bluuebunny/crossref_metadata_2025 huggingface dataset:
Pertussis toxin directly activates endothelial cell p42/p44 MAP kinases via a novel signaling pathway
Authors: Garcia Joe G. N. , Wang Peiyi , Liu Feng , Hershenson Marc B. , Borbiev Talaibek , Verin Alexander D.
Published: December 2017

Bordetella pertussisgenerates a bacterial toxin utilized in signal transduction investigation because of its ability to ADP ribosylate specific G proteins. We previously noted that pertussis toxin (PTX) directly activates endothelial cells, resulting in disruption of monolayer integrity and intercellular gap formation via a signaling pathway that involves protein kinase C (PKC). We studied the effect of PTX on the activity of the 42- and 44-kDa extracellular signal-regulated kinases (ERK), members of a kinase family known to be activated by PKC. PTX caused a rapid time-dependent increase in bovine pulmonary artery endothelial cell ERK activity that was significantly attenuated by 1) pharmacological inhibition of MEK, the upstream ERK activating kinase, 2) an MEK dominant-negative construct, and 3) PKC inhibition with bisindolylmaleimide. There was little evidence for the involvement of either Gβγ-subunits, Ras GTPases, Raf-1, p60src, or phosphatidylinositol 3′-kinases in PTX-mediated ERK activation. Both the purified β-oligomer binding subunit of the PTX holotoxin and a PTX holotoxin mutant genetically engineered to eliminate intrinsic ADP ribosyltransferase activity completely reproduced PTX effects on ERK activation, suggesting that PTX-induced ERK activation involves a novel PKC-dependent signaling mechanism that is independent of either Ras or Raf-1 activities and does not require G protein ADP ribosylation.
Arachidonic acid reversibly enhances N-type calcium current at an extracellular site
Authors: Barrett Curtis F. , Liu Liwang , Rittenhouse Ann R.
Published: December 2017

We examined the effects of arachidonic acid (AA) on whole cell Ca2+ channel activity in rat superior cervical ganglion neurons. Our companion paper (Liu L, Barrett CF, and Rittenhouse AR. Am J Physiol Cell Physiol 280: C1293–C1305, 2001) demonstrates that AA induces several effects, including enhancement of current amplitude at negative voltages, and increased activation kinetics. This study examines the mechanisms underlying these effects. First, enhancement is rapidly reversible by bath application of BSA. Second, enhancement appears to occur extracellularly, since intracellular albumin was without effect on enhancement, and bath-applied arachidonoyl coenzyme A, an amphiphilic AA analog that cannot cross the cell membrane, mimicked enhancement. In addition, enhancement is voltage dependent, in that currents were enhanced to the greatest degree at −10 mV, whereas virtually no enhancement occurred positive of +30 mV. We also demonstrate that AA-induced increases in activation kinetics are correlated with enhancement of current amplitude. An observed increase in the voltage sensitivity may underlie these effects. Finally, the majority of enhancement is mediated through N-type current, thus providing the first demonstration that this current type can be enhanced by AA.
Activation of P2Y but not P2X4 nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts
Authors: Weidema A. Frederik , Dixon S. Jeffrey , Sims Stephen M.
Published: December 2017

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of [Ca2+]ipersisted on removal of extracellular Ca2+(Ca[Formula: see text]), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca2+-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of [Ca2+]i. After store depletion in the absence of Ca[Formula: see text], addition of Ca[Formula: see text] led to a rise of [Ca2+]i consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La3+. In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X4 nucleotide receptors, but with no rise of [Ca2+]i. We conclude that nucleotide-induced elevation of [Ca2+]i in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.
Etk/Bmx activation modulates barrier function in epithelial cells
Authors: Hamm-Alvarez Sarah F. , Chang Allen , Wang Yanru , Jerdeva Galina , Lin H. Helen , Kim Kwang-Jin , Ann David K.
Published: December 2017

Etk/Bmx is a member of the Tec family of cytoplasmic non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased transepithelial resistance (TER). After 24 h of hypoxic (1% O2) exposure, the TER and equivalent active ion transport rate ( I eq) were reduced to <5% of the normoxia control in Pa-4 cells, whereas both TER and I eqwere maintained at comparable and 60% levels, respectively, relative to their normoxic controls in cells with Etk activation. Moreover, Pa-4 cells exhibited an abundant actin stress fiber network with a diffuse distribution of β-catenin at the cell periphery. By contrast, Etk-activated cells displayed a redistribution of actin to an exclusively peripheral network, with a discrete band of β-catenin also concentrated at the cell periphery, and an altered occludin distribution profile. On the basis of these findings, we propose that Etk may be a novel regulator of epithelial junctions during physiological and pathophysiological conditions.
[Ca2+]i as a potential downregulator of α2β1-integrin-mediated A2058 tumor cell migration to type IV collagen
Authors: Hodgson Louis , Dong Cheng
Published: December 2017

We have investigated cellular Ca2+ regulation during A2058 human melanoma cell chemotaxis to type IV collagen (CIV). We have identified α2β1-integrin as the primary mediator of A2058 cell response to CIV in vitro. Integrin ligation initiated a characteristic intracellular Ca2+ concentration ([Ca2+]i) response consisting of an internal release and a receptor-mediated Ca2+ entry. Thapsigargin (TG) pretreatment drained overlapping and CIV-inducible internal Ca2+ stores while initiating a store-operated Ca2+ release (SOCR). CIV-mediated Ca2+ entry was additive to TG-SOCR, suggesting an independent signaling mechanism. Similarly, ionophore application in a basal medium containing Ca2+ initiated a sustained influx. Elevated [Ca2+]i from TG-SOCR or ionophore significantly attenuated cell migration to CIV by recruiting the Ca2+/calcineurin-mediated signaling pathway. Furthermore, low [Ca2+]i induced by EGTA application in the presence of ionophore fully restored cell motility to CIV. Together, these results suggest that [Ca2+]isignaling accompanying A2058 cell response to α2β1-integrin ligation is neither necessary nor sufficient and that elevated [Ca2+]idownregulates cell motility via a calcineurin-mediated mechanism in A2058 cell chemotaxis to CIV.
p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 MAPK
Authors: Dechert Melissa A. , Holder Jennifer M. , Gerthoffer William T.
Published: December 2017

Cell migration contributes to many physiological processes and requires dynamic changes in the cytoskeleton. These migration-dependent cytoskeletal changes are partly mediated by p21-activated protein kinases (PAKs). At least four closely related isoforms, PAK1, PAK2, PAK3, and PAK4, exist in mammalian cells. In smooth muscle cells, little is known about the expression, activation, or ability of PAKs to regulate migration. Our study revealed the existence of three PAK isoforms in cultured tracheal smooth muscle cells (TSMCs). Additionally, we constructed adenoviral vectors encoding wild type and a catalytically inactive PAK1 mutant to investigate PAK activation and its role in TSMC migration. Stimulation of TSMCs with platelet-derived growth factor (PDGF) increased the activity of PAK1 over time. Overexpression of mutant PAK1 blocked PDGF-induced chemotactic cell migration. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) in cells overexpressing wild-type PAK1 was similar to vector controls; however, p38 MAPK phosphorylation was severely reduced by overexpression of the PAK1 mutant. Collectively, these results suggest a role for PAK1 in chemotactic TSMC migration that involves catalytic activity and may require signaling to p38 MAPK among other pathways.
Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats
Authors: Hornberger Troy A. , Hunter R. Bridge , Kandarian Susan C. , Esser Karyn A.
Published: December 2017

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 α-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).
COX-2 expression and cell cycle progression in human fibroblasts
Authors: Gilroy Derek W. , Saunders Michael A. , Wu Kenneth K.
Published: December 2017

Cyclooxygenase-2 (COX-2) is continuously expressed in most cancerous cells where it appears to modulate cellular proliferation and apoptosis. However, little is known about the contribution of transient COX-2 induction to cell cycle progression or programmed cell death in primary cells. In this study we determined whether COX-2 regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E2(PGE2) were not detected in quiescent cells but were expressed during the G0/G1 phase of the cell cycle induced by serum. Inhibition of COX-2 did not alter G0/G1 to S phase transition or induce apoptosis at concentrations that diminished PGE2. Addition of interleukin-1β to serum enhanced COX-2 expression and PGE2 synthesis over that by serum alone but had no effect on the progression of these cells into S phase. Furthermore, platelet-derived growth factor drove the G0 fibroblasts into the cell cycle without inducing detectable levels of COX-2 or PGE2. Collectively, these data show that transient COX-2 expression in primary human fibroblasts does not influence cell cycle progression.
Anti-adrenergic effects of nitric oxide donor SIN-1 in rat cardiac myocytes
Authors: Stojanovic Miroslav O. , Ziolo Mark T. , Wahler Gordon M. , Wolska Beata M.
Published: December 2017

We studied how the nitric oxide (NO · ) donor 3-morpholinosydnonimine (SIN-1) alters the response to β-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1 H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO · , are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of β-adrenergic stimulation.
p38 MAPK mediates renal tubular cell TNF-α production and TNF-α-dependent apoptosis during simulated ischemia
Authors: Meldrum K. K. , Meldrum D. R. , Hile K. L. , Yerkes E. B. , Ayala A. , Cain M. P. , Rink R. C. , Casale A. J. , Kaefer M. A.
Published: December 2017

Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK1, we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-α (TNF-α) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-α mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-α mRNA production and TNF-α bioactivity, and both p38 MAPK inhibition and TNF-α neutralization (anti-porcine TNF-α antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-α mRNA and biologically active TNF-α and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-α production and TNF-α-dependent apoptosis after SI.
ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia
Authors: Blaug Sasha , Hybiske Kevin , Cohn Jonathan , Firestone Gary L. , Machen Terry E. , Miller Sheldon S.
Published: December 2017

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].
Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes
Authors: Mercado Adriana , de los Heros Paola , Vázquez Norma , Meade Patricia , Mount David B. , Gamba Gerardo
Published: December 2017

The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here that Xenopus laevis oocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl−-dependent86Rb+ uptake mechanism after activation by the cysteine-reactive compounds N-ethylmaleimide (NEM) and mercuric chloride (HgCl2). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 ± 3.0 and 15.4 ± 4.7 mM for Rb+ and Cl−, respectively, with an anion selectivity for K+ transport of Cl− = PO[Formula: see text] = Br−> I− > SCN− > gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 μM for furosemide and bumetanide, respectively. A partial cDNA encoding the Xenopus K-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. J Biol Chem 275: 30326–30334, 2000).
Contrasting effects of thiol-modulating agents on endothelial NO bioactivity
Authors: Huang Annong , Xiao Hui , Samii Jason M. , Vita Joseph A. , Keaney John F.
Published: December 2017

The bioactivity of endothelium-derived nitric oxide (NO) is an important component of vascular homeostasis that is sensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define its role in modulating endothelial NO bioactivity. In porcine aortic endothelial cells (PAECs), we depleted intracellular GSH (>70%) using 1) buthionine-( S,R)-sulfoximine (BSO), which inhibits GSH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putatively depletes GSH through glutathione S-transferase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-induced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatment of either PAECs, PAEC membrane fractions, or purified endothelial nitric oxide synthase (eNOS) resulted in significant inhibition (∼75%) of eNOS catalytic activity measured asl-[3H]arginine-to-l-[3H]citrulline conversion. This effect appeared related to oxidation of eNOS thiols as it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP response to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evidence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pleiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity.
Cloning and characterization of a functional P2X receptor from larval bullfrog skin
Authors: Jensik Philip J. , Holbird Doyle , Collard Michael W. , Cox Thomas C.
Published: December 2017

ATP activates an apical-to-basolateral nonselective cation current across the skin of larval bullfrogs ( Rana catesbeiana) with similarities to currents carried by some P2X receptors. A functional P2X receptor was cloned from tadpole skin RNA that encodes a 409-amino acid protein with highest protein homology to cP2X8. RT-PCR showed that this transcript was found in skin, heart, eye, brain, and skeletal muscle of tadpoles but not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP activated a transient current similar to other P2X receptors and the ATP-activated transient in short-circuit current ( I sc) across intact skin. The agonists 2-methylthio-ATP and adenosine-5′- O-(thiotriphoshate) also activated transient currents. α,β-Methylene-ATP and ADP were poor agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor to agonist activation. Under external Na+-free, Ca2+/Mg2+-free conditions ( N-methyl-d-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated I sc in the tadpole skin. Because the clone is not found in adult skin, it may have some exclusive role in the tadpole such as sensory reception by the skin or triggering apoptosis at metamorphosis.
Roles of tumor necrosis factor p55 and p75 receptors in TNF-α-induced vascular permeability
Authors: Ferrero Elisabetta , Zocchi Maria Raffaella , Magni Elena , Panzeri Maria Carla , Curnis Flavio , Rugarli Claudio , Ferrero Maria Elena , Corti Angelo
Published: December 2017

We have investigated the role of p55 and p75 tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2, respectively) in TNF-induced alteration of endothelial permeability in vitro and in vivo. Stimulation of TNFR1 with an agonist antibody or a receptor-selective TNF mutein increased the flux of125I-albumin through endothelial cell monolayers. An antagonist anti-TNFR1 antibody, but not antagonist anti-TNFR2 antibodies, blocked the activity of TNF in vitro. Stimulation of TNFR1, but not TNFR2, induced cytoskeletal reorganization associated with increased permeability. SB-203580, a p38 mitogen-activated protein kinase inhibitor, blocked TNFR1-induced cytoskeletal reorganization and permeability. A selective mouse TNFR1 agonist and human TNF, which binds to murine TNFR1, increased the leakage of trypan blue-albumin from liver vessels in mice. These results indicate that stimulation of TNFR1 is necessary and sufficient to increase endothelial permeability in vitro and in vivo. However, an antagonist anti-murine TNFR2 antibody partially inhibited the effect of murine TNF on liver vessels, suggesting that TNFR2 also plays a role in the regulation of TNF-induced vascular permeability in vivo.
Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K+ channels in the TAL
Authors: Gu Rui-Min , Wei Yuan , Falck John R. , Krishna U. Murali , Wang Wen-Hui
Published: December 2017

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165–F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP o, a product of channel number ( N) and open probability ( P o), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP o of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP o of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.
Agonist-specific cross talk between ERKs and p38mapk regulates PGI2 synthesis in endothelium
Authors: Houliston Rebecca A. , Pearson Jeremy D. , Wheeler-Jones Caroline P. D.
Published: December 2017

We have examined the mechanisms regulating prostacyclin (PGI2) synthesis after acute exposure of human umbilical vein endothelial cells (HUVEC) to interleukin-1α (IL-1α). IL-1α evoked an early (30 min) release of PGI2 and [3H]arachidonate that was blocked by the cytosolic phospholipase A2α (cPLA2α) inhibitor arachidonyl trifluoromethyl ketone. IL-1α-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44mapk) coincided temporally with phosphorylation of cPLA2α and with the onset of PGI2synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors, PD-98059 and U-0126, blocked IL-1α-induced ERK activation and partially attenuated cPLA2α phosphorylation and PGI2 release, suggesting that ERK-dependent and -independent pathways regulate cPLA2α phosphorylation. SB-203580 treatment enhanced IL-1α-induced MEK, p42/44mapk, and cPLA2α phosphorylation but reduced thrombin-stimulated MEK and p42/44mapk activation. IL-1α, but not thrombin, activated Raf-1 as assessed by immune-complex kinase assay, as did SB-203580 alone. These results show that IL-1α causes an acute upregulation of PGI2generation in HUVEC, establish a role for the MEK/ERK/cPLA2α pathway in this early release, and provide evidence for an agonist-specific cross talk between p38mapkand p42/44mapk that may reflect receptor-specific differences in the signaling elements proximal to MAPK activation.
PAF receptor antagonist modulates neutrophil responses with thermal injury in vivo
Authors: Fazal Nadeem , Al-Ghoul Walid M. , Choudhry Mashkoor A. , Sayeed Mohammed M.
Published: December 2017

The role of platelet-activating factor (PAF) in Ca2+ signaling and Ca2+-related enhancement of reactive oxygen intermediate (ROI) generation in neutrophils of burn-injured rats was ascertained by evaluating the effect of treatment of the rats with a PAF receptor antagonist. The treatment of rats with the antagonist also allowed us to evaluate the role of PAF in the priming of neutrophil ROI response with burn in vivo. A full skin thickness burn injury was produced in anesthetized rats by exposing 30% of total body surface area to 98°C water for 10 s. Sham and burn rats were killed 1 day later, and their blood was collected to obtain neutrophils. Fluorescence-activated cell sorter analysis was used to quantify ROI production by the neutrophils. Cytosolic-free Ca2+ concentration ([Ca2+]i) imaging technique was employed to measure neutrophil [Ca2+]i in individual cells and microfluorometry for the assessment of [Ca2+]i responses in suspensions of neutrophils. There was an overt enhancement of ROI generation by burn rat neutrophils. ROI release was accompanied by a marked elevation of [Ca2+]i signaling. The treatment of rats with PAF receptor antagonist before burn prevented the upregulation of both [Ca2+]i and ROI generation in neutrophils. These studies indicate that enhanced ROI production in neutrophils in the early stages after burn injury results from a PAF-mediated priming of the [Ca2+]i signaling pathways in vivo.
Hypotonicity induces L-selectin shedding in human neutrophils
Authors: Kaba Nubia K. , Knauf Philip A.
Published: December 2017

Expression levels of adhesion molecules on neutrophils are affected under various conditions, including ischemia, possibly because of associated increases in cell volume. We examined the effects of cell swelling in hypotonic media on the level of L-selectin (CD62L) and β2-integrin (CD18) on human neutrophils. In hypotonic media, neutrophils shed L-selectin. The shedding was greatly reduced by 30 μM RO31-9790, the metalloprotease (sheddase) inhibitor. Hypotonicity-induced L-selectin shedding was also time and tonicity dependent. Decreasing tonicity caused increased shedding. In 0.6× medium (0.6× the normal tonicity of 300 mosmol/kgH2O), shedding increased over a 2-h period, after which >70% of the neutrophils had lost L-selectin. In contrast to L-selectin, the level of β2-integrin on the neutrophil surface was not significantly affected. Thus L-selectin shedding, which occurs on neutrophil activation and is usually accompanied by β2-integrin upregulation, was selectively induced by hypotonicity without a corresponding effect on β2-integrin.
Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation
Authors: Dérand Renaud , Bulteau-Pignoux Laurence , Mettey Yvette , Zegarra-Moran Olga , Howell L. Daniel , Randak Christoph , Galietta Luis J. V. , Cohn Jonathan A. , Norez Caroline , Romio Leila , Vierfond Jean-Michel , Joffre Michel , Becq Frédéric
Published: December 2017

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC50 = 85 μM) and activated CFTR in Calu-3 cells (EC50 = 47 μM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.
Inhibition of epithelial chloride secretion by butyrate: role of reduced adenylyl cyclase expression and activity
Authors: Resta-Lenert Silvia , Truong Francis , Barrett Kim E. , Eckmann Lars
Published: December 2017

Butyrate and other short-chain fatty acids (SCFAs) are found at high concentrations in the colonic lumen and affect multiple epithelial cell functions. To better understand how SCFAs regulate ion transport, we investigated the effects of SCFAs on Cl− secretion in human colonic epithelial cell line T84. Butyrate inhibited Cl− secretory responses to prostaglandin E2, forskolin, and cholera toxin. Other SCFAs were less effective or inactive. Reduced secretion was associated with decreased synthesis of the second messenger cAMP rather than increased degradation. Expression and activity of adenylyl cyclase were decreased by butyrate, whereas phosphodiesterase activity was unaffected and phosphodiesterase inhibition did not reverse the effects of butyrate on Cl−secretion. Furthermore, butyrate decreased expression of the basolateral Na-K-2Cl cotransporter, indicating that it might modulate the secretory capacity of the cells. However, butyrate did not affect secretory responses to the calcium-dependent secretagogue carbachol, cAMP analogs, or uroguanylin, indicating that normal secretory responses to adequate levels of second messengers in butyrate-treated T84 cells are possible. These results show that butyrate affects several aspects of epithelial Cl− secretion, including second messenger generation and expression of key ion transporters. However, these effects may not all be equally important in determining Cl− secretion in response to physiologically relevant secretagogues.
Combined modulation of the mesangial machinery for monocyte recruitment by inhibition of NF-κB
Authors: Zernecke Alma , Weber Kim S. C. , Weber Christian
Published: December 2017

The activation of nuclear factor-κB (NF-κB) is required for the induction of many of the adhesion molecules and chemokines involved in the inflammatory leukocyte recruitment to the kidney. Here we studied the effects of NF-κB inhibition on the machinery crucial for monocyte infiltration of the glomerulus during inflammation. In mesangial cells (MC), the protease inhibitors MG-132 and N-α-tosyl-l-lysine chloromethyl ketone or adenoviral overexpression of IκB-α prevented the complete IκB-α degradation following tumor necrosis factor-α (TNF-α) stimulation. This resulted in a marked inhibition of TNF-α-induced expression of mRNA and protein for the immunoglobulin molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 and the chemokines growth-related oncogene-α, monocyte chemoattractant protein-1, interleukin-8, or fractalkine in MC. Finally, the inhibition of IκB-α degradation or IκB-α overexpression suppressed the chemokine-induced transendothelial monocyte chemotaxis toward MC and the chemokine-triggered firm adhesion of monocytic cells to MC. The inhibition of NF-κB by pharmacological intervention or gene transfer may present a multimodal approach to control the machinery propagating inflammatory recruitment of monocytes during glomerular disease.
Trafficking of GFP-tagged ΔF508-CFTR to the plasma membrane in a polarized epithelial cell line
Authors: Loffing-Cueni Dominique , Loffing Jan , Shaw Collin , Taplin Amilyn M. , Govindan Malu , Stanton Caitlin R. , Stanton Bruce A.
Published: December 2017

The ΔF508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and “chemical chaperones” allow ΔF508-CFTR to traffic to the plasma membrane and increase Cl− permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce ΔF508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing ΔF508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl− secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl− current across the apical plasma membrane. Thus butyrate increased the amount of functional ΔF508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl− secretion because of inhibitory effects on Cl− uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on ΔF508-CFTR will allow investigation of ΔF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.
Gap junctions and fluid flow response in MC3T3-E1 cells
Authors: Saunders M. M. , You J. , Trosko J. E. , Yamasaki H. , Li Z. , Donahue H. J. , Jacobs C. R.
Published: December 2017

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca2+concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E2 (PGE2) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca2+ (Ca[Formula: see text]) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE2 but not to the Ca[Formula: see text] response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.
H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity
Authors: Kevil Christopher G. , Okayama Naotsuka , Alexander J. Steven
Published: December 2017

We previously reported that exposure of endothelial cells to H2O2results in a loss of cell-cell apposition and increased endothelial solute permeability. The purpose of this study was to determine how tyrosine phosphorylation and tyrosine phosphatases contribute to oxidant-mediated disorganization of endothelial cell junctions. We found that H2O2 caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues. H2O2 exposure also results in increased endothelial monolayer permeability, which was attenuated by pp60, an inhibitor of src kinase. Inhibition of protein tyrosine phosphatase activity by phenylarsine oxide (PAO) demonstrated a similar permeability profile compared with H2O2, suggesting that tyrosine phosphatase activity is important in maintaining a normal endothelial solute barrier. Immunofluorescence shows that H2O2 exposure caused a loss of pan-reactive cadherin and β-catenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H2O2 also caused β-catenin to dissociate from the endothelial cytoskeleton, which was not prevented by PP1. Finally, we determined that PP1 did not prevent cadherin internalization. These data suggest that oxidants like H2O2 produce biological effects through protein phosphotyrosine modifications by decreasing total cellular phosphatase activity combined with increased src kinase activity, resulting in increased endothelial solute permeability.
Half-lives of plasma membrane Na+/H+ exchangers NHE1–3: plasma membrane NHE2 has a rapid rate of degradation
Authors: Cavet Megan E. , Akhter Shafinaz , Murtazina Rakhilya , Sanchez de Medina Fermin , Tse Chung-Ming , Donowitz Mark
Published: December 2017

The Na+/H+ exchangers NHE2 and NHE3 are involved in epithelial Na+ and HCO[Formula: see text]absorption. To increase insights into the functions of NHE2 vs. NHE3, we compared their cellular processing with each other and with the housekeeping isoform NHE1. Using biotinylated exchanger, we determined that the half-life of plasma membrane NHE2 was short (3 h) compared with that of NHE1 (24 h) and NHE3 (14 h) in both PS120 fibroblasts and Caco-2 cells. NHE2 transport and plasma membrane levels were reduced by 3 h of Brefeldin A treatment, whereas NHE1 was unaffected. NHE2 was degraded by the lysosomes but not proteosomes, as demonstrated by increasing levels of endocytosed NHE2 protein after inhibition of the lysosomes, but not with proteosome inhibition. Unlike that of NHE3, basal NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized in the juxtanuclear recycling endosome. Therefore, for NHE2, protein degradation and/or protein synthesis probably play important roles in its basal and regulated states. These results suggest fundamental differences in the cellular processing and trafficking of NHE2 and NHE3. These differences may underlie the specialized roles that these exchangers play in epithelial cells.
Basal lamina of ovarian follicle regulates an inward Cl− current in differentiated granulosa cells
Authors: Qin Wuxuan , Rane Stanley G. , Asem Elikplimi K.
Published: December 2017

Patch-clamp experiments were conducted to study the effects of basal lamina (basement membrane) of preovulatory chicken ovarian follicle on membrane currents in differentiated chicken granulosa cells in a homologous system. The membrane capacitance (measure of total membrane area) was smaller in cells cultured on intact basal lamina than that of control cells. The granulosa cells expressed outward and two inward currents. A small fraction of the cells (3%) expressed only a transient fast-activating and -inactivating inward current carried by Ca2+. The majority of the cells, however, expressed a slowly activating and inactivating inward current (carried by Cl−) that was superimposed on the transient Ca2+ current. All cells expressed an outward current characteristic of the delayed-rectifier K+ current. The removal of extracellular Ca2+ led to elimination of the slow inward Cl− current, indicating that it is a Ca2+-dependent Cl− current. Both peak amplitude and current density of the inward Cl− current were significantly lower in cells cultured on freshly isolated intact basal lamina (or basal lamina stored at 4°C for 12 mo) than those of control cells; however, basal lamina had no significant effect on the density of the outward current. Similar to the observations made for intact basal lamina, solubilized basal lamina suppressed the inward Cl− current in differentiated granulosa cells. These data show that homologous basal lamina modulates a Ca2+-dependent Cl− current in differentiated granulosa cells. These findings provide a partial explanation for the mechanisms that subserve the reported effects of basal lamina (basement membrane) on the metabolic functions of differentiated granulosa cells.
Effect of atropine and vagotomy on pancreatic polypeptide response to a meal in dogs.
Authors: Taylor I L , Impicciatore M , Carter D C , Walsh J H
Published: December 2017

In four conscious dogs the pancreatic polypeptide (PP) response to a standard beef-liver meal was measured by specific radioimmunoassay and compared with the response seen after the intravenous injection of 25 or 100 microgram/kg atropine. All three tests were performed twice in each animal and then repeated after truncal vagotomy. The mean prevagotomy postprandial PP increment was 85 +/- 16 pmol/liter in the first 2-h period and 54.5 +/- 13 pmol/liter in the second. After the injection of 25 microgram/kg atropine there was significant reduction in the early response (mean delta PP = 39 +/- 17 pmol/liter, P less than 0.05), but not the late (mean delta PP = 62 +/- 18 pmol/liter). After 100 microgram/kg atropine sulfate, the response was significantly reduced during both periods (mean delta PP = 5.5 +/- 5.2 and 20 +/- 8.8 pmol/liter, respectively, P less than 0.01). Truncal vagotomy significantly (P less than 0.01) reduced the PP response over both time periods (mean delta PP = 6.4 +/- 2.2 and 5.8 +/- 3.8 pmol/liter), and the small residual response was completely abolished by atropine. In five additional dogs an infusion of bethanechol (100 microgram . kg-1.h-1) caused a significant increase (P less than 0.05) in the plasma concentration (mean delta PP = 40.9 +/- 11.8 pmol/liter), which was abolished by pretreatment with atropine (mean delta PP = -2.9 +/- 2.1 pmol/liter). These studies suggest that PP release in response to a meal in the dog is largely under vagal-cholinergic control.
Folic acid transport by mammalian small intestine.
Authors: Rose R C , Koch M J , Nahrwold D L
Published: December 2017

The unidirectional influx of folic acid across the mucosal border of hamster duodenum and rat jejunum was determined. Influx follows saturation kinetics, is sodium-dependent, and is inhibited by methotrexate and is sodium-dependent, and is inhbited by methotrexate and folinic acid in the mucosal bathing solution. In hamster duodenum, the maximal influx is 1.2 nmol/(cm2.h), and the folic acid concentration required to give a half maximal influx (Km) is 7.2 micron.. At mucosal folic acid concentration of 1.5 micron, influx is reduced at least 65% by removal of sodium from the bathing solution. The influx process is significantly inhibited by cyanide and 2,4-dinitrophenol. The possibility was evaluated that the acidic microclimate at the brush border regulates the rate of folic acid transport and that inhibition of transmural transport by sodium-free media, ouabain, and methotrexate is brought about indirectly by an increase in pH of the microclimate. The data favor the alternative concept of a sodium-dependent carrier mechanism for entry of folic acid into the cells. The information presented is consistent with active transport of folic acid by a sodium-gradient mechanism, but additional information will be necessary to substantiate such a model.
Role of ligandin in transfer of bilirubin from plasma into liver.
Authors: Wolkoff A W , Goresky C A , Sellin J , Gatmaitan Z , Arias I M
Published: December 2017

Multiple-indicator dilution studies of labeled bilirubin uptake were carried out on isolated perfused rat livers with variable ligandin concentrations (from normal and thyroidectomized animals with and without phenobarbital pretreatment). Ligandin concentrations, measured immunologically, increased 25% after thyroidectomy and approximately doubled after phenobarbital pretreatment but decreased to normal during perfusion in the thyroidectomized nonpretreated group. A distributed two-compartment model was fitted to the dilution data and estimates of influx, efflux, and sequestration coefficients were obtained. Influx and sequestration coefficients did not vary significantly between the groups. Efflux coefficients were significantly smaller (P less than 0.001), and hepatic ligandin concentrations were significantly larger (p less than 0.001) in phenobarbital-treated rats than in other groups. The efflux coefficient varied inversely with ligandin concentration and the volume of distribution in tissue, as perceived from the plasma space, increased in proportion to the concentration of ligandin. The increased net uptake of tracer bilirubin by the liver of phenobarbital-pretreated animals is due to decreased tracer efflux secondary to the increase in intracellular binding of bilirubin by ligandin.