bluuebunny/crossref_metadata_2025 huggingface dataset:
K+ current inhibition by amphiphilic fatty acid metabolites in rat ventricular myocytes
Authors: Xu Zhi , Rozanski George J.
Published: December 2017

Fatty acid metabolites accumulate in the heart under pathophysiological conditions that affect β-oxidation and can elicit marked electrophysiological changes that are arrhythmogenic. The purpose of the present study was to determine the impact of amphiphilic fatty acid metabolites on K+currents that control cardiac refractoriness and excitability. Transient outward ( I to) and inward rectifier ( I K1) K+ currents were recorded by the whole cell voltage-clamp technique in rat ventricular myocytes, and the effects of two major fatty acid metabolites were examined: palmitoylcarnitine and palmitoyl-coenzyme A (palmitoyl-CoA). Palmitoylcarnitine (0.5–10 μM) caused a concentration-dependent decrease in I todensity in myocytes internally dialyzed with the amphiphile; 10 μM reduced mean I todensity at +60 mV by 62% compared with control ( P < 0.05). In contrast, external palmitoylcarnitine at the same concentrations had no effect, nor did internal dialysis significantly alter I K1. Dialysis with palmitoyl-CoA (1–10 μM) produced a smaller decrease in I to density compared with that produced by palmitoylcarnitine; 10 μM reduced mean I to density at +60 mV by 37% compared with control ( P < 0.05). Both metabolites delayed recovery of I tofrom inactivation but did not affect voltage-dependent properties. Moreover, the effects of palmitoylcarnitine were relatively specific, as neither palmitate (10 μM) nor carnitine (10 μM) alone significantly influenced I to when added to the pipette solution. These data therefore suggest that amphiphilic fatty acid metabolites downregulate I to channels by a mechanism confined to the cytoplasmic side of the membrane. This decrease in cardiac K+ channel activity may delay repolarization under pathophysiological conditions in which amphiphile accumulation is postulated to occur, such as diabetes mellitus or myocardial infarction.
ABC transporter-facilitated ATP conductive transport
Authors: Schwiebert Erik M.
Published: December 2017

The concept that the cystic fibrosis (CF) transmembrane conductance regulator, the protein product of the CF gene, can conduct larger multivalent anions such as ATP as well as Cl− is controversial. In this review, I examine briefly past findings that resulted in controversy. It is not the goal of this review to revisit these disparate findings in detail. Rather, I focus intently on more recent studies, current studies in progress, and possible future directions that arose from the controversy and that may reconcile this issue. Important questions and hypotheses are raised as to the physiological roles that ATP-binding cassette (ABC) transporter-facilitated ATP transport and signaling may play in the control of epithelial cell function. Perhaps the identification of key biological paradigms for ABC transporter-mediated extracellular nucleotide signaling may unify and guide the CF research community and other research groups interested in ABC transporters toward understanding why ABC transporters facilitate ATP transport.
Modulation of the sarcolemmal L-type current by alteration in SR Ca2+ release
Authors: Balog Edward M. , Gallant Esther M.
Published: December 2017

Modulation of the L-type current by sarcoplasmic reticulum (SR) Ca2+ release has been examined in patch-clamped mouse myotubes. Inhibition of SR Ca2+ release by inclusion of ryanodine in the internal solution shifted the half-activating voltage ( V 0.5) of the L-type current from 1.1 ± 2.1 to −7.7 ± 1.7 mV. Ruthenium red in the internal solution shifted V 0.5 from 5.4 ± 1.9 to −3.2 ± 4.1 mV. Chelation of myoplasmic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid perfusion shifted V 0.5 from 4.4 ± 1.7 to −3.5 ± 3.3 mV and increased the peak current. Extracellular caffeine (1 mM), which should enhance SR Ca2+ release, significantly decreased the peak Ca2+ current. In low (0.1 mM) internal EGTA, myotube contraction was abolished by internal perfusion with ryanodine or ruthenium red, whereas addition of caffeine to the extracellular solution lowered the contractile threshold, indicating that these modulators of SR Ca2+ release had the expected effects on contraction. Therefore, SR Ca2+ release appears to modulate the sarcolemmal L-type current, suggesting a retrograde communication from the SR to the sarcolemmal L-type channels in excitation-contraction coupling.
Effects of ramp shortening during linear phase of relaxation on [Ca2+]i in intact skeletal muscle fibers
Authors: Jiang Yandong , Julian Fred J.
Published: December 2017

The effects of shortening distance at V u, the unloaded shortening speed, and filament overlap on the amount of extra Ca2+ released during relaxation in muscle, as indicated by the bump area, were studied. Single, intact frog skeletal muscle fibers at 3°C were used. The myoplasmic free Ca2+ concentration ([Ca2+]i) was estimated by using fura 2 salt injected into the myoplasm. Ramps were applied, either at full overlap with different sizes or at varying overlaps with a fixed size, in the linear phase of relaxation. At full overlap, a plot of bump area vs. ramp size was fit by using a sigmoidal curve with one-half of the bump area equal to 25.9 nm. With a fixed ramp size of 100 nm/half-sarcomere, the plot of bump area vs. mean sarcomere length (SLm) was fit by a straight line intersecting the SLm axis at ∼3.5 μm, close to just no overlap. The results suggest that the transition in the distribution of attached cross bridges from the isometric case to one appropriate for unloaded shortening at V u is completed within 50 nm/half-sarcomere and support the view that attached cross bridges in the overlap zone influence the affinity of Ca2+for troponin C in the thin filament.
Swelling activation of transport pathways in erythrocytes: effects of Cl−, ionic strength, and volume changes
Authors: Guizouarn Hélène , Motais René
Published: December 2017

If swelling of a cell is induced by a decrease in external medium tonicity, the regulatory response is more complex than if swelling of similar magnitude is due to salt uptake. The present results provide an explanation. In fish erythrocytes, two distinct transport pathways were swelling activated: a channel of broad specificity and a K+-Cl−cotransporter. Each was activated by a specific signal: the channel by a decrease in intracellular ionic strength and the K+-Cl−cotransporter by cell enlargement. A decrease in ionic strength also affected K+-Cl−cotransport activity, but by acting as a negative modulator of the cotransport. Thus cells swollen by salt accumulation respond by activating exclusively the K+-Cl−cotransport, leading to a Cl−-dependent K+ loss. By contrast, cells swollen by electrolyte dilution respond by activating both pathways, leading to a reduced loss of electrolytes and a large loss of taurine. Thus two swelling-sensitive pathways, differently regulated, would allow control of the ionic composition of a cell exposed to different volume perturbations.
Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells
Authors: Fomin Victor P. , Cox Blair E. , Word R. Ann
Published: December 2017

Although it is well known that progesterone alters uterine contractility and plays an important role in maintenance of pregnancy, the biochemical mechanisms by which progesterone alters uterine contractility in human gestation are less clear. In this investigation we sought to identify progesterone-induced adaptations in human myometrial smooth muscle cells that may alter Ca2+signaling in response to contractile agents. Cells were treated with vehicle or the progesterone analog medroxyprogesterone acetate (MPA) for 5 days, and intracellular free Ca2+ concentration ([Ca2+]i) was quantified after treatment with oxytocin (OX) or endothelin (ET)-1. OX- and ET-1-induced increases in [Ca2+]iwere significantly attenuated in cells pretreated with MPA in a dose-dependent manner. Progesterone receptor antagonists prevented the attenuated Ca2+ transients induced by MPA. ETA and ETB receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted in significant downregulation of ETAand ETB receptor binding. MPA did not alter ionomycin-stimulated increases in [Ca2+]iand had no effect on inositol trisphosphate-dependent or -independent release of Ca2+ from internal Ca2+ stores. We conclude that adaptations of Ca2+ homeostasis in myometrial cells during pregnancy may include progesterone-induced modification of receptor-mediated increases in [Ca2+]i.
Phenotypic abnormalities in macrophages from leptin-deficient, obese mice
Authors: Lee Fung-Yee Janet , Li Yunbo , Yang Eun K. , Yang Shi Qi , Lin Hui Zhi , Trush Michael A. , Dannenberg Andrew J. , Diehl Anna Mae
Published: December 2017

Obesity is a complex syndrome that involves defective signaling by a number of different factors that regulate appetite and energy homeostasis. Treatment with exogenous leptin reverses hyperphagia and obesity in ob/ obmice, which have a mutation that causes leptin deficiency, proving the importance of this factor and its receptors in the obesity syndrome. Cells with leptin receptors have been identified outside of the appetite regulatory centers in the brain. Thus leptin has peripheral targets. Because macrophages express signaling-competent leptin receptors, these cells may be altered during chronic leptin deficiency. Consistent with this concept, the present study identifies several phenotypic abnormalities in macrophages from ob/ obmice, including decreased steady-state levels of uncoupling protein-2 mRNA, increased mitochondrial production of superoxide and hydrogen peroxide, constitutive activation of CCAAT enhancer binding protein (C/EBP)-β, an oxidant-sensitive transcription factor, increased expression of interleukin-6 and cyclooxygenase (COX)-2, two C/EBP-β target genes, and increased COX-2-dependent production of PGE2. Given the importance of macrophages in the general regulation of inflammation and immunity, these alterations in macrophage function may contribute to obesity-related pathophysiology.
EGF induces nuclear translocation of STAT2 without tyrosine phosphorylation in intestinal epithelial cells
Authors: Johnson Leonard R. , McCormack Shirley A. , Yang Chuan-He , Pfeffer Susan R. , Pfeffer Lawrence M.
Published: December 2017

Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that bind to activated membrane receptors, undergo ligand-dependent phosphorylation on tyrosine residues, and translocate to the nucleus, where they induce transcription of specific genes in response to a variety of ligands, including cytokines and some growth factors. Using immunocytochemical and biochemical techniques, we investigated the localization and responses of STAT1 and STAT2 to epidermal growth factor (EGF) stimulation in IEC-6 intestinal epithelial cells and HeLa cells. These studies provide the first description of STAT activation and localization in response to EGF in intestinal epithelial cells and some novel findings regarding the activation and localization of STATs in general. These include the following. First, EGF promoted the tyrosine phosphorylation of STAT1 in IEC-6 cells and caused its translocation to the nucleus. Second, in the absence of EGF stimulation both STAT1 and STAT2 were localized to the Golgi apparatus in IEC-6 cells. Third, EGF caused the translocation of STAT2 to the nucleus in both IEC-6 and HeLa cells without inducing the tyrosine phosphorylation of STAT2.
Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland
Authors: Aller Stephen G. , Lombardo Ilise D. , Bhanot Sumeet , Forrest John N.
Published: December 2017

In the shark, C-type natriuretic peptide (CNP) is the only cardiac natriuretic hormone identified and is a potent activator of Cl− secretion in the rectal gland, an epithelial organ of this species that contains cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channels. We have cloned an ancestral CNP receptor (NPR-B) from the shark rectal gland that has an overall amino acid identity to the human homologue of 67%. The shark sequence maintains six extracellular Cys present in other NPR-B but lacks a glycosylation site and a Glu residue previously considered important for CNP binding. When shark NPR-B and human CFTR were coexpressed in Xenopusoocytes, CNP increased the cGMP content of oocytes (EC50 12 nM) and activated CFTR Cl− channels (EC50 8 nM). Oocyte cGMP increased 36-fold (from 0.11 ± 0.03 to 4.03 ± 0.45 pmol/oocyte) and Cl− current increased 37-fold (from −34 ± 14 to −1,226 ± 151 nA) in the presence of 50 nM CNP. These findings identify the specific natriuretic peptide receptor responsible for Cl− secretion in the shark rectal gland and provide the first evidence for activation of CFTR Cl− channels by a cloned NPR-B receptor.
GAIP, a Gαi-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking
Authors: Wylie Fiona , Heimann Kirsten , Le Tam Luan , Brown Darren , Rabnott Glenn , Stow Jennifer L.
Published: December 2017

Proteins of the regulators of G protein signaling (RGS) family bind to Gα subunits to downregulate their signaling in a variety of systems. Gα-interacting protein (GAIP) is a mammalian RGS protein that shows high affinity for the activated state of Gαi-3, a protein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP in epithelial cells and to investigate its potential role in the regulation of membrane trafficking. LLC-PK1 cells were stably transfected with a c- myc-tagged GAIP cDNA. In the transfected and untransfected cells, GAIP was found in the cytosol and on cell membranes. Immunogold labeling showed that membrane-bound GAIP was localized on budding vesicles around Golgi stacks. When an in vitro assay was used to generate vesicles from isolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgi vesicles. Finally, the constitutive trafficking and secretion of sulfated proteoglycans was measured in cell lines overexpressing GAIP. We show evidence for GAIP regulation of secretory trafficking before the level of the trans-Golgi network but not in post-Golgi secretion. The location and functional effects of GAIP overlap only partially with those of Gαi-3 and suggest multiple roles for GAIP in epithelial cells.
NADPH oxidase inhibition does not interfere with low P O 2 transduction in rat and rabbit CB chemoreceptor cells
Authors: Obeso A. , Gómez-Niño A. , Gonzalez C.
Published: December 2017

The aim of the present work was to elucidate the role of NADPH oxidase in hypoxia sensing and transduction in the carotid body (CB) chemoreceptor cells. We have studied the effects of several inhibitors of NADPH oxidase on the normoxic and hypoxia-induced release of [3H]catecholamines (CA) in an in vitro preparation of intact CB of the rat and rabbit whose CA deposits have been labeled by prior incubation with the natural precursor [3H]tyrosine. It was found that diphenyleneiodonium (DPI; 0.2–25 μM), an inhibitor of NADPH oxidase, caused a dose-dependent release of [3H]CA from normoxic CB chemoreceptor cells. Contrary to hypoxia, DPI-evoked release was only partially Ca2+ dependent. Concentrations of DPI reported to produce full inhibition of NADPH oxidase in the rat CB did not prevent the hypoxic release response in the rat and rabbit CB chemoreceptor cells, as stimulation with hypoxia in the presence of DPI elicited a response equaling the sum of that produced by DPI and hypoxia applied separately. Neopterin (3–300 μM) and phenylarsine oxide (0.5–2 μM), other inhibitors of NADPH oxidase, did not promote release of [3H]CA in normoxic conditions or affect the response elicited by hypoxia. On the basis of effects of neopterin and phenylarsine oxide, it is concluded that NADPH oxidase does not appear to play a role in oxygen sensing or transduction in the rat and rabbit CB chemoreceptor cells in vitro and, in the context of the present study, that DPI effects are not related to NADPH oxidase inhibition.
Expression and localization of aquaporins in rat gastrointestinal tract
Authors: Koyama Yu , Yamamoto Tadashi , Tani Tatsuo , Nihei Kouei , Kondo Daisuke , Funaki Haruko , Yaoita Eishin , Kawasaki Katsutoshi , Sato Nobuaki , Hatakeyama Katsuyoshi , Kihara Itaru
Published: December 2017

A family of water-selective channels, aquaporins (AQP), has been demonstrated in various organs and tissues. However, the localization and expression of the AQP family members in the gastrointestinal tract have not been entirely elucidated. This study aimed to demonstrate the expression and distribution of several types of the AQP family and to speculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1–5 and AQP8 was examined in various portions through the gastrointestinal tract. AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon, and their expression was relatively intense in the small intestine and colon. In contrast, AQP4 mRNA was selectively expressed in the stomach and small intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated cellular localization of these AQP in these portions. AQP1 was localized on endothelial cells of lymphatic vessels in the submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma membranes of stratified squamous epithelial cells in the esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in the colon. However, AQP3 was not apparently detected in the small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland cells in the small intestine. AQP8 mRNA expression was demonstrated in the absorptive columnar epithelial cells of the jejunum and colon by in situ hybridization. These findings may indicate that water crosses the epithelial layer through these water channels, suggesting a possible role of the transcellular route for water intake or outlet in the gastrointestinal tract.
Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation
Authors: Wiederkehr Michael R. , Zhao Hui , Moe Orson W.
Published: December 2017

Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.
Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6
Authors: Berry Susan A. , Bergad Pearl L. , Stolz Allison M. , Towle Howard C. , Schwarzenberg Sarah Jane
Published: December 2017

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a γ-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the −319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5.4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.
Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage
Authors: Angele Martin K. , Knöferl Markus W. , Schwacha Martin G. , Ayala Alfred , Cioffi William G. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

Studies indicate that macrophage immune responses in males are depressed after trauma-hemorrhage, whereas they are enhanced in females under such conditions. Nonetheless, the involvement of male and female sex steroids in this gender-dependent dimorphic immune response after trauma-hemorrhage remains unclear. To study this, male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5α-dihydrotestosterone (DHT), 17β-estradiol, or a combination of both steroid hormones for 14 days before soft tissue trauma (i.e., laparotomy) and hemorrhagic shock (35 ± 5 mmHg for 90 min followed by adequate fluid resuscitation) or a sham operation. Twenty-four hours later the animals were killed, plasma was obtained, and Kupffer cell and splenic and peritoneal macrophage cultures were established. For DHT-treated mice, we observed significantly decreased releases of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-6 by splenic macrophage (−50 and −57%, respectively) and peritoneal macrophage (−51 and −52%, respectively) cultures after trauma-hemorrhage compared with releases by cultures of cells from mice subjected to a sham operation; in contrast, responses of splenic and peritoneal macrophage cultures from other groups subjected to trauma-hemorrhage did not change significantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release of IL-10 in DHT-treated hemorrhaged animals was increased compared with that in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and female sex steroids exhibit divergent immunomodulatory properties with respect to cell-mediated immune responses after trauma-hemorrhage.
Expression of PKA inhibitor (PKI) gene abolishes cAMPmediated protection to endothelial barrier dysfunction
Authors: Lum Hazel , Jaffe H. Ari , Schulz Irena T. , Masood Anwar , RayChaudhury Amlan , Green Richard D.
Published: December 2017

We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1−, E3−, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor ( PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 μM thrombin increased transendothelial albumin clearance rate (0.012 ± 0.003 and 0.035 ± 0.005 μl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.
Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2
Authors: Said Hamid M. , Ortiz Alvaro , Kumar Chandira K. , Chatterjee Nabendu , Dudeja Pradeep K. , Rubin Stanley
Published: December 2017

The present study examined the intestinal uptake of thiamine (vitamin B1) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) Na+ independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 μM and maximal velocity of 13.37 ± 0.94 pmol ⋅ mg protein−1 ⋅ 3 min−1; 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a Ca2+/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is Na+ independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells.
Endothelins activate Ca2+-gated K+ channels via endothelin B receptors in CD-1 mouse erythrocytes
Authors: Rivera Alicia , Rotter Michelle A. , Brugnara Carlo
Published: December 2017

Cell dehydration mediated by Ca2+-activated K+ channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca2+-activated K+ channel (Gardos channel) with maximal velocity ( V max) of 0.154 ± 0.02 mmol ⋅ l cells−1 ⋅ min−1and an affinity constant ( K 0.5) for Ca2+ of 286 ± 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V max by 88 ± 9% ( n = 8) and decreased their K 0.5 for Ca2+ to 139 ± 63 nM ( P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC50 of 75 ± 20 nM for ET-1 and 374 ± 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 ± 51 and 785 ± 143 nM and with capacity of binding sites of 298 ± 38 and 1,568 ± 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ETB) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ETB receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ETB receptors and a calphostin-sensitive mechanism.
Effects of macromolecular transport and stochastic fluctuations on dynamics of genetic regulatory systems
Authors: Smolen Paul , Baxter Douglas A. , Byrne John H.
Published: December 2017

To predict the dynamics of genetic regulation, it may be necessary to consider macromolecular transport and stochastic fluctuations in macromolecule numbers. Transport can be diffusive or active, and in some cases a time delay might suffice to model active transport. We characterize major differences in the dynamics of model genetic systems when diffusive transport of mRNA and protein was compared with transport modeled as a time delay. Delays allow for history-dependent, non-Markovian responses to stimuli (i.e., “molecular memory”). Diffusion suppresses oscillations, whereas delays tend to create oscillations. When simulating essential elements of circadian oscillators, we found the delay between transcription and translation necessary for oscillations. Stochastic fluctuations tend to destabilize and thereby mask steady states with few molecules. This computational approach, combined with experiments, should provide a fruitful conceptual framework for investigating the function and dynamic properties of genetic regulatory systems.
Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes
Authors: Bize Isabel , Güvenç Birol , Robb Aeisha , Buchbinder Guido , Brugnara Carlo
Published: December 2017

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 ± 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 ± 10% per 100 meq/l). Increases in membrane PP2A activity (270 ± 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 ± 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.
Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between Gq and Gs
Authors: Meszaros J. Gary , Gonzalez Annette M. , Endo-Mochizuki Yuka , Villegas Sonia , Villarreal Francisco , Brunton Laurence L.
Published: December 2017

Cardiac fibroblasts (CFs) are an important cellular component of myocardial responses to injury and to hypertrophic stimuli. We studied G protein-coupled receptors to understand how CFs integrate signals that activate Gq, Gs, and Gi. We predicted that the second messenger pathways present in CFs were distinct from those in cardiac myocytes and that unique signaling interactions existed in the CFs. ANG II, bradykinin, ATP, and UTP stimulated inositol phosphate (IP) production 2.2- to 7-fold. Each of these agonists elevated intracellular Ca2+ concentration ([Ca2+]i) via release from the intracellular Ca2+ storage compartment. Endothelin-1 (ET-1), carbachol, and norepinephrine failed to increase either IP production or [Ca2+]i. Although agonists that activated IP and Ca2+ transients had no effect on cAMP production when administered alone, these agents potentiated the β2-adrenergic response two- to fourfold. Hormones known to inhibit adenylyl cyclase activity in cardiac myocytes, such as ET-1 and carbachol, failed to lower the β-adrenergic response in fibroblasts. Order of potency and inhibitor data indicate that the functional receptor subtypes in these cells are β2, P2Y2, and AT1 for isoproterenol, ATP, and ANG II, respectively. We conclude that CFs express functional G protein-linked receptors that couple to Gq and Gs, with little or no coupling to Gi. The expression of receptors and their coupling to Gq- but not to Gi-linked responses distinguishes the signaling in CFs from that in myocytes. Furthermore, agonists that activate Gq in CFs potentiate stimulation of Gs, an example of signaling cross talk not observed in adult myocytes. These data suggest that G protein-mediated signaling in CFs is unique and may contribute to the specificity of hormone and drug action on individual cell types within the heart.
Riboflavin uptake by human-derived colonic epithelial NCM460 cells
Authors: Said Hamid M. , Ortiz Alvaro , Moyer Mary Pat , Yanagawa Norimoto
Published: December 2017

Normal microflora of the large intestine synthesize a number of water-soluble vitamins including riboflavin (RF). Recent studies have shown that colonic epithelial cells posses an efficient carrier-mediated mechanism for absorbing some of these micronutrients. The aim of the present study was to determine whether colonic cells also posses a carrier-mediated mechanism for RF uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed colonic epithelial cells NCM460 and [3H]RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na+ independent; 3) saturable as a function of concentration with an apparent K mof 0.14 μM and V max of 3.29 pmol ⋅ mg protein−1 ⋅ 3 min−1; 4) inhibited by the structural analogs lumiflavin and lumichrome ( K i of 1.8 and 14.1 μM, respectively) but not by the unrelated biotin; 5) inhibited in a competitive manner by the membrane transport inhibitor amiloride ( K i = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca2+/calmodulin-mediated pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellular model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca2+/calmodulin-mediated pathway. It is suggested that the identified transport system may be involved in the absorption of bacterially synthesized RF in the large intestine and that this source of RF may contribute toward RF homeostasis, especially that of colonocytes.
Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of NOS
Authors: Buras Jon A. , Stahl Gregory L. , Svoboda Kathy K. H. , Reenstra Wende R.
Published: December 2017

Hyperbaric oxygen (HBO) is being studied as a therapeutic intervention for ischemia/reperfusion (I/R) injury. We have developed an in vitro endothelial cell model of I/R injury to study the impact of HBO on the expression of intercellular adhesion molecule-1 (ICAM-1) and polymorphonuclear leukocyte (PMN) adhesion. Human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell (BAEC) induction of ICAM-1 required simultaneous exposure to both hypoxia and hypoglycemia as determined by confocal laser scanning microscopy, ELISA, and Western blot. HBO treatment reduced the expression of ICAM-1 to control levels. Adhesion of PMNs to BAECs was increased following hypoxia/hypoglycemia exposure (3.4-fold, P < 0.01) and was reduced to control levels with exposure to HBO ( P = 0.67). Exposure of HUVECs and BAECs to HBO induced the synthesis of endothelial cell nitric oxide synthase (eNOS). The NOS inhibitor nitro-l-arginine methyl ester attenuated HBO-mediated inhibition of ICAM-1 expression. Our findings suggest that the beneficial effects of HBO in treating I/R injury may be mediated in part by inhibition of ICAM-1 expression through the induction of eNOS.
Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during muscle fatigue
Authors: Matar W. , Nosek T. M. , Wong D. , Renaud J.-M.
Published: December 2017

The effects of 10 μM glibenclamide, an ATP-sensitive K+ (KATP) channel blocker, and 100 μM pinacidil, a channel opener, were studied to determine how the KATP channel affects mouse extensor digitorum longus (EDL) and soleus muscle during fatigue. Fatigue was elicited with 200-ms-long tetanic contractions every second. Glibenclamide did not affect rate and extent of fatigue, force recovery, or 86Rb+ fractional loss. The only effects of glibenclamide during fatigue were: an increase in resting tension (EDL and soleus), a depolarization of the cell membrane, a prolongation of the repolarization phase of action potential, and a greater ATP depletion in soleus. Pinacidil, on the other hand, increased the rate but not the extent of fatigue, abolished the normal increase in resting tension during fatigue, enhanced force recovery, and increased 86Rb+ fractional loss in both the EDL and soleus. During fatigue, the decreases in ATP and phosphocreatine of soleus muscle were less in the presence of pinacidil. The glibenclamide effects suggest that fatigue, elicited with intermittent contractions, activates few KATP channels that affect resting tension and membrane potentials but not tetanic force, whereas opening the channel with pinacidil causes a faster decrease in tetanic force, improves force recovery, and helps in preserving energy.
Polyamine depletion delays apoptosis of rat intestinal epithelial cells
Authors: Ray Ramesh M. , Viar Mary Jane , Yuan Qing , Johnson L. R.
Published: December 2017

The polyamines spermidine, spermine, and their precursor putrescine are essential for cell growth and the regulation of the cell cycle. Recent studies suggest that excessive accumulation of polyamines favors either malignant transformation or apoptosis, depending on the cell type and the stimulus. This study examines the involvement of polyamines in the induction of apoptosis by the DNA topoisomerase I inhibitor, camptothecin. In IEC-6 cells, camptothecin induced apoptosis within 6 h, accompanied by detachment of cells. Detached cells showed DNA laddering and caspase 3 induction, characteristic features of apoptosis. Depletion of putrescine, spermidine, and spermine bydl-α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC) that is the first rate-limiting enzyme for polyamine biosynthesis, decreased the apoptotic index. Delayed apoptosis was accompanied by a decrease in caspase 3 activity in polyamine-depleted cells. Addition of putrescine restored the induction of apoptosis as indicated by an increase in the number of detached cells and caspase 3 activity. Polyamine depletion did not change the level of caspase 3 protein. Inhibition of S-adenosylmethionine decarboxylase by a specific inhibitor [diethylglyoxal bis-(guanylhydrazone); DEGBG] led to depletion of spermidine and spermine with a significant accumulation of putrescine and induction of ODC. The DEGBG-treated cells showed an increase in apoptosis, suggesting the importance of putrescine in the apoptotic process. Addition of putrescine to DFMO-treated cell extracts did not increase caspase 3 activity. The above results indicate that polyamine depletion delays the onset of apoptosis in IEC-6 cells and confers protection against DNA damaging agents, suggesting that polyamines might be involved in the caspase activating signal cascade.
ERK signaling mediates the induction of inflammatory cytokines by bufalin in human monocytic cells
Authors: Kurosawa Masahiro , Numazawa Satoshi , Tani Yoshihiro , Yoshida Takemi
Published: December 2017

Treatment of human leukemia THP-1 cells with bufalin, a specific inhibitor of Na+-K+-ATPase, sequentially induces c- fos and inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) gene expressions before the appearance of mature phenotypes of monocytic cells. In this study we examined the signal transduction leading to bufalin-induced gene expressions. Bufalin selectively activated extracellular signal-regulated kinase (ERK), compared with other mitogen-activated protein (MAP) kinase family members. Pretreatment of THP-1 cells with PD-98059, an inhibitor of the ERK-kinase cascade, abolished bufalin-induced c- fos and IL-1β gene expressions, indicating that the ERK-kinase cascade mediates the induction of inflammatory cytokines by bufalin. Inhibition of the Na+/Ca2+ exchanger by KB-R7943 and of protein kinase C (PKC) by Ro-31-8220 suppressed ERK activation and gene expressions of c- fos and IL-1β. These findings suggest that Na+-K+-ATPase inhibition by bufalin induces calcium influx and thereby activates PKC and ERK. In cells treated with an inhibitor of p38 MAP kinases, SB-203580, bufalin-mediated ERK activation became persistent and the induction of IL-1β and TNF-α expressions was significantly augmented. These results suggest that cross talk in bufalin-mediated ERK activation is negatively regulated by endogenous p38 MAP kinase activations.
Effect of lactate on depolarization-induced Ca2+ release in mechanically skinned skeletal muscle fibers
Authors: Dutka T. L. , Lamb G. D.
Published: December 2017

It is unclear whether accumulation of lactate in skeletal muscle fibers during intense activity contributes to muscle fatigue. Using mechanically skinned fibers from rat and toad muscle, we were able to examine the effect of l(+)-lactate on excitation-contraction coupling independently of other metabolic changes. We investigated the effects of lactate on the contractile apparatus, caffeine-induced Ca2+ release from the sarcoplasmic reticulum, and depolarization-induced Ca2+release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable (20–35%) inhibition of caffeine-induced Ca2+ release, seemingly by a direct effect on the Ca2+ release channels. However, 15 mM lactate had no detectable effect on Ca2+release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect on normal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.
Interaction of PIP2 with the XIP region of the cardiac Na/Ca exchanger
Authors: He Zhaoping , Feng Siyi , Tong Qiusheng , Hilgemann Donald W. , Philipson Kenneth D.
Published: December 2017

The sarcolemmal Na/Ca exchanger undergoes an inactivation process in which exchange activity decays over several seconds following activation by the application of Na to the intracellular surface of the protein. Inactivation is eliminated by an increase in membrane phosphatidylinositol 4,5-bisphosphate (PIP2). Inactivation is also strongly affected by mutations to a basic 20-amino acid segment of the exchanger known as the endogenous XIP region. The hypothesis that PIP2 directly interacts with the XIP region of the exchanger was tested. First, we investigated the ability of a peptide with the same sequence as the XIP region to bind to immobilized phospholipid vesicles.125I-labeled XIP bound avidly to vesicles containing only a low concentration (<3%) of PIP2. The binding was specific, in that binding was not displaced by other basic peptides. The effects of altering the sequence of XIP peptides also indicated binding specificity. Second, we examined the functional response to PIP2 of exchangers with mutated XIP regions. Outward Na/Ca exchange currents were measured using the giant excised patch technique. The mutated exchangers either had no inactivation or accelerated inactivation. In both cases, the exchangers no longer responded to PIP2 or to PIP2 antibodies. Overall, the data indicate that the affinity of the endogenous XIP region for PIP2 is an important determinant of the inactivation process.
VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism
Authors: Steinbrech Douglas S. , Mehrara Babak J. , Saadeh Pierre B. , Greenwald Joshua A. , Spector Jason A. , Gittes George K. , Longaker Michael T.
Published: December 2017

Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.