bluuebunny/crossref_metadata_2025 huggingface dataset:
HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells
Authors: Schäfer Claus , Clapp Peter , Welsh Michael J. , Benndorf Rainer , Williams John A.
Published: December 2017

We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.
Involvement of JAK2 and MAPK on type II nitric oxide synthase expression in skin-derived dendritic cells
Authors: Cruz M. T. , Duarte C. B. , Gonçalo M. , Carvalho A. P. , Lopes M. C.
Published: December 2017

In this report, we demonstrate that a fetal mouse skin-derived dendritic cell line produces nitric oxide (NO) in response to the endotoxin [lipopolysaccharide (LPS)] and to cytokines [tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Expression of the inducible isoform of NO synthase (iNOS) was confirmed by immunofluorescence with an antibody against iNOS. The tyrosine kinase inhibitor genistein decreased LPS- and GM-CSF-induced nitrite ([Formula: see text]) production. The effect of LPS and cytokines on [Formula: see text] production was inhibited by the Janus kinase 2 (JAK2) inhibitor tyrphostin B42. The p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB-203580 also reduced the [Formula: see text] production evoked by LPS, TNF-α, or GM-CSF, but it was not as effective as tyrphostin B42. Inhibition of MAPK kinase with PD-098059 also slightly reduced the effect of TNF-α or GM-CSF on [Formula: see text]production. Immunocytochemistry studies revealed that the transcription factor nuclear factor-κB was translocated from the cytoplasm into the nuclei of fetal skin-derived dendritic cells (FSDC) stimulated with LPS, and this translocation was inhibited by tyrphostin B42. Our results show that JAK2 plays a major role in the induction of iNOS in FSDC.
CFTR upregulates the expression of the basolateral Na+-K+-2Cl−cotransporter in cultured pancreatic duct cells
Authors: Shumaker Holli , Soleimani Manoocher
Published: December 2017

The purpose of the current experiments was 1) to assess basolateral Na+-K+-2Cl−cotransporter (NKCC1) expression and 2) to ascertain the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of this transporter in a prototypical pancreatic duct epithelial cell line. Previously validated human pancreatic duct cell lines (CFPAC-1), which exhibit physiological features prototypical of cystic fibrosis, and normal pancreatic duct epithelia (stable recombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown to confluence before molecular and functional studies. High-stringency Northern blot hybridization, utilizing specific cDNA probes, confirmed that NKCC1 was expressed in both cell lines and its mRNA levels were twofold higher in CFPAC-WT cells than in CFPAC-1 cells ( P < 0.01, n = 3). Na+-K+-2Cl−cotransporter activity, assayed as the bumetanide-sensitive, Na+- and Cl−-dependent[Formula: see text] entry into the cell (with[Formula: see text] acting as a substitute for K+), increased by ∼115% in CFPAC-WT cells compared with CFPAC-1 cells ( P < 0.01, n = 6). Reducing the intracellular Cl− by incubating the cells in a Cl−-free medium increased Na+-K+-2Cl−cotransporter activity by twofold ( P< 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells and mediates the entry of Cl−. NKCC1 activity is enhanced in the presence of an inward Cl− gradient. The results further indicate that the presence of functional CFTR enhances the expression of NKCC1. We speculate that CFTR regulates this process in a Cl−-dependent manner.
Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat
Authors: Carroll Stefanie , Nicotera Pierluigi , Pette Dirk
Published: December 2017

Ca2+transients were investigated in single fibers isolated from rat extensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca2+ concentration ([Ca2+]) were observed 2 h after stimulation onset and persisted throughout the stimulation period. The elevated [Ca2+] levels were in the range characteristic of slow-twitch fibers from soleus muscle. In addition, we noticed a transitory elevation of the integral [Ca2+] per pulse with a maximum (∼5-fold) after 1 day. Steep decreases in rate constant of [Ca2+] decay could be explained by an immediate impairment of Ca2+ uptake and, with longer stimulation periods, by an additional loss of cytosolic Ca2+ binding capacity resulting from a decay in parvalbumin content. A partial recovery of the rate constant of [Ca2+] decay in 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca2+ sequestration.
Heterogeneity of pig intestinald-glucose transport systems
Authors: Halaihel Nabil , Gerbaud Daniele , Vasseur Monique , Alvarado Francisco
Published: December 2017

Heterogeneity of intestinald-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two d-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a d-glucose and d-mannose transporter exhibiting no preference for Na+over K+. A nonsaturabled-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker l-glucose. It is also shown that, in this kinetic work, 1) use of d-glucose-contaminatedd-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range (≥50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion.
Folic acid inhibition of EGFR-mediated proliferation in human colon cancer cell lines
Authors: Jaszewski Richard , Khan Ahmed , Sarkar Fazlul H. , Kucuk Omer , Tobi Martin , Zagnoon Abbas , Dhar Ravi , Kinzie Joseph , Majumdar Adhip P. N.
Published: December 2017

Although accumulating evidence suggests a chemopreventive role for folic acid in colon cancer, the regulation of this process in unknown. We hypothesize that supplemental folic acid exerts its chemopreventive role by inhibiting mucosal hyperproliferation, an event considered to be central to the initiation of carcinogenesis in the gastrointestinal tract. The present investigation examines the effect of supplemental folic acid on proliferation of Caco-2 and HCT-116 colon cancer cell lines. Furthermore, because certain tyrosine kinases, particularly epidermal growth factor receptor (EGFR), play a role in regulating cell proliferation, we also examined the folic acid-induced changes in tyrosine kinase activity and expression of EGFR. In Caco-2 and HCT-116 cells, maintained in RPMI 1640 medium containing 1 μg/ml folic acid, we observed that the supplemental folic acid inhibited proliferation in a dose-dependent manner. Pretreatment of HCT-116 and Caco-2 cell lines with supplemental folic acid (1.25 μg/ml) completely abrogated transforming growth factor-α (TGF-α)-induced proliferation in both cell lines. Tyrosine kinase activity and the relative concentration of EGFR were markedly diminished in both cell lines following a 24-h exposure to supplemental folic acid. The folic acid-induced inhibition of EGFR tyrosine kinase activity in colon cancer cell lines was also associated with a concomitant reduction in the relative concentration of the 14-kDa membrane-bound precursor form of TGF-α. In conclusion, our data suggest that supplemental folic acid is effective in reducing proliferation in two unrelated colon cancer cell lines and that EGFR tyrosine kinase appears to be involved in regulating this process.
Myosin molecular motor dysfunction in dystrophic mouse diaphragm
Authors: Coirault Catherine , Lambert Francine , Marchand-Adam Sylvain , Attal Pierre , Chemla Denis , Lecarpentier Yves
Published: December 2017

Cross-bridge properties and myosin heavy chain (MHC) composition were investigated in isolated diaphragm from 6-mo-old control ( n = 12) and mdx( n = 12) mice. Compared with control, peak tetanic tension fell by 50% in mdx mice ( P < 0.001). The total number of cross bridges per square millimeter (×109), the elementary force per cross bridge, and the peak mechanical efficiency were lower in mdx than in control mice (each P < 0.001). The duration of the cycle and the rate constant for cross-bridge detachment were significantly lower in mdx than in control mice. In the overall population, there was a linear relationship between peak tetanic tension and either total number of cross bridges per square millimeter or elementary force per cross bridge ( r = 0.996 and r = 0.667, respectively, each P < 0.001). The mdx mice presented a higher proportion of type IIA MHC ( P < 0.001) than control mice and a reduction in type IIX MHC ( P < 0.001) and slow myosin isoforms ( P < 0.01) compared with control mice. We concluded that, in mdx mice, impaired diaphragm strength was associated with qualitative and quantitative changes in myosin molecular motors. It is proposed that reduced force generated per cross bridge contributed to diaphragm weakness in mdx mice.
Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells
Authors: Nadler Laurie S. , Kumar Geetha , Hinds Thomas R. , Migeon Jacques C. , Nathanson Neil M.
Published: December 2017

We have characterized the muscarinic ACh receptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK) strain II epithelial cells. Binding studies with the membrane-impermeable antagonist N-[3H]methylscopolamine demonstrated that mAChRs are ∼2.5 times more abundant on the basolateral than on the apical surface. Apical, but not basolateral, mAChRs inhibited forskolin-stimulated adenylyl cyclase activity in response to the agonist carbachol. Neither apical nor basolateral mAChRs exhibited detectable carbachol-stimulated phospholipase C activity. Carbachol application to the apical or the basolateral membrane resulted in a threefold increase in intracellular Ca2+ concentration, which was completely inhibited by pertussis toxin on the apical side and partially inhibited on the basolateral side. RT-PCR analysis showed that MDCK cells express the M4 and M5 receptor mRNAs. These data suggest that M4 receptors reside on the apical and basolateral membranes of polarized MDCK strain II cells and that the M5 receptor may reside in the basolateral membrane of a subset of cells.
PKC-ε regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS
Authors: Song Jaekyung Cecilia , Hrnjez Bruce J. , Farokhzad Omid C. , Matthews Jeffrey B.
Published: December 2017

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-ε. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-ε, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.
Role of microtubules in the regulation of metabolism in isolated cerebral microvessels
Authors: Lloyd Pamela G. , Hardin Christopher D.
Published: December 2017

We used13C-labeled substrates and nuclear magnetic resonance spectroscopy to examine carbohydrate metabolism in vascular smooth muscle of freshly isolated pig cerebral microvessels (PCMV). PCMV utilized [2-13C]glucose mainly for glycolysis, producing [2-13C]lactate. Simultaneously, PCMV utilized the glycolytic intermediate [1-13C]fructose 1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing [1-13C]glucose with only minor [3-13C]lactate production. The dissimilarity in metabolism of [2-13C]FBP derived from [2-13C]glucose breakdown and metabolism of exogenous [1-13C]FBP demonstrates that carbohydrate metabolism is compartmented in PCMV. Because glycolytic enzymes interact with microtubules, we disrupted microtubules with vinblastine. Vinblastine treatment significantly decreased [2-13C]lactate peak intensity (87.8 ± 3.7% of control). The microtubule-stabilizing agent taxol also reduced [2-13C]lactate peak intensity (90.0 ± 2.4% of control). Treatment with both agents further decreased [2-13C]lactate production (73.3 ± 4.0% of control). Neither vinblastine, taxol, or the combined drugs affected [1-13C]glucose peak intensity (gluconeogenesis) or disrupted the compartmentation of carbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest that they produce their effects on glycolytic rate by competing with glycolytic enzymes for binding, not by affecting the overall assembly state of the microtubule network. Glycolysis, but not gluconeogenesis, may be regulated in part by glycolytic enzyme-microtubule interactions.
Skeletal muscle reperfusion injury is mediated by neutrophils and the complement membrane attack complex
Authors: Kyriakides Constantinos , Austen William , Wang Yong , Favuzza Joanne , Kobzik Lester , Moore Francis D. , Hechtman Herbert B.
Published: December 2017

The relative inflammatory roles of neutrophils, selectins, and terminal complement components are investigated in this study of skeletal muscle reperfusion injury. Mice underwent 2 h of hindlimb ischemia followed by 3 h of reperfusion. The role of neutrophils was defined by immunodepletion, which reduced injury by 38%, as did anti-selectin therapy with recombinant soluble P-selectin glycoprotein ligand-immunoglobulin (Ig) fusion protein. Injury in C5-deficient and soluble complement receptor type 1-treated wild-type mice was 48% less than that of untreated wild-type animals. Injury was restored in C5-deficient mice reconstituted with wild-type serum, indicating the effector role of C5–9. Neutropenic C5-deficient animals showed additive reduction in injuries (71%), which was lower than C5-deficient neutrophil-replete mice, indicating neutrophil activity without C5a. Hindlimb histological injury was worse in ischemic wild-type and C5-deficient animals reconstituted with wild-type serum. In conclusion, the membrane attack complex and neutrophils act additively to mediate skeletal muscle reperfusion injury. Neutrophil activity is independent of C5a but is dependent on selectin-mediated adhesion.
Na+ entry via store-operated channels modulates Ca2+signaling in arterial myocytes
Authors: Arnon Assaf , Hamlyn John M. , Blaustein Mordecai P.
Published: December 2017

In many nonexcitable cells, hormones and neurotransmitters activate Na+ influx and mobilize Ca2+ from intracellular stores. The stores are replenished by Ca2+influx via “store-operated” Ca2+ channels (SOC). The main routes of Na+ entry in these cells are unresolved, and no role for Na+ in signaling has been recognized. We demonstrate that the SOC are a major Na+ entry route in arterial myocytes. Unloading of the Ca2+stores with cyclopiazonic acid (a sarcoplasmic reticulum Ca2+ pump inhibitor) and caffeine induces a large external Na+-dependent rise in the cytosolic Na+ concentration. One component of this rise in cytosolic Na+ concentration is likely due to Na+/Ca2+exchange; it depends on elevation of cytosolic Ca2+ and is insensitive to 10 mM Mg2+ and 10 μM La3+. Another component is inhibited by Mg2+ and La3+, blockers of SOC; this component persists in cells preloaded with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid to buffer Ca2+ transients and prevent Na+/Ca2+exchange-mediated Na+ entry. This Na+ entry apparently is mediated by SOC. The Na+ entry influences Na+ pump activity and Na+/Ca2+exchange and has unexpectedly large effects on cell-wide Ca2+ signaling. The SOC pathway may be a general mechanism by which Na+ participates in signaling in many types of cells.
Rapid entry of bitter and sweet tastants into liposomes and taste cells: implications for signal transduction
Authors: Peri Irena , Mamrud-Brains Hanna , Rodin Sergey , Krizhanovsky Valery , Shai Yechiel , Nir Shlomo , Naim Michael
Published: December 2017

Some amphipathic bitter tastants and non-sugar sweeteners are direct activators of G proteins and stimulate transduction pathways in cells not related to taste. We demonstrate that the amphipathic bitter tastants quinine and cyclo(Leu-Trp) and the non-sugar sweetener saccharin translocate rapidly through multilamellar liposomes. Furthermore, when rat circumvallate (CV) taste buds were incubated with the above tastants for 30 s, their intracellular concentrations increased by 3.5- to 7-fold relative to their extracellular concentrations. The time course of this dramatic accumulation was also monitored in situ in rat single CV taste buds under a confocal laser-scanning microscope. Tastants were clearly localized to the taste cell cytosol. It is proposed that, due to their rapid permeation into taste cells, these amphipathic tastants may be available for activation of signal transduction components (e.g., G proteins) directly within the time course of taste sensation. Such activation may occur in addition to the action of these tastants on putative G protein-coupled receptors. This phenomenon may be related to the slow taste onset and lingering aftertaste typically produced by many bitter tastants and non-sugar sweeteners.
Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange
Authors: Scott Daryl A. , Karniski Lawrence P.
Published: December 2017

Pendred syndrome, characterized by congenital sensorineural hearing loss and goiter, is one of the most common forms of syndromic deafness. The gene causing Pendred syndrome ( PDS) encodes a protein designated pendrin, which is expressed in the thyroid, kidney, and fetal cochlea. Pendrin functions as an iodide and chloride transporter, but its role in the development of hearing loss and goiter is unknown. In this study, we examined the mechanism of pendrin-mediated anion transport in Xenopus laevis oocytes. Unlabeled formate added to the uptake medium inhibited pendrin-mediated 36Cl uptake in X. laevis oocytes. In addition, the uptake of [14C]formate was stimulated in oocytes injected with PDS cRNA compared with water-injected controls. These results indicate that formate is a substrate for pendrin. Furthermore, chloride stimulated the efflux of [14C]formate and formate stimulated the efflux of 36Cl in oocytes expressing pendrin, results consistent with pendrin-mediated chloride/formate exchange. These data demonstrate that pendrin is functionally similar to the renal chloride/formate exchanger, which serves as an important mechanism of chloride transport in the proximal tubule. A similar process could participate in the development of ion gradients within the inner ear.
Secretagogue response of goblet cells and columnar cells in human colonic crypts1
Authors: Halm Dan R. , Halm Susan Troutman
Published: December 2017

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 μM) or histamine (10 μM) released contents from goblet granules. Stimulation with prostaglandin E2(PGE2, 5 μM) or adenosine (10 μM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine (∼160 fl/cell), but not with PGE2 or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE2 stimulated a prolonged fluid secretion that attained a rate of ∼350 pl/min. Columnar cells lost ∼50% of apical volume during maximal PGE2 stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.
Calcium sparks in smooth muscle
Authors: Jaggar Jonathan H. , Porter Valerie A. , Lederer W. Jonathan , Nelson Mark T.
Published: December 2017

Local intracellular Ca2+transients, termed Ca2+ sparks, are caused by the coordinated opening of a cluster of ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum of smooth muscle cells. Ca2+ sparks are activated by Ca2+ entry through dihydropyridine-sensitive voltage-dependent Ca2+ channels, although the precise mechanisms of communication of Ca2+ entry to Ca2+ spark activation are not clear in smooth muscle. Ca2+ sparks act as a positive-feedback element to increase smooth muscle contractility, directly by contributing to the global cytoplasmic Ca2+ concentration ([Ca2+]) and indirectly by increasing Ca2+ entry through membrane potential depolarization, caused by activation of Ca2+ spark-activated Cl− channels. Ca2+ sparks also have a profound negative-feedback effect on contractility by decreasing Ca2+ entry through membrane potential hyperpolarization, caused by activation of large-conductance, Ca2+-sensitive K+ channels. In this review, the roles of Ca2+sparks in positive- and negative-feedback regulation of smooth muscle function are explored. We also propose that frequency and amplitude modulation of Ca2+ sparks by contractile and relaxant agents is an important mechanism to regulate smooth muscle function.
Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of ΔF508-CFTR
Authors: Rubenstein Ronald C. , Zeitlin Pamela L.
Published: December 2017

The most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), ΔF508, is a trafficking mutant that has prolonged associations with molecular chaperones and is rapidly degraded, at least in part by the ubiquitin-proteasome system. Sodium 4-phenylbutyrate (4PBA) improves ΔF508-CFTR trafficking and function in vitro in cystic fibrosis epithelial cells and in vivo. To further understand the mechanism of action of 4PBA, we tested the hypothesis that 4PBA modulates the targeting of ΔF508-CFTR for ubiquitination and degradation by reducing the expression of Hsc70 in cystic fibrosis epithelial cells. IB3-1 cells (genotype ΔF508/W1282X) that were treated with 0.05–5 mM 4PBA for 2 days in culture demonstrated a dose-dependent reduction in Hsc70 protein immunoreactivity and mRNA levels. Immunoprecipitation with Hsc70-specific antiserum demonstrated that Hsc70 and CFTR associated under control conditions and that treatment with 4PBA reduced these complexes. Levels of immunoreactive Hsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBA treatment. These data suggest that 4PBA may improve ΔF508-CFTR trafficking by allowing a greater proportion of mutant CFTR to escape association with Hsc70.
Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration
Authors: Wang Jian-Ying , Wang Jian , Golovina Vera A. , Li Li , Platoshyn Oleksandr , Yuan Jason Xiao-Jian
Published: December 2017

Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K+ channels (Kv) controls membrane potential ( E m) that regulates cytoplasmic free Ca2+ concentration ([Ca2+]cyt) by governing the driving force for Ca2+ influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K+ channel gene expression, E m, and [Ca2+]cyt in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, α-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca2+ channels, the depolarized E m in DFMO-treated cells decreased [Ca2+]cyt as a result of reduced driving force for Ca2+ influx through capacitative Ca2+ entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E m, and [Ca2+]cyt but also restored cell migration to normal. Removal of extracellular Ca2+ or blockade of Kv channels (by 4-aminopyridine, 1–5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca2+]cyt by increasing the driving force (the electrochemical gradient) for Ca2+ influx and thus stimulates cell migration.
Cyclic nucleotide-gated cation channels mediate sodium and calcium influx in rat colon
Authors: Qiu W. , Lee B. , Lancaster M. , Xu W. , Leung S. , Guggino S. E.
Published: December 2017

We found mRNA for the three isoforms of the cyclic nucleotide-gated nonselective cation channel expressed in the mucosal layer of the rat intestine from the duodenum to the colon and in intestinal epithelial cell lines in culture. Because these channels are permeable to sodium and calcium and are stimulated by cGMP or cAMP, we measured 8-bromo-cGMP-stimulated sodium-mediated short-circuit current ( I sc) in proximal and distal colon and unidirectional45Ca2+fluxes in proximal colon to determine whether these channels could mediate transepithelial sodium and calcium absorption across the colon. Sodium-mediated I sc, stimulated by 8-bromo-cGMP, were inhibited by dichlorobenzamil and l-cis-diltiazem, blockers of cyclic nucleotide-gated cation channels, suggesting that these ion channels can mediate transepithelial sodium absorption. Sodium-mediated I sc and net transepithelial45Ca2+absorption were stimulated by heat-stable toxin from Escherichia coli that increases cGMP. Addition of l-cis-diltiazem inhibited the enhanced transepithelial absorption of both ions. These results suggest that cyclic nucleotide-gated cation channels simultaneously increase net sodium and calcium absorption in the colon of the rat.
Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes
Authors: Kong In Deok , Koh Sang Don , Sanders Kenton M.
Published: December 2017

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to −50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca2+ with equimolar Mn2+ caused STOCs to “run down.” Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude “mini-STOCs” remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca2+-activated K+ channels (SK channels). Application of ATP or 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid, a P2 receptor blocker. Similarly, pretreatment of cells with U-73122 (1 μM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP3) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P2Y receptors results in localized Ca2+ release via PLC- and IP3-dependent mechanisms. Release of Ca2+ is coupled to STOCs, which are composed of currents mediated by large-conductance Ca2+-activated K+ channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.
Endoplasmic reticulum Ca2+-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells
Authors: Gukovskaya Anna S. , Gukovsky Sofiya , Pandol Stephen J.
Published: December 2017

In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca2+-ATPase thapsigargin, 2,5-di-( t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca2+-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca2+-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca2+-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.
Ca2+ activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase
Authors: Territo Paul R. , Mootha Vamsi K. , French Stephanie A. , Balaban Robert S.
Published: December 2017

Ca2+ has been postulated as a cytosolic second messenger in the regulation of cardiac oxidative phosphorylation. This hypothesis draws support from the well-known effects of Ca2+ on muscle activity, which is stimulated in parallel with the Ca2+-sensitive dehydrogenases (CaDH). The effects of Ca2+ on oxidative phosphorylation were further investigated in isolated porcine heart mitochondria at the level of metabolic driving force (NADH or Δψ) and ATP production rates (flow). The resulting force-flow (F-F) relationships permitted the analysis of Ca2+ effects on several putative control points within oxidative phosphorylation, simultaneously. The F-F relationships resulting from additions of carbon substrates alone provided a model of pure CaDH activation. Comparing this curve with variable Ca2+ concentration ([Ca2+]) effects revealed an approximate twofold higher ATP production rate than could be explained by a simple increase in NADH or Δψ via CaDH activation. The half-maximal effect of Ca2+ at state 3 was 157 nM and was completely inhibited by ruthenium red (1 μM), indicating matrix dependence of the Ca2+ effect. Arsenate was used as a probe to differentiate between F0/F1-ATPase and adenylate translocase activity by a futile recycling of ADP-arsenate within the matrix, catalyzed by the F0/F1-ATPase. Ca2+increased the ADP arsenylation rate more than twofold, suggesting a direct effect on the F0/F1-ATPase. These results suggest that Ca2+ activates cardiac aerobic respiration at the level of both the CaDH and F0/F1-ATPase. This type of parallel control of both intermediary metabolism and ATP synthesis may provide a mechanism of altering ATP production rates with minimal changes in the high-energy intermediates as observed in vivo.
Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle
Authors: Rokolya Aniko , Singer Harold A.
Published: December 2017

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) has been implicated in the regulation of smooth muscle contractility. The goals of this study were to determine: 1) to what extent CaM kinase II is activated by contractile stimuli in intact arterial smooth muscle, and 2) the effect of a CaM kinase II inhibitor (KN-93) on CaM kinase II activation, phosphorylation of myosin regulatory light chains (MLC20), and force. Both histamine (1 μM) and KCl depolarization activated CaM kinase II with a time course preceding maximal force development, and suprabasal CaM kinase II activation was sustained during tonic contractions. CaM kinase II activation was inhibited by KN-93 pretreatment (IC50 ∼1 μM). KN-93 inhibited histamine-induced tonic force maintenance, whereas early force development and MLC20 phosphorylation responses during the entire time course were unaffected. Both force development and maintenance in response to KCl were inhibited by KN-93. Rapid increases in KCl-induced MLC20 phosphorylation were also inhibited by KN-93, whereas steady-state MLC20 phosphorylation responses were unaffected. In contrast, phorbol 12,13-dibutyrate (PDBu) did not activate CaM kinase II and PDBu-stimulated force development was unaffected by KN-93. Thus KN-93 appears to target a step(s) essential for force maintenance in response to physiological stimuli, suggesting a role for CaM kinase II in regulating tonic contractile responses in arterial smooth muscle. Pharmacological activation of protein kinase C bypasses the KN-93 sensitive step.
Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes
Authors: McLean Lee Anne , Roscoe Jane , Jørgensen Nanna K. , Gorin Fredric A. , Cala Peter M.
Published: December 2017

Malignant gliomas exhibit alkaline intracellular pH (pHi) and acidic extracellular pH (pHe) compared with nontransformed astrocytes, despite increased metabolic H+ production. The acidic pHe limits the availability of[Formula: see text], thereby reducing both passive and dynamic [Formula: see text]-dependent buffering. This implies that gliomas are dependent upon dynamic[Formula: see text]-independent H+buffering pathways such as the type 1 Na+/H+exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pHi(pHi = 7.31–7.48) than normal astrocytes (pHi = 6.98), and increased rates of recovery from acidification, under nominally CO2/[Formula: see text]-free conditions. Inhibition of NHE1 in the absence of CO2/[Formula: see text] resulted in pronounced acidification of gliomas, whereas normal astrocytes were unaffected. When suspended in CO2/[Formula: see text] medium astrocyte pHi increased, yet glioma pHi unexpectedly acidified, suggesting the presence of an[Formula: see text]-dependent acid loading pathway. Nucleotide sequencing of NHE1 cDNA from the gliomas demonstrated that genetic alterations were not responsible for this altered NHE1 function. The data suggest that NHE1 activity is significantly elevated in gliomas and may provide a useful target for the development of tumor-selective therapies.
Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle
Authors: Hedges Jason C. , Oxhorn Brian C. , Carty Michael , Adam Leonard P. , Yamboliev Ilia A. , Gerthoffer William T.
Published: December 2017

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.
Interaction of α- and β-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase β-subunit
Authors: Okamoto Curtis T. , Chow Dar C. , Forte And John G.
Published: December 2017

The assembly of the β-subunit of the gastric H-K-ATPase (HKβ) with the α-subunit of the H-K-ATPase or the Na-K-ATPase (NaKα) was characterized with two anti-HKβ monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKβ, MAb 2/2E6 was observed to bind to HKβ only when interactions between α- and β-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S226LHY229 of the extracellular domain of HKβ. The epitope for MAb 2G11 was mapped to the eight NH2-terminal amino acids of the cytoplasmic domain of HKβ. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKβ with α-subunits of the endogenous cell surface NaKα, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKβ. In HKβ-transfected LLC-PK1 cells, significant immunofluorescent labeling of HKβ at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKβ could also be coimmunoprecipitated with NaKα. Thus assembly of HKβ with NaKα does not appear to be a stringent requirement for cell surface delivery of HKβ in LLC-PK1 cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid α-β heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKβ that is associated with α-β interaction.
Cellular basis for contractile dysfunction in the diaphragm from a rabbit infarct model of heart failure
Authors: MacFarlane Niall G. , Darnley G. Murray , Smith Godfrey L.
Published: December 2017

Abnormal respiratory muscle function is thought to contribute to breathlessness and exercise intolerance in heart failure but little is known about possible alterations in the function of such muscle. We have measured tetanic force and intracellular Ca2+ concentration ([Ca2+]i) in isolated, arterially perfused hemidiaphragm preparations from a rabbit coronary artery ligation model of heart failure. Increasing stimulation frequency (10–100 Hz) caused a progressive increase of force and [Ca2+]i in control preparations, whereas force and [Ca2+]i only increased between 10 and 25 Hz stimulation (decreasing at higher frequencies) in preparations from ligated animals. Cyclopiazonic acid produced a dose-dependent shift in the relationship between stimulation frequency and [Ca2+]i in control preparations that was similar to the shift observed in the diaphragm of coronary-ligated animals. These data indicate that the in vitro contractile characteristics of the diaphragm are significantly altered in our model and that altered [Ca2+]i regulation contributes to the reduced diaphragm strength observed in heart failure.
Proliferative activity and tumorigenic conversion: impact on cellular metabolism in 3-D culture
Authors: Kunz-Schughart Leoni A. , Doetsch Joerg , Mueller-Klieser Wolfgang , Groebe Karlfried
Published: December 2017

Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of[Formula: see text] distributions with microelectrodes, and determination of cellular oxygen uptake rates (Q˙[Formula: see text]) have been applied. In the ras-transfected, tumorigenic spheroid types, the size dependencies of some of the measured parameters exhibited sharp breaks at diameters of ∼830 μm for Rat1-T1 and ∼970 μm for MR1 spheroids, respectively, suggesting that some fundamental change in cell metabolism occurred at these characteristic diameters (denoted as “metabolic switch”).Q˙[Formula: see text]decreased and lactate concentration increased as functions of size below the characteristic diameters. Concomitantly, glucose and lactate turnover rates decreased in MR1 spheroids and increased in Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting cell quiescence and lactate accumulation) showed an enhancement ofQ˙[Formula: see text]with size. Systematic variations in the ATP and glucose levels in the viable cell rim were observed for Rat1-T1 spheroids only. Proliferative activity, Q˙[Formula: see text], and ATP levels in small, nontumorigenic Rat1 and M1 aggregates did not differ systematically from those recorded in the largest spheroids of the corresponding ras transfectants. Unexpectedly, respiratory activity was present not only in viable but also in the morphologically disintegrated core regions of M1 aggregates. Our data suggest that myc but not rastransfection exerts major impacts on cell metabolism. Moreover, some kind of switch has been detected that triggers profound readjustment of tumor cell metabolism when proliferative activity begins to stagnate, and that is likely to initiate some other, yet unidentified energy-consuming process.
Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl− secretion by T84 cells
Authors: Dickson Jeffrey L. , Conner Tracy D. , Ecay Tom W.
Published: December 2017

Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl−secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl− secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl− secretion measured as short-circuit current ( I SC). However, 500 nM wortmannin significantly inhibited forskolin-stimulated I SC. cAMP activation of apical membrane CFTR Cl− channels in α-toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters accounts for the observed reduction in T84 Cl− secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.
Chlorpromazine modulates the morphological macro- and microstructure of endothelial cells
Authors: Hueck I. S. , Hollweg H. G. , Schmid-Schönbein G. W. , Artmann G. M.
Published: December 2017

Chlorpromazine (CP), an amphipathic, antipsychotic agent, causes concave membrane bending in red blood cells with formation of stomatocytic shapes by modulation of the phospholipid bilayer. This study was designed to investigate the effects of CP on the shape of bovine aortic endothelial cells (BAEC) and their membranes in confluent monolayers with phase-contrast and transmission electron microscopy. Exposure of BAECs to nanomolar levels of CP leads to membrane curvature changes. With increasing CP concentrations, the membrane assumed a shape with enhanced numbers of intracellular caveolae and projection of pseudopodia at all junctions. At higher CP concentrations (up to 150 μM), the endothelial cells assumed almost spherical shapes. The evidence suggests that CP may affect lipid bilayer bending of BAECs in analogy with previous observations on erythrocytes, supporting the formation of caveolae and pseudopodia in BAECs due to the induction of concave membrane bending, as well as an effect on endothelial cell membrane adhesion at higher CP concentrations with loss of cellular attachment at junctions.