditions, sup (+) cells cease proliferating and undergo apoptosis; in contrast, sup (−) cells continue slow growth and undergo necrosis. In sup (+) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serum treatment of sup (+) cells but are not found in sup (−) cells. Inhibiting ER calcium depletion in low serum-treated sup (+) cells by treating with high levels of calcium prevents both ceramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup (−) cells by treating with low serum plus thapsigargin results in elevated ceramide levels and apoptosis. Furthermore, C6-ceramide treatment induced apoptosis of sup (−) cells in low serum, a condition that does not normally cause apoptosis. C6-ceramide treatment did not induce apoptosis in either sup (+) or sup (−) cells in 10% serum but did cause G2/M arrest. These studies show that ceramide production is downstream of ER calcium release.
Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation
Authors: Liang Tony W. , DeMarco Richard A. , Mrsny Randy J. , Gurney Austin , Gray Alane , Hooley Jeffery , Aaron Holly L. , Huang Arthur , Klassen Toni , Tumas Daniel B. , Fong Sherman
Published: December 2017

Cell-cell interactions of the mucosal epithelia are important for the maintenance and establishment of epithelial barrier function. During events of inflammation, such cell-cell interactions are often disrupted, resulting in a leaky epithelial barrier, which in turn can lead to various inflammatory and infective dysfunctions. Human junctional adhesion molecule (huJAM), found on the mucosal epithelia and vascular endothelia of many major organ systems, is a membrane glycoprotein which resolves to a doublet band of ∼40 and ∼37 kDa under SDS-PAGE analysis, representing differentially glycosylated forms of the same protein. huJAM was localized to the lateral membrane of Caco-2 cells (a human colonic epithelial cell line) monolayers, in an area basolateral of the epithelial tight junctions (TJ). Through functional and biochemical assays, we show huJAM to be able to homotypically associate and to participate in TJ restitution after trypsin-EDTA disruption. Furthermore, we also observed a migration of huJAM expression toward areas of cell-cell contacts during events of cell adhesion and monolayer formation. These qualities makes huJAM a likely player in the regulation of cell-cell contacts and the subsequent formation of TJs.
Role of aquaporin water channels in pleural fluid dynamics
Authors: Song Yuanlin , Yang Baoxue , Matthay Michael A. , Ma Tonghui , Verkman A. S.
Published: December 2017

Continuous movement of fluid into and out of the pleural compartment occurs in normal chest physiology and in pathophysiological conditions associated with pleural effusions. RT-PCR screening and immunostaining revealed expression of water channel aquaporin-1 (AQP1) in microvascular endothelia near the visceral and parietal pleura and in mesothelial cells in visceral pleura. Comparative physiological measurements were done on wild-type vs. AQP1 null mice. Osmotically driven water transport was measured in anesthetized, mechanically ventilated mice from the kinetics of pleural fluid osmolality after instillation of 0.25 ml of hypertonic or hypotonic fluid into the pleural space. Osmotic equilibration of pleural fluid was rapid in wild-type mice (50% equilibration in <2 min) and remarkably slowed by greater than fourfold in AQP1 null mice. Small amounts of AQP3 transcript were also detected in pleura by RT-PCR, but osmotic water transport was not decreased in AQP3 null mice. In spontaneously breathing mice, the clearance of isosmolar saline instilled in the pleural space (∼4 ml · kg−1 · h−1) was not affected by AQP1 deletion. In a fluid overload model produced by intraperitoneal saline administration and renal artery ligation, the accumulation of pleural fluid (∼0.035 ml/h) and was not affected by AQP1 deletion. Finally, in a thiourea toxicity model of acute endothelial injury causing pleural effusions and lung interstitial edema, pleural fluid accumulation in the first 3 h (∼4 ml · kg−1 · h−1) was not affected by AQP1 deletion. These results indicate rapid osmotic equilibration across the pleural surface that is facilitated by AQP1 water channels. However, AQP1 does not appear to play a role in clinically relevant mechanisms of pleural fluid accumulation or clearance.
15-Deoxy-Δ12,14-prostaglandin J2facilitates thyroglobulin production by cultured human thyrocytes
Authors: Kasai Kikuo , Banba Nobuyuki , Hishinuma Akira , Matsumura Michiko , Kakishita Hirofumi , Matsumura Mihoko , Motohashi Satoshi , Sato Noriyuki , Hattori Yoshiyuki
Published: December 2017

A cyclopentenone-type prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2(15-d-PGJ2), has been shown to induce the cellular stress response and to be a ligand for the peroxisome proliferator-activated receptor (PPAR)-γ. We studied its effect on the basal and thyrotropin (TSH)-induced production of thyroglobulin (TG) by human thyrocytes cultured in the presence of 10% FBS. In 15-d-PGJ2-treated cells in which the agent itself did not stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated, including the production of TG and cAMP, whereas such production was decreased in untreated cells according to duration of culture. PGD2 and PGJ2, which are precursors to 15-d-PGJ2, exhibited an effect similar to 15-d-PGJ2. However, the antidiabetic thiazolidinediones known to be specific ligands for PPAR-γ, and WY-14643, a specific PPAR-α ligand, lacked this effect. 15-d-PGJ2 and its precursors, but not the thiazolidinediones, induced gene expression for heme oxygenase-1 (HO-1), a stress-related protein, and strongly inhibited interleukin-1 (IL-1)-induced nitric oxide (NO) production. Cyclopentenone-type PGs have been recently shown to inhibit nuclear factor-κB (NF-κB) activation via a direct and PPAR-independent inhibition of inhibitor-κB kinase, suggesting that, in human thyrocytes, such PGs may inhibit IL-1-induced NO production, possibly via an inhibition of NF-κB activation. On the other hand, sodium arsenite, a known activator of the stress response pathway, induced HO-1 mRNA expression but lacked a promoting effect on TG production. Thus 15-d-PGJ2 and its precursors appear to facilitate TG production via a PPAR-independent mechanism and through a different pathway from the cellular stress response that is available to cyclopentenone-type PGs. Our findings reveal a novel role of these PGs associated with thyrocyte differentiation.
Nitrite uptake and metabolism and oxidant stress in human erythrocytes
Authors: May James M. , Qu Zhi-Chao , Xia Li , Cobb Charles E.
Published: December 2017

Nitric oxide, when released into the bloodstream, is quickly scavenged by Hb in erythrocytes or oxidized to nitrite. Nitrite can also enter erythrocytes and oxidize Hb. The goals of this work were to determine the mechanism of erythrocyte nitrite uptake and whether this uptake causes oxidant stress in these cells. Erythrocytes took up 0.8 mM nitrite with a half-time of 11 min. Nitrite uptake was sensitive to temperature and to the pH and ionic composition of the medium but was not inhibited by the specific anion-exchange inhibitor DIDS. About 25% of nitrite uptake occurred on the sodium-dependent phosphate transporter and the rest as diffusion of nitrous acid or other species across the plasma membrane. Methemoglobin formation increased in proportion to the intracellular nitrite concentration. Nitrite reacted with erythrocyte ascorbate, but ascorbate loading of cells decreased nitrite-induced methemoglobin formation only at high nitrite concentrations. In conclusion, nitrite rapidly enters erythrocytes and reacts with oxyhemoglobin but does not exert a strong oxidant stress on these cells.
cGMP-dependent ADP depolymerization of actin mediates estrogen increase in cervical epithelial permeability
Authors: Gorodeski George I.
Published: December 2017

Estrogen increases secretion of cervical mucus in women, and the effect depends on fragmentation of the cytoskeleton. The objective of the present study was to understand the molecular mechanism of estrogen action. Treatment of human cervical epithelial cells with 17β-estradiol, sodium nitroprusside (SNP), or 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased cellular monomeric G-actin and decreased polymerized F-actin. The effects of estradiol were blocked by tamoxifen, by the guanylate cyclase inhibitor LY-83583, and by the cGMP-dependent protein kinase inhibitor KT-5823. The effects of SNP were blocked by LY-83583 and KT-5823, while the effects of 8-Br-cGMP were blocked only by KT-5823. Treatment with phalloidin decreased paracellular permeability and G-actin. Treatment with 17β-estradiol, SNP, or 8-Br-cGMP attenuated SNP-induced phosphorylation of [32P]adenylate NAD in vitro: tamoxifen blocked the effect of estrogen; LY-83583 blocked the effect of SNP but not that of 8-Br-cGMP, while KT-5823 blocked effects of both SNP and 8-Br-cGMP. These results indicate that estrogen, nitric oxide (NO), and cGMP stimulate actin depolymerization. A possible mechanism is NO-induced, cGMP-dependent protein kinase augmentation of ADP-ribosylation of monomeric actin.
Telokin expression is restricted to smooth muscle tissues during mouse development
Authors: Herring B. Paul , Lyons Gary E. , Hoggatt April M. , Gallagher Patricia J.
Published: December 2017

Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3′ region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and α-actin.
Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers
Authors: Bebök Zsuzsanna , Tousson Albert , Schwiebert Lisa M. , Venglarik Charles J.
Published: December 2017

Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl− current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O2 produced similar improvements in Cl− current and CFTR protein as air-liquid interface culture, while increasing Po 2 from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.
Properties of ATP-dependent K+ channels in adrenocortical cells
Authors: Xu Lin , Enyeart John J.
Published: December 2017

Bovine adrenocortical zona fasciculata (AZF) cells express a novel ATP-dependent K+-permeable channel ( I AC). Whole cell and single-channel recordings were used to characterize I AC channels with respect to ionic selectivity, conductance, and modulation by nucleotides, inorganic phosphates, and angiotensin II (ANG II). In outside-out patch recordings, the activity of unitary I AC channels is enhanced by ATP in the patch pipette. These channels were K+ selective with no measurable Na+ or Ca2+ conductance. In symmetrical K+ solutions with physiological concentrations of divalent cations (M2+), I ACchannels were outwardly rectifying with outward and inward chord conductances of 94.5 and 27.0 pS, respectively. In the absence of M2+, conductance was nearly ohmic. Hydrolysis-resistant nucleotides including AMP-PNP and NaUTP were more potent than MgATP as activators of whole cell I AC currents. Inorganic polytriphosphate (PPPi) dramatically enhanced I AC activity. In current-clamp recordings, nucleotides and PPPi produced resting potentials in AZF cells that correlated with their effectiveness in activating I AC. ANG II (10 nM) inhibited whole cell I AC currents when patch pipettes contained 5 mM MgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP. Inhibition by ANG II was not reduced by selective kinase antagonists. These results demonstrate that I AC is a distinctive K+-selective channel whose activity is increased by nucleotide triphosphates and PPPi. Furthermore, they suggest a model for I AC gating that is controlled through a cycle of ATP binding and hydrolysis.
State-dependent modification of ATP-sensitive K+ channels by phosphatidylinositol 4,5-bisphosphate
Authors: Okamura Midori , Kakei Masafumi , Ichinari Koutaro , Miyamura Akihiro , Oketani Naoya , Koriyama Nobuyuki , Tei Chuwa
Published: December 2017

With inside-out patch recordings in ventricular myocytes from the hearts of guinea pigs, we studied ATP-sensitive K+ (KATP) channels activated by phosphatidylinositol 4,5-bisphosphate (PIP2) with respect to sensitivity to ATP when in either a rundown state (RS) or a non-rundown state (NRS). Rundown of KATP channels was induced by exposure either to ATP-free solution or to ATP-free solution containing 19 μM Ca2+. Exposure of membrane patches to 10 μM PIP2 reactivated channels with both types of rundown. The reactivation by PIP2 did not require ATP in the bath. The IC50 of channels recovered from RS and before the rundown was 37.1 and 31.1 μM, respectively. PIP2irreversibly increased the mean current when the channel was in the NRS. This was associated with a shift of IC50 to 250.6 μM after PIP2 exposure. PIP2 activates NRS KATP channels by decreasing their sensitivity to ATP, whereas PIP2 reactivates RS-KATP channels independently of ATP without changing ATP sensitivity.
CCK independently activates intracellular trypsinogen and NF-κB in rat pancreatic acinar cells
Authors: Han Bing , Ji Baoan , Logsdon Craig D.
Published: December 2017

In the cholecystokinin (CCK) hyperstimulation model of acute pancreatitis, two early intracellular events, activation of trypsinogen and activation of nuclear factor-κB (NF-κB), are thought to be important in the development of the disease. In this study, the relationship between these two events was investigated. NF-κB activity was monitored by using a DNA binding assay and mob-1 chemokine gene expression. Intracellular trypsin activity was measured by using a fluorogenic substrate. Protease inhibitors including FUT-175, Pefabloc, and E-64d prevented CCK stimulation of intracellular trypsinogen and NF-κB activation. Likewise, the NF-κB inhibitors pyrrolidine dithiocarbamate and N-acetyl-l-cysteine inhibited CCK stimulation of NF-κB and intracellular trypsinogen activation. These results suggested a possible codependency of these two events. However, CCK stimulated NF-κB activation in Chinese hamster ovary-CCKAcells, which do not express trypsinogen, indicating that trypsin is not necessary for CCK activation of NF-κB. Furthermore, adenovirus-mediated expression in acinar cells of active p65 subunits to stimulate NF-κB, or of inhibitory κB-α molecules to inhibit NF-κB, did not affect either basal or CCK-mediated trypsinogen activation. Thus trypsinogen and NF-κB activation are independent events stimulated by CCK.
Alteration of Cx43:Cx40 expression ratio in A7r5 cells
Authors: Burt Janis M. , Fletcher Anna M. , Steele Timothy D. , Wu Yan , Cottrell G. Trevor , Kurjiaka David T.
Published: December 2017

Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.
Glucocorticoids increase sodium pump α2- and β1-subunit abundance and mRNA in rat skeletal muscle
Authors: Thompson Curtis B. , Dorup Inge , Ahn Julie , Leong Patrick K. K. , McDonough Alicia A.
Published: December 2017

Fourteen-day adrenal steroid treatment increases [3H]ouabain binding sites 22–48% in muscle biopsies from patients treated with adrenal steroids for chronic obstructive lung disease and in rats treated with dexamethasone (Dex). Ouabain binding measures plasma membrane sodium pumps (Na+-K+-ATPase) with isoform-dependent affinity. In this study we have established the specific pattern of Dex regulation of sodium pump isoform protein and mRNA levels in muscle. Rats were infused with Dex (0.1 mg/kg per day) or vehicle for 14 days. Abundance of sodium pump catalytic α1- and α2-subunits and glycoprotein β1- and β2-subunits was determined by immunoblot in soleus, extensor digitorum longus, whole gastrocnemius, and diaphragm and was normalized to the mean vehicle control value. Dex increased α2 and β1 protein in all muscle types by 53–78% and ∼50%, respectively. Dex increased α1protein only in diaphragm (65 ± 7%). At the mRNA level in whole hindlimb muscle, Dex increased α2 (6.4 ± 0.5-fold) and β1 (1.54 ± 0.15-fold) and decreased β2 (to 0.36 ± 0.6 of control). In summary, α2β1 is the Dex-responsive pump in all skeletal muscles, and changes in α2 and β1mRNA levels can drive the 50% change in α2β1-subunits, which can account for the reported increase in [3H]ouabain binding.
Regulation of the mitochondrial permeability transition by matrix Ca2+ and voltage during anoxia/reoxygenation
Authors: Korge Paavo , Honda Henry M. , Weiss James N.
Published: December 2017

We studied the interplay between matrix Ca2+ concentration ([Ca2+]) and mitochondrial membrane potential (Δψ) in regulation of the mitochondrial permeability transition (MPT) during anoxia and reoxygenation. Without Ca2+loading, anoxia caused near-synchronous Δψ dissipation, mitochondrial Ca2+ efflux, and matrix volume shrinkage when a critically low Po 2 was reached, which was rapidly reversible upon reoxygenation. These changes were related to electron transport inhibition, not MPT. Cyclosporin A-sensitive MPT did occur when extramitochondrial [Ca2+] was increased to promote significant Ca2+ uptake during anoxia, depending on the Ca2+ load size and ability to maintain Δψ. However, when [Ca2+] was increased after complete Δψ dissipation, MPT did not occur until reoxygenation, at which time reactivation of electron transport led to partial Δψ regeneration. In the setting of elevated extramitochondrial Ca2+, this enhanced matrix Ca2+ uptake while promoting MPT because of less than full recovery of Δψ. The interplay between Δψ and matrix [Ca2+] in accelerating or inhibiting MPT during anoxia/reoxygenation has implications for preventing reoxygenation injury associated with MPT.
Stimulation of cardiac L-type calcium channels by extracellular ATP
Authors: Liu Qi-Yi , Rosenberg Robert L.
Published: December 2017

The co-release of ATP with norepinephrine from sympathetic nerve terminals in the heart may augment adrenergic stimulation of cardiac Ca2+ channel activity. To test for a possible direct effect of extracellular ATP on L-type Ca2+ channels, single channels were reconstituted from porcine sarcolemma into planar lipid bilayers so that intracellular signaling pathways could be controlled. Extracellular ATP (2–100 μM) increased the open probability of the reconstituted channels, with a maximal increase of ∼2.6-fold and an EC50 of 3.9 μM. The increase in open probability was due to an increase in channel availability and a decrease in channel inactivation rate. Other nucleotides displayed a rank order of effectiveness of ATP > α,β-methylene-ATP > 2-methylthio-ATP > UTP > adenosine 5′- O-(3-thiotriphosphate) >> ADP; adenosine had no effect. Several antagonists of P2 receptors had no impact on the ATP-dependent increase in open probability, indicating that receptor activation was not required. These results suggest that extracellular ATP and other nucleotides can stimulate the activity of cardiac L-type Ca2+ channels via a direct interaction with the channels.
Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with 15N
Authors: Welbourne Tomas , Nissim Itzhak
Published: December 2017

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK1-F+ cell line. We used high-affinity glutamate transport inhibitors d-aspartate (d-Asp) and dl-threo-β-hydroxyaspartate (THA) to block extracellular uptake and then used [15N]glutamate or [2-15N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-15N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mMd-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% ( P < 0.05) as the extracellular enrichment increased by 39% ( P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% ( P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH[Formula: see text] formed from [2-15N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both d-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.
IFN-γ downregulates expression of Na+/H+ exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells
Authors: Rocha Flavio , Musch Mark W. , Lishanskiy Leonid , Bookstein Cres , Sugi Kazunori , Xie Yue , Chang Eugene B.
Published: December 2017

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-γ (IFN-γ) affects expression and function of the apical membrane Na+/H+ exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-γ for 2, 24, and 48 h. Adult rats were injected with IFN-γ intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional 22Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-γ treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-γ. Chronic exposure of intestinal epithelial cells to IFN-γ results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.