bluuebunny/crossref_metadata_2025 huggingface dataset:
Human cytomegalovirus infection stimulates Cl−/ HCO 3 − exchanger activity in human fibroblasts
Authors: Maglova Lilia M. , Crowe William E. , Altamirano Aníbal A. , Russell John M.
Published: December 2017

The effects of human cytomegalovirus (HCMV) infection on Cl−/[Formula: see text]exchanger activity in human lung fibroblasts (MRC-5 cells) were studied using fluorescent, ion-sensitive dyes. The intracellular pH (pHi) of mock- and HCMV-infected cells bathed in a solution containing 5% CO2-25 mM[Formula: see text] were nearly the same. However, replacement of external Cl−with gluconate caused an H2DIDS-inhibitable (100 μM) increase in the pHi of HCMV-infected cells but not in mock-infected cells. Continuous exposure to hyperosmotic external media containing CO2/[Formula: see text]caused the pHi of both cell types to increase. The pHi remained elevated in mock-infected cells. However, in HCMV-infected cells, the pHi peaked and then recovered toward control values. This pHirecovery phase was completely blocked by 100 μM H2DIDS. In the presence of CO2/[Formula: see text], there was an H2DIDS-sensitive component of net Cl− efflux (external Cl− was substituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl− (in the nominal absence of CO2/[Formula: see text]), the H2DIDS-sensitive net Cl− efflux was much greater from HCMV- than from mock-infected cells. In mock-infected cells, H2DIDS-sensitive, net Cl− efflux decreased as pHi increased, whereas for HCMV-infected cells, efflux increased as pHi increased. All these results are consistent with an HCMV-induced enhancement of Cl−/[Formula: see text]exchanger activity.
A role for MAP kinase in differentiated smooth muscle contraction evoked by α-adrenoceptor stimulation
Authors: Dessy Chantal , Kim Inkyeom , Sougnez Carrie L. , Laporte Regent , Morgan Kathleen G.
Published: December 2017

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in smooth muscle contraction by monitoring MAP kinase activation, caldesmon phosphorylation, and contractile force during agonist stimulation. Isometric tension in response to KCl and phenylephrine (PE) was measured from strips of ferret aorta. MAP kinase activation was monitored by Western blot using a phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmon antibodies. We report here that treatment of smooth muscle strips with PD-098059, a specific inhibitor of MAP kinase kinase, did not detectably modify the KCl-evoked contraction but significantly inhibited the contraction to PE in the absence of extracellular Ca2+. In this experimental condition, where the contraction occurs in the absence of increases in 20-kDa myosin light chain phosphorylation, PD-098059 also inhibited significantly MAP kinase and caldesmon phosphorylation. Collectively, these results demonstrate a direct cause-and-effect relationship between MAP kinase activation and Ca2+-independent smooth muscle contraction and support the concept of caldesmon phosphorylation as the missing link between both events.
Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells
Authors: Funaki Haruko , Yamamoto Tadashi , Koyama Yu , Kondo Daisuke , Yaoita Eishin , Kawasaki Katsutoshi , Kobayashi Hideyuki , Sawaguchi Shoichi , Abe Haruki , Kihara Itaru
Published: December 2017

Aquaporin (AQP) 5 gene was recently isolated from salivary gland and identified as a member of the AQP family. The mRNA expression and localization have been examined in several organs. The present study was focused on elucidation of AQP5 expression and localization in the eye, salivary gland, and lung in rat. RNase protection assay confirmed intense expression of AQP5 mRNA in these organs but negligible expression in other organs. To examine the mRNA expression sites in the eye, several portions were microdissected for total RNA isolation. AQP5 mRNA was enriched in cornea but not in other portions (retina, lens, iris/ciliary body, conjunctiva, or sclera). AQP5 was selectively localized on the surface of corneal epithelium in the eye by immunohistochemistry and immunoelectron microscopy using an affinity-purified anti-AQP5 antibody. AQP5 was also localized on apical membranes of acinar cells in the lacrimal gland and on the microvilli protruding into intracellular secretory canaliculi of the serous salivary gland. In the lung, apical membranes of type I pulmonary epithelial cells were also immunostained with the antibody. These findings suggest a role of AQP5 in water transport to prevent dehydration or to secrete watery products in these tissues.
Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid
Authors: Said Hamid M. , Ortiz Alvaro , McCloud Eric , Dyer David , Moyer Mary Pat , Rubin Stanley
Published: December 2017

Previous studies showed that the normal microflora of the large intestine synthesizes biotin and that the colon is capable of absorbing intraluminally introduced free biotin. Nothing, however, is known about the mechanism of biotin absorption in the large intestine and its regulation. To address these issues, we used the human-derived, nontransformed colonic epithelial cell line NCM460. The initial rate of biotin uptake was found to be 1) temperature and energy dependent, 2) Na+ dependent (coupling ratio of 1:1), 3) saturable as a function of concentration [apparent Michaelis constant ( K m) of 19.7 μM], 4) inhibited by structural analogs with a free carboxyl group at the valeric acid moiety, and 5) competitively inhibited by the vitamin pantothenic acid (inhibition constant of 14.4 μM). Pretreatment with the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl- sn-glycerol significantly inhibited biotin uptake. In contrast, pretreatment with the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated via a marked decrease in maximal uptake velocity and a slight increase in apparent K m. Pretreatment of cells with modulators of the protein kinase A-mediated pathway, on the other hand, showed no significant effect on biotin uptake. These results demonstrate, for the first time, the functional existence of a Na+-dependent, specialized carrier-mediated system for biotin uptake in colonic epithelial cells. This system is shared with pantothenic acid and appears to be under the regulation of an intracellular PKC-mediated pathway.
Bioluminescence detection of ATP release mechanisms in epithelia
Authors: Taylor Amanda L. , Kudlow Brian A. , Marrs Kevin L. , Gruenert Dieter C. , Guggino William B. , Schwiebert Erik M.
Published: December 2017

Autocrine and paracrine release of and extracellular signaling by ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles of cellular ATP release are unknown. We tested the hypothesis that epithelia release ATP under basal and stimulated conditions by using a newly designed and highly sensitive assay for bioluminescence detection of ATP released from polarized epithelial monolayers. This bioluminescence assay measures ATP released from cystic fibrosis (CF) and non-CF human epithelial monolayers in a reduced serum medium through catalysis of the luciferase-luciferin reaction, yielding a photon of light collected by a luminometer. This novel assay measures ATP released into the apical or basolateral medium surrounding epithelia. Of relevance to CF, CF epithelia fail to release ATP across the apical membrane under basal conditions. Moreover, hypotonicity is an extracellular signal that stimulates ATP release into both compartments of non-CF epithelia in a reversible manner; the response to hypotonicity is also lost in CF epithelia. The bioluminescence detection assay for ATP released from epithelia and other cells will be useful in the study of extracellular nucleotide signaling in physiological and pathophysiological paradigms. Taken together, these results suggest that extracellular ATP may be a constant regulator of epithelial cell function under basal conditions and an autocrine regulator of cell volume under hypotonic conditions, two functions that may be lost in CF and contribute to CF pathophysiology.
G protein-dependent activation of smooth muscle eNOS via natriuretic peptide clearance receptor
Authors: Murthy K. S. , Teng B.-Q. , Jin J.-G. , Makhlouf G. M.
Published: December 2017

In gastrointestinal smooth muscle, the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting with VIP2/PACAP3receptors coupled via Gs to adenylyl cyclase and with distinct receptors coupled via Gi1 and/or Gi2 to a smooth muscle endothelial nitric oxide synthase (eNOS). The present study identifies the receptor as the single-transmembrane natriuretic peptide clearance receptor (NPR-C). RT-PCR and Northern analysis demonstrated expression of the natriuretic peptide receptors NPR-C and NPR-B but not NPR-A in rabbit gastric muscle cells. In binding studies using125I-labeled atrial natriuretic peptide (125I-ANP) and125I-VIP as radioligands, VIP, ANP, and the selective NPR-C ligand cANP(4–23) bound with high affinity to NPR-C. ANP, cANP-(4–23), and VIP initiated identical signaling cascades consisting of Ca2+ influx, activation of eNOS via Gi1 and Gi2, stimulation of cGMP formation, and muscle relaxation. NOS activity and cGMP formation were abolished (93 ± 3 to 96 ± 2% inhibition) by nifedipine, pertussis toxin, the NOS inhibitor, N G-nitro-l-arginine, and the antagonists ANP-(1–11) and VIP-(10–28). NOS activity stimulated by all three ligands in muscle membranes was additively inhibited by Gi1 and Gi2 antibodies (82 ± 2 to 84 ± 1%). In reconstitution studies, VIP, cANP-(4–23), and guanosine 5′- O-(3-thiotriphosphate) stimulated NOS activity in membranes of COS-1 cells cotransfected with NPR-C and eNOS. The results establish a unique mechanism for G protein-dependent activation of a constitutive NOS expressed in gastrointestinal smooth muscle involving interaction of the relaxant neuropeptides VIP and PACAP with a single-transmembrane natriuretic peptide receptor, NPR-C.
Effect of PCMBS on CO2permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant
Authors: Cooper Gordon J. , Boron Walter F.
Published: December 2017

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 ( Cell Physiol. 43): C543–548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ∼40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane’s lipid, or 4) increasing the expression of a native “gas channel.” The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule out options 2–4 listed above. Thus our results suggest that CO2passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.
Functional demonstration of Na+-K+-2Cl−cotransporter activity in isolated, polarized choroid plexus cells
Authors: Wu Qiang , Delpire Eric , Hebert Steven C. , Strange Kevin
Published: December 2017

The function of the apical Na+-K+-2Cl−cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl−cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl−/[Formula: see text](anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 μM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 μM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 μM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood.
Taxol inhibits endosomal-lysosomal membrane trafficking at two distinct steps in CV-1 cells
Authors: Sonee Manisha , Barrón Ernesto , Yarber Francie A. , Hamm-Alvarez Sarah F.
Published: December 2017

Although taxol inhibits membrane trafficking, the nature of this inhibition has not been well defined. In this study, we define the effects of taxol on endocytosis in CV-1 cells using density gradient centrifugation of membranes over sorbitol density gradients. After taxol treatment, resident endosomal enzymes and the epidermal growth factor (EGF) receptor (EGFR) showed significant ( P ≤ 0.05) enrichment in membranes with properties of early endosomes ( fractions 4 and 5); the EGFR and Na+-K+-ATPase were also significantly ( P ≤ 0.05) depleted in lysosomal fractions ( fractions 10 and 11). The suggestion that taxol specifically reduces movement of endosomal constituents to lysosomes was supported by fluorescence microscopy studies revealing restriction of EGF to the peripheries of taxol-treated cells, in contrast to the perinuclear lysosomal-like distribution of EGF seen in controls. Kinetic studies with 125I-labeled EGF were also consistent with a taxol-induced block in traffic from endosomes and lysosomes after 15 min of uptake but also suggested an additional taxol-sensitive step in trafficking that involved redistribution of 125I-EGF within high-density compartments after 150 min. Related changes in cytoplasmic dynein distribution were observed within high-density compartments from taxol-treated cells, suggesting that this motor might participate in this later taxol-sensitive trafficking event. Electron microscopic examination of high-density membranes ( fraction 12) showed that taxol increased the numbers of small (<500 nm) dense vesicles, with a relative depletion of the larger (>500 nm) vesicles found in controls. These data demonstrate that disruption of endocytic events by taxol includes the early accumulation of protein and endocytic markers in endosomes and the later accumulation in a dense compartment that we propose is a subdomain of the lysosomes.
Endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by tyrosine kinases
Authors: Freedman Steven D. , Katz Mark H. , Parker Eliza M. , Gelrud Andres
Published: December 2017

We have shown that endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by the pH of the acinar lumen and is associated with cleavage of GP2, a glycosyl phosphatidylinositol-anchored protein. The aim of this study was to determine the transduction pathway by which endocytosis is activated. Apical endocytosis was studied in rat pancreatic acini by prestimulation with cholecystokinin followed by measurement of horseradish peroxidase (HRP) uptake. Lanthanum, staurosporine, and forskolin had no effect on HRP uptake. Cytochalasin D significantly inhibited endocytosis, indicating a dependence on actin filament integrity. Genistein and the specific tyrphostin inhibitor B42 also inhibited HRP uptake, implicating tyrosine kinases in the regulation of HRP uptake. With the use of an Src kinase-specific substrate, Src kinase activity was temporally related to activation of endocytosis. The tyrosine-dependent phosphorylation of an 85-kDa substrate in both rat and mouse pancreatic acini correlated with Src kinase activation and pH-dependent regulation of HRP uptake. These results indicate that apical endocytosis in acinar cells is associated with tyrosine kinase activation and is dependent on the actin cytoskeleton.
Cloning and characterization of a human electrogenic Na+- HCO 3 − cotransporter isoform (hhNBC)
Authors: Choi Inyeong , Romero Michael F. , Khandoudi Nassirah , Bril Antoine , Boron Walter F.
Published: December 2017

Our group recently cloned the electrogenic Na+-[Formula: see text]cotransporter (NBC) from salamander kidney and later from mammalian kidney. Here we report cloning an NBC isoform (hhNBC) from a human heart cDNA library. hhNBC is identical to human renal NBC (hkNBC), except for the amino terminus, where the first 85 amino acids in hhNBC replace the first 41 amino acids of hkNBC. About 50% of the amino acid residues in this unique amino terminus are charged, compared with ∼22% for the corresponding 41 residues in hkNBC. Northern blot analysis, with the use of the unique 5′ fragment of hhNBC as a probe, shows strong expression in pancreas and expression in heart and brain, although at much lower levels. In Xenopus oocytes expressing hhNBC, adding 1.5% CO2/10 mM[Formula: see text] hyperpolarizes the membrane and causes a rapid fall in intracellular pH (pHi), followed by a pHi recovery. Subsequent removal of Na+ causes a depolarization and a reduced rate of pHi recovery. Removal of Cl− from the bath does not affect the pHi recovery. The stilbene derivative DIDS (200 μM) greatly reduces the hyperpolarization caused by adding CO2/[Formula: see text]. In oocytes expressing hkNBC, the effects of adding CO2/[Formula: see text]and then removing Na+ were similar to those observed in oocytes expressing hhNBC. We conclude that hhNBC is an electrogenic Na+-[Formula: see text]cotransporter and that hkNBC is also electrogenic.
Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion
Authors: Cortese Jorge Daniel
Published: December 2017

The variety of mitochondrial morphology in healthy and diseased cells can be explained by regulated mitochondrial fusion. Previously, a mitochondrial outer membrane fraction containing fusogenic, aluminum fluoride (AlF4)-sensitive GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese, Exp. Cell Res. 240: 122–133, 1998). Quantitative confocal microscopy now reveals that mtg transiently increases mitochondrial membrane potential (ΔΨ) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced ΔΨ increment is blocked by fusogenic GTPase-specific modulators such as guanosine 5′- O-(3-thiotriphosphate), excess GTP (>100 μM), and AlF4, suggesting a linkage between ΔΨ and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing ΔΨ or ATP synthesis with respiratory inhibitors limits mtg- and AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor ( Bordetella pertussis toxin) hyperpolarizes mitochondria and leads to a loss of AlF4-dependent mitochondrial fusion. These results place mtg-induced ΔΨ changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert ΔΨ-dependent control of the fusion process. Mammalian mitochondrial morphology thus can be modulated by cellular energetics.
Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels
Authors: Li Hongfei , Marshall Zermeena M. , Whorton A. Richard
Published: December 2017

Nitric oxide (NO) is known to produce some of its biological activity through modification of cellular thiols. Return of cellular thiols to their basal state requires the activity of the GSH redox cycle, suggesting important interactions between NO signaling and regulation of cellular redox status. Because continuous exposure to NO may lead to adaptive responses in cellular redox systems, we investigated the effects of NO on cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mM S-nitroso- N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure to lower concentrations of SNAP (≤1 mM) led to a progressive increase in cytosolic GSH, reaching fourfold above basal by 16 h. The mechanism may involve an increase in GSH biosynthesis through effects on biosynthetic enzymes or through increased supply of cysteine, the limiting substrate. In this regard, we report that chronic exposure to SNAP led to a concentration-dependent increase in cystine uptake over a time course similar to that seen for elevation of GSH. The effect of SNAP on cystine uptake was inhibitable by either cycloheximide or actinomycin D, suggesting a requirement for both RNA and protein synthesis. Furthermore, uptake was Na+independent and was blocked by extracellular glutamate. Extracellular glutamate also blocked SNAP-mediated elevation of cytosolic GSH. Finally, in a coculture model, NO produced by cytokine-pretreated RAW 264.7 cells increased both GSH levels and cystine uptake in naive endothelial cells. These findings strongly suggest that NO leads to adaptive induction of the[Formula: see text] amino acid transport system, increased cystine uptake, and elevation of intracellular GSH levels.
In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer
Authors: Wright Carola E. , Haddad F. , Qin A. X. , Bodell P. W. , Baldwin K. M.
Published: December 2017

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.
SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton
Authors: Foster Leonard J. , Yaworsky Karen , Trimble William S. , Klip Amira
Published: December 2017

The acute stimulation of glucose uptake by insulin in fat and muscle cells is primarily the result of translocation of facilitative glucose transporter 4 (GLUT-4) from an internal compartment to the plasma membrane. Here, we investigate the role of SNAP23 (a 23-kDa molecule resembling the 25-kDa synaptosome associated protein) in GLUT-4 translocation and glucose uptake in 3T3-L1 adipocytes. Microinjection of a polyclonal antibody directed to the carboxy terminus of SNAP23 inhibited GLUT-4 incorporation into the membrane in response to insulin, whereas microinjection of full-length recombinant SNAP23 enhanced the insulin effect. Introduction of recombinant SNAP23 into chemically permeabilized cells also enhanced insulin-stimulated glucose transport. These results indicate that SNAP23 is required for insulin-dependent, functional incorporation of GLUT-4 into the plasma membrane and that the carboxy terminus of the protein is essential for this process. SNAP23 is therefore likely to be a fusion catalyst along with syntaxin-4 and vesicle-associated membrane protein (VAMP)-2. Furthermore, the endogenous content of SNAP23 appears to be limiting for insulin-dependent GLUT-4 exposure at the cell surface. A measurable fraction of SNAP23 was sedimented with cytoskeletal elements when extracted with Triton X-100, unlike VAMP-2 and syntaxin-4, which were exclusively soluble in detergent. We hypothesize that SNAP23 and its interaction with the cytoskeleton may be targets for regulation of GLUT-4 traffic.
Unloading induces transcriptional activation of the sarco(endo)plasmic reticulum Ca2+-ATPase 1 gene in muscle
Authors: Peters David G. , Mitchell-Felton Heather , Kandarian Susan C.
Published: December 2017

Previous work showed that protein and mRNA levels of the “fast” isoform of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) are markedly increased in unloaded slow-twitch soleus muscles, suggesting pretranslational control of gene expression [L. M. Schulte, J. Navarro, and S. C. Kandarian. Am. J. Physiol. 264 ( Cell Physiol. 33): C1308–C1315, 1993]. However, because of the difficulty of measuring transcription rates from whole muscle, transcriptional activation of the SERCA1 gene with unloading has not been confirmed. Because SERCA1 pre-mRNA levels can reflect transcriptional activity, in the present study SERCA1 introns were sequenced to allow intron-directed RT-PCR measurement of SERCA1 pre-mRNA. These data were then compared with changes in SERCA1 mRNA expression in control and unloaded soleus muscles. After 2, 4, and 10 days of unloading, SERCA1 pre-mRNA and mRNA transcript levels increased significantly by two-, three-, and sevenfold, respectively ( P < 0.01). Parallel increases in SERCA1 pre-mRNA and mRNA suggest transcriptional activation of the endogenous SERCA1 gene by muscle unloading. SERCA2, the cardiac/slow-twitch skeletal muscle isoform, was not markedly increased by unloading, and RNase protection assays showed no change in alternative splicing of SERCA1 or SERCA2 primary transcripts. With use of in vivo plasmid injection, the activity of a reporter gene driven by 3.6 kb of the SERCA1 5′-flanking region increased fivefold in 7-day-unloaded soleus muscles. Comparison of the magnitude of transcriptional activation of endogenous and constructed SERCA1 genes by unloading confirms the fidelity of using intronic RT-PCR to examine muscle gene transcription rates and suggests that cis-acting elements sufficient for regulating unloading-induced transcriptional activation are contained in this promoter construct.
Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy
Authors: Nakaura Hiroyuki , Morimoto Sachio , Yanaga Fumi , Nakata Masashi , Nishi Hirofumi , Imaizumi Tsutomu , Ohtsuki Iwao
Published: December 2017

A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM). In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50–55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca2+-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI. Both these two truncated TnTs conferred a lower cooperativity and a higher Ca2+ sensitivity on the Ca2+-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase. The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca2+ sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM.
Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions
Authors: Underwood Johnnie L. , Murphy Collin G. , Chen Janet , Franse-Carman Linda , Wood Irmgard , Epstein David L. , Alvarado Jorge A.
Published: December 2017

The regulation of transendothelial fluid flow by glucocorticoids was studied in vitro with use of human endothelial cells cultured from Schlemm’s canal (SCE) and the trabecular meshwork (TM) in conjunction with computer-linked flowmeters. After 2–7 wk of 500 nM dexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-fold increase in fluid flow resistance, a 2-fold increase in the representation of tight junctions, a 10- to 30-fold reduction in the mean area occupied by interendothelial “gaps” or preferential flow channels, and a 3- to 5-fold increase in the expression of the junction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, its expression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed with the use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase in resistance and the accompanying alterations in cell junctions and gaps. These results support the hypothesis that intercellular junctions are necessary for the development and maintenance of transendothelial flow resistance in cultured SCE and TM cells and are likely involved in the mechanism of increased resistance associated with glucocorticoid exposure.
Innate mechanisms of epithelial host defense: spotlight on intestine
Authors: Hecht Gail
Published: December 2017

The single layer of epithelial cells lining the intestinal tract is charged with a most difficult task: protecting the underlying biological compartments from both the normal commensal flora that reside within the intestinal lumen as well as the uninvited pathogens. To such an end, the intestinal epithelial cells are equipped with a panoply of defense mechanisms, both constitutive and inducible. This review focuses only on those defense mechanisms that are initiated and executed by the intestinal epithelial cell. Fitting these strict criteria are three major categories of epithelial host defense: enhanced salt and water secretion, expression of antimicrobial proteins and peptides, and production of intestinal mucins. Each of these areas is discussed in this review.
Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase
Authors: Duman Joseph G. , Tyagarajan Kamala , Kolsi Michelle S. , Moore Hsiao-Ping H. , Forte John G.
Published: December 2017

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 ± 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 ± 0.9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 ± 3.1 and 9.6 ± 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.
Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway
Authors: Kobuchi Hirotsugu , Roy Sashwati , Sen Chandan K. , Nguyen Hao G. , Packer Lester
Published: December 2017

The cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in inflammatory responses. Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a naturally occurring dietary flavonol, has potent anti-inflammatory properties. The effect of quercetin on ICAM-1 expression induced by agonists phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-α (TNF-α) in human endothelial cell line ECV304 (ECV) was investigated. Quercetin treatment downregulated both PMA- and TNF-α-induced surface expression, as well as the ICAM-1 mRNA levels, in ECV cells in a dose-dependent (10–50 μM) manner. Quercetin had no effect on PMA- or TNF-α-induced nuclear factor-κB (NF-κB) activation. However, under similar conditions a remarkable dose-dependent downregulation of activator protein-1 (AP-1) activation was observed. This decrease in AP-1 activation was observed to be associated with the inhibitory effects of quercetin on the c-Jun NH2-terminal kinase (JNK) pathway. These results suggest that quercetin downregulates both PMA- and TNF-α-induced ICAM-1 expression via inhibiting both AP-1 activation and the JNK pathway.
Molecular mechanism of the intestinal biotin transport process
Authors: Chatterjee Nabendu S. , Kumar Chandira K. , Ortiz Alvaro , Rubin Stanley A. , Said Hamid M.
Published: December 2017

Previous studies have characterized different aspects of the cellular/membrane mechanism and regulation of the intestinal uptake process of the water-soluble vitamin biotin. Little, however, is known about the molecular mechanisms of the uptake process. In this study, we have identified a cDNA from rat small intestine that appears to be involved in biotin transport. The open reading frame of this cloned cDNA consisted of 1,905 bases and was identical to that identified for the vitamin transporter in placental tissue. Significant heterogeneity, however, was found in the 5′ untranslated region of this clone, with three distinct variants (II, III, IV) being identified in the small intestine; the placental variant (variant I), however, was not present in the small gut. Variant II was found to be the predominant form expressed in the rat small and large intestines. Functional identity of the cloned intestinal cDNA was confirmed by stable expression in COS-7 cells, which showed a four- to fivefold increase in biotin uptake in transfected COS-7 cells compared with controls. The induced biotin uptake in transfected COS-7 cells was found to be 1) Na+ dependent, 2) saturable as a function of concentration with an apparent K m of 8.77 μM and a V max of 779.7 pmol ⋅ mg protein−1 ⋅ 3 min−1, and 3) inhibited by unlabeled biotin and pantothenic acid and their structural analogs. The distribution of complementary mRNA transcripts of the cloned cDNA along the vertical and longitudinal axes of the intestinal tract was also determined. Results of this study describe the molecular characteristics of the intestinal biotin absorption process and report the identification of a cDNA that encodes a Na+-dependent biotin uptake carrier that appears to exist in the form of multiple variants.
Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A4
Authors: Titos Esther , Chiang Nan , Serhan Charles N. , Romano Mario , Gaya Joan , Pueyo Gloria , Clària Joan
Published: December 2017

Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise new potent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during the interaction of leukocytes with either endothelial or epithelial cells. Here, we examined ATL biosynthesis in rat hepatocytes either alone or in coincubation with nonparenchymal liver cells (NPC) and in liver homogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, a rat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expression and predominantly produced thromboxane A2(TXA2) and 15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shifted the arachidonic acid metabolism from TXA2 to 15-HETE in a concentration-dependent manner. In contrast, neither indomethacin, ibuprofen, valeryl salicylate, nor nimesulide was able to trigger 15-HETE biosynthesis. SKF-525A, a cytochrome P-450 inhibitor, significantly reduced the effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital, a potent inducer of cytochrome P-450 activity, further increased ASA-induced 15-HETE production. ASA treatment of hepatocyte-NPC coincubations resulted in the generation of significant amounts of ATL. In addition, in vivo experiments demonstrated augmented hepatic levels of 15-epi-lipoxin A4 in ASA-treated rats. Taken together and considering that ASA is hydrolyzed on its first pass through the portal circulation, these data indicate that, during ASA's consumption, liver tissue generates biologically relevant amounts of ATL by COX-2-independent mechanisms.
Na+/H+ exchangers (NHE1–3) have similar turnover numbers but different percentages on the cell surface
Authors: Cavet Megan E. , Akhter Shafinaz , de Medina Fermin Sanchez , Donowitz Mark , Tse Chung-Ming
Published: December 2017

NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity ( V max) by22Na+uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s−1, when V max was determined using 22Na uptake at 22°C and 742 ± 47, 459 ± 16, and 609 ± 39 s−1 when V max was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.
Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle
Authors: Miller Kristy J. , Thaloor Deepa , Matteson Sarah , Pavlath Grace K.
Published: December 2017

Hepatocyte growth factor (HGF) is the only known growth factor that activates quiescent satellite cells in skeletal muscle. We hypothesized that local delivery of HGF may enhance regeneration after trauma by increasing the number of myoblasts available for restoring normal tissue architecture. Injection of HGF into muscle at the time of injury increases myoblast number but does not enhance tissue repair as determined using quantitative histological analyses. Rather, depending on the dose and the timing of HGF administration relative to the injury, regeneration can be inhibited. The greatest inhibitory effect is observed when HGF is administered on the day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and early differentiation normally occur. To establish a mechanism for this inhibition, we show that HGF can act directly on primary muscle cells to block differentiation. These results demonstrate that 1) exogenous HGF synergizes with factors in damaged muscle to increase myoblast number, 2) regeneration is not regulated solely by myoblast number, and 3) HGF inhibits muscle differentiation both in vitro and in vivo.
Age-dependent response of the electrocardiogram to K+ channel blockers in mice
Authors: Wang Li , Swirp Shauni , Duff Henry
Published: December 2017

Developmental changes in electrocardiogram (ECG) and response to selective K+ channel blockers were assessed in conscious, unsedated neonatal ( days 1, 7, 14) and adult male mice (>60 days of age). Mean sinus R-R interval decreased from 120 ± 3 ms in day 1 to 110 ± 3 ms in day 7, 97 ± 3 ms in day 14, and 81 ± 1 ms in adult mice ( P < 0.001 by ANOVA; all 3 groups different from day 1). In parallel, the mean P-R interval progressively decreased during development. Similarly, the mean Q-T interval decreased from 62 ± 2 ms in day 1 to 50 ± 2 ms in day 7, 47 ± 8 ms in day 14 neonatal mice, and 46 ± 2 ms in adult mice ( P < 0.001 by ANOVA; all 3 groups are significantly different from day 1). Q-Tc was calculated as Q-[Formula: see text] interval. Q-Tc significantly shortened from 179 ± 4 ms in day 1 to 149 ± 5 ms in day 7 mice ( P < 0.001). In addition, the J junction-S-T segment elevation observed in day 1 neonatal mice resolved by day 14. Dofetilide (0.5 mg/kg), the selective blocker of the rapid component of the delayed rectifier ( I Kr) abolished S-T segment elevation and prolonged Q-T and Q-Tc intervals in day 1 neonates but not in adult mice. In contrast, 4-aminopyridine (4-AP, 2.5 mg/kg) had no effect on day 1 neonates but in adults prolonged Q-T and Q-Tc intervals and specifically decreased the amplitude of a transiently repolarizing wave, which appears as an r′ wave at the end of the apparent QRS in adult mice. In conclusion, ECG intervals and configuration change during normal postnatal development in the mouse. K+ channel blockers affect the mouse ECG differently depending on age. These data are consistent with the previous findings that the dofetilide-sensitive I Kr is dominant in day 1 mice, whereas 4-AP-sensitive currents, the transiently repolarizing K+ current, and the rapidly activating, slowly inactivating K+current are the dominant K+currents in adult mice. This study provides background information useful for assessing abnormal development in transgenic mice.
The IκB/NF-κB system: a key determinant of mucosal inflammation and protection
Authors: Jobin Christian , Sartor R. Balfour
Published: December 2017

The ubiquitous transcription factor NF-κB is a central regulator of the transcriptional activation of a number of genes involved in cell adhesion, immune and proinflammatory responses, apoptosis, differentiation, and growth. Induction of these genes in intestinal epithelial cells (IECs) by activated NF-κB profoundly influences mucosal inflammation and repair. NF-κB activation requires the removal of IκB from NF-κB by inducible proteolysis, which liberates this transcription factor for migration to the nucleus, where it binds to κB-regulatory elements and induces transcription. IκBα degradation is incomplete and delayed in IECs, resulting in buffered responses to luminal stimuli. The stimulatory environment partially determines whether the effect of NF-κB is protective or deleterious for the host. κB-dependent proinflammatory gene expression, particularly chemokines, major histocompatibility complex class II antigens, and adhesion molecules may be extremely important in early protective responses to mucosal pathogens but, when dysregulated, could lead to the development of chronic inflammation, as seen in inflammatory bowel diseases. The key role of NF-κB in regulating expression of a number of proinflammatory genes makes this protein an attractive target for selective therapeutic intervention.
Sensitivity of myometrium to CGRP varies during mouse estrous cycle and in response to progesterone
Authors: Naghashpour M. , Dahl G.
Published: December 2017

Calcitonin gene-related peptide (CGRP) inhibits contractions of the myometrium. Isometric force measurements on myometrial strips were carried out to monitor the inhibitory capacity of CGRP in the myometrium during the estrous cycle and in response to estrogen and progesterone in ovariectomized mice. CGRP inhibition of KCl-induced contractions was lowest at estrus and significantly increased during metestrus and diestrus. Progesterone treatment of ovariectomized mice resulted in a significant increase in the responsiveness of the myometrium to CGRP. Expression of CGRP-receptor component protein (CGRP-RCP), a marker of CGRP-receptor expression, was quantitated by Western and Northern blot analyses. The levels of inhibition exerted by CGRP during the various stages of the estrous cycle and in response to steroid hormone treatment correlated with the protein levels of CGRP-RCP. The mRNA levels did not change significantly during the estrous cycle or in response to hormone treatment, indicating that the regulation of CGRP-RCP protein does not occur at the transcriptional level. CGRP had an inhibitory effect both when applied before the stimulus for contraction and when applied during a sustained contracture induced by KCl. This suggests that CGRP-induced generation of second messengers can influence late events in electro-/chemomechanical coupling and/or the contractile machinery directly.