bluuebunny/crossref_metadata_2025 huggingface dataset:
KATP channels depress force by reducing action potential amplitude in mouse EDL and soleus muscle
Authors: Gong B. , Legault D. , Miki T. , Seino S. , Renaud J. M.
Published: May 2013

Although ATP-sensitive K+ (KATP) channel openers depress force, channel blockers have no effect. Furthermore, the effects of channel openers on single action potentials are quite small. These facts raise questions as to whether 1) channel openers reduce force via an activation of KATP channels or via some nonspecific effects and 2) the reduction in force by KATP channels operates by changes in amplitude and duration of the action potential. To answer the first question we tested the hypothesis that pinacidil, a channel opener, does not affect force during fatigue in muscles of Kir6.2-/- mice that have no cell membrane KATP channel activity. When wild-type extensor digitorum longus (EDL) and soleus muscles were stimulated to fatigue with one tetanus per second, pinacidil increased the rate at which force decreased, prevented a rise in resting tension, and improved force recovery. Pinacidil had none of these effects in Kir6.2-/- muscles. To answer the second question, we tested the hypothesis that the effects of KATP channels on membrane excitability are greater during action potential trains than on single action potentials, especially during metabolic stress such as fatigue. During fatigue, M wave areas of control soleus remained constant for 90 s, suggesting no change in action potential amplitude for half of the fatigue period. In the presence of pinacidil, the decrease in M wave areas became significant within 30 s, during which time the rate of fatigue also became significantly faster compared with control muscles. It is therefore concluded that, once activated, KATP channels depress force and that this depression involves a reduction in action potential amplitude.
Regulation of xanthine oxidoreductase by intracellular iron
Authors: Martelin Eeva , Lapatto Risto , Raivio Kari O.
Published: May 2013

Xanthine oxidoreductase (XOR) may produce reactive oxygen species and play a role in ischemia-reperfusion injury. Because tissue iron levels increase after ischemia, and because XOR contains functionally critical iron-sulfur clusters, we studied the effects of intracellular iron on XOR expression. Ferric ammonium citrate and FeSO4elevated intracellular iron levels and increased XOR activity up to twofold in mouse fibroblast and human bronchial epithelial cells. Iron increased XOR protein and mRNA levels, whereas protein and RNA synthesis inhibitors abolished the induction of XOR activity. A human XOR promoter construct (nucleotides +42 to −1937) was not induced by iron in human embryonic kidney cells. Hydroxyl radical scavengers did not block induction of XOR activity by iron. Iron chelation by deferoxamine (DFO) decreased XOR activity but did not lower endogenous XOR protein or mRNA levels. Furthermore, DFO reduced the activity of overexpressed human XOR but not the amount of immunoreactive protein. Our data show that XOR activity is transcriptionally induced by iron but posttranslationally inactivated by iron chelation.
Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells
Authors: Gendron Fernand-Pierre , Neary Joseph T. , Theiss Patty M. , Sun Grace Y. , Gonzalez Fernando A. , Weisman Gary A.
Published: May 2013

Astrocytes are involved in normal and pathological brain functions, where they become activated and undergo reactive gliosis. Astrocytes have been shown to respond to extracellular nucleotides via the activation of P2 receptors, either G protein-coupled P2Y receptors or P2X receptors that are ligand-gated ion channels. In this study, we have examined the manner in which activation of the P2X7 nucleotide receptor, an extracellular ATP-gated ion channel expressed in astrocytes, can lead to the phosphorylation of ERK1/2. Results showed that the P2X7 receptor agonist 2′,3′- O-(4-benzoyl)benzoyl-ATP induced ERK1/2 phosphorylation in human astrocytoma cells overexpressing the recombinant rat P2X7 receptor (rP2X7-R), a response that was inhibited by the P2X7 receptor antagonist, oxidized ATP. Other results suggest that rP2X7-R-mediated ERK1/2 phosphorylation was linked to the phosphorylation of the proline-rich/Ca2+-activated tyrosine kinase Pyk2, c-Src, phosphatidylinositol 3′-kinase, and protein kinase Cδ activities and was dependent on the presence of extracellular Ca2+. These results support the hypothesis that the P2X7 receptor and its signaling pathways play a role in astrocyte-mediated inflammation and neurodegenerative disease.
Reactive oxygen species are important mediators of taurine release from skeletal muscle cells
Authors: Ørtenblad Niels , Young Jette Feveile , Oksbjerg Niels , Nielsen Jacob Holm , Lambert Ian Henry
Published: May 2013

The present study illustrates elements of the signal cascades involved in the activation of taurine efflux pathways in myotubes derived from skeletal muscle cells. Exposing primary skeletal muscle cells, loaded with 14C-taurine, to 1) hypotonic media, 2) the phospholipase A2 (PLA2) activator melittin, 3) anoxia, or 4) lysophosphatidyl choline (LPC) causes an increase in 14C-taurine release and a concomitant production of reactive oxygen species (ROS). The antioxidants butulated hydroxy toluene and vitamin E inhibit the taurine efflux after cell swelling, anoxia, and addition of LPC. The muscle cells possess two separate taurine efflux pathways, i.e., a swelling- and melittin-induced pathway that requires 5-lipoxygenase activity for activation and a LPC-induced pathway. The two pathways are distinguished by their opposing sensitivity toward the anion channel blocker DIDS and cholesterol. These data provide evidence for PLA2 products and ROS as key mediators of the signal cascade leading to taurine efflux in muscle.
Distribution of ClC-2 chloride channel in rat and human epithelial tissues
Authors: Lipecka Joanna , Bali Moëz , Thomas Annick , Fanen Pascale , Edelman Aleksander , Fritsch Janine
Published: May 2013

The ubiquitous ClC-2 Cl−channel is thought to contribute to epithelial Cl−secretion, but the distribution of the ClC-2 protein in human epithelia has not been investigated. We have studied the distribution of ClC-2 in adult human and rat intestine and airways by immunoblotting and confocal microscopy. In the rat, ClC-2 was present in the lateral membranes of villus enterocytes and was predominant at the basolateral membranes of luminal colon enterocytes. The expression pattern of ClC-2 in the human intestine differed significantly, because ClC-2 was mainly detected in a supranuclear compartment of colon cells. We found significant expression of ClC-2 at the apex of ciliated cells in both rat and human airways. These results show that the distribution of ClC-2 in airways is consistent with participation of ClC-2 channels in Cl− secretion and indicate that extrapolation of results from studies of ClC-2 function in rat intestine to human intestine is not straightforward.
VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells
Authors: Chen Jun , Braet Filip , Brodsky Sergey , Weinstein Talia , Romanov Victor , Noiri Eisei , Goligorsky Michael S.
Published: May 2013

Glomerular epithelial cells (GEC) are a known site of vascular endothelial growth factor (VEGF) production. We established immortalized rat GEC, which retained the ability to produce VEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells cultured on GEC-conditioned matrix, an indicator of the permeability of monolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased by VEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showed that VEGF results in a rapid appearance of transcellular elongated structures decorated with caveolin. Transmission electron microscopy of endothelial cells showed that caveolae undergo rapid internalization and fusion 30 min after application of VEGF-165. Later (36 h), endothelial cells pretreated with VEGF developed fenestrae and showed a decrease in electrical resistance. Immunoelectron microscopy of glomeruli confirmed VEGF localization to podocytes and in the basement membrane. In summary, immortalized GEC retain the ability to synthesize VEGF. Matrix-deposited and soluble VEGF leads to the enhancement of caveolae expression, their fission and fusion, formation of elongated caveolin-decorated structures, and eventual formation of fenestrae, both responsible for the increase in endothelial permeability.
Peroxynitrite increases iNOS through NF-κB and decreases prostacyclin synthase in endothelial cells
Authors: Cooke Christy-Lynn M. , Davidge Sandra T.
Published: May 2013

Peroxynitrite, a marker of oxidative stress, is elevated in conditions associated with vascular endothelial cell dysfunction, such as atherosclerosis, preeclampsia, and diabetes. However, the effects of peroxynitrite on endothelial cell function are not clear. The endothelium-derived enzymes nitric oxide synthase (NOS) and prostaglandin H synthase (PGHS) mediate vascular reactivity and contain oxidant-sensitive isoforms (iNOS and PGHS-2) that can be induced by nuclear factor (NF)-κB activation. We investigated the effect(s) of peroxynitrite on NOS and PGHS pathways in endothelial cells. We hypothesized that peroxynitrite will increase levels of iNOS and PGHS-2 through activation of NF-κB. Western immunoblots of endothelial cells show that 3-morpholinosydnonimine (SIN-1; 0.5 mM), a peroxynitrite donor, increased iNOS protein mass, which can be inhibited by pyrroline dithiocarbamate (an NF-κB inhibitor) (167 ± 24.2 vs. 78 ± 19%, P < 0.05, n = 6). SIN-1 treatment also significantly increased NF-κB translocation into endothelial cell nuclei (135 ± 10%, P < 0.05). Endothelial NOS, PGHS-1, and PGHS-2 protein levels were not altered by SIN-1. However, prostacyclin synthase protein mass, but not mRNA, was significantly reduced in SIN-1-treated endothelial cells (78 ± 8.9%, P < 0.05). Our results illustrate novel mechanisms through which peroxynitrite may modulate vascular endothelial function.
Calcium dependence of exocytosis in lacrimal gland acinar cells
Authors: Sundermeier Thomas , Matthews Gary , Brink Peter R. , Walcott Benjamin
Published: May 2013

Simultaneous measurements of membrane capacitance and intracellular calcium concentration were used to examine the calcium dependence of exocytosis in single acinar cells from mouse lacrimal gland and to establish the quantitative relation between calcium concentration and rate of exocytosis. Application of adrenergic or muscarinic agonists elevated intracellular calcium and evoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation was eliminated by internal dialysis with the calcium buffer EGTA, which demonstrated that the increase in intracellular calcium was necessary for agonist-evoked exocytosis. When internal calcium was elevated by application of the calcium ionophore ionomycin, exocytosis was evoked in the absence of agonist stimulation. Thus an increase in intracellular calcium was necessary and sufficient for exocytosis in single acinar cells. The rate of change of membrane capacitance increased as approximately the third power of the calcium concentration, which is similar to the dependence of exocytosis rate on calcium concentration in other secretory cells.
Adhesion of flowing monocytes to hypoxia-reoxygenation-exposed endothelial cells: role of Rac1, ROS, and VCAM-1
Authors: Ng C. K. Domingos , Deshpande Shailesh S. , Irani Kaikobad , Alevriadou B. Rita
Published: May 2013

Production of reactive oxygen species (ROS) by ischemic tissue after ischemia-reperfusion (I/RP) is an important factor that contributes to tissue injury. The small GTPase Rac1 mediates the oxidative burst, and ROS act on signaling pathways involved in expression of inflammatory genes. Because there is evidence implicating monocytes in the pathogenesis of I/RP injury, our objective was to determine the molecular mechanisms that regulate adhesive interactions between monocytes and hypoxia-reoxygenation (H/RO)-exposed cultured endothelial cells (ECs). When U937 cells were perfused over human umbilical vein ECs at 1 dyn/cm2, H (1 h at 1% O2)/RO (13 h) significantly increased the fluxes of rolling and stably adherent U937 cells. Either EC treatment with the antioxidant pyrrolidine dithiocarbamate (PDTC) or infection with AdRac1N17, which results in expression of the dominant-negative form of Rac1, abolished H/RO-induced ROS production, attenuated rolling, and abolished stable adhesion of U937 cells to H/RO-exposed ECs. Infection with AdRac1N17 also abolished H/RO-induced upregulation of vascular cell adhesion molecule (VCAM)-1. In turn, blocking VCAM-1 abolished U937 cell stable adhesion and slightly increased rolling. We concluded that the Rac1-dependent ROS partially regulate rolling and exclusively regulate stable adhesion of monocytic cells to ECs after H/RO and that stable adhesion, but not rolling, is mediated by ROS-induced expression of VCAM-1.
NF-κB induced by IL-1β inhibits elastin transcription and myofibroblast phenotype
Authors: Kuang Ping-Ping , Berk John L. , Rishikof David C. , Foster Judith A. , Humphries Donald E. , Ricupero Dennis A. , Goldstein Ronald H.
Published: May 2013

Interleukin (IL)-1β released after lung injury regulates the production of extracellular matrix components. We found that IL-1β treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibroblasts. Deletion analysis of the rat elastin promoter detected a cis-acting element located at −118 to −102 bp that strongly bound Sp1 and Sp3 but not nuclear factor (NF)-κB. This element mediated IL-1β-induced inhibition of the elastin promoter. IL-1β treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-κB. Overexpression of p65 decreased elastin promoter activity and markedly reduced elastin mRNA. Immunoprecipitation studies indicated an interaction between the p65 subunit and Sp1 protein. Microarray analysis of mRNA isolated after overexpression of p65 or treatment with IL-1β revealed downregulation of α-smooth muscle actin and calponin mRNAs. Expression of these genes is associated with the myofibroblast phenotype. These results indicate that IL-1β activates the nuclear localization of NF-κB that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype.
Angiotensin II AT2 receptor inhibits smooth muscle cell migration via fibronectin cell production and binding
Authors: Chassagne Catherine , Adamy Christophe , Ratajczak Philippe , Gingras Bruno , Teiger Emmanuel , Planus Emmanuelle , Oliviero Patricia , Rappaport Lydie , Samuel Jane-Lise , Meloche Sylvain
Published: May 2013

To explore the vascular function of the angiotensin II (ANG II) AT2receptor subtype (AT2R), we generated a vascular smooth muscle cell (SMC) line expressing the AT2R (SMC-vAT2). The involvement of AT2R in the motility response of SMCs was examined in SMC-vAT2 cells and their controls (SMC-v) cultured on either laminin or fibronectin matrix proteins with the agarose drop technique. All experiments were conducted in the presence of a saturating concentration of losartan to inactivate the AT1R subtype. Under basal conditions, both cell lines migrated outside drops, but on laminin only. Treatment with ANG II significantly inhibited the migration of SMC-vAT2but not SMC-v cells, and this effect was prevented by the AT2R antagonist CGP-42112A. The decreased migration of SMC-vAT2 was not associated with changes in cell growth, cytoskeleton stiffness, or smooth muscle actin, desmin, and tenascin expression. However, it was correlated with increased synthesis and binding of fibronectin. Both responses were prevented by incubation with selective AT2R antagonists. Addition of GRGDTP peptide, which prevents cell attachment of fibronectin, reversed the AT2R inhibitory effect on SMC-vAT2 migration. These results suggest that activated ANG II AT2R inhibits SMC migration via cellular fibronectin synthesis and associated cell binding.
CFTR in K562 human leukemic cells
Authors: Assef Yanina A. , Damiano Alicia E. , Zotta Elsa , Ibarra Cristina , Kotsias Basilio A.
Published: May 2013

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 μM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.
Activation of gene expression in human neutrophils by high mobility group box 1 protein
Authors: Park Jong Sung , Arcaroli John , Yum Ho-Kee , Yang Huan , Wang Haichao , Yang Kuang-Yao , Choe Kang-Hyeon , Strassheim Derek , Pitts Todd M. , Tracey Kevin J. , Abraham Edward
Published: May 2013

High mobility group box 1 (HMGB1) protein, a DNA binding protein that stabilizes nucleosomes and facilitates transcription, was recently identified as a late mediator of endotoxin lethality. High serum HMGB1 levels in patients with sepsis are associated with increased mortality, and administration of HMGB1 produces acute inflammation in animal models of lung injury and endotoxemia. Neutrophils occupy a critical role in mediating the development of endotoxemia-associated acute lung injury, but previously it was not known whether HMGB1 could influence neutrophil activation. In the present experiments, we demonstrate that HMGB1 increases the nuclear translocation of NF-κB and enhances the expression of proinflammatory cytokines in human neutrophils. These proinflammatory effects of HMGB1 in neutrophils appear to involve the p38 MAPK, phosphatidylinositol 3-kinase/Akt, and ERK1/2 pathways. The mechanisms of HMGB1-induced neutrophil activation are distinct from endotoxin-induced signals, because HMGB1 leads to a different profile of gene expression, pattern of cytokine expression, and kinetics of p38 activation compared with LPS. These findings indicate that HMGB1 is an effective stimulus of neutrophil activation that can contribute to development of a proinflammatory phenotype in diseases characterized by excessively high levels of HMGB1.
ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca2+, PKC, and Rho kinase
Authors: Wu Steven S. , Chiu Terence , Rozengurt Enrique
Published: May 2013

The G protein-coupled receptor agonists angiotensin II (ANG II) and lysophosphatidic acid (LPA) rapidly induce tyrosine phosphorylation of the cytosolic proline-rich tyrosine kinase 2 (Pyk2) in IEC-18 intestinal epithelial cells. The combined Pyk2 tyrosine phosphorylation induced by phorbol 12,13-dibutyrate, a direct agonist of protein kinase C (PKC), and ionomycin, a Ca2+ionophore, was equal to that induced by ANG II. Inhibition of either PKC or Ca2+ signaling attenuated the effect of ANG II and LPA, although simultaneous inhibition of both pathways failed to completely abolish Pyk2 tyrosine phosphorylation. Cytochalasin D, which disrupts stress fibers, strongly inhibited the response of Pyk2 to ANG II or LPA. The distinct Rho-associated kinase (ROK) inhibitors HA-1077 and Y-27632, as well as the Rho inhibitor Clostridium botulinum C3 exoenzyme, also significantly attenuated ANG II- and LPA-stimulated Pyk2 tyrosine phosphorylation. Simultaneous inhibition of PKC, Ca2+, and either actin assembly or ROK completely abolished the Pyk2 response. Together, these results show that ANG II and LPA rapidly induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells via separate Ca2+-, PKC-, and Rho-mediated pathways.
Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-κB pathways
Authors: Zhao Dezheng , Kuhnt-Moore Sabina , Zeng Huiyan , Wu Jack S. , Moyer Mary P. , Pothoulakis Charalabos
Published: May 2013

Neurotensin (NT), a neuropeptide highly expressed in the gastrointestinal tract, participates in the pathophysiology of intestinal inflammation. We recently showed that NT stimulates interleukin-8 (IL-8) expression in NCM460 nontransformed human colonic epithelial cells via both mitogen-activating protein kinase (MAPK)- and NF-κB-dependent pathways. However, the molecular mechanism by which NT induces expression of proinflammatory cytokines such as IL-8 has not been investigated. In this study we show that inhibition of endogenous Rho family proteins (RhoA, Rac1, and Cdc42) by their respective dominant negative mutants inhibits NT-induced IL-8 protein production and promoter activity. Western blot experiments demonstrated that NT strongly activated RhoA, Rac1, and Cdc42. Overexpression of the dominant negative mutants of RhoA, Rac1, and Cdc42 significantly inhibited NT-induced NF-κB-dependent reporter gene expression and NF-κB DNA binding activity. NT also stimulated p38 MAPK phosphorylation, and overexpression of dominant negative mutants of RhoA, Rac1, and Cdc42 did not significantly alter p38 and ERK1/2 phosphorylation in response to NT. Together, our findings indicate that NT-stimulated IL-8 expression is mediated via a Rho-dependent NF-κB-mediated pathway.
Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways
Authors: Machinal-Quélin F. , Dieudonné M. N. , Leneveu M. C. , Pecquery R. , Giudicelli Y.
Published: May 2013

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([3H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-γ2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.
Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells
Authors: Kreft M. , Kuster V. , Grilc S. , Rupnik M. , Milisav I. , Zorec R.
Published: May 2013

Synaptotagmin I (Syt I), a low-affinity Ca2+-binding protein, is thought to serve as the Ca2+ sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca2+], suggesting that Syt I may play a more complex role in determining the high-affinity Ca2+dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca2+-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca2+-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca2+-dependent exocytosis by cytosol dialysis with solutions containing 1 μM [Ca2+] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca2+-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca2+].
CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle
Authors: Ginnan Roman , Singer Harold A.
Published: May 2013

In vascular smooth muscle (VSM) and many other cells, G protein receptor-coupled activation of mitogen-activated protein kinases has been linked, in part, to increases in free intracellular Ca2+. Previously, we demonstrated that ionomycin-, angiotensin II-, and thrombin-induced activation of extracellular signal-regulated kinase (ERK)1/2 in VSM cells was attenuated by pretreatment with KN-93, a selective inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II). In the present study, we show that the Ca2+-dependent pathway leading to activation of ERK1/2 is preceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation and is attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibits Ca2+-dependent PYK2 activation and EGF receptor tyrosine phosphorylation in response to ionomycin, ATP, and platelet-derived growth factor but has no effect on phorbol 12,13-dibutyrate- or EGF-induced responses. The results implicate CaM kinase II as an intermediate in the Ca2+/calmodulin-dependent activation of PYK2.
Satellite cell proliferation and differentiation during postnatal growth of porcine skeletal muscle
Authors: Mesires N. T , Doumit M. E.
Published: May 2013

Age-related changes in satellite cell proliferation and differentiation during rapid growth of porcine skeletal muscle were examined. Satellite cells were isolated from hindlimb muscles of pigs at 1, 7, 14, and 21 wk of age (4 animals/age group). Satellite cells were separated from cellular debris by using Percoll gradient centrifugation and were adsorbed to glass coverslips for fluorescent immunostaining. Positive staining for neural cell adhesion molecule (NCAM) distinguished satellite cells from nonmyogenic cells. The proportion of NCAM-positive cells (satellite cells) in isolates decreased from 1 to 7 wk of age. Greater than 77% of NCAM-positive cells were proliferating cell nuclear antigen positive at all ages studied. Myogenin-positive satellite cells decreased from 30% at 1 wk to 14% at 7 wk of age and remained at constant levels thereafter. These data indicate that a high percentage of satellite cells remain proliferative during rapid postnatal muscle growth. The reduced proportion of myogenin-positive cells during growth may reflect a decrease in the proportion of differentiating satellite cells or accelerated incorporation of myogenin-positive cells into myofibers.
Heterologous desensitization of EGF receptors and PDGF receptors by sequestration in caveolae
Authors: Matveev Sergey V. , Smart Eric J.
Published: May 2013

Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors have been reported to signal via caveolin-containing membranes called caveolae. In contrast, others report that EGF and PDGF receptors are exclusively associated with caveolin-devoid membranes called rafts. Our subcellular fractionation and coimmunoprecipitation studies demonstrate that, in the absence of ligand, EGF and PDGF receptors are associated with rafts. However, in the presence of ligand, EGF and PDGF receptors transiently associate with caveolae. Surprisingly, pretreatment of cells with EGF prevents PDGF-dependent phosphorylation of PDGF receptors and extracellular signal-regulated kinase (ERK) 1/2 kinase activation. Furthermore, cells pretreated with PDGF prevent EGF-dependent phosphorylation of EGF receptors and ERK1/2 kinase activation. Radioligand binding studies demonstrate that incubation of cells with EGF or PDGF causes both EGF and PDGF receptors to be reversibly sequestered from the extracellular space. Experiments with methyl-β-cyclodextrin, filipin, and antisense caveolin-1 demonstrate that sequestration of the receptors is dependent on cholesterol and caveolin-1. We conclude that ligand-induced stimulation of EGF or PDGF receptors can cause the heterologous desensitization of the other receptor by sequestration in cholesterol-rich, caveolin-containing membranes or caveolae.
Activation of K+ channels and increased migration of differentiated intestinal epithelial cells after wounding
Authors: Rao Jaladanki N. , Platoshyn Oleksandr , Li Li , Guo Xin , Golovina Vera A. , Yuan Jason X.-J. , Wang Jian-Ying
Published: May 2013

Early mucosal restitution occurs by epithelial cell migration to reseal superficial wounds after injury. Differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene migrate over the wounded edge much faster than undifferentiated parental cells in an in vitro model. This study determined whether these differentiated intestinal epithelial cells exhibit increased migration by altering voltage-gated K+ (Kv) channel expression and cytosolic free Ca2+ concentration ([Ca2+]cyt). Stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 and Kv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1 cells nor parental IEC-6 cells expressed voltage-dependent Ca2+ channels. The increased expression of Kv channels in differentiated IEC-Cdx2L1 cells was associated with an increase in whole cell K+ currents, membrane hyperpolarization, and a rise in [Ca2+]cyt. The migration rates in differentiated IEC-Cdx2L1 cells were about four times those of parental IEC-6 cells. Inhibition of Kv channel expression by polyamine depletion decreased [Ca2+]cyt, reduced myosin stress fibers, and inhibited cell migration. Elevation of [Ca2+]cyt by ionomycin promoted myosin II stress fiber formation and increased cell migration. These results suggest that increased migration of differentiated intestinal epithelial cells is mediated, at least partially, by increasing Kv channel activity and Ca2+ influx during restitution.
Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells
Authors: Lurtz Monica M. , Louis Charles F.
Published: May 2013

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+.
Expression of glucocorticoid receptor α- and β-isoforms in human cells and tissues
Authors: Pujols Laura , Mullol Joaquim , Roca-Ferrer Jordi , Torrego Alfons , Xaubet Antoni , Cidlowski John A. , Picado César
Published: May 2013

Alternative splicing of the human glucocorticoid receptor (GR) primary transcript generates two protein isoforms: GR-α and GR-β. We investigated the expression of both GR isoforms in healthy human cells and tissues. GR-α mRNA abundance (×106 cDNA copies/μg total RNA) was as follows: brain (3.83 ± 0.80) > skeletal muscle > macrophages > lung > kidney > liver > heart > eosinophils > peripheral blood mononuclear cells (PBMCs) > nasal mucosa > neutrophils > colon (0.33 ± 0.04). GR-β mRNA was much less expressed than GR-α mRNA. Its abundance (×103 cDNA copies/μg total RNA) was as follows: eosinophils (1.55 ± 0.58) > PBMCs > liver ≥ skeletal muscle > kidney > macrophages > lung > neutrophils > brain ≥ nasal mucosa > heart (0.15 ± 0.08). GR-β mRNA was not found in colon. While GR-α protein was detected in all cells and tissues, GR-β was not detected in any specimen. Our results suggest that, in physiological conditions, the default splicing pathway is the one leading to GR-α. The alternative splicing event leading to GR-β is minimally activated.
Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts
Authors: Cohen Alex W. , Park David S. , Woodman Scott E. , Williams Terrence M. , Chandra Madhulika , Shirani Jamshid , Pereira de Souza Andrea , Kitsis Richard N. , Russell Robert G. , Weiss Louis M. , Tang Baiyu , Jelicks Linda A. , Factor Stephen M. , Shtutin Vitaliy , Tanowitz Herbert B. , Lisanti Michael P.
Published: May 2013

Recently, development of a caveolin-1-deficient (Cav-1 null) mouse model has allowed the detailed analysis of caveolin-1's function in the context of a whole animal. Interestingly, we now report that the hearts of Cav-1 null mice are markedly abnormal, despite the fact that caveolin-1 is not expressed in cardiac myocytes. However, caveolin-1 is abundantly expressed in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelia. Quantitative imaging studies of Cav-1 null hearts demonstrate a significantly enlarged right ventricular cavity and a thickened left ventricular wall with decreased systolic function. Histological analysis reveals myocyte hypertrophy with interstitial/perivascular fibrosis. Because caveolin-1 is thought to act as a negative regulator of the p42/44 MAP kinase cascade, we performed Western blot analysis with phospho-specific antibodies that only recognize activated ERK1/2. As predicted, the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (i.e., interstitial fibrotic lesions) and isolated cardiac fibroblasts. In addition, endothelial and inducible nitric oxide synthase levels are dramatically upregulated. Thus loss of caveolin-1 expression drives p42/44 MAP kinase activation and cardiac hypertrophy.
Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes
Authors: Cavalli Amy L. , O'Brien Nicole W. , Barlow Steven B. , Betto Romeo , Glembotski Christopher C. , Palade Philip T. , Sabbadini Roger A.
Published: May 2013

Calcium channels are important in a variety of cellular events including muscle contraction, signaling, proliferation, and apoptosis. Sphingolipids have been recognized as mediators of intracellular calcium release through their actions on a calcium channel, sphingolipid calcium release-mediating protein of the endoplasmic reticulum (SCaMPER). The current study investigates the expression and function of SCaMPER in cardiomyocytes. Northern analyses and RT-PCR cloning and sequencing revealed SCaMPER expression in both human and rat cardiac tissue. Immunofluorescence and Western blot analyses demonstrated that SCaMPER is abundant in cardiac tissue and is localized to the sarcotubular junction. This was confirmed by the colocalization of SCaMPER with dihydropyridine and ryanodine receptors by confocal microscopy. Purified T tubules were shown to contain SCaMPER and immunoelectron micrographs suggested that SCaMPER is located to the junctional T tubules, but a junctional SR localization cannot be ruled out. The sphingolipid ligand for SCaMPER, sphingosylphosphorylcholine (SPC), initiated calcium release from the cardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNA produced a substantial reduction of sphingolipid-induced calcium release, suggesting that SCaMPER is a potentially important calcium channel of cardiomyocytes.
Mechanisms of normal and tumor-derived angiogenesis
Authors: Papetti Michael , Herman Ira M.
Published: May 2013

Often those diseases most evasive to therapeutic intervention usurp the human body's own cellular machinery or deregulate normal physiological processes for propagation. Tumor-induced angiogenesis is a pathological condition that results from aberrant deployment of normal angiogenesis, an essential process in which the vascular tree is remodeled by the growth of new capillaries from preexisting vessels. Normal angiogenesis ensures that developing or healing tissues receive an adequate supply of nutrients. Within the confines of a tumor, the availability of nutrients is limited by competition among actively proliferating cells, and diffusion of metabolites is impeded by high interstitial pressure (Jain RK. Cancer Res 47: 3039–3051, 1987). As a result, tumor cells induce the formation of a new blood supply from the preexisting vasculature, and this affords tumor cells the ability to survive and propagate in a hostile environment. Because both normal and tumor-induced neovascularization fulfill the essential role of satisfying the metabolic demands of a tissue, the mechanisms by which cancer cells stimulate pathological neovascularization mimic those utilized by normal cells to foster physiological angiogenesis. This review investigates mechanisms of tumor-induced angiogenesis. The strategies used by cancer cells to develop their own blood supply are discussed in relation to those employed by normal cells during physiological angiogenesis. With an understanding of blood vessel growth in both normal and abnormal settings, we are better suited to design effective therapeutics for cancer.
Changes of action potentials and force at lowered [Na+]o in mouse skeletal muscle: implications for fatigue
Authors: Cairns Simeon P. , Buller Sarah J. , Loiselle Denis S. , Renaud Jean-Marc
Published: May 2013

We examined 1) whether the effects of lowered trans-sarcolemmal Na+ gradient on force differed between nonfatigued fast- and slow-twitch muscles of mice and 2) whether effects on action potentials could explain the decrease of force. The Na+ gradient was reduced by lowering the extracellular [Na+] ([Na+]o). The peak force-[Na+]o relationships for the twitch and tetanus were the same in nonfatigued extensor digitorum longus and soleus muscles: force was maintained over a large range of [Na+]o and then decreased abruptly over a much smaller range. However, fatigue was significantly exacerbated at a lowered [Na+]o that had little effect in nonfatigued soleus muscle. This finding suggests that substantial differences exist in the Na+ effect on force between nonfatigued and fatigued muscle. The reduced contractility in nonfatigued muscles at lowered [Na+]o was largely due to 1) an increased number of inexcitable fibers and threshold for action potentials, 2) a reduction of action potential amplitude, and 3) a reduced capacity to generate action potentials throughout trains.
Effect of reactive oxygen species on NH 4 + permeation in Xenopus laevis oocytes
Authors: Cougnon Marc , Benammou Samia , Brouillard Franck , Hulin Philippe , Planelles Gabrielle
Published: May 2013

To investigate the effects of reactive oxygen species (ROS) on NH[Formula: see text]permeation in Xenopus laevis oocytes, we used intracellular double-barreled microelectrodes to monitor the changes in membrane potential ( V m) and intracellular pH (pHi) induced by a 20 mM NH4Cl-containing solution. Under control conditions, NH4Cl exposure induced a large membrane depolarization (to V m = 4.0 ± 1.5 mV; n = 21) and intracellular acidification [reaching a change in pHi(ΔpHi) of 0.59 ± 0.06 pH units in 12 min]; the initial rate of cell acidification (dpHi/d t) was 0.06 ± 0.01 pH units/min. Incubation of the oocytes in the presence of H2O2 or β-amyloid protein had no marked effect on the NH4Cl-induced ΔpHi. By contrast, in the presence of photoactivated rose bengal (RB), tert-butyl-hydroxyperoxide ( t-BHP), or xanthine/xanthine oxidase (X/XO), the same experimental maneuver induced significantly greater ΔpHi and dpHi/d t. These increases in ΔpHiand dpHi/d t were prevented by the ROS scavengers histidine and desferrioxamine, suggesting involvement of the reactive species 1ΔgO2 and ·OH. Using the voltage-clamp technique to identify the mechanism underlying the ROS-measured effects, we found that RB induced a large increase in the oocyte membrane conductance ( G m). This RB-induced G m increase was prevented by 1 mM diphenylamine-2-carboxylate (DPC) and by a low Na+concentration in the bath. We conclude that RB, t-BHP, and X/XO enhance NH[Formula: see text] influx into the oocyte via activation of a DPC-sensitive nonselective cation conductance pathway.
TGF-β effects on epithelial ion transport and barrier: reduced Cl− secretion blocked by a p38 MAPK inhibitor
Authors: Howe Kathryn , Gauldie Jack , McKay Derek M.
Published: May 2013

Growth factors affect a variety of epithelial functions. We examined the ability of TGF-β to modulate epithelial ion transport and permeability. Filter-grown monolayers of human colonic epithelia, T84 and HT-29 cells, were treated with TGF-β (0.1–100 ng/ml, 15 min–72 h) or infected with an adenoviral vector encoding TGF-β (Ad-TGFβ) for 144 h. Ion transport (i.e., short-circuit current, I sc) and transepithelial resistance (TER) were assessed in Ussing chambers. Neither recombinant TGF-β nor Ad-TGFβ infection affected baseline I sc; however, exposure to ≥1 ng/ml TGF-β led to a significant (30–50%) reduction in the I sc responses to forskolin, vasoactive intestinal peptide, and cholera toxin (agents that evoke Cl− secretion via cAMP mobilization) and to the cell-permeant dibutyryl cAMP. Pharmacological analysis of signaling pathways revealed that the inhibition of cAMP-driven epithelial Cl− secretion by TGF-β was blocked by pretreatment with SB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors of JNK, ERK1/2 MAPK, or phosphatidylinositol 3′-kinase. TGF-β enhanced the barrier function of the treated monolayers by up to threefold as assessed by TER; however, this event was temporally displaced from the altered I sc response, being statistically significant only at 72 h posttreatment. Thus, in addition to TGF-β promotion of epithelial barrier function, we show that this growth factor also reduces responsiveness to cAMP-dependent secretagogues in a chronic manner and speculate that this serves as a braking mechanism to limit secretory enteropathies.
Stiffness changes in cultured airway smooth muscle cells
Authors: An Steven S. , Laudadio Rachel E. , Lai Jean , Rogers Rick A. , Fredberg Jeffrey J.
Published: May 2013

Airway smooth muscle (ASM) cells in culture stiffen when exposed to contractile agonists. Such cell stiffening may reflect activation of the contractile apparatus as well as polymerization of cytoskeletal biopolymers. Here we have assessed the relative contribution of these mechanisms in cultured ASM cells stimulated with serotonin (5-hydroxytryptamine; 5-HT) in the presence or absence of drugs that inhibit either myosin-based contraction or polymerization of filamentous (F) actin. Magnetic twisting cytometry was used to measure cell stiffness, and associated changes in structural organization of actin cytoskeleton were evaluated by confocal microscopy. We found that 5-HT increased cell stiffness in a dose-dependent fashion and also elicited rapid formation of F-actin as marked by increased intensity of FITC-phalloidin staining in these cells. A calmodulin antagonist (W-7), a myosin light chain kinase inhibitor (ML-7) and a myosin ATPase inhibitor (BDM) each ablated the stiffening response but not the F-actin polymerization induced by 5-HT. Agents that inhibited the formation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, and Y-27632) attenuated both baseline stiffness and the extent of cell stiffening in response to 5-HT. Together, these data suggest that agonist-evoked stiffening of cultured ASM cells requires actin polymerization as well as myosin activation and that neither actin polymerization nor myosin activation by itself is sufficient to account for the cell stiffening response.
Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization
Authors: Lewis Alison , Di Ciano Caterina , Rotstein Ori D. , Kapus András
Published: May 2013

Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ∼2.5- and ∼7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.