bluuebunny/crossref_metadata_2025 huggingface dataset:
H2O2 and ethanol act synergistically to gate ryanodine receptor/calcium-release channel
Authors: Oba Toshiharu , Ishikawa Tatsuya , Murayama Takashi , Ogawa Yasuo , Yamaguchi Mamoru
Published: December 2017

We examined the effect of low concentrations of H2O2 on the Ca2+-release channel/ryanodine receptor (RyR) to determine if H2O2 plays a physiological role in skeletal muscle function. Sarcoplasmic reticulum vesicles from frog skeletal muscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle were incorporated into lipid bilayers. Channel activity of the frog RyR was not affected by application of 4.4 mM (0.02%) ethanol. Open probability ( P o) of such ethanol-treated RyR channels was markedly increased on subsequent addition of 10 μM H2O2. Increase of H2O2to 100 μM caused a further increase in channel activity. Application of 4.4 mM ethanol to 10 μM H2O2-treated RyRs activated channel activity. Exposure to 10 or 100 μM H2O2 alone, however, failed to increase P o. Synergistic action of ethanol and H2O2 was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12). H2O2 at 100–500 μM had no effect on purified channel activity. Application of FKBP12 to the purified RyR1 drastically decreased channel activity but did not alter the effects of ethanol and H2O2. These results suggest that H2O2 may play a pathophysiological, but probably not a physiological, role by directly acting on skeletal muscle RyRs in the presence of ethanol.
Mechanical signals and mechanosensitive modulation of intracellular [Ca2+] in smooth muscle
Authors: An Steven S. , Hai Chi-Ming
Published: December 2017

We tested the hypothesis that strain is the primary mechanical signal in the mechanosensitive modulation of intracellular Ca2+concentration ([Ca2+]i) in airway smooth muscle. We found that [Ca2+]i was significantly correlated with muscle length during isotonic shortening against 20% isometric force (Fiso). When the isotonic load was changed to 50% Fiso, data points from the 20 and 50% Fiso experiments overlapped in the length-[Ca2+]i relationship. Similarly, data points from the 80% Fiso experiments clustered near those from the 50% Fiso experiments. Therefore, despite 2.5- and 4-fold differences in external load, [Ca2+]idid not deviate much from the length-[Ca2+]irelation that fitted the 20% Fiso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca2+ uptake by 10 μM cyclopiazonic acid (CPA) did not significantly change [Ca2+]i in carbachol-induced isometric contractions and isotonic shortening. CPA also did not significantly change myosin light-chain phosphorylation or force redevelopment when carbachol-activated muscle strips were quickly released from optimal length ( L o) to 0.5 L o. These results are consistent with the hypothesis and suggest that SR Ca2+ uptake is not the underlying mechanism.
Expression of nucleotide-regulated Cl−currents in CF and normal mouse tracheal epithelial cell lines
Authors: Thomas E. J. , Gabriel S. E. , Makhlina M. , Hardy S. P. , Lethem M. I.
Published: December 2017

The dominant route for Cl− secretion in mouse tracheal epithelium is via Cl− channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(−/−)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/−)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10−4 M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current ( I sc) and resistance of both cell lines, with larger increases in I scbeing elicited by ionomycin (10−6 M). Both cell lines expressed P2Y2 receptors and responded to the purinergic agonists ATP, UTP, and 5′-adenylylimidodiphosphate (10−4 M) with an increase in I sc. This response could be inhibited by DIDS and was abolished in the presence of Cl−-free Ringer solution. Reducing the mucosal Cl− concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(−/−) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I sc and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl− currents and may prove valuable tools in studying the properties of this pathway.
Regulation of Na+-K+-Cl−cotransporter in primary astrocytes by dibutyryl cAMP and high [K+]o
Authors: Su Gui , Haworth Robert A. , Dempsey Robert J. , Sun Dandan
Published: December 2017

In this study, we examined the Na+-K+-Cl− cotransporter activity and expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for 7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes ( P < 0.05). Expression of an ∼161-kDa cotransporter protein was increased by 91% in the DBcAMP-treated astrocytes. Moreover, the specific [3H]bumetanide binding was increased by 67% in the DBcAMP-treated astrocytes. Inhibition of protein synthesis by cyclohexamide (2–3 μg/ml) significantly attenuated the DBcAMP-mediated upregulation of the cotransporter activity and expression. The Na+-K+-Cl−cotransporter in astrocytes has been suggested to play a role in K+ uptake. In 75 mM extracellular K+concentration, the cotransporter-mediated K+ influx was stimulated by 147% in nontreated cells and 79% in DBcAMP-treated cells ( P < 0.05). To study whether this high K+-induced stimulation of the cotransporter is attributed to membrane depolarization and Ca2+ influx, the role of the L-type voltage-dependent Ca2+ channel was investigated. The high-K+-mediated stimulation of the cotransporter activity was abolished in the presence of either 0.5 or 1.0 μM of the L-type channel blocker nifedipine or Ca2+-free HEPES buffer. A rise in intracellular free Ca2+ in astrocytes was observed in high K+. These results provide the first evidence that the Na+-K+-Cl− cotransporter protein expression can be regulated selectively when intracellular cAMP is elevated. The study also demonstrates that the cotransporter in astrocytes is stimulated by high K+ in a Ca2+-dependent manner.
Riboflavin transport by isolated perfused rabbit renal proximal tubules
Authors: Yanagawa Norimoto , Shih Remi N. G. , Jo Oak D. , Said Hamid M.
Published: December 2017

Rabbit renal proximal tubular transport of riboflavin (RF) was examined by using the in vitro isolated tubule perfusion technique. We found that proximal tubules actively reabsorbed ( J lb) and secreted ( J bl) RF. At 0.1 μM RF concentration, J bl was significantly higher than J lb, resulting in a net secretion. This net secretion of RF was decreased at 0.01 μM RF concentration and increased at 1 μM RF concentration. Both J lb and J bl were inhibited by lowering temperature or by adding iodoacetate, a metabolic inhibitor, and lumichrome, an RF analog, suggesting the involvement of carrier-mediated transport mechanisms. J bl was inhibited by probenecid, an anion transport inhibitor, and by para-aminohippuric acid, an organic anion, suggesting the relevance of RF secretion to renal organic anion transport. J bl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca2+/calmodulin-dependent pathway on RF secretion. Finally, we found that addition of chlorpromazine, a phenothiazine derivative, inhibited both J lb and J bl, raising the concern about the nutritional status in patients receiving such a type of medication.
Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells
Authors: Nowicki Susana , Kruse Maria Sol , Brismar Hjalmar , Aperia Anita
Published: December 2017

Short-term regulation of sodium metabolism is dependent on the modulation of the activity of sodium transporters by first and second messengers. In understanding diseases associated with sodium retention, it is necessary to identify the coupling between these messengers. We have examined whether dopamine, an important first messenger in tubular cells, activates and translocates various protein kinase C (PKC) isoforms. We used a proximal tubular-like cell line, LLCPK-1 cells, in which dopamine was found to inhibit Na+-K+-ATPase in a PKC-dependent manner. Translocation of PKC isoforms was studied with both subcellular fractionation and confocal microscopy. Both techniques revealed a dopamine-induced translocation from cytosol to plasma membrane of PKC-α and -ε, but not of PKC-δ, -γ, and -ζ. The process of subcellular fractionation resulted in partial translocation of PKC-ε. This artifact was eliminated in confocal studies. Confocal imaging permitted detection of translocation within 20 s. Translocation was abolished by a phospholipase C inhibitor and by an antagonist against the dopamine 1 subtype (D1) but not the 2 subtype of receptor (D2). In conclusion, this study visualizes in renal epithelial cells a very rapid activation of the PKC-α and -ε isoforms by the D1 receptor subtype.
Adaptive regulation of intestinal folate uptake: effect of dietary folate deficiency
Authors: Said Hamid M. , Chatterjee Nabendu , Haq Riaz Ul , Subramanian Veedamali S. , Ortiz Alvaro , Matherly Larry H. , Sirotnak F. M. , Halsted Charles , Rubin Stanley A.
Published: December 2017

Folate is an essential micronutrient that, in mammals, must be obtained from exogenous sources via intestinal absorption. Previous studies have characterized different aspects of the mechanism of the intestinal folate uptake process. Much less, however, is known about regulation of this process. In this study, we examined the effect of dietary folate deficiency on intestinal folate uptake using the rat as an animal model. The results showed that dietary folate deficiency leads to a significant ( P < 0.01) and specific upregulation in the transepithelial transport of folic acid. The upregulation in transepithelial folate transport 1) was found to be due to an induction in carrier-mediated folate uptake across the brush-border membrane (BBM) and was mediated via a significant ( P < 0.01) increase in the maximal velocity but not the apparent Michaelis constant of the uptake process, 2) was associated with a marked increase in the steady-state mRNA level of reduced folate carrier-1 and in the level of the expressed protein at the intestinal BBM, and 3) was associated with a marked (>10-fold) increase in the activity of the intestinal BBM form of folate hydrolase. Results of this study demonstrate, for the first time, that dietary folate deficiency leads to a marked upregulation in intestinal folate uptake and in the activity of folate hydrolase. Furthermore, the upregulation in folate uptake is associated with an increase in mRNA and protein levels of folate carrier, suggesting possible involvement of a transcriptional regulatory mechanism(s) in the upregulation.
Regulation of the epithelial Na+ channel by extracellular acidification
Authors: Awayda Mouhamed S. , Boudreaux Michael J. , Reger Roxanne L. , Hamm L. Lee
Published: December 2017

The effect of extracellular acidification was tested on the native epithelial Na+ channel (ENaC) in A6 epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-induced fluctuation analysis in A6 epithelia and dual electrode voltage clamp in oocytes. In A6 cells, a decrease of extracellular pH (pHo) from 7.4 to 6.4 caused a slow stimulation of the amiloride-sensitive short-circuit current ( I Na) by 68.4 ± 11% ( n = 9) at 60 min. This increase of I Na was attributed to an increase of open channel and total channel ( N T) densities. Similar changes were observed with pHo 5.4. The effects of pHo were blocked by buffering intracellular Ca2+ with 5 μM 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid. In oocytes, pHo 6.4 elicited a small transient increase of the slope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min) followed by a decrease to 83.7 ± 11.7% of control at 60 min ( n = 6). Thus small decreases of pHostimulate the native ENaC by increasing N T but do not appreciably affect ENaC expressed in Xenopus oocytes. These effects are distinct from those observed with decreasing intracellular pH with permeant buffers that are known to inhibit ENaC.
Inhibition of growth hormone action in models of inflammation
Authors: Bergad Pearl L. , Schwarzenberg Sarah Jane , Humbert Jeffrey T. , Morrison Michelle , Amarasinghe Sherani , Towle Howard C. , Berry Susan A.
Published: December 2017

Growth hormone (GH) action is attenuated during the hepatic acute-phase response (APR). To understand this attenuation, we asked whether GH and cytokine-signaling pathways intersect during an APR. In hypophysectomized rats treated with lipopolysaccharide (LPS), accumulation of activated signal transducer and transcription activator 5 (Stat5) in hepatic nuclei in response to GH and its binding to a GH response element (GHRE) from the serine protease inhibitor (Spi) 2.1 promoter are diminished in a time-dependent manner. Similarly, accumulation of activated Stat3 in hepatic nuclei in response to LPS and its binding to a high-affinity sis-inducible element (SIE) are also diminished by the simultaneous administration of GH. In functional assays with primary hepatocytes, LPS-stimulated monocyte-conditioned medium (MoCM) inhibits the GH response of Stat5-dependent Spi 2.1 reporter activity but induces Stat3-dependent Spi 2.2 reporter activity, as in an APR. Similar results are obtained when hepatocytes are treated with either tumor necrosis factor-α (TNF-α) or interleukin (IL)-1β. TNF-α, IL-1β, and IL-6 also inhibit GH-induced Spi 2.1 mRNA expression in hepatocytes. Thus inhibition of the GH signaling pathway during an APR results in reduced expression of GH-responsive genes.
CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking
Authors: Rao Rammohan V. , Holicky Eileen L. , Kuntz Susan M. , Miller Laurence J.
Published: December 2017

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.
Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle
Authors: Hypolite Joseph A. , DiSanto Michael E. , Zheng Yongmu , Chang Shaohua , Wein Alan J. , Chacko Samuel
Published: December 2017

Urinary bladder filling and emptying requires coordinated control of bladder body and urethral smooth muscles. Bladder dome, midbladder, base, and urethra showed significant differences in the percentage of 20-kDa myosin light chain (LC20) phosphorylation (35.45 ± 4.6, 24.7 ± 2.2, 13.6± 2.1, and 12.8 ± 2.7%, respectively) in resting muscle. Agonist-mediated force was associated with a rise in LC20 phosphorylation, but the extent of phosphorylation at all levels of force was less for urethral than for bladder body smooth muscle. RT-PCR and quantitative competitive RT-PCR analyses of total RNA from bladder body and urethral smooth muscles revealed only a slight difference in myosin heavy chain mRNA copy number per total RNA, whereas mRNA copy numbers for NH2-terminal isoforms SM-B (inserted) and SM-A (noninserted) in these muscles showed a significant difference (2.28 × 108 vs. 1.68 × 108 for SM-B and 0.12 × 108 vs. 0.42 × 108 for SM-A, respectively), which was also evident at the protein level. The ratio of COOH-terminal isoforms SM2:SM1 in the urethra was moderately but significantly lower than that in other regions of the bladder body. A high degree of LC20phosphorylation and SM-B in the bladder body may help to facilitate fast cross-bridge cycling and force generation required for rapid emptying, whereas a lower level of LC20 phosphorylation and the presence of a higher amount of SM-A in urethral smooth muscle may help to maintain the high basal tone of urethra, required for urinary continence.
Muscle LIM protein is upregulated in fast skeletal muscle during transition toward slower phenotypes
Authors: Willmann Raffaella , Kusch Justine , Sultan Karim R. , Schneider Achim G. , Pette Dirk
Published: December 2017

Muscle LIM protein (MLP) is constitutively expressed in slow, but undetectable in fast, muscles of the rat. Here we show that MLP was upregulated at both the mRNA and protein levels under experimental conditions leading to transitions from fast to slower phenotypes. Chronic low-frequency stimulation and mechanical overloading by synergist removal both induced fast-to-slow shifts in myosin heavy chain (MHC) isoforms and expression of MLP in fast muscles. High amounts of MLP mRNA and protein were also present in fast muscles of the myotonic, hyperactive ADR mouse. Hypothyroidism evoked shifts in myosin composition toward slower isoforms and increased the MLP protein content of soleus (SOL) muscle but failed to induce MLP in fast muscles. Unweighting by hindlimb suspension elicited slow-to-fast transitions in MHC expression without altering MLP levels in SOL muscle. Hyperthyroidism shifted the MHC pattern toward faster isoforms but did not affect MLP content in SOL muscle. We conclude that alterations in MLP expression are associated with transitions from fast to slower phenotypes but not with slow-to-fast muscle fiber transitions.
Regulatory pathways and uptake ofl-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells
Authors: Sampaio-Maia B. , Serrão M. P. , Soares-da-Silva P.
Published: December 2017

We examined the nature and regulation of the inwardl-3,4-dihydroxyphenylalanine (l-DOPA) transporter in rat capillary cerebral endothelial (RBE4) cells, type 1 astrocytes (DI TNC1), and Neuro-2a neuroblastoma cells. In all three cell types, the inward transfer of l-DOPA was largely promoted through the 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid-sensitive and sodium-independent L-type amino acid transporter. Only in DI TNC1 cells was the effect of maneuvers that increase intracellular cAMP levels accompanied by increases inl-DOPA uptake. Also, only in DI TNC1 cells was the effect of the guanylyl cyclase inhibitor LY-83583 accompanied by a 65% increase in l-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease inl-DOPA accumulation. In all three cell types, the Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited l-DOPA uptake in a noncompetitive manner. Thapsigargin (1 and 3 μM) and A-23187 (1 and 3 μM) failed to alter l-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased l-DOPA uptake in DI TNC1cells. We concluded that l-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of l-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.
NH2-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion
Authors: Broughman James R. , Mitchell Kathy E. , Sedlacek Roger L. , Iwamoto Takeo , Tomich John M. , Schultz Bruce D.
Published: December 2017

A synthetic, channel-forming peptide, derived from the α-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH2 terminus (N-K4-M2GlyR). In Ussing chamber experiments, apical N-K4-M2GlyR (250 μM) increased transepithelial short-circuit current ( I sc) by 7.7 ± 1.7 and 10.6 ± 0.9 μA/cm2 in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672–C1679, 1997). N-K4-M2GlyR caused a concentration-dependent increase in I sc ( k [1/2] = 190 μM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K4-M2GlyR-mediated increases in I sc were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl− conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-dinitrostilben-2,2′-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K4-M2GlyR-mediated I sc. Whole cell membrane patch voltage-clamp studies revealed an N-K4-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K4-M2GlyR forms a 50-pS anion conductance with a k [1/2] for Cl−of 290 meq. These results indicate that N-K4-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.
Redistribution and abnormal activity of phospholipase A2 isoenzymes in postinfarct congestive heart failure
Authors: McHowat Jane , Tappia Paramjit S. , Liu Song-Yan , McCrory Raetreal , Panagia Vincenzo
Published: December 2017

Cardiac sarcolemmal (SL) cis-unsaturated fatty acid sensitive phospholipase D ( cis-UFA PLD) is modulated by SL Ca2+-independent phospholipase A2(iPLA2) activity via intramembrane release of cis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca2+ concentration and contractile performance of the cardiomyocyte, changes in iPLA2 activity may contribute to abnormal function of the failing heart. We examined PLA2 immunoprotein expression and activity in the SL and cytosol from noninfarcted left ventricular (LV) tissue of rats in an overt stage of congestive heart failure (CHF). Hemodynamic assessment of CHF animals showed an increase of the LV end-diastolic pressure with loss of contractile function. In normal hearts, immunoblot analysis revealed the presence of cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) in the cytosol, with cPLA2 and iPLA2 in the SL. Intracellular PLA2 activity was predominantly Ca2+independent, with minimal sPLA2 activity. CHF increased cPLA2 immunoprotein and PLA2 activity in the cytosol and decreased SL iPLA2 and cPLA2immunoprotein and SL PLA2 activity. sPLA2activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA2isoenzymes, suggesting that PLA2-mediated bioprocesses are altered in CHF.
Progesterone treatment abolishes exogenously expressed ionic currents in Xenopus oocytes
Authors: Shcherbatko Anatoly D. , Davenport Christopher M. , Speh Joan C. , Levinson Simon R. , Mandel Gail , Brehm Paul
Published: December 2017

Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na+ and K+ channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na+ and K+ channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na+ currents temporally coincided with development of germinal vesicle breakdown, while elimination of K+currents was delayed by ∼2 h. Coexpression of human β1-subunit with rat skeletal muscle α-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na+ channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na+ and K+ channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na+ channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.
Depletion of focal adhesion kinase by antisense depresses contractile activation of smooth muscle
Authors: Tang Dale D. , Gunst Susan J.
Published: December 2017

Focal adhesion kinase (FAK) undergoes tyrosine phosphorylation in response to the contractile stimulation of tracheal smooth muscle. We hypothesized that FAK may play an important role in signaling pathways that regulate smooth muscle contraction. FAK antisense or FAK sense was introduced into muscle strips by reversible permeabilization, and strips were incubated with antisense or sense for 7 days. Antisense decreased FAK expression compared with that in untreated and sense-treated tissues, but it did not affect the expression of vinculin or myosin light chain kinase. Increases in force, intracellular free Ca2+, and myosin light chain phosphorylation in response to stimulation with ACh or KCl were depressed in FAK-depleted tissues, but FAK depletion did not affect the activation of permeabilized tracheal muscle strips with Ca2+. The tyrosine phosphorylation of paxillin, a substrate for FAK, was also significantly reduced in FAK-depleted strips. We conclude that FAK is a necessary component of the signaling pathways that regulate smooth muscle contraction and that FAK plays a role in regulating intracellular free Ca2+ and myosin light chain phosphorylation.
PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC
Authors: Chiu Terence , Rozengurt Enrique
Published: December 2017

Protein kinase C (PKC) is implicated in the regulation of multiple important functions in intestinal epithelial cells, but the downstream signaling targets of PKCs in these cells remain poorly characterized. Here we report that treatment of normal rat intestinal cell lines IEC-6 and IEC-18 with phorbol 12,13-dibutyrate (PDBu) led to a rapid and striking PKC-dependent activation of protein kinase D (PKD; also known as PKCμ). Unlike conventional and novel PKCs, PKD did not undergo downregulation in response to prolonged (24 h) exposure of IEC-6 or IEC-18 cells to PDBu. PKD was also rapidly activated in these cells by lysophosphatidic acid (LPA) or angiotensin in a concentration-dependent fashion via a PKC-dependent pathway. EC50 values were 0.1 μM and 2 nM for LPA and angiotensin II, respectively. LPA-induced PKD activation was prevented selectively by treatment with pertussis toxin. PKD activation was tightly associated with an increase in PKD autophosphorylation at serine 916. Our results identify PKD as a novel early point of convergence and integration of Gi and Gq signaling in intestinal epithelial cells.
Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration
Authors: Rao Jaladanki N. , Li Li , Golovina Vera A. , Platoshyn Oleksandr , Strauch Eric D. , Yuan Jason Xiao-Jian , Wang Jian-Ying
Published: December 2017

Expression of voltage-gated K+ (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential ( E m) and cytosolic free Ca2+concentration ([Ca2+]cyt). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated [Ca2+]cyt following activation of K+ channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by α-difluoromethylornithine (DFMO) reduced whole cell K+ currents [ I K(v)] through Kv channels and caused membrane depolarization, which was associated with decreases in [Ca2+]cyt, RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I K(v ) , E m, [Ca2+]cyt, and RhoA protein and restored cell migration to normal. Elevation of [Ca2+]cytinduced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C3 transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.
Evaluation of ovarian POMC mRNA through quantitative RT-PCR analysis in Rana esculenta
Authors: Nabissi M. , Soverchia L. , Lihrmann I. , Vaudry H. , Mosconi G. , Polzonetti-Magni A. M.
Published: December 2017

The evaluation of changes in the expression of specific genes requires accurate measurement of the corresponding mRNA concentration, especially when the gene is expressed at a very low level. We previously showed that the proopiomelanocortin (POMC) gene is expressed in the ovary of the frog Rana esculenta, and, to evaluate its mRNA content in frog ovary, we have now developed a sensitive quantitative RT-PCR method. This study provides evidence for the validation of this method and for the effects of captivity and hypophysectomy on POMC gene expression in the ovary of this anuran. Our data indicate that ovarian POMC gene is involved in short-term captivity stress response and seems not influenced by pituitary. These results are discussed taking into account the knowledge of the role played by opioids in stress response; moreover, a local control of POMC gene expression is also suggested.
Transcriptional regulation of rat Na+/H+exchanger isoform-2 (NHE-2) gene by Sp1 transcription factor
Authors: Bai Liqun , Collins James F. , Xu Hua , Ghishan Fayez K.
Published: December 2017

The rat Na+/H+ exchanger isoform-2 ( NHE-2) gene promoter lacks a TATA box and is very GC rich. A minimal promoter extending from bp −36 to +116 directs high-level expression of NHE-2 in mouse inner medullary collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found in this region. The introduction of mutations within these Sp1 consensus elements and DNA footprinting revealed that only two of them were utilized and are critical for basal transcriptional activation in mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 100-fold, suggesting that Sp1 is critical for transcriptional regulation. However, neither Sp3 nor Sp4 was able to activate transcription in these cells. Furthermore, in mIMCD-3 cells, Sp1-mediated transcriptional activation was repressed by expression of Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may repress transcriptional activation by competing with Sp1 for binding to core cis-elements.
Molecular identification of a TTX-sensitive Ca2+current
Authors: Guatimosim Silvia , Sobie Eric A. , dos Santos Cruz Jader , Martin Laura A. , Lederer W. J.
Published: December 2017

The TTX-sensitive Ca2+ current [ I Ca(TTX)] observed in cardiac myocytes under Na+-free conditions was investigated using patch-clamp and Ca2+-imaging methods. Cs+ and Ca2+were found to contribute to I Ca(TTX), but TEA+ and N-methyl-d-glucamine (NMDG+) did not. HEK-293 cells transfected with cardiac Na+ channels exhibited a current that resembled I Ca(TTX) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na+ channel itself gives rise to I Ca(TTX). Furthermore, repeated activation of I Ca(TTX) led to a 60% increase in intracellular Ca2+ concentration, confirming Ca2+ entry through this current. Ba2+ permeation of I Ca(TTX), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na+channels under our experimental conditions. The report of block of I Ca(TTX) in guinea pig heart by mibefradil (10 μM) was supported in transfected HEK-293 cells, but Na+current was also blocked (half-block at 0.45 μM). We conclude that I Ca(TTX) reflects current through cardiac Na+ channels in Na+-free (or “null”) conditions. We suggest that the current be renamed I Na(null) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I Na(null)and Ca2+ flux through slip-mode conductance of cardiac Na+ channels is discussed in the context of ion channel biophysics and “permeation plasticity.”
Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells
Authors: Morse Diane M. , Smullen Jennifer L. , Davis C. William
Published: December 2017

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y2 purinoceptor (P2Y2-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC50 = 4.7 μM), ATP, and adenosine-5′- O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 ± 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 ± 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y6-R, with a maximal effect approximately one-half that elicited by P2Y2-R stimulation. Not indicated were P2Y1-R-, P2Y4-R-, or P2Y11-R-mediated effects. A2B-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5′-( N-ethylcarboxamido)adenosine, EC50 = 0.09 μM; adenosine, EC50 = 0.7 μM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A2-R antagonist 8-( p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y2-R and, after an apparent ectohydrolysis to adenosine, through A2BAR.
Expression and regulation of ClC-5 chloride channels: effects of antisense and oxidants
Authors: Weng T. X. , Mo L. , Hellmich H. L. , Yu A. S. L. , Wood T. , Wills N. K.
Published: December 2017

Genetic mutations of the Cl− channel ClC-5 cause Dent's disease in humans. We recently cloned an amphibian ortholog of Xenopus ClC-5 (xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl− > I− anion sensitivity, and were inhibited at low pH, similar to previous results in oocytes. In oocytes, antisense xClC-5 cRNA injection had no effect on endogenous membrane currents or the heterologous expression of human ClC-5. Activators of cAMP and protein kinase C inhibitors had no significant effects on ClC-5 currents expressed in either COS-7 cells or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA) inhibitor, and hydrogen peroxide decreased the currents. We conclude that the basic properties of ClC-5 currents were independent of the host cell type used for expression. In addition, ClC-5 channels may be modulated by PKA and reactive oxygen species.
Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line
Authors: Payne John A. , Ferrell Christina , Chung Chee Yeun
Published: December 2017

A low K-resistant mutant Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown previously to lack functional Na-K-Cl cotransporter (NKCC) activity, indicating that it may be a useful NKCC “knockout” cell line for structure-function studies. Using immunological probes, we first characterized the defect in the endogenous NKCC protein of the LK-C1 cells and then fully restored NKCC activity in these cells by stably expressing the human secretory NKCC1 protein (hNKCC1). The endogenous NKCC protein of the LK-C1 cells was expressed at significantly lower levels than in wild-type MDCK cells and was not properly glycosylated. This latter finding indicated that the lack of functional NKCC activity in the LK-C1 cells may be due to the inability to process the protein to the plasma membrane. In contrast, exogenously expressed hNKCC1 protein was properly processed and fully functional at the plasma membrane. Significantly, the exogenous hNKCC1 protein was regulated in a manner similar to the protein in native secretory cells as it was robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on permeable supports, the exogenous hNKCC1 protein was properly polarized and functional at the basolateral membrane. The low levels of endogenous NKCC protein expression, the absence of any endogenous NKCC transport activity, and the ability to form a polarized epithelium indicate that the LK-C1 cells offer an excellent expression system with which to study the molecular physiology of the cation Cl cotransporters.
Tumor necrosis factor-α inhibits store-mediated Ca2+ entry in the human hepatocellular carcinoma cell line HepG2
Authors: Rosado Juan A. , Rosenzweig Ivana , Harding Susanne , Sage Stewart O.
Published: December 2017

Tumor necrosis factor-α (TNF-α) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-α on thapsigargin (TG)-induced store-mediated Ca2+ entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF-α slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-α significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF-α abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-α was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-α inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-α.
Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts
Authors: Katoh Kazuo , Kano Yumiko , Amano Mutsuki , Kaibuchi Kozo , Fujiwara Keigi
Published: December 2017

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.
Apical P2Y4 purinergic receptor controls K+ secretion by vestibular dark cell epithelium
Authors: Marcus Daniel C. , Scofield Margaret A.
Published: December 2017

It was previously shown that K+ secretion by vestibular dark cell epithelium is under control of G protein-coupled receptors of the P2Y family in the apical membrane that are activated by both purine and uridine nucleotides (P2Y2, P2Y4, or P2Y6). The present study was conducted to determine the subtype of purinergic receptor and to test whether these receptors undergo desensitization. The transepithelial short-circuit current represents electrogenic K+ secretion and was found to be reduced by UTP, ATP, and diadenosine tetraphosphate, but not UDP. Neither pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 30 μM) nor suramin (100 μM) inhibited the effect of UTP. The potencies of the agonists were consistent with rodent P2Y4and P2Y2, but not P2Y6, receptors. The ineffectiveness of suramin was consistent with P2Y4, but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in vestibular labyrinth. Sustained exposure to ATP or UTP for 15 min caused a constant depression of short-circuit current with no apparent desensitization. The results support the conclusion that regulation of K+ secretion across vestibular dark cell epithelium occurs by P2Y4 receptors without desensitization of the response.
Smad4 mediates activation of mitogen-activated protein kinases by TGF-β in pancreatic acinar cells
Authors: Simeone Diane M. , Zhang Lizhi , Graziano Kathleen , Nicke Barbara , Pham Trinh , Schaefer Claus , Logsdon Craig D.
Published: December 2017

Transforming growth factor-β (TGF-β) inhibits pancreatic acinar cell growth. In many cell types, TGF-β mediates its growth inhibitory effects by activation of Smad proteins. Recently, it has been reported that Smad proteins may interact with the mitogen-activated protein (MAP) kinase signaling pathways. In this study, we report on the interactions between the TGF-β and MAP kinase signaling pathways in isolated rat pancreatic acinar cells. TGF-β activated the MAP kinases extracellular signal-related kinases (ERKs) and p38 in pancreatic acinar cells, but had no effect on c- junNH2-terminal kinase activity. Activation of MAP kinase by TGF-β was maximal 4 h after treatment. The ability of TGF-β to activate ERKs was concentration dependent and dependent on protein synthesis. TGF-β's stimulation of ERK activation was blocked by PD-98059, an inhibitor of MAP kinase kinase 1, and by adenoviral transfer of dominant negative RasN17. Furthermore, adenoviral-mediated expression of dominant negative Smad4 blocked the ability of TGF-β to activate acinar cell MAP kinase, demonstrating that this activation is downstream of Smads. The biological relevance of ERK activation by TGF-β was indicated by demonstrating that inhibition of ERK signaling by PD-98059 blocked the ability of TGF-β to activate the transcription factor activator protein-1. These studies provide new insight into the signaling mechanisms by which TGF-β mediates biological actions in pancreatic acinar cells.
ClC-2 Cl− channels in human lung epithelia: activation by arachidonic acid, amidation, and acid-activated omeprazole
Authors: Cuppoletti John , Tewari Kirti P. , Sherry Ann M. , Kupert Elena Y. , Malinowska Danuta H.
Published: December 2017

ClC-2 Cl− channels represent a potential target for therapy in cystic fibrosis. Key questions regarding the feasibility of using ClC-2 as a therapeutic target are addressed in the present studies, including whether the channels are present in human lung epithelia and whether activators of the channel can be identified. Two new mechanisms of activation of human recombinant ClC-2 Cl− channels expressed in HEK-293 cells were identified: amidation with glycine methyl ester catalyzed by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and treatment with acid-activated omeprazole. ClC-2 mRNA was detected by RT-PCR. Channel function was assessed by measuring Cl− currents by patch clamp in the presence of a cAMP-dependent protein kinase (PKA) inhibitor, myristoylated protein kinase inhibitor, to prevent PKA-activated Cl− currents. Calu-3, A549, and BEAS-2B cell lines derived from different human lung epithelia contained ClC-2 mRNA, and Cl− currents were increased by amidation, acid-activated omeprazole, and arachidonic acid. Similar results were obtained with buccal cells from healthy individuals and cystic fibrosis patients. The ClC-2 Cl− channel is thus a potential target for therapy in cystic fibrosis.