bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional regulation of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb
Authors: Xu Hua , Inouye Michael , Hines Eric R. , Collins James F. , Ghishan Fayez K.
Published: May 2013

The type IIb sodium-phosphate (NaPi-IIb) cotransporter mediates intestinal phosphate absorption. Previous work in our laboratory has shown that EGF inhibited NaPi-IIb cotransporter expression through transcriptional regulation. To understand this regulation, progressively shorter human NaPi-IIb promoter constructs were used to define the EGF response region, and gel mobility shift assays (GMSAs) were used to characterize DNA-protein interactions. Promoter analysis determined that the EGF response region was located between −784 and −729 base pair (bp) of the promoter. GMSAs and overexpression studies revealed an interaction between this promoter region and c-myb transcription factor. Inhibition of EGF receptor activation restored promoter function. Further studies suggested that MAPK, PKC, and/or PKA pathways are involved in this regulation. In conclusion, these studies suggest that EGF decreases human NaPi-IIb gene expression by modifying the c-myb protein such that it inhibits transcriptional activation. We further conclude that this downregulation of promoter function is mediated by EGF-activated PKC/PKA and MAPK pathways. This is the first study that demonstrates involvement of c-myb in the regulation of intestinal nutrient absorption.
Influence of ANG II on cytoplasmic sodium in cultured rabbit nonpigmented ciliary epithelium
Authors: Hou Yining , Delamere Nicholas A.
Published: May 2013

Angiotensin (ANG) II receptors have been reported in the nonpigmented ciliary epithelium (NPE) of the eye. In cultured NPE, we found ANG II caused a dose-dependent rise of cytoplasmic sodium. The sodium increase was inhibited by the AT1-AT2 receptor antagonist saralasin (IC50 = 3.7 nM) and the AT1 antagonist losartan (IC50 = 0.6 nM) but not by the AT2 antagonist PD-123319. ANG II also caused a dose-dependent increase in the rate of ouabain-sensitive86Rb uptake. The ANG II-induced cell sodium increase and86Rb uptake increase were reduced by dimethylamiloride (DMA; 10 μM). On the basis of this finding, we propose that Na+/H+ exchange is stimulated by ANG II. Simultaneously, ANG II appears to inhibit H+-ATPase-mediated proton export. Thus Ang II (10 nM) did not alter the baseline cytoplasmic pH (pHi) but reduced pHi in cells that were also exposed to 10 μM DMA. Consistent with the notion of H+-ATPase inhibition in ANG II-treated NPE, bafilomycin A1 (100 nM) (BAF) and ANG II were both observed to suppress the pHi increase that occurs upon exposure to a mixture of epinephrine (1 μM) and acetylcholine (10 μM) and the pHi increase elicited by depolarization. In ATP hydrolysis measurements, H+-ATPase activity (bafilomycin A1-sensitive ATP hydrolysis) was reduced significantly in cells that had been pretreated 10 min with 10 nM ANG II. In summary, these studies suggest that ANG II causes H+-ATPase inhibition and an increase of cell sodium due to activation of Na+/H+ exchange.
IκBα-dependent regulation of low-shear flow-induced NF-κB activity: role of nitric oxide
Authors: Mohan Sumathy , Hamuro Masao , Sorescu George P. , Koyoma Koichi , Sprague Eugene A. , Jo Hanjoong , Valente Anthony J. , Prihoda Thomas J. , Natarajan Mohan
Published: May 2013

We have investigated the role of inhibitor κBα (IκBα) in the activation of nuclear factor κB (NF-κB) observed in human aortic endothelial cells (HAEC) undergoing a low shear stress of 2 dynes/cm2. Low shear for 6 h resulted in a reduction of IκBα levels, an activation of NF-κB, and an increase in κB-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion. Overexpression of IκBα in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of IκBα is the major factor in the low shear-induced activation of NF-κB in HAEC. We then investigated the role of nitric oxide (NO) in the regulation of IκBα/NF-κB. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-κB activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 μM) or sodium nitroprusside (1 mM) before low shear stress significantly increased cytoplasmic IκBα and concomitantly reduced NF-κB binding activity and κB-dependent VCAM-1 promoter activity. Together, these data suggest that NO may play a major role in the regulation of IκBα levels in HAEC and that the application of low shear flow increases NF-κB activity by attenuating NO generation and thus IκBα levels.
Uptake of histamine by mouse peritoneal macrophages and a macrophage cell line, RAW264.7
Authors: Tanaka Satoshi , Deai Katsuya , Inagaki Mariko , Ichikawa Atsushi
Published: May 2013

We have previously demonstrated that dietary histamine is accumulated in the spleens of l-histidine decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis. To characterize the clearance system for dietary histamine in mice, we investigated the cell type and mechanism responsible for histamine uptake in the spleens of HDC-deficient mice. Immunohistochemical analyses using an antihistamine antibody indicated that a portion of the CD14+ cells in the spleen is involved in histamine storage. Peritoneal macrophages obtained from Balb/c mice and a mouse macrophage cell line, RAW264.7, had potential for histamine uptake, which was characterized by a low affinity and high capacity for histamine. The histamine uptake by RAW264.7 cells was observed at physiological temperature and was potently inhibited by pyrilamine, chlorpromazine, quinidine, and chloroquine, moderately inhibited by Nα-methylhistamine, dopamine, and serotonin, and not affected by tetraethylammonium and 1-methyl-4-phenylpyridinium. Intracellular histamine was not metabolized in RAW264.7 cells and was released at physiological temperature in the absence of extracellular histamine. These results suggest that histamine uptake by macrophages may be involved in the clearance of histamine in the local histamine-enriched environment.
The converter domain modulates kinetic properties ofDrosophila myosin
Authors: Littlefield Kimberly Palmiter , Swank Douglas M. , Sanchez Becky M. , Knowles Aileen F. , Warshaw David M. , Bernstein Sanford I.
Published: May 2013

Recently the converter domain, an integral part of the “mechanical element” common to all molecular motors, was proposed to modulate the kinetic properties of Drosophila chimeric myosin isoforms. Here we investigated the molecular basis of actin filament velocity ( V actin) changes previously observed with the chimeric EMB-IC and IFI-EC myosin proteins [the embryonic body wall muscle (EMB) and indirect flight muscle isoforms (IFI) with genetic substitution of the IFI and EMB converter domains, respectively]. In the laser trap assay the IFI and IFI-EC myosins generate the same unitary step displacement (IFI = 7.3 ± 1.0 nm, IFI-EC = 5.8 ± 0.9 nm; means ± SE). Thus converter-mediated differences in the kinetics of strong actin-myosin binding, rather than the mechanical capabilities of the protein, must account for the observed V actin values. Basal and actin-activated ATPase assays and skinned fiber mechanical experiments definitively support a role for the converter domain in modulating the kinetic properties of the myosin protein. We propose that the converter domain kinetically couples the Pi and ADP release steps that occur during the cross-bridge cycle.
Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells
Authors: Bouley Richard , Sun Tian-Xiao , Chenard Melissa , McLaughlin Margaret , McKee Mary , Lin Herbert Y. , Brown Dennis , Ausiello Dennis A.
Published: May 2013

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.
Temporal alterations in protein signaling cascades during recovery from muscle atrophy
Authors: Childs Thomas E. , Spangenburg Espen E. , Vyas Dharmesh R. , Booth Frank W.
Published: May 2013

Currently, the repertoire of cellular and molecular pathways that control skeletal muscle atrophy and hypertrophy are not well defined. It is possible that intracellular regulatory signaling pathways are active at different times during the muscle hypertrophy process. The hypothesis of the given experiments was that cellular signals related to protein translation would be active at early time points of skeletal muscle regrowth, whereas transcriptional signals would be active at later time points of skeletal muscle regrowth. The phosphorylation status of p38 MAPK and JNK increased at the end of limb immobilization but returned to control values at recovery day 3. Transient increases in phosphorylation and in protein concentration occurred during recovery of soleus muscle mass. Phosphorylation of Akt, p70S6k, and signal transducer and activator of transcription 3 (STAT3) peaked on recovery day 3 compared with day 0. Glycogen synthase kinase (GSK)-3β phosphorylation was increased on the sixth and fifteenth recovery day. In addition, transient peaks in seven protein concentrations occurred at different times of recovery: STAT3, calcineurin A (CaNA), CaNB, and β4E-BP1 protein concentrations peaked on the third recovery day; p70S6k, STAT3, Akt, and GSK3-β peaked on the sixth recovery day; and GSK3-β peaked on the fifteenth recovery day. The apexes of STAT3 and GSK3-β protein concentrations remained elevated for two recovery time points. Thus the time course of increase in molecules of signaling pathways differed as the young rat soleus muscle regrew from an atrophied state.
DMT1, a physiologically relevant apical Cu1+transporter of intestinal cells
Authors: Arredondo Miguel , Muñoz Patricia , Mura Casilda V. , Núñez Marco T.
Published: May 2013

Despite important advances in the understanding of copper secretion and excretion, the molecular components of intestinal copper absorption remain a mystery. DMT1, also known as Nramp2 and DCT1, is the transporter responsible for intestinal iron uptake. Electrophysiological evidence suggests that DMT1 can also be a copper transporter. Thus we examined the potential role of DMT1 as a copper transporter in intestinal Caco-2 cells. Treatment of cells with a DMT1 antisense oligonucleotide resulted in 80 and 48% inhibition of iron and copper uptake, respectively. Cells incorporated considerable amounts of copper as Cu1+, whereas Cu2+ transport was about 10-fold lower. Cu1+inhibited apical Fe2+ transport. Fe2+, but not Fe3+, effectively inhibited Cu1+ uptake. The iron content of the cells influenced both copper and iron uptake. Cells with low iron content transported fourfold more iron and threefold more copper than cells with high iron content. These results demonstrate that DMT1 is a physiologically relevant Cu1+ transporter in intestinal cells, indicating that intestinal absorption of copper and iron are intertwined.
Clustering of very late antigen-4 integrins modulates K+ currents to alter Ca2+-mediated monocyte function
Authors: Colden-Stanfield Margaret
Published: May 2013

Endothelial cell vascular cell adhesion molecule-1 (VCAM-1) activates adherent monocytes by clustering their very late antigen-4 (VLA-4) receptors, resulting in the modulation of the inwardly rectifying ( I ir) and delayed rectifying ( I dr) K+ currents, hyperpolarization of the cells, and enhanced Ca2+ influx (Colden-Stanfield M and Gallin EK. Am J Physiol Cell Physiol 275: C267–C277, 1998; Colden-Stanfield M and Scanlon M. Am J Physiol Cell Physiol 279: C488–C494, 2000). The present study was undertaken to test the hypothesis that monoclonal antibodies (MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4 integrins, which play a key role in signaling an increase in the secretion of the proinflammatory cytokine interleukin-8 (IL-8). Whole cell ionic currents and IL-8 secretion from THP-1 monocytes that were incubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or an isotype-matched MAb against CD45 (MAbCD45) were measured. Clustering of VLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalent MAbVLA-4, modulated the K+ currents in an identical manner to incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 or VCAM-1 augmented Ca2+-mediated IL-8 secretion from THP-1 monocytes and was completely abolished by exposure to CsCl, an I ir blocker. Thus VLA-4 integrin clustering by cross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently to be associated with events leading to monocyte differentiation, enhanced Ca2+-mediated macrophage function, and possibly atherosclerotic plaque formation.
Cx40 and Cx43 expression ratio influences heteromeric/ heterotypic gap junction channel properties
Authors: Cottrell G. Trevor , Wu Yan , Burt Janis M.
Published: May 2013

In cells that coexpress connexin (Cx)40 and Cx43, the ratio of expression can vary depending on the cellular environment. We examined the effect of changing Cx40:Cx43 expression ratio on functional gap junction properties. Rin cells transfected with Cx40 or Cx43 (Rin40, Rin43) were cocultured with 6B5n, A7r5, A7r540C1, or A7r540C3 cells for electrophysiological and dye coupling analysis. Cx40:Cx43 expression ratio in 6B5n, A7r5, A7r540C1, and A7r540C3 cells was ∼1:1, 3:1, 5:1, and 10:1, respectively. When Rin43 cells were paired with coexpressing cells, there was an increasing asymmetry of voltage-dependent gating and a shift toward smaller conductance events as Cx40:Cx43 ratio increased in the coexpressing cell. These observations could not be predicted by linear combinations of Cx40 and Cx43 properties in proportion to the expressed ratios of the two Cxs. When Rin40 cells were paired with coexpressing cells, the net voltage gating and single-channel conductance behavior were similar to those of Rin40/Rin40 cell pairs. Dye permeability properties of cell monolayers demonstrated that as Cx40:Cx43 expression ratio increased in coexpressing cells the charge and size selectivity of dye transfer reflected that of Rin40 cells, as would be predicted. These data indicate that the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents expressed in the cell; however, the dye permeability of these same channels can be predicted by the relative Cx contributions.
Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis
Authors: Kimmich George A. , Roussie James A. , Randles Joan
Published: May 2013

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor—oxaloacetate (OA) or α-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) “protects” the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.
NHE blockade inhibits chemokine production and NF-κB activation in immunostimulated endothelial cells
Authors: Németh Zoltán H. , Deitch Edwin A. , Lu Qi , Szabó Csaba , Haskó György
Published: May 2013

Na+/H+exchanger (NHE) activation has been documented to contribute to endothelial cell injury caused by inflammatory states. However, the role of NHEs in regulation of the endothelial cell inflammatory response has not been investigated. The present study tested the hypothesis that NHEs contribute to endothelial cell inflammation induced by endotoxin or interleukin (IL)-1β. NHE inhibition using amiloride, 5-( N-ethyl- N-isopropyl)-amiloride, and 5-( N-methyl- N-isobutyl)amiloride as well as the non-amiloride NHE inhibitors cimetidine, clonidine, and harmaline suppressed endotoxin-induced IL-8 and monocyte chemoattractant protein (MCP)-1 production by human umbilical endothelial vein cells (HUVECs). The suppressive effect of amiloride on endotoxin-induced IL-8 production was associated with a decreased accumulation of IL-8 mRNA. NHE inhibitors suppressed both inhibitory (I)κB degradation and nuclear factor (NF)-κB DNA binding, suggesting that a decrease in activation of the IκB-NF-κB system contributed to the suppression of HUVEC inflammatory response by NHE blockade. NHE inhibition decreased also the IL-1β-induced HUVEC inflammatory response, because amiloride suppressed IL-1β-induced E-selectin expression on HUVECs. These results demonstrate that maximal activation of the HUVEC inflammatory response requires a functional NHE.
Functional and molecular evidence for Na+-HCO 3 − cotransporter in porcine vas deferens epithelia
Authors: Carlin Ryan W. , Quesnell Rebecca R. , Zheng Ling , Mitchell Kathy E. , Schultz Bruce D.
Published: May 2013

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na+, HCO[Formula: see text], and Cl− for a full response. HCO[Formula: see text]-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Na+-driven, HCO[Formula: see text]-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4′-dinitrostilbene-2,2′-disulfonate-sensitive stoichiometry of 2 base equivalents per Na+. Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338–391 and 928–1035) identified a single band of ∼145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO[Formula: see text] and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.
Enhancement of L-type Ca2+ current from neonatal mouse ventricular myocytes by constitutively active PKC-βII
Authors: Alden Kris J. , Goldspink Paul H. , Ruch Stuart W. , Buttrick Peter M. , Garcı́a Jesús
Published: May 2013

The cardiac L-type calcium current ( I Ca) can be modified by activation of protein kinase C (PKC). However, the effect of PKC activation on I Ca is still controversial. Some studies have shown a decrease in current, whereas other studies have reported a biphasic effect (an increase followed by a decrease in current or vice versa). A possible explanation for the conflicting results is that several isoforms of PKC with opposing effects on I Ca were activated simultaneously. Here, we examined the influence of a single PKC isoform (PKC-βII) on L-type calcium channels in isolation from other cardiac isoforms, using a transgenic mouse that conditionally expresses PKC-βII. Ventricular cardiac myocytes were isolated from newborn mice and examined for expression of the transgene using single cell RT-PCR after I Ca recording. Cells expressing PKC-βII showed a twofold increase in nifedipine-sensitive I Ca. The PKC-βII antagonist LY-379196 returned I Caamplitude to levels found in non-PKC-βII-expressing myocytes. The increase in I Ca was independent of Cav1.2-subunit mRNA levels as determined by quantitative RT-PCR. Thus these data demonstrate that PKC-β is a potent modulator of cardiac L-type calcium channels and that this specific isoform increases I Ca in neonatal ventricular myocytes.
Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187
Authors: Kalra Seema , Li Ning , Seetharam Shakuntla , Alpers David H. , Seetharam Bellur
Published: May 2013

The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to ∼25–30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.
Glutathione reverses early effects of glycation on myosin function
Authors: Ramamurthy B. , Jones A. Daniel , Larsson L.
Published: May 2013

Nonenzymatic glycosylation (glycation) has been recognized as an important posttranslational modification underlying alterations of structure and function of extracellular proteins during aging and diabetes. Intracellular proteins may also be affected by this modification, and glycation has been suggested to contribute to aging-related impairment in skeletal muscle function. Glycation is the chemical reaction of reducing sugars with primary amino groups resulting in the formation of irreversible advanced glycation end products. Glutathione is an abundant tripeptide in skeletal muscle. To understand the effect of glutathione on glycated myosin function, we used a single-fiber in vitro motility assay in which myosin is extracted from a single muscle fiber segment to propel fluorescent-labeled actin filaments. Myosin function responded to glucose exposure in a dose-dependent manner, i.e., motility speeds were reduced by 10, 34, and 90% of preincubation values after 30-min exposure to 1, 3, and 6 mM glucose, respectively. The 30-min 6 mM glucose incubation was followed by a 20-min 10 mM glutathione incubation. Glutathione treatment restored motility (0.98 ± 0.06 μm/s, n = 3; P < 0.001) after glucose exposure (0.10 ± 0.07 μm/s, n = 3), close to preincubation levels (1.12 ± 0.06 μm/s, n = 3). It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function.
Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion
Authors: Kabakov Alexander E. , Budagova Karina R. , Latchman David S. , Kampinga Harm H.
Published: May 2013

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.
Lateral membrane LXA4 receptors mediate LXA4's anti-inflammatory actions on intestinal epithelium
Authors: Kucharzik Torsten , Gewirtz Andrew T. , Merlin Didier , Madara James L. , Williams Ifor R.
Published: May 2013

Lipoxin A4 (LXA4) and its stable analogs downregulate chemokine secretion in polarized epithelia. This anti-inflammatory effect has been suggested to be mediated by the LXA4receptor (LXA4R), a G protein-coupled receptor. To determine whether LXA4R is expressed on the apical, basolateral, or both poles of intestinal epithelia, an NH2-terminal c-myc epitope tag was added to the human LXA4R cDNA and recombinant retroviruses were used to transduce polarized epithelial cells. In polarized T84 intestinal epithelial cells, c-myc-LXA4R was preferentially expressed on the basolateral surface as indicated by cell surface-selective biotinylation and confocal microscopy. Furthermore, expression of c-myc-LXA4R and a truncation mutant lacking the cytoplasmic terminus was primarily confined to the lateral subdomain. We also observed that the expression of myc-LXA4 conferred enhanced downregulation of IL-8 expression in response to LXA4analog and that blockade of the CysLT1 receptor by montelukast did not prevent this response to LXA4 analog. Thus LXA4generated in or near the paracellular space via neutrophil-epithelial interactions can rapidly act on epithelial LXA4R to downregulate epithelial promotion of intestinal inflammation.
Cell volume increase and extracellular Ca2+ are needed for hyposmotically induced prolactin release in tilapia
Authors: Seale A. P. , Richman N. H. , Hirano T. , Cooke I. , Grau E. G.
Published: May 2013

In the tilapia ( Oreochromis mossambicus), as in many euryhaline teleost fish, prolactin (PRL) plays a central role in freshwater adaptation, acting on osmoregulatory surfaces to reduce ion and water permeability and increase solute retention. Consistent with these actions, PRL release is stimulated as extracellular osmolality is reduced both in vivo and in vitro. In the current experiments, a perfusion system utilizing dispersed PRL cells was developed for permitting the simultaneous measurement of cell volume and PRL release. Intracellular Ca2+ was monitored using fura 2-loaded cells under the same conditions. When PRL cells were exposed to hyposmotic medium, an increase in PRL cell volume preceded the increase in PRL release. Cell volume increased in proportion to decreases of 15 and 30% in osmolality. However, regulatory volume decrease was clearly seen only after a 30% reduction. The hyposmotically induced PRL release was sharply reduced in Ca2+-deleted hyposmotic medium, although cell volume changes were identical to those observed in normal hyposmotic medium. In most cells, a rise in intracellular Ca2+ concentration ([Ca2+]i) during hyposmotic stimulation was dependent on the availability of extracellular Ca2+, although small transient increases in [Ca2+]i were sometimes observed upon introduction of Ca2+-deleted media of the same or reduced osmolality. These results indicate that an increase in cell size is a critical step in the transduction of an osmotic signal into PRL release and that the hyposmotically induced increase in PRL release is greatly dependent on extracellular Ca2+.
Calcium homeostasis in rat cardiomyocytes during chronic hypoxia: a time course study
Authors: Pei Jian-Ming , Kravtsov Gennadi M. , Wu Song , Das Rapti , Fung Man Lung , Wong Tak Ming
Published: May 2013

The present study determined Ca2+ handling in the hearts of rats subjected to chronic hypoxia (CH). Spectrofluorometry was used to measure intracellular Ca2+ concentration ([Ca2+]i) and its responses to electrical stimulation, caffeine, and isoproterenol in myocytes from the right ventricle of rats breathing 10% oxygen for 1, 3, 7, 14, 21, 28, and 56 days and age-matched controls. The protein expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and its ryanodine receptor (RyR) were measured. The uptake of 45Ca2+ by SERCA, release by RyR, and extrusion by Na+/Ca2+ exchange (NCX) were determined. It was found that Ca2+ homeostasis and Ca2+ responses to β-adrenoceptor stimulation reached a new equilibrium after 4 wk of CH. Ca2+ content in the sarcoplasmic reticulum (SR) was reduced, but cytosolic Ca2+ remained unchanged after CH. Expression of SERCA and its Ca2+ uptake, Ca2+ release via RyR, and NCX activity were suppressed by CH. The results indicate impaired Ca2+ handling, which may be responsible for the attenuated Ca2+ responses to β-adrenoceptor stimulation in CH.
Quantification of cytoskeletal deformation in living cells based on hierarchical feature vector matching
Authors: Delhaas Tammo , van Engeland Saskia , Broers Jos , Bouten Carlijn , Kuijpers Nico , Ramaekers Frans , Snoeckx Luc H. E. H.
Published: May 2013

The cytoskeleton is a dynamic scaffold in living cells even in the absence of externally imposed forces. In this study on cytoskeletal deformation, the applicability of hierarchical feature vector matching (HFVM), a new matching method, currently applied in space research and three-dimensional surface reconstruction, was investigated. Stably transfected CHO-K1 cells expressing green fluorescent protein (GFP) coupled to vimentin were used to visualize spontaneous movement of the vimentin cytoskeleton of individual cells using a confocal laser scanning system. We showed that, with proper parameter and configuration settings, HFVM could recognize and trace 60–70% of all image points in artificially translated, rotated, or deformed images. If only points belonging to the cytoskeleton were selected for matching purposes, the percentage of matched points increased to 98%. This high percentage of recognition also could be reached in a time series of images, in which a certain degree of bleaching of the fluorescence over the recording time of 30 min was inevitable. In these images, HFVM allowed the detection as well as the quantification of spontaneous cytoskeletal movements of up to 10% of the cell width. Therefore, HFVM appears to be a reliable method of quantifying dynamic cytoskeletal behavior in living cells.
The third intracellular loop and carboxyl tail of neurokinin 1 and 3 receptors determine interactions with β-arrestins
Authors: Schmidlin Fabien , Roosterman Dirk , Bunnett Nigel W.
Published: May 2013

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with β-arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of β-arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only β-arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with β-arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca2+ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with β-arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with β-arrestin 1 and determine the rate of resensitization.
Acridine orange induces translocation of phosphatidylserine to red blood cell surface
Authors: Koshkaryev Alexander , Yedgar Saul , Relevy Hanna , Fibach Eithan , Barshtein Gregory
Published: May 2013

Clustering of band-3 on red blood cell (RBC) surface has been assumed to catalyze RBC phagocytosis. In studying this subject, acridine orange (AO) has commonly been employed on the assumption that it specifically induces band-3 clustering. In the present study, we show that AO strongly induces translocation of phosphatidylserine (PS) to RBC surface. Because surface PS is well known to induce RBC intercellular interaction, these findings suggest that the use of AO as a specific inducer of band-3 clustering is questionable. It is possible that band-3 clustering and PS translocation are interdependent, and this interrelationship has yet to be explored.
Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells
Authors: Zhao Rongbao , Gao Feng , Goldman I. David
Published: May 2013

Although the reduced folate carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share ∼40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ∼50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia.
Transforming growth factor-β inhibition of proteasomal activity: a potential mechanism of growth arrest
Authors: Tadlock Laura , Yamagiwa Yoko , Hawker James , Marienfeld Carla , Patel Tushar
Published: May 2013

Although the proteasome plays a critical role in the controlled degradation of proteins involved in cell cycle control, the direct modulation of proteasomal function by growth regulatory signaling has not yet been demonstrated. We assessed the effect of transforming growth factor (TGF)-β, a potent inhibitor of cell growth, on proteasomal function. TGF-β selectively decreased hydrolysis of the proteasomal substrate Cbz-Leu-Leu-Leu-7-amido-4-methyl-coumarin (z-LLL-AMC) in a concentration-dependent manner but did not inhibit hydrolysis of other substrates Suc-Leu-Leu-Val-Tyr-AMC (suc-LLVY-AMC) or Cbz-Leu-Leu-Glu-AMC (z-LLE-AMC). An increase in intracellular oxidative injury occurred during incubation with TGF-β. Furthermore, in vitro hydrolysis of z-LLL-AMC, but not suc-LLVY-AMC, was decreased by hydrogen peroxide. TGF-β did not increase cellular expression of heat shock protein (HSP)90, a potent inhibitor of z-LLL-AMC hydrolysis in vitro. The physiological relevance of TGF-β inhibition of proteasomal activity was studied by assessing the role of z-LLL-AMC hydrolysis on cyclin-dependent kinase inhibitor expression and cell growth. TGF-β increased expression of p27KIP1 but did not alter expression of p21WAF1 or p16INK4A. The peptide aldehyde Cbz-Leu-Leu-leucinal (LLL-CHO or MG132) potently inhibited z-LLL-AMC hydrolysis in cell extracts as well as increasing p27KIP1 and decreasing cell proliferation. Thus growth inhibition by TGF-β decreases a specific proteasomal activity via an HSP90-independent mechanism that may involve oxidative inactivation or modulation of proteasomal subunit composition and results in altered cellular expression of key cell cycle regulatory proteins such as p27KIP1.
Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells
Authors: Wang Xiuqiong , Kaetzel Marcia A. , Yoo Sung E. , Kim Paul S. , Dedman John R.
Published: May 2013

The exposure of anionic phospholipids on the external surface of injured endothelial cells and activated platelets is a primary biological signal to initiate blood coagulation. Disease conditions that promote the formation of ectopic thrombi result in tissue ischemia. Annexins, Ca2+-dependent anionic phospholipid binding proteins, are potential therapeutic agents for the inhibition of coagulation. We have designed a transgene that targets secretion of annexin V from cultured thyroid cells under the control of doxycycline. Our results indicate that annexin V in the endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis, processing, and secretion of thyroglobulin. ER luminal Ca2+was moderately increased and can be released by inositol 1,4,5-trisphosphate. Our study demonstrates that targeting and secretion of annexin V through the secretory pathway of mammalian cells does not adversely affect cellular function. Regulated synthesis and release of annexin V may exert anticoagulatory and anti-inflammatory effects systemically and may prove useful in further developing therapeutic strategies for conditions including antiphospholipid syndrome.
Myosin light chain phosphorylation facilitates in vivo myosin filament reassembly after mechanical perturbation
Authors: Qi D. , Mitchell R. W. , Burdyga T. , Ford L. E. , Kuo K.-H. , Seow C. Y.
Published: May 2013

Phosphorylation of the 20-kDa regulatory myosin light chain (MLC) of smooth muscle is known to cause monomeric myosins in solution to self-assemble into thick filaments. The role of MLC phosphorylation in thick filament formation in intact muscle, however, is not clear. It is not known whether the phosphorylation is necessary to initiate thick filament assembly in vivo. Here we show, by using a potent inhibitor of MLC kinase (wortmannin), that the MLC phosphorylation and isometric force in trachealis muscle could be abolished without affecting calcium transients. By measuring cross-sectional densities of the thick filaments electron microscopically, we also show that inhibition of MLC phosphorylation alone did not cause disassembly of the filaments. The unphosphorylated thick filaments, however, partially dissolved when the muscle was subjected to oscillatory strains (which caused a 25% decrease in the thick filament density). The postoscillation filament density recovered to the preoscillation level only when wortmannin was removed and the muscle was stimulated. The data suggest that in vivo thick filament reassembly after mechanical perturbation is facilitated by the cyclic MLC phosphorylation associated with repeated stimulation.
Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase
Authors: Liu Lijun , Mohammadi Kamiar , Aynafshar Behrouz , Wang Haojie , Li Daxiang , Liu Jiang , Ivanov Alexander V. , Xie Zijian , Askari Amir
Published: May 2013

Ouabain binding to Na+/K+-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca2+ concentration ([Ca2+]i) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na+/K+-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20–30% of cellular contents of α1- and α2-isoforms of Na+/K+-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and α-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar α1-isoform and caveolin-3; and 3) 50–60% increases in caveolar Src and α2-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na+/K+-ATPase to ERK1/2 and [Ca2+]i are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and α2-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.
Determinants of cardiac Na+/Ca2+exchanger temperature dependence: NH2-terminal transmembrane segments
Authors: Marshall Christian , Elias Chadwick , Xue Xiao-Hua , Le Hoa Dinh , Omelchenko Alexander , Hryshko Larry V. , Tibbits Glen F.
Published: May 2013

The cardiac Na+/Ca2+ exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30°C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH2-terminal region of the molecule. Specifically, we found that ∼75% of the Na+/Ca2+ exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.
Protein kinase C regulates endocytosis and recycling of E-cadherin
Authors: Le Tam Luan , Joseph Shannon R. , Yap Alpha S. , Stow Jennifer L.
Published: May 2013

E-cadherin is a major component of adherens junctions in epithelial cells. We showed previously that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. In the present study, we investigated the potential role of protein kinase C (PKC) in regulating the trafficking of surface E-cadherin in Madin-Darby canine kidney cells. Using surface biotinylation and immunofluorescence, we found that treatment of cells with phorbol esters increased the rate of endocytosis of E-cadherin, resulting in accumulation of E-cadherin in apically localized early or recycling endosomes. The recycling of E-cadherin back to the surface was also decreased in the presence of phorbol esters. Phorbol ester-induced endocytosis of E-cadherin was blocked by specific inhibitors, implicating novel PKC isozymes, such as PKC-ε in this pathway. PKC activation led to changes in the actin cytoskeleton facilitating E-cadherin endocytosis. Depolymerization of actin increased endocytosis of E-cadherin, whereas the PKC-induced uptake of E-cadherin was blocked by the actin stabilizer jasplakinolide. Our findings show that PKC regulates vital steps of E-cadherin trafficking, its endocytosis, and its recycling.
Coordinated expression of UT-A and UT-B urea transporters in rat testis
Authors: Fenton R. A. , Cooper G. J. , Morris I. D. , Smith C. P.
Published: May 2013

The blood-seminiferous tubule barrier is responsible for maintaining the unique microenvironment conducive to spermatogenesis. A key feature of the blood-testis barrier is selective permeability to solutes and water transport, conferred by the Sertoli cells of the seminiferous tubules (SMTs). Movement of fluid into the lumen of the seminiferous tubule is crucial to spermatogenesis. By Northern analysis, we have shown that 4.0-, 3.3-, 2.8-, and ∼1.7-kb UT-A mRNA transcripts and a 3.8-kb UT-B mRNA transcript are detected within rat testis. Western analysis revealed the expression of both characterized and novel UT-A and UT-B proteins within the testis. Immunolocalization studies determined that UT-A and UT-B protein expression are coordinated with the developmental stage of the SMT. UT-A proteins were detected in Sertoli cell nuclei at all stages of tubule development and in residual bodies of stage VIII tubules. UT-B protein was expressed on Sertoli cell membranes of stage II–III tubules. Using in vitro perfusion, we determined that a phloretin-inhibitable urea pathway exists across the SMTs of rat testis and conclude that UT-B is likely to participate in this pathway.