bluuebunny/crossref_metadata_2025 huggingface dataset:
NF-κB inactivation converts a hepatocyte cell line TNF-α response from proliferation to apoptosis
Authors: Xu Yang , Bialik Shani , Jones Brett E. , Iimuro Yuji , Kitsis Richard N. , Srinivasan Anu , Brenner David A. , Czaja Mark J.
Published: December 2017

Toxins convert the hepatocellular response to tumor necrosis factor-α (TNF-α) stimulation from proliferation to cell death, suggesting that hepatotoxins somehow sensitize hepatocytes to TNF-α toxicity. Because nuclear factor-κB (NF-κB) activation confers resistance to TNF-α cytotoxicity in nonhepatic cells, the possibility that toxin-induced sensitization to TNF-α killing results from inhibition of NF-κB-dependent gene expression was examined in the RALA rat hepatocyte cell line sensitized to TNF-α cytotoxicity by actinomycin D (ActD). ActD did not affect TNF-α-induced hepatocyte NF-κB activation but decreased NF-κB-dependent gene expression. Expression of an IκB superrepressor rendered RALA hepatocytes sensitive to TNF-α-induced apoptosis in the absence of ActD. Apoptosis was blocked by caspase inhibitors, and TNF-α treatment led to activation of caspase-2, caspase-3, and caspase-8 only when NF-κB activation was blocked. Although apoptosis was blocked by the NF-κB-dependent factor nitric oxide (NO), inhibition of endogenous NO production did not sensitize cells to TNF-α-induced cytotoxicity. Thus NF-κB activation is the critical intracellular signal that determines whether TNF-α stimulates hepatocyte proliferation or apoptosis. Although exogenous NO blocks RALA hepatocyte TNF-α cytotoxicity, endogenous production of NO is not the mechanism by which NF-κB activation inhibits this death pathway.
Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells
Authors: Kuwada Scott K. , Lund Kirk A. , Li Xiu Fen , Cliften Peter , Amsler Kurt , Opresko Lee K. , Wiley H. Steven
Published: December 2017

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, β-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.
K-Cl cotransporter expression in the human kidney
Authors: Liapis Helen , Nag M. , Kaji Deepak M.
Published: December 2017

The K-Cl cotransporter protein KCC1 is a membrane transport protein that mediates the coupled, electroneutral transport of K and Cl across plasma membranes. The precise cell type(s) in the kidney that express the K-Cl cotransporter have remained unknown. The aim of the present investigation was to define the distribution of KCC1 mRNA in the human kidney. We used in situ hybridization with a nonradioactive digoxigenin-labeled riboprobe. We identified abundant KCC1 mRNA expression in the epithelial cells throughout the distal and proximal renal tubular epithelium. The transporter was also expressed in glomerular mesangial cells and endothelial cells of the renal vessels. These findings suggest that the K-Cl cotransporter may have an important role in transepithelial K and Cl reabsorption.
Zinc deficiency decreases plasma level and hepatic mRNA abundance of apolipoprotein A-I in rats and hamsters
Authors: Wu John Y. J. , Reaves Scott K. , Wang Yi Ran , Wu Yan , Lei Polin P. , Lei Kai Y.
Published: December 2017

The influence of Zn deficiency on the plasma level as well as the hepatic and intestinal gene expression of apolipoprotein (apo) A-I was examined in rats and hamsters. Male Sprague-Dawley rats (8 wk old) and Golden Syrian hamsters (7 wk old) were assigned to three dietary treatments: Zn adequate (ZA, 30 mg Zn/kg diet), Zn deficient (ZD, <0.5 mg Zn/kg diet), and Zn replete (ZDA, ZD animals fed the ZA diet for the last 2 days). The dietary treatments lasted for 18 days for rats or 6 wk for hamsters. For the measurement of apoA-I mRNA abundance, hamster apoA-I cDNA was cloned from the small intestine. The full-length 905-base pair cDNA shared ∼80% similarity with the human, rat, and mouse apoA-I cDNAs. Hepatic and plasma Zn levels were reduced in ZD animals but normalized in ZDA rats and increased in ZDA hamsters compared with ZA animals. Zn deficiency reduced plasma apoA-I and hepatic apoA-I mRNA levels 13 and 38%, respectively, in ZD rats. The 2 days of Zn replenishment raised plasma apoA-I and hepatic apoA-I mRNA levels in ZDA rats by 34 and 28%, respectively, higher than ZA rats. Similarly, these levels were decreased by 18 and 25%, respectively, in ZD hamsters but normalized in ZDA hamsters compared with ZA hamsters. In contrast to the alterations of hepatic apoA-I mRNA levels, neither Zn deficiency nor subsequent Zn repletion produced alterations in the intestinal apoA-I mRNA abundance. Data from this study demonstrated that Zn deficiency specifically decreases hepatic apoA-I gene expression, which may at least be partly responsible for the reduction of plasma apoA-I levels.
Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel
Authors: Rojas Cecilia V. , Neely Alan , Velasco-Loyden Gabriela , Palma Verónica , Kukuljan Manuel
Published: December 2017

Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant α-subunits with the β1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near −40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.
Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-α and IFN-γ
Authors: Satriano Joseph , Ishizuka Shunji , Archer D. Clay , Blantz Roland C. , Kelly Carolyn J.
Published: December 2017

Nitric oxide (NO) has been described to exert cytostatic effects on cellular proliferation; however the mechanisms responsible for these effects have yet to be fully resolved. Polyamines, conversely, are required components of cellular proliferation. In experimental models of inflammation, a relationship between these two pathways has been suggested by the temporal regulation of a common precursor, arginine. This study was undertaken to determine the effects NO and the NO synthase (NOS)-inducing cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), exert on polyamine regulation. The transformed kidney proximal tubule cell line, MCT, maintains high constitutive levels of the first polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). NO donors markedly suppressed ODC activity in MCT and all other cell lines examined. TNF-α and IFN-γ induction of NO generation resulted in suppressed ODC activity, an effect prevented by the inducible NOS inhibitorl- N 6-(1-iminoethyl)lysine (l-NIL). Dithiothreitol reversal of NO-mediated ODC suppression supports nitrosylation as the mechanism of inactivation. We also evaluated polyamine uptake, inasmuch as inhibition of ODC can result in a compensatory induction of polyamine transporters. Administration of NO donors, or TNF-α and IFN-γ, suppressed [3H]putrescine uptake, thereby preventing transport-mediated reestablishment of intracellular polyamine levels. This study demonstrates the capacity of NO and inflammatory cytokines to regulate both polyamine biosynthesis and transport.
Purinergic and cholinergic agonists induce exocytosis from the same granule pool in HT29-Cl.16E monolayers
Authors: Bertrand C. A. , Laboisse C. L. , Hopfer U.
Published: December 2017

Several secretagogues induce mucin secretion in epithelial monolayers, as determined by measuring released granule contents. To assess whether different agonists act on the same granule pool, capacitance changes in intact monolayers of the goblet cell line HT29-Cl.16E were measured by a novel impedance method. Apical ATP (purinergic agonist) and basolateral carbachol (cholinergic agonist) induce rapid exocytosis with maximal capacitance changes within 3 min. The maximal levels of exocytosis that can be induced by optimal concentrations of either agonist are the same and produce a 30–40% increase in total monolayer capacitance. When ATP and carbachol are applied simultaneously, the magnitude of exocytosis is unchanged from the single-secretagogue level. The recovery of capacitance to baseline (endocytosis) is significantly faster after ATP stimulation than after carbachol stimulation. When ATP and carbachol are applied sequentially at doses that give maximal exocytosis, the magnitude of the capacitance increase produced by the second secretagogue is less than or equal to that of the capacitance decrease during the recovery period. Together, these data suggest that purinergic and cholinergic agonists act on the same granule pool.
Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells
Authors: Lin Hung-Yun , Davis Faith B. , Gordinier Jennifer K. , Martino Leon J. , Davis Paul J.
Published: December 2017

Thyroid hormone [l-thyroxine (T4)] rapidly induced phosphorylation and nuclear translocation (activation) of mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the absence of cytokine or growth factor. A pertussis toxin-sensitive and guanosine 5′- O-(3-thiotriphosphate)-sensitive cell surface mechanism responsive to T4 and agarose-T4, suggesting a G protein-coupled receptor, was implicated. Cells depleted of MAPK or treated with MAPK pathway inhibitors showed reduced activation of MAPK and of the signal transducer and activator of transcription STAT1α by T4; they also showed reduced T4 potentiation of the antiviral action of interferon-γ (IFN-γ). T4 treatment caused tyrosine-phosphorylated MAPK-STAT1α nuclear complex formation and enhanced Ser-727 phosphorylation of STAT1α, in the presence or absence of IFN-γ. STAT1α-deficient cells transfected with STAT1α containing an alanine-for-serine substitution at residue 727 (STAT1αA727) showed minimal T4-stimulated STAT1α activation. IFN-γ induced the antiviral state in cells containing wild-type STAT1α (STAT1αwt) or STAT1αA727; T4 potentiated IFN-γ action in STAT1αwt cells but not in STAT1αA727 cells. T4-directed STAT1α Ser-727 phosphorylation is MAPK mediated and results in potentiated STAT1α activation and enhanced IFN-γ activity.
Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting
Authors: Mitch William E. , Bailey James L. , Wang Xiaonan , Jurkovitz Claudine , Newby David , Price S. Russ
Published: December 2017

The ubiquitin-proteasome proteolytic system is stimulated in conditions causing muscle atrophy. Signals initiating this response in these conditions are unknown, although glucocorticoids are required but insufficient to stimulate muscle proteolysis in starvation, acidosis, and sepsis. To identify signals that activate this system, we studied acutely diabetic rats that had metabolic acidosis and increased corticosterone production. Protein degradation was increased 52% ( P < 0.05), and mRNA levels encoding ubiquitin-proteasome system components, including the ubiquitin-conjugating enzyme E214k, were higher (transcription of the ubiquitin and proteasome subunit C3 genes in muscle was increased by nuclear run-off assay). In diabetic rats, prevention of acidemia by oral NaHCO3 did not eliminate muscle proteolysis. Adrenalectomy blocked accelerated proteolysis and the rise in pathway mRNAs; both responses were restored by administration of a physiological dose of glucocorticoids to adrenalectomized, diabetic rats. Finally, treating diabetic rats with insulin for ≥24 h reversed muscle proteolysis and returned pathway mRNAs to control levels. Thus acidification is not necessary for these responses, but glucocorticoids and a low insulin level in tandem activate the ubiquitin-proteasome proteolytic system.
ATP dependence is not an intrinsic property of Na+/H+exchanger NHE1: requirement for an ancillary factor
Authors: Aharonovitz Orit , Demaurex Nicolas , Woodside Michael , Grinstein Sergio
Published: December 2017

Na+/H+exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibition of the antiport. No evidence has been found for direct binding of nucleotide to exchangers or alteration in their state of phosphorylation, suggesting ancillary factors may be involved. This possibility was tested by comparing the activity of dog red blood cells (RBC) and their resealed ghosts. Immunoblotting experiments using isoform-specific polyclonal and monoclonal antibodies indicated RBC membranes express Na+/H+exchanger isoform 1 (NHE1). In intact RBC, uptake of Na+ was greatly stimulated when the cytosol was acidified. The stimulated uptake was largely eliminated by amiloride and by submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also be elicited by acidification in resealed ghosts containing ATP, the absolute rate of transport was markedly diminished at comparable pH. Dissipation of the pH gradient was ruled out as the cause of diminished transport rate in ghosts. This was accomplished by a “pH clamping” procedure based on continued export of base equivalents by the endogenous anion exchanger. These observations suggest a critical factor required to maintain optimal Na+/H+exchange activity is lost or inactivated during preparation of ghosts. Depletion of ATP, achieved by incubation with 2-deoxy-d-glucose, inhibited Na+/H+exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate of Na+/H+exchange in ATP-depleted but otherwise intact cells was similar to the transport rate of ghosts. Therefore, we tentatively conclude that full activation of NHE1 requires both ATP and an additional regulatory factor, which may mediate the action of the nucleotide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) that is inactivated or missing in ghosts.
Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification
Authors: Wu Mei-Lin , Chen Jeng-Haur , Chen Wei-Hao , Chen Yu-Jen , Chu Kuan-Chou
Published: December 2017

The mechanism involved in N-methyl-d-glucamine (NMDA)-induced Ca2+-dependent intracellular acidosis is not clear. In this study, we investigated in detail several possible mechanisms using cultured rat cerebellar granule cells and microfluorometry [fura 2-AM or 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM]. When 100 μM NMDA or 40 mM KCl was added, a marked increase in the intracellular Ca2+ concentration ([Ca2+]i) and a decrease in the intracellular pH were seen. Acidosis was completely prevented by the use of Ca2+-free medium or 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca2+. The following four mechanisms that could conceivably have been involved were excluded: 1) Ca2+ displacement of intracellular H+ from common binding sites; 2) activation of an acid loader or inhibition of acid extruders; 3) overproduction of CO2 or lactate; and 4) collapse of the mitochondrial membrane potential due to Ca2+uptake, resulting in inhibition of cytosolic H+ uptake. However, NMDA/KCl-induced acidosis was largely prevented by glycolytic inhibitors (iodoacetate or deoxyglucose in glucose-free medium) or by inhibitors of the Ca2+-ATPase (i.e., Ca2+/H+exchanger), including La3+, orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca2+-ATPase is involved in NMDA-induced intracellular acidosis in granule cells. We also provide new evidence that NMDA-evoked intracellular acidosis probably serves as a negative feedback signal, probably with the acidification itself inhibiting the NMDA-induced [Ca2+]iincrease.
Anchoring protein is required for cAMP-dependent stimulation of L-type Ca2+ channels in rabbit portal vein
Authors: Zhong Juming , Hume Joseph R. , Keef Kathleen D.
Published: December 2017

Stimulation of cardiac L-type Ca2+ channels by cAMP-dependent protein kinase (PKA) requires anchoring of PKA to a specific subcellular environment by A-kinase anchoring proteins (AKAP). This study evaluated the possible requirement of AKAP in PKA-dependent regulation of L-type Ca2+ channels in vascular smooth muscle cells using the conventional whole cell patch-clamp technique. Peak Ba2+current in freshly isolated rabbit portal vein myocytes was significantly increased by superfusion with either 0.5 μM isoproterenol (131 ± 3% of the control value, n = 11) or 10 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP; 114 ± 1%, n = 8). The PKA-induced stimulatory effects of both isoproterenol and 8-BrcAMP were completely abolished by a specific PKA inhibitor KT-5720 (0.2 μM) or by dialyzing cells with Ht 31 (100 μM), a peptide that inhibits the binding of PKA to AKAP. In contrast, Ht 31 did not block the excitatory effect of the catalytic subunit of PKA when dialyzed into the cells. These data suggest that stimulation of Ca2+ channels in vascular myocytes by endogenous PKA requires localization of PKA through binding to AKAP.
Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells
Authors: Jayaraman Sujatha , Teitler Leah , Skalski Bohdan , Verkman A. S.
Published: December 2017

Limitations of available indicators [such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl− are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl− and/or I− sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(β-d-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400–470 and 490–560 nm, molar extinction 11,100 M−1 ⋅ cm−1(424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I− by a collisional mechanism (Stern-Volmer constant 60 M−1) and is not affected by other halides, nitrate, cations, or pH changes (pH 5–8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I− (50% quenching at 38 mM I−). LZQ was used to measure forskolin-stimulated I−/Cl−and I−/[Formula: see text]exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype.
Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter
Authors: Race Joanne E. , Makhlouf Fadi N. , Logue Paul J. , Wilson Frederick H. , Dunham Philip B. , Holtzman Eli J.
Published: December 2017

We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75–76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and ∼20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH2 and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.
Expression of antioxidant enzymes in human inflammatory cells
Authors: Pietarinen-Runtti Petra , Lakari Essi , Raivio Kari O. , Kinnula Vuokko L.
Published: December 2017

Because antioxidant enzymes may have an important role in the oxidant resistance of inflammatory cells, we investigated the mRNA levels and specific activities of manganese and copper-zinc superoxide dismutases (Mn SOD and Cu,Zn SOD), catalase (Cat), and glutathione peroxidase, as well as the concentrations of glutathione (GSH) in human neutrophils, monocytes, monocyte-derived macrophages, and alveolar macrophages. Levels of GSH and glutathione peroxidase activity in monocytes were three times higher than in neutrophils, whereas the mRNA of Cat was 50-fold and its specific activity 4-fold higher in neutrophils. Although Mn SOD mRNA levels were higher in neutrophils, enzyme activities, as well as those of Cu,Zn SOD, were similar in all phagocytic cells. Neutrophils lost their viability, assessed by adenine nucleotide depletion, within 24 h ex vivo and more rapidly if GSH was depleted. However, neutrophils were the most resistant cell type to exogenous H2O2. In conclusion, high Cat activity of neutrophils appears to explain their high resistance against exogenous H2O2, whereas low GSH content and GSH-related enzymes seem to account for the poor survival of human neutrophils.
Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway
Authors: Paolucci Lina , Sinnett-Smith James , Rozengurt Enrique
Published: December 2017

Protein kinase D (PKD) is a serine-threonine protein kinase with distinct structural features and enzymological properties. Herein we demonstrate that lysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss 3T3 and Rat-1 cells. LPA induced PKD activation in a concentration-dependent fashion with maximal stimulation (7.6-fold) achieved at 5 μM. Treatment of Swiss 3T3 cells with the protein kinase C (PKC) inhibitors GF-I, Ro-31–8220, and Gö-7874 completely abrogated PKD activation induced by LPA at concentrations that did not inhibit PKD activity when added directly to the in vitro kinase assays. PKD activation induced by LPA was attenuated markedly and selectively by prior exposure of either Swiss 3T3 or Rat-1 cells to pertussis toxin (PTx) in a concentration-dependent manner. In contrast, treatment with the protein tyrosine kinase inhibitor genistein, the MEK inhibitor PD-098059, or the phosphoinositide 3-kinase inhibitor wortmannin did not affect PKD activation in response to LPA. These results provide the first example of PTx-sensitive and PKC-dependent PKD activation and identify a novel Gi-dependent event in the action of LPA.
PKA holoenzyme is functionally coupled to CFTR by AKAPs
Authors: Huang P. , Trotter K. , Boucher R. C. , Milgram S. L. , Stutts M. J.
Published: December 2017

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 ± 0.12 to 1.23 ± 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.
Coordinate expression of secretory phospholipase A2 and cyclooxygenase-2 in activated human keratinocytes
Authors: Rys-Sikora Krystyna E. , Konger Raymond L. , Schoggins John W. , Malaviya Rama , Pentland Alice P.
Published: December 2017

PGE2 levels are altered in human epidermis after in vivo wounding; however, mechanisms modulating PGE2 production in activated keratinocytes are unclear. In previous studies, we showed that PGE2 is a growth-promoting autacoid in human primary keratinocyte cultures, and its production is modulated by plating density, suggesting that regulated PGE2 synthesis is an important component of wound healing. Here, we examine the role of phospholipase A2(PLA2) and cyclooxygenase (COX) enzymes in modulation of PGE2 production. We report that the increased PGE2 production that occurs in keratinocytes grown in nonconfluent conditions is also observed after in vitro wounding, indicating that similar mechanisms are involved. This increase was associated with coordinate upregulation of both COX-2 and secretory PLA2 (sPLA2) proteins. Increased sPLA2 activity was also observed. By RT-PCR, we identified the presence of type IIA and type V sPLA2, along with the M-type sPLA2 receptor. Thus the coordinate expression of sPLA2 and COX-2 may be responsible for the increased prostaglandin synthesis in activated keratinocytes during wound repair.
Aldosterone induces Ras methylation in A6 epithelia
Authors: Al-Baldawi N. F. , Stockand J. D. , Al-Khalili O. K. , Yue G. , Eaton D. C.
Published: December 2017

Aldosterone increases Na+ reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/μg protein. Aldosterone stimulates protein methylation by increasing methyltransferase activity from 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/μg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the aldosterone-induced increase in methyltransferase activity. One of these inhibitors, the isoprenyl-cysteine methyltransferase-specific inhibitor, S- trans, trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces protein methylation in two molecular weight ranges: near 90 kDa and around 20 kDa. The lower molecular weight range is the weight of small G proteins, and aldosterone does increase both Ras protein 1.6-fold and Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides reduce the activity of Na+ channels by about fivefold. We conclude that 1) protein methylation is essential for aldosterone-induced increases in Na+ transport; 2) one target for methylation is p21ras; and 3) inhibition of Ras expression or Ras methylation inhibits Na+ channel activity.
Selective mobilization of CD14+CD16+ monocytes by exercise
Authors: Steppich Birgit , Dayyani Farshid , Gruber Rudolf , Lorenz Reinhard , Mack Matthias , Ziegler-Heitbrock H. W. Löms
Published: December 2017

Strenuous, anaerobic exercise leads to an increase of leukocytes that are mobilized from the marginal pool. We have analyzed in human peripheral blood the effect of exercise on the number of CD14+CD16+ monocytes as determined by two-color immunofluorescence and flow cytometry. We show herein that this type of monocyte responds with a dramatic up to 4.8-fold increase. Mobilization does not occur after 1 min at 100 or 200 W but 1 min at 400 W leads to a twofold increase of the CD14+CD16+ monocytes immediately after exercise. The numbers remain high at 5 min and gradually decrease to reach the initial level at 20 min postexercise. After 20 min of rest, the CD14+CD16+ monocytes can be mobilized again by a second exercise. The CD14+CD16+ monocytes appear to be mobilized from the marginal pool where they preferentially home because of a higher expression of adhesion molecules like CD11d and very late antigen-4. Exercise goes along with an increase of catecholamines, and mobilization of the CD14+CD16+ monocytes can be substantially reduced by treatment of donors with the β-adrenergic receptor blocker propranolol. Mobilization of CD14+CD16+monocytes by a catecholamine-dependent mechanism may contribute to the increase of these cells in various clinical conditions.
Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling
Authors: Rizoli Sandro B. , Rotstein Ori D. , Parodo Jean , Phillips M. James , Kapus Andras
Published: December 2017

Hypertonicity suppresses neutrophil functions by unknown mechanisms. We investigated whether osmotically induced cytoskeletal changes might be related to the hypertonic inhibition of exocytosis. Hyperosmolarity abrogated the mobilization of all four granule types induced by diverse stimuli, suggesting that it blocks the process of exocytosis itself rather than individual signaling pathways. Concomitantly, osmotic stress provoked a twofold increase in F-actin, induced the formation of a submembranous F-actin ring, and abolished depolymerization that normally follows agonist-induced actin assembly. Several observations suggest a causal relationship between actin polymerization and inhibition of exocytosis: 1) prestimulus actin levels were inversely proportional to the stimulus-induced degranulation, 2) latrunculin B (LB) prevented the osmotic actin response and restored exocytosis, and 3) actin polymerization induced by jasplakinolide inhibited exocytosis under isotonic conditions. The shrinkage-induced tyrosine phosphorylation and the activation of the Na+/H+ exchanger were not affected by LB. Inhibition of osmosensitive kinases failed to prevent the F-actin change, suggesting that the osmotic tyrosine phosphorylation and actin polymerization are independent phenomena. Thus cytoskeletal remodeling appears to be a key component in the neutrophil-suppressive, anti-inflammatory effects of hypertonicity.
gp120-induced alterations of human astrocyte function: Na+/H+ exchange, K+conductance, and glutamate flux
Authors: Patton Holly K. , Zhou Zhen-Hong , Bubien James K. , Benveniste Etty N. , Benos Dale J.
Published: December 2017

Many human immunodeficiency virus (HIV)-infected patients suffer from impaired neurological function and dementia. This facet of the disease has been termed acquired immunodeficiency syndrome (AIDS)-associated dementia complex (ADC). Several cell types, including astrocytes and neurons, are not productively infected by virus but are involved in ADC pathophysiology. Previous studies of rat astrocytes showed that an HIV coat protein (gp120) accelerated astrocyte Na+/H+ exchange and that the resultant intracellular alkalinization activated a pH-sensitive K+ conductance. The present experiments were conducted to determine whether gp120 affected human astrocytes in the same fashion. It was found that primary human astrocytes express a pH-sensitive K+ conductance that was activated on intracellular alkalinization. Also, gp120 treatment of whole cell clamped human astrocytes activated this conductance specifically. Furthermore, gp120 inhibited glutamate uptake by primary human astrocytes. These altered physiological processes could contribute to pathophysiological changes in HIV-infected brains. Because the gp120-induced cell physiological changes were partially inhibited by dimethylamiloride (an inhibitor of Na+/H+exchange), our findings suggest that modification of human astrocyte Na+/H+ exchange activity may provide a means of addressing some of the neurological complications of HIV infection.
The role of hydroxyl radical as a messenger in Cr(VI)-induced p53 activation
Authors: Wang Suwei , Leonard Stephen S. , Ye Jianping , Ding Min , Shi Xianglin
Published: December 2017

The present study investigates whether reactive oxygen species (ROS) are involved in p53 activation, and if they are, which species is responsible for the activation. Our hypothesis is that hydroxyl radical (·OH) functions as a messenger for the activation of this tumor suppressor protein. Human lung epithelial cells (A549) were used to test this hypothesis. Cr(VI) was employed as the source of ROS due to its ability to generate a whole spectrum of ROS inside the cell. Cr(VI) is able to activate p53 by increasing the protein levels and enhancing both the DNA binding activity and transactivation ability of the protein. Increased cellular levels of superoxide radicals (O2 −·), hydrogen peroxide (H2O2), and ·OH radicals were detected on the addition of Cr(VI) to the cells. Superoxide dismutase, by enhancing the production of H2O2 from O2 −· radicals, increased p53 activity. Catalase, an H2O2 scavenger, eliminated ·OH radical generation and inhibited p53 activation. Sodium formate and aspirin, ·OH radical scavengers, also suppressed p53 activation. Deferoxamine, a metal chelator, inhibited p53 activation by chelating Cr(V) to make it incapable of generating radicals from H2O2. NADPH, which accelerated the one-electron reduction of Cr(VI) to Cr(V) and increased ·OH radical generation, dramatically enhanced p53 activation. Thus ·OH radical generated from Cr(VI) reduction in A549 cells is responsible for Cr(VI)-induced p53 activation.
Transport and function of syntaxin 3 in human epithelial intestinal cells
Authors: Breuza Lionel , Fransen Jack , Le Bivic André
Published: December 2017

To follow the transport of human syntaxin (Syn) 3 to the apical surface of intestinal cells, we produced and expressed in Caco-2 cells a chimera made of the entire Syn3 coding sequence and the extracellular domain of the human transferrin receptor (TfR). This chimera (Syn3TfR) was localized to the apical membrane and was transported along the direct apical pathway, suggesting that this is also the case for endogenous Syn3. To test the potential role of Syn3 in apical transport, we overexpressed it in Caco-2 cells and measured the efficiency of apical and basolateral delivery of several endogenous markers. We observed a strong inhibition of apical delivery of sucrase-isomaltase (SI), an apical transmembrane protein, and of α-glucosidase, an apically secreted protein. No effect was observed on the basolateral delivery of Ag525, a basolateral antigen, strongly suggesting that Syn3 is necessary for efficient delivery of proteins to the apical surface of intestinal cells.
Effect of antioxidant protection by p-coumaric acid on low-density lipoprotein cholesterol oxidation
Authors: Zang Lun-Yi , Cosma Greg , Gardner Henry , Shi Xianglin , Castranova Vince , Vallyathan Val
Published: December 2017

Mechanisms in which p-coumaric acid (CA) acts as an antioxidant are not well understood. This study investigated whether CA can act as a direct scavenger of reactive oxygen species (ROS) and whether it minimizes the oxidation of low-density lipoprotein (LDL). Rats were administered CA in drinking water at low or high doses for 10, 21, and 30 days (uptakes were 29 and 317 mg/day, respectively). Blood levels of 8-epiprostaglandin F2α were monitored as a marker of LDL oxidation. Oral administration of CA (317 mg/day) for 30 days significantly inhibited LDL oxidation. CA also reduced LDL cholesterol levels in serum but had no effect on levels of high-density lipoprotein cholesterol. In vitro studies that used electron spin resonance in combination with spin trapping techniques were used to determine the ability of CA to scavenge ROS and alter LDL oxidation. CA effectively scavenged ·OH in a dose-dependent manner. IC50 and maximum velocity for CA scavenging of ·OH were 4.72 μM and 1.2 μM/s, respectively, with a rate constant of 1.8 × 1011 M−1 · s−1. Our studies suggest that the antioxidant properties of CA may involve the direct scavenging of ROS such as ·OH.
Substrate flexibility regulates growth and apoptosis of normal but not transformed cells
Authors: Wang Hong-Bei , Dembo Micah , Wang Yu-Li
Published: December 2017

One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H- ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.
Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat
Authors: Stevens Laurence , Firinga Carole , Gohlsch Bärbel , Bastide Bruno , Mounier Yvonne , Pette Dirk
Published: December 2017

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the β2-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb (“fast-to-faster transitions”). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.
Postnatal expression and activity of the mitochondrial 2-oxoglutarate-malate carrier in intact hearts
Authors: Griffin J. L. , O'Donnell J. M. , White L. T. , Hajjar R. J. , Lewandowski E. D.
Published: December 2017

This study examines the functional implications of postnatal changes in the expression of the mitochondrial transporter protein, 2-oxoglutarate-malate carrier (OMC). Online 13C nuclear magnetic resonance (13C NMR) measurements of isotope kinetics in hearts from neonate (3–4 days) and adult rabbits provided tricarboxylic acid cycle flux rates and flux rates through OMC. Neonate and adult hearts oxidizing 2.5 mM [2,4-13C2]butyrate were subjected to either normal or high cytosolic redox state (2.5 mM lactate) conditions to evaluate the recruitment of malate-aspartate activity and the resulting OMC flux. During development from neonate (3–4 days) to adult, mitochondrial protein density in the heart increased from 19 ± 3% to 31 ± 2%, whereas OMC expression decreased by 65% per mitochondrial protein content ( P < 0.05). Correspondingly, OMC flux was lower in adults hearts than in neonates by 73% (neonate = 7.4 ± 0.4, adult = 2.0 ± 0.1 μmol/min per 100 mg mitochondrial protein; P < 0.05). Despite clear changes in OMC content and flux, the responsiveness of the malate-aspartate shuttle to increased cytosolic NADH was similar in both adults and neonates with an approximate threefold increase in OMC flux (in densitometric units/100 mg mitochondrial protein: neonate = 25.8 ± 2.5, adult = 6.0 ± 0.2; P < 0.05). The13C NMR data demonstrate that OMC activity is a principal component of the rate of labeling of glutamate.
Integrin mobilizes intracellular Ca2+ in renal vascular smooth muscle cells
Authors: Chan Wah-Lun , Holstein-Rathlou N.-H. , Yip Kay-Pong
Published: December 2017

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca2+ signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca2+]i rapidly increased from 91 ± 4 to 287 ± 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca2+mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca2+]i, which propagated as Ca2+ waves traveling across the cell and induced a rapid elevation of nuclear [Ca2+]i. Spontaneous recurrence of smaller-amplitude Ca2+ waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca2+ channels with nifedipine or removal of extracellular Ca2+ did not inhibit the RGD-induced Ca2+mobilization. However, pretreatment of 20 μM ryanodine completely eliminated the RGD-induced Ca2+ mobilization. Anti-β1 and anti-β3-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca2+]i. Incubation with anti-β1- or β3-integrin antibodies inhibited the increase in [Ca2+]i induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca2+ from ryanodine-sensitive Ca2+stores in renal VSMC via integrins, which can trigger cytoplasmic Ca2+ waves propagating throughout the cell.
Bitter taste transduced by PLC-β2-dependent rise in IP3 and α-gustducin-dependent fall in cyclic nucleotides
Authors: Yan Wentao , Sunavala Gulshan , Rosenzweig Sophia , Dasso Max , Brand Joseph G. , Spielman Andrew I.
Published: December 2017

Current evidence points to the existence of multiple processes for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an α-gustducin (Gαgust)-mediated stimulation of phosphodiesterase in bitter taste transduction. Additionally, a taste-enriched G protein γ-subunit, Gγ13, colocalizes with Gαgustand mediates the denatonium-stimulated production of inositol 1,4,5-trisphosphate (IP3). Using quench-flow techniques, we show here that the bitter stimuli, denatonium and strychnine, induce rapid (50–100 ms) and transient reductions in cAMP and cGMP and increases in IP3 in murine taste tissue. This decrease of cyclic nucleotides is inhibited by Gαgust antibodies, whereas the increase in IP3 is not affected by antibodies to Gαgust. IP3 production is inhibited by antibodies specific to phospholipase C-β2(PLC-β2), a PLC isoform known to be activated by Gβγ-subunits. Antibodies to PLC-β3 or to PLC-β4 were without effect. These data suggest a transduction mechanism for bitter taste involving the rapid and transient metabolism of dual second messenger systems, both mediated through a taste cell G protein, likely composed of Gαgust/β/γ13, with both systems being simultaneously activated in the same bitter-sensitive taste receptor cell.
Circulating monocytes are not the source of elevations in plasma IL-6 and TNF-α levels after prolonged running
Authors: Starkie R. L. , Rolland J. , Angus D. J. , Anderson M. J. , Febbraio M. A.
Published: December 2017

The present study was undertaken to examine the effect of prolonged running on monocyte intracellular cytokine production and plasma cytokine concentration. Blood samples were collected 1 h before, immediately after, 2 h after, and 24 h after a competitive marathon run. There was no change in the number of cells spontaneously producing tumor necrosis factor (TNF)-α; however, there was a decrease in the number of cells producing interleukin (IL)-1α and IL-6 ( P < 0.01) postexercise. In contrast, there was an increase in the number of monocytes that responded to lipopolysaccharide stimulation by producing IL-1α, TNF-α, and IL-6 ( P < 0.01) immediately and 2 h postexercise; however, these cells contained less cytokine ( P < 0.05). Plasma IL-6, TNF-α, epinephrine, norepinephrine, and cortisol concentrations were markedly increased ( P < 0.01) postexercise. These data demonstrate that circulating monocytes are not the source of elevated levels of plasma IL-6 and TNF-α after prolonged running. In addition, it is likely that stress hormones result in a decrease in the amount of cytokine produced by LPS-stimulated cells postexercise.