bluuebunny/crossref_metadata_2025 huggingface dataset:
Aldosterone potentiates 1,25-dihydroxyvitamin D3 action in renal thick ascending limb via a nongenomic, ERK-dependent pathway
Authors: Good David W. , George Thampi , Watts Bruns A.
Published: May 2013

Recently, we demonstrated that aldosterone inhibits HCO3- absorption in the rat medullary thick ascending limb (MTAL) via a nongenomic pathway blocked by inhibitors of extracellular signal-regulated kinase (ERK) activation. Here we examined the effects on the MTAL of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which regulates cell functions through nongenomic mechanisms in nonrenal systems. Addition of 1 nM 1,25(OH)2D3 to the bath decreased HCO3- absorption by 24%, from 15.0 ± 0.3 to 11.4 ± 0.5 pmol· min-1· mm-1 ( P < 0.001). This inhibition was maximal within 60 min and was eliminated by pretreatment with actinomycin D, cycloheximide, or inhibitors of protein kinase C. In MTAL bathed with 1 nM aldosterone [added 15-20 min before 1,25(OH)2D3], the absolute (5.6 ± 0.3 vs. 3.6 ± 0.3 pmol· min-1· mm-1) and fractional (49 ± 2 vs. 24 ± 2%) decreases in HCO3- absorption induced by 1,25(OH)2D3 were significantly greater than those in the absence of aldosterone ( P < 0.05). The effect of aldosterone to potentiate inhibition by 1,25(OH)2D3 was not affected by spironolactone but was eliminated by the MAPK kinase/ERK inhibitor U-0126. U-0126 did not affect inhibition of HCO3- absorption by 1,25(OH)2D3 alone. Aldosterone induced rapid activation of ERK via a transcription-independent pathway. We conclude that 1) 1,25(OH)2D3 inhibits HCO3- absorption in the MTAL via a genomic pathway involving protein kinase C, which may contribute to 1,25(OH)2D3-induced regulation of urinary net acid and/or Ca2+ excretion and 2) aldosterone potentiates inhibition by 1,25(OH)2D3 through an ERK-dependent, nongenomic pathway. These results identify a novel regulatory interaction whereby aldosterone acts via nongenomic mechanisms to enhance the genomic response to 1,25(OH)2D3. Aldosterone may influence a broad range of biological processes, including epithelial transport, by modifying the response of target tissues to 1,25(OH)2D3 stimulation.
IK channels are involved in the regulatory volume decrease in human epithelial cells
Authors: Wang Jun , Morishima Shigeru , Okada Yasunobu
Published: May 2013

Parallel activation of Ca2+-dependent K+ channels and volume-sensitive Cl− channels is known to be responsible for KCl efflux during regulatory volume decrease (RVD) in human epithelial Intestine 407 cells. The present study was performed to identify the K+ channel type. RT-PCR demonstrated mRNA expression of Ca2+-activated, intermediate conductance K+(IK), but not small conductance K+ (SK1) or large conductance K+ (BK) channels in this cell line. Whole cell recordings showed that ionomycin or hypotonic stress activated inwardly rectifying K+ currents that were reversibly blocked by IK channel blockers [clotrimazole (CLT) and charybdotoxin] but not by SK and BK channel blockers (apamin and iberiotoxin). Inside-out recordings revealed the existence of CLT-sensitive single K+-channel activity, which exhibited an intermediate unitary conductance (30 pS at −100 mV). The channel was activated by cytosolic Ca2+ in inside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin or iberiotoxin. Thus we conclude that the IK channel is involved in the RVD process in these human epithelial cells.
Mitochondrial oxidant production by a pollutant dust and NO-mediated apoptosis in human alveolar macrophage
Authors: Huang Yuh-Chin T. , Soukup Joleen , Harder Shirley , Becker Susanne
Published: May 2013

Residual oil fly ash (ROFA) is a pollutant dust that stimulates production of reactive oxygen species (ROS) from mitochondria and apoptosis in alveolar macrophages (AM), but the relationship between these two processes is unclear. In this study, human AM were incubated with ROFA or vanadyl sulfate (VOSO4), the major metal constituent in ROFA, with or without nitro-l-arginine methyl ester (l-NAME), diphenyleneiodonium (DPI), and mitochondrial electron transport inhibitors. Interactions among production of ROS, nitric oxide (NO), and apoptosis of AM were determined. ROFA-stimulated ROS production was attenuated by DPI, rotenone, antimycin, and NaN3, but not by l-NAME, a pattern mimicked by VOSO4. ROFA-induced apoptosis was inhibited byl-NAME and a caspase-3-like protease inhibitor, but not by mitochondrial inhibitors. ROFA enhanced NO-mediated increase in caspase-3-like activity. VOSO4 had minor effects on apoptosis. Thus ROFA-stimulated production of ROS from mitochondria was independent of apoptosis of AM, which was mediated by activation of caspase-3-like proteases and NO. The pro-oxidant effect but not the proapoptotic effect of ROFA was mediated by vanadium.
PDGF upregulates delayed rectifier via Src family kinases and sphingosine kinase in oligodendroglial progenitors
Authors: Soliven Betty , Ma Lan , Bae Hyun , Attali Bernard , Sobko Alexander , Iwase Tamaki
Published: May 2013

An increase in the expression of the delayed rectifier current ( I K) has been shown to correlate with mitogenesis in many cell types. However, pathways involved in the upregulation of I K by growth factors in oligodendroglial progenitors (OPs) have not been well-elucidated. In this study, we found that treatment with platelet-derived growth factor (PDGF) and basic fibroblast growth factor but not ciliary neurotrophic factor resulted in increased I K density and upregulation of Kv1.5 and Kv1.6 mRNA transcripts. The effect of PDGF on I K was blocked by mimosine, a cell cycle inhibitor, and by genistein, a tyrosine kinase inhibitor. Using inhibitors of PDGF-activated pathways, we found that PDGF-induced upregulation of Kv1.5 and I K density involves Src family tyrosine kinases, sphingosine kinase, and intracellular Ca2+ but not ERK1/2 or phosphatidylinositol 3-kinase pathways. Furthermore, agents that were effective inhibitors of PDGF-induced I Kupregulation also attenuated OP proliferation, supporting the concept that I K is an important link between PDGF-activated signaling cascades and cell cycle progression.
On the mechanism of thrombin-induced angiogenesis: involvement of αvβ3-integrin
Authors: Tsopanoglou Nikos E. , Andriopoulou Paraskevi , Maragoudakis Michael E.
Published: May 2013

Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of αvβ3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with αvβ3 as demonstrated by direct binding of αvβ3 protein to immobilized thrombin. This interaction of thrombin with αvβ3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to αvβ3 or a specific peptide antagonist to αvβ3 can abolish all these αvβ3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to αvβ3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi.
Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli
Authors: Gartzke Joachim , Lange Klaus
Published: May 2013

The interaction of weak electromagnetic fields (EMF) with living cells is a most important but still unresolved biophysical problem. For this interaction, thermal and other types of noise appear to cause severe restrictions in the action of weak signals on relevant components of the cell. A recently presented general concept of regulation of ion and substrate pathways through microvilli provides a possible theoretical basis for the comprehension of physiological effects of even extremely low magnetic fields. The actin-based core of microfilaments in microvilli is proposed to represent a cellular interaction site for magnetic fields. Both the central role of F-actin in Ca2+ signaling and its polyelectrolyte nature eliciting specific ion conduction properties render the microvillar actin filament bundle an ideal interaction site for magnetic and electric fields. Ion channels at the tip of microvilli are connected with the cytoplasm by a bundle of microfilaments forming a diffusion barrier system. Because of its polyelectrolyte nature, the microfilament core of microvilli allows Ca2+ entry into the cytoplasm via nonlinear cable-like cation conduction through arrays of condensed ion clouds. The interaction of ion clouds with periodically applied EMFs and field-induced cation pumping through the cascade of potential barriers on the F-actin polyelectrolyte follows well-known physical principles of ion-magnetic field (MF) interaction and signal discrimination as described by the stochastic resonance and Brownian motor hypotheses. The proposed interaction mechanism is in accord with our present knowledge about Ca2+signaling as the biological main target of MFs and the postulated extreme sensitivity for coherent excitation by very low field energies within specific amplitude and frequency windows. Microvillar F-actin bundles shielded by a lipid membrane appear to function like electronic integration devices for signal-to-noise enhancement; the influence of coherent signals on cation transduction is amplified, whereas that of random noise is reduced.
Nuclear factor-κB activation promotes restitution of wounded intestinal epithelial monolayers
Authors: Egan Laurence J. , de Lecea Ana , Lehrman Evan D. , Myhre Gennett M. , Eckmann Lars , Kagnoff Martin F.
Published: May 2013

Epithelial restitution, the movement of wound-edge cells into an area of epithelial cell denudation, is an important early step in the ulcer healing process. Growth factors regulate epithelial restitution, yet little is known about the transcriptional pathways that mediate their effects on cell migration. The transcription factor nuclear factor (NF)-κB is a master regulator of the host inflammatory response that is activated in the epithelium in intestinal inflammation, which often accompanies epithelial injury. We hypothesized that NF-κB may be an important transcriptional regulator of epithelial restitution. In an in vitro model of scrape-wounded monolayers of nontransformed rat intestinal epithelial (RIE-1) cells, NF-κB was activated in epithelial cells at the wound edge. Blocking of NF-κB activation by either pharmacological or genetic approaches inhibited intestinal epithelial restitution. Moreover, scrape wounding activated the epidermal growth factor receptor (EGFR) in cells at the wound edge, and, importantly, inhibiting EGFR tyrosine kinase activity decreased scrape wound-induced NF-κB activation and cell migration. These results indicate a novel role of NF-κB activation in a signaling pathway important for restitution and healing of intestinal epithelia. To the extent NF-κB may have parallel functions in vivo, they also suggest a need for caution in the proposed use of NF-κB inhibitors for the treatment of conditions associated with inflammation and injury of intestinal and other mucosal surfaces.
Rho GTPase signaling modulates cell shape and contractile phenotype in an isoactin-specific manner
Authors: Kolyada Alexey Y. , Riley Kathleen N. , Herman Ira M.
Published: May 2013

Rho family small GTPases (Rho, Rac, and Cdc42) play an important role in cell motility, adhesion, and cell division by signaling reorganization of the actin cytoskeleton. Here, we report an isoactin-specific, Rho GTPase-dependent signaling cascade in cells simultaneously expressing smooth muscle and nonmuscle actin isoforms. We transfected primary cultures of microvascular pericytes, cells related to vascular smooth muscle cells, with various Rho-related and Rho-specific expression plasmids. Overexpression of dominant positive Rho resulted in the formation of nonmuscle actin-containing stress fibers. At the same time, α-vascular smooth muscle actin (αVSMactin) containing stress fibers were disassembled, resulting in a dramatic reduction in cell size. Rho activation also yielded a disassembly of smooth muscle myosin and nonmuscle myosin from stress fibers. Overexpression of wild-type Rho had similar but less dramatic effects. In contrast, dominant negative Rho and C3 exotransferase or dominant positive Rac and Cdc42 expression failed to alter the actin cytoskeleton in an isoform-specific manner. The loss of smooth muscle contractile protein isoforms in pericyte stress fibers, together with a concomitant decrease in cell size, suggests that Rho activation influences “contractile” phenotype in an isoactin-specific manner. This, in turn, should yield significant alteration in microvascular remodeling during developmental and pathologic angiogenesis.
Distribution and functional properties of human KCNH8 (Elk1) potassium channels
Authors: Zou Anruo , Lin Zhixin , Humble Margaret , Creech Christopher D. , Wagoner P. Kay , Krafte Douglas , Jegla Timothy J. , Wickenden Alan D.
Published: May 2013

The Elk subfamily of the Eag K+ channel gene family is represented in mammals by three genes that are highly conserved between humans and rodents. Here we report the distribution and functional properties of a member of the human Elk K+ channel gene family, KCNH8. Quantitative RT-PCR analysis of mRNA expression patterns showed that KCNH8, along with the other Elk family genes, KCNH3 and KCNH4, are primarily expressed in the human nervous system. KCNH8 was expressed at high levels, and the distribution showed substantial overlap with KCNH3. In Xenopus oocytes, KCNH8 gives rise to slowly activating, voltage-dependent K+ currents that open at hyperpolarized potentials (half-maximal activation at -62 mV). Coexpression of KCNH8 with dominant-negative KCNH8, KCNH3, and KCNH4 subunits led to suppression of the KCNH8 currents, suggesting that Elk channels can form heteromultimers. Similar experiments imply that KCNH8 subunits are not able to form heteromultimers with Eag, Erg, or Kv family K+ channels.
Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells
Authors: Bannon Desmond I. , Abounader Roger , Lees Peter S. J. , Bressler Joseph P.
Published: May 2013

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller V max for iron uptake compared with control cell lines. One clone was further characterized and found to display an ∼50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.
PepT1-mediated fMLP transport induces intestinal inflammation in vivo
Authors: Buyse Marion , Tsocas Annick , Walker Francine , Merlin Didier , Bado Andre
Published: May 2013

In the present study, the effect of H+/peptide transporter (PepT1)-mediated N-formylmethionyl-leucyl-phenylalanine (fMLP) transport on inflammation in vivo in the rat small intestine, which expresses high PepT1 levels, and in the rat colon, which does not express PepT1, were investigated using myeloperoxidase (MPO) activity and histological analysis. We found that 10 μM fMLP perfusion in the jejunum for 4 h significantly increased MPO activity and altered the architecture of jejunal villi. In contrast, 10 μM fMLP perfusion in the colon for 4 h did not induce any inflammation. In addition, we have shown that 50 mM Gly-Gly alone did not affect basal MPO activity but completely inhibited the MPO activity induced by 10 μM fMLP in the jejunum. Together, these experiments demonstrate that 1) the differential expression of PepT1 between the small intestine and the colon plays an important role in epithelial-neutrophil interactions and 2) the inhibition of fMLP uptake by jejunal epithelial cells (expressing PepT1) reduces the neutrophil ability to move across the epithelium, in agreement with our previously published in vitro study. This report constitutes the first in vivo study showing the implication of a membrane transporter (PepT1) in intestinal inflammation.
β-Adrenergic potentiation of endoplasmic reticulum Ca2+ release in brown fat cells
Authors: Leaver Eric V. , Pappone Pamela A.
Published: May 2013

We find that the adrenergic agonist isoproterenol increases intracellular Ca2+ concentration ([Ca2+]i) in cultured rat brown adipocytes. At the concentration used (10 μM), isoproterenol-induced Ca2+ responses were sensitive to block by either α1- or β-adrenergic antagonists, suggesting an interaction between these receptor subtypes. Despite reliance on β-adrenoceptor activation, the Ca2+ response was not due solely to increases in cAMP because, administered alone, the selective β3-adrenergic agonist BRL-37344 or forskolin did not increase [Ca2+]i. However, increased cAMP elicited vigorous [Ca2+]i increases in the presence of barely active concentrations of the α-adrenergic agonist phenylephrine or the P2Y receptor agonist UTP. Consistent with isoproterenol recruiting only inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, endoplasmic reticulum store depletion by thapsigargin blocked isoproterenol-induced Ca2+ increases, but removal of external Ca2+did not. These results argue that increases in cAMP sensitize the IP3-mediated Ca2+ release system in brown adipocytes.
A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway
Authors: Said Hamid M. , Ortiz Alvaro , Ma Thomas Y.
Published: May 2013

Vitamin B6 is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B6 and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B6 homeostasis. Due to the water-soluble nature of vitamin B6 and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B6 in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na+ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant ( Km) of 11.99 ± 1.41 μM and a maximal velocity ( Vmax) of 67.63 ± 3.87 pmol · mg protein-1 · 3 min-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant ( Ki) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca+2/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the Vmax, but not the apparent Km, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway.
Skeletal muscle LIM protein 1 (SLIM1/FHL1) induces α5β1-integrin-dependent myocyte elongation
Authors: McGrath Meagan J. , Mitchell Christina A. , Coghill Imogen D. , Robinson Paul A. , Brown Susan
Published: May 2013

Skeletal muscle LIM protein 1 (SLIM1/FHL1) contains four and a half LIM domains and is highly expressed in skeletal and cardiac muscle. Elevated SLIM1 mRNA expression has been associated with postnatal skeletal muscle growth and stretch-induced muscle hypertrophy in mice. Conversely, SLIM1 mRNA levels decrease during muscle atrophy. Together, these observations suggest a link between skeletal muscle growth and increased SLIM1 expression. However, the precise function of SLIM1 in skeletal muscle, specifically the role of SLIM1 during skeletal muscle differentiation, is not known. This study investigated the effect of increased SLIM1 expression during skeletal muscle differentiation. Western blot analysis showed an initial decrease followed by an increase in SLIM1 expression during differentiation. Overexpression of SLIM1 in Sol8 or C2C12 skeletal muscle cell lines, at levels observed during hypertrophy, induced distinct effects in differentiating myocytes and undifferentiated reserve cells, which were distinguished by differential staining for two markers of differentiation, MyoD and myogenin. In differentiating skeletal myocytes, SLIM1 overexpression induced hyperelongation, which, by either plating cells on poly-l-lysine or using a series of peptide blockade experiments, was shown to be specifically dependent on ligand binding to the α5β1-integrin, whereas in reserve cells, SLIM1 overexpression induced the formation of multiple cytoplasmic protrusions (branching), which was also integrin mediated. These results suggest that SLIM1 may play an important role during the early stages of skeletal muscle differentiation, specifically in α5β1-integrin-mediated signaling pathways.
Mechanism of v-Src- and mitogen-activated protein kinase-induced reduction of gap junction communication
Authors: Cottrell G. Trevor , Lin Rui , Warn-Cramer Bonnie J. , Lau Alan F. , Burt Janis M.
Published: May 2013

Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.
Apoptosis by Cd2+ or CdMT in proximal tubule cells: different uptake routes and permissive role of endo/lysosomal CdMT uptake
Authors: Erfurt Cornelia , Roussa Eleni , Thévenod Frank
Published: May 2013

The mechanisms of cadmium-metallothionein (CdMT) uptake and toxicity in proximal tubule (PT) cells are not well understood. The effects of 10 μM CdCl2 or Cd7MT-1 (MT-1 saturated with 10 μM CdCl2) on 109Cd2+ uptake, viability, and MT levels of cultured rat PT cells were investigated. Apical 109Cd2+ uptake was measured in confluent monolayers, apoptosis was assessed with Hoechst 33342, and intracellular MT levels were monitored by immunofluorescence and quantitative morphometry. 109Cd2+ uptake into PTC increased over time and plateaued at 24 h. 109Cd7MT-1 uptake was delayed but reached a similar magnitude after 40 h. With Cd2+, apoptosis occurred within 4 h, peaked at 24 h, and declined at 48-72 h. Cd7MT-1 induced apoptosis after 24-36 h, reaching similar levels as with Cd2+ after 48 h. Cd2+ and Cd7MT-1 significantly increased intracellular MT immunoreactivity after 20 and 4 h, respectively. The weak base chloroquine and the inhibitor of phosphatidylinositol 3-kinases, LY-294002, selectively inhibited the effects of Cd7MT-1 on MT immunoreactivity and apoptosis. PT cells accumulated 109Cd7MT-1 in membrane vesicles associated with the late endo/lysosomal marker LAMP1 but less with the early endosomal marker Rab5a, which was abolished by chloroquine or LY-294002. Thus development of apoptosis followed the uptake kinetics of Cd2+ and Cd7MT-1. Endo/lysosomal inhibitors prevented uptake of Cd7MT-1 into endo/lysosomes and apoptosis but had no effect on these parameters with Cd2+, suggesting that apoptosis of PT cells is triggered by free cytosolic Cd2+, either by direct apical transport or by translocation of free Cd2+ from endo/lysosomes after endocytosis of Cd7MT-1.
Interplay between integrins and FLK-1 in shear stress-induced signaling
Authors: Wang Yingxiao , Miao Hui , Li Song , Chen Kuang-Den , Li Yi-Shuan , Yuan Suli , Shyy John Y.-J. , Chien Shu
Published: May 2013

Blood flow can modulate vascular cell functions. We studied interactions between integrins and Flk-1 in transducing the mechanical shear stress due to flow. This application of a step shear stress caused Flk-1 · Casitas B-lineage lymphoma (Cbl) activation (Flk-1 · Cbl association, tyrosine phosphorylation of the Cbl-bound Flk-1, and tyrosine phosphorylation of Cbl) in bovine aortic endothelial cells (BAECs). The activation of integrins by plating BAECs on vitronectin or fibronectin also induced this Flk-1 · Cbl activation. The shear-induced Flk-1 · Cbl activation was blocked by inhibitory antibodies for αvβ3- or β1-integrin, suggesting that it is mediated by integrins. Inhibition of Flk-1 by SU1498 also abolished this shear-induced Flk-1 · Cbl activation. In contrast to the requirement of integrins for Flk-1 · Cbl activation, the Flk-1 blocker SU1498 had no detectable effect on the shear-induced integrin activation, suggesting that integrins and Flk-1 play sequential roles in the signal transduction hierarchy induced by shear stress. Integrins are essential for the mechanical activation of Flk-1 by shear stress but not for the chemical activation of Flk-1 by VEGF.
Neuroprotective role of δ-opioid receptors in cortical neurons
Authors: Zhang Junhui , Gibney Geoffrey Thomas , Zhao Peng , Xia Ying
Published: May 2013

We recently demonstrated that δ-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons ( days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([d-Ala2,d-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) μ- or κ-opioid receptor inhibition (β-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.
PKCϵ is involved in JNK activation that mediates LPS-induced TNF-α, which induces apoptosis in macrophages
Authors: Comalada Mònica , Xaus Jordi , Valledor Annabel F. , López-López Carlos , Pennington Daniel J. , Celada Antonio
Published: May 2013

Lipopolysaccharide (LPS) is a powerful stimulator of macrophages and induces apoptosis in these cells. Using primary cultures of bone marrow-derived macrophages, we found that the autocrine production of tumor necrosis factor-α (TNF-α) has a major function in LPS-induced apoptosis. LPS activates PKC and regulates the different mitogen-activated protein kinases (MAPK). We aimed to determine its involvement either in the secretion of TNF-α or in the induction of apoptosis. Using specific inhibitors and mice with the gene for PKCϵ disrupted, we found that LPS-induced TNF-α-dependent apoptosis is mostly mediated by PKCϵ, which is not directly involved in the signaling mechanism of apoptosis but rather in the process of TNF-α secretion. In our cell model, all three MAPKs were involved in the regulation of TNF-α secretion, but at different levels. JNK mainly regulates TNF-α transcription and apoptosis, whereas ERK and p38 contribute to the regulation of TNF-α production, probably through posttranscriptional mechanisms. Only JNK activity is mediated by PKCϵ in response to LPS and so plays a major role in TNF-α secretion and LPS-induced apoptosis. We demonstrated in macrophages that LPS involving PKCϵ regulates JNK activity and produces TNF-α, which induces apoptosis.
Rac regulates cardiovascular superoxide through diverse molecular interactions: more than a binary GTP switch
Authors: Gregg David , Rauscher Frederick M. , Goldschmidt-Clermont Pascal J.
Published: May 2013

The small G protein Rac has been implicated in multiple cardiovascular processes. Rac has two major functions: 1) it regulates the organization of the actin cytoskeleton, and 2) it controls the activity of the key enzyme complex NADPH oxidase to control superoxide production in both phagocytes and nonphagocytic cells. In phagocytes, superoxide derived from NADPH has a bactericidal function, whereas Rac-derived superoxide in the cardiovascular system has a diverse array of functions that have recently been a subject of intense interest. Rac is differentially activated by cellular receptors coupled to distinct Rac-activating adapter molecules, with each leading to pathway-specific arrays of downstream effects. Thus it may be important to investigate not just whether Rac is activated but also where, how, and for what effector. An understanding of the biochemical functions of Rac and its effectors lays the groundwork for a dissection of the exact array of effects produced by Rac in common cardiovascular processes, including cardiac and vascular hypertrophy, hypertension, leukocyte migration, platelet biology, and atherosclerosis. In addition, investigation of the spatiotemporal regulation of both Rac activation and consequent superoxide generation may produce new insights into the development of targeted antioxidant therapies for cardiovascular disease and enhance our understanding of important cardiovascular drugs, including angiotensin II antagonists and statins, that may depend on Rac modulation for their effect.
Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation
Authors: Hossain M. Moazzem , Hwang Daw-Yang , Huang Qi-Quan , Sasaki Yasuharu , Jin Jian-Ping
Published: May 2013

h2-Calponin is found in both smooth muscle and nonmuscle cells, and its function remains to be established. Western blots with specific monoclonal antibodies detected significant expression of h2-calponin in the growing embryonic stomach and urinary bladder and the early pregnant uterus. Although the expression of h1-calponin is upregulated in the stomach and bladder during postnatal development, the expression of h2-calponin is decreased to low levels in quiescent smooth muscle cells. To investigate a hypothesis that h2-calponin regulates the function of the actin cytoskeleton during cytokinesis, a smooth muscle-originated cell line (SM3) lacking calponin was transfected to express either sense or antisense h2-calponin cDNA and the effects on the rates of cell proliferation were examined. Both stable and transient sense cDNA-transfected cells had a significantly decreased proliferation rate compared with the antisense cDNA-transfected or nontransfected cells. Immunofluorescence microscopy showed that the force-expressed h2-calponin was associated with actin-tropomyosin microfilaments. The number of binuclear cells was significantly greater in the sense cDNA-transfected culture, in which h2-calponin was concentrated in a nuclear ring structure formed by actin filaments. The results suggest that h2-calponin may regulate cytokinesis by inhibiting the activity of the actin cytoskeleton.
Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone
Authors: Noble Brendon S. , Peet Nicky , Stevens Hazel Y. , Brabbs Alex , Mosley John R. , Reilly Gwendolen C. , Reeve Jonathan , Skerry Timothy M. , Lanyon Lance E.
Published: May 2013

Bone is removed or replaced in defined locations by targeting osteoclasts and osteoblasts in response to its local history of mechanical loading. There is increasing evidence that osteocytes modulate this targeting by their apoptosis, which is associated with locally increased bone resorption. To investigate the role of osteocytes in the control of loading-related modeling or remodeling, we studied the effects on osteocyte viability of short periods of mechanical loading applied to the ulnae of rats. Loading, which produced peak compressive strains of −0.003 or −0.004, was associated with a 78% reduction in the resorption surface at the midshaft. The same loading regimen resulted in a 40% relative reduction in osteocyte apoptosis at the same site 3 days after loading compared with the contralateral side ( P = 0.01). The proportion of osteocytes that were apoptotic was inversely related to the estimated local strain ( P < 0.02). In contrast, a single short period of loading resulting in strains of −0.008 engendered both tissue microdamage and subsequent bone remodeling and was associated with an eightfold increase in the proportion of apoptotic osteocytes ( P = 0.02) at 7 days. This increase in osteocyte apoptosis was transient and preceded both intracortical remodeling and death of half of the osteocytes ( P < 0.01). The data suggest that osteocytes might use their U-shaped survival response to strain as a mechanism to influence bone remodeling. We hypothesize that this relationship reflects a causal mechanism by which osteocyte apoptosis regulates bone's structural architecture.
Vasopressin-mediated mitogenic signaling in intestinal epithelial cells
Authors: Chiu Terence , Wu Steven S. , Santiskulvong Chintda , Tangkijvanich Pisit , Yee Hal F. , Rozengurt Enrique
Published: May 2013

The role of G protein-coupled receptors and their ligands in intestinal epithelial cell signaling and proliferation is poorly understood. Here, we demonstrate that arginine vasopressin (AVP) induces multiple intracellular signal transduction pathways in rat intestinal epithelial IEC-18 cells via a V1A receptor. Addition of AVP to these cells induces a rapid and transient increase in cytosolic Ca2+concentration and promotes protein kinase D (PKD) activation through a protein kinase C (PKC)-dependent pathway, as revealed by in vitro kinase assays and immunoblotting with an antibody that recognizes autophosphorylated PKD at Ser916. AVP also stimulates the tyrosine phosphorylation of the nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinase phosphorylation at Tyr418, indicative of Src activation. AVP induces extracellular signal-related kinase (ERK)-1 (p44mapk) and ERK-2 (p42mapk) activation, a response prevented by treatment with mitogen-activated protein kinase kinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors (GF-I and Ro-31-8220), depletion of Ca2+ (EGTA and thapsigargin), selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or the selective Src family kinase inhibitor PP-2. Furthermore, AVP acts as a potent growth factor for IEC-18 cells, inducing DNA synthesis and cell proliferation through ERK-, Ca2+-, PKC-, EGFR tyrosine kinase-, and Src-dependent pathways.
Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle
Authors: Stevens Laurence , Bastide Bruno , Kischel Philippe , Pette Dirk , Mounier Yvonne
Published: May 2013

This study focuses on the effects of mechanical unloading of rat soleus muscle on the isoform patterns of the three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). Mechanical unloading was achieved by hindlimb unloading (HU) for time periods of 7, 15, and 28 days. Relative concentrations of slow and fast TnT, TnI, and TnC isoforms were assessed by electrophoretic and immunoblot analyses. HU induced profound slow-to-fast isoform transitions of all Tn subunits, although to different extents and with different time courses. The effectiveness of the isoform transitions was higher for TnT than for TnI and TnC. Indeed, TnI and TnC encompassed minor partial exchanges of slow isoforms with their fast counterparts, whereas the expression pattern of fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in the same manner by HU. This suggests that the slow and fast counterparts of the Tn subunit isoforms are regulated independently in response to HU. The changes in TnTf composition occurred in parallel with previously demonstrated transitions within the pattern of the fast myosin heavy chains in the same muscles.
Role of reactive oxygen species and NAD(P)H oxidase in α1-adrenoceptor signaling in adult rat cardiac myocytes
Authors: Xiao Lei , Pimentel David R. , Wang Jing , Singh Krishna , Colucci Wilson S. , Sawyer Douglas B.
Published: May 2013

We recently reported that α1-adrenoceptor (α1-AR) stimulation induces hypertrophy via activation of the mitogen/extracellular signal-regulated kinase (MEK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 pathway and generates reactive oxygen species (ROS) in adult rat ventricular myocytes (ARVM). Here we investigate the intracellular source of ROS in ARVM and the mechanism by which ROS activate hypertrophic signaling after α1-AR stimulation. Pretreatment of ARVM with the ROS scavenger Mn(III)terakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) completely inhibited the α1-AR-stimulated activation of Ras-MEK1/2-ERK1/2. Direct addition of H2O2 or the superoxide generator menadione activated ERK1/2, which is also prevented by MnTMPyP pretreatment. We found that ARVM express gp91phox, p22phox, p67phox, and p47phox, four major components of NAD(P)H oxidase, and that α1-AR-stimulated ERK1/2 activation was blocked by four structurally unrelated inhibitors of NAD(P)H oxidase [diphenyleneiodonium, phenylarsine oxide, 4-(2-aminoethyl)benzenesulfonyl fluoride, and cadmium]. Conversely, inhibitors for other potential ROS-producing systems, including mitochondrial electron transport chain, nitric oxide synthase, xanthine oxidase, and cyclooxygenase, had no effect on α1-AR-stimulated ERK1/2 activation. Taken together, our results show that ventricular myocytes express components of an NAD(P)H oxidase that appear to be involved in α1-AR-stimulated hypertrophic signaling via ROS-mediated activation of Ras-MEK1/2-ERK1/2.
Vascular endothelial cells express isoforms of protein kinase A inhibitor
Authors: Lum Hazel , Hao Zengping , Gayle Dave , Kumar Priyadarsini , Patterson Carolyn E. , Uhler Michael D.
Published: May 2013

First published September 5, 2001; 10.1152/ ajpcell.00256.2001.—The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKIα, human PKIγ, and mouse PKIβ sequences detected PKIα and PKIγ mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKIα and PKIγ were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKIα mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKIγ mRNA. Western blot analysis indicated that PKIγ protein was detected in all three cell types, whereas PKIα was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKIα and PKIγ, which may be physiologically important in endothelial barrier function.
Inhibition of interferon-γ expression by osmotic shrinkage of peripheral blood lymphocytes
Authors: Lang K. S. , Weigert C. , Braedel S. , Fillon S. , Palmada M. , Schleicher E. , Rammensee H.-G. , Lang F.
Published: May 2013

A hypertonic environment, as it prevails in renal medulla or in hyperosmolar states such as hyperglycemia of diabetes mellitus, has been shown to impair the immune response, thus facilitating the development of infection. The present experiments were performed to test whether hypertonicity influences activation of T lymphocytes. To this end, peripheral blood lymphocytes (PBL) of cytomegalovirus (CMV)-positive donors were stimulated by human leukocyte antigen (HLA)-A2-restricted CMV epitope NLVPMVATV to produce interferon (IFN)-γ at varying extracellular osmolarity. As a result, increasing extracellular osmolarity during exposure to the CMV antigen indeed decreased IFN-γ formation. Addition of NaCl was more effective than urea. A 50% inhibition was observed at 350 mosM by addition of NaCl. The combined application of the Ca2+ ionophore ionomycin (1 μg/ml) and the phorbol ester phorbol 12-myristate 13-acetate (PMA; 5 μg/ml) stimulated IFN-γ production, an effect again reversed by hyperosmolarity. Moreover, hyperosmolarity abrogated the stimulating effect of ionomycin (1 μg/ml) and PMA (5 μg/ml) on the transcription factors activator protein (AP)-1, nuclear factor of activated T cells (NFAT), and NF-κB but not Sp1. In conclusion, osmotic cell shrinkage blunts the stimulatory action of antigen exposure on IFN-γ production, an effect explained at least partially by suppression of transcription factor activation.
EGF receptor transactivation is obligatory for protein synthesis stimulation by G protein-coupled receptors
Authors: Voisin Laure , Foisy Sylvain , Giasson Edith , Lambert Chantal , Moreau Pierre , Meloche Sylvain
Published: May 2013

The epidermal growth factor receptor (EGFR) was recently identified as a signal transducer of G protein-coupled receptors (GPCRs). In this study, we have examined the contribution of EGFR transactivation to the growth-promoting effect of GPCRs on vascular smooth muscle cells. Activation of the Gq-coupled ANG II receptor or Gi-coupled lysophosphatidic acid receptor resulted in increased tyrosine phosphorylation and activation of EGFR. Specific inhibition of EGFR kinase activity by tyrphostin AG-1478 or expression of a dominant-negative EGFR mutant abolished this response. Importantly, inhibition of EGFR function strongly attenuated the global stimulation of protein synthesis by GPCR agonists in vitro in cultured aortic smooth muscle cells and in vivo in the rat aorta and in small resistance arteries. The growth inhibition was associated with a marked reduction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathway activity and the resulting suppression of eukaryotic translation initiation factor 4E and 4E binding protein 1 phosphorylation. Our results demonstrate that EGFR transactivation is a physiologically relevant action of GPCRs linked to translational control and protein synthesis.
Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells
Authors: Wang Ning , Tolić-Nørrelykke Iva Marija , Chen Jianxin , Mijailovich Srboljub M. , Butler James P. , Fredberg Jeffrey J. , Stamenović Dimitrije
Published: May 2013

The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness ( G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ( [Formula: see text] t ) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18 [Formula: see text] t + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.