bluuebunny/crossref_metadata_2025 huggingface dataset:
Peroxynitrite attenuates hepatic ischemia-reperfusion injury
Authors: Liu Peitan , Xu Baohuan , Quilley John , Wong Patrick Y.-K.
Published: December 2017

In the present study, we examined the effects of peroxynitrite on reperfusion injury using a rat model of hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia, followed by 4 h of reperfusion. Groups A and B rats were sham-operated controls that received vehicle or peroxynitrite; groups C and D rats were subjected to HI/R and received peroxynitrite or vehicle, respectively. A dose of 2 μmol/kg body wt of peroxynitrite, diluted in saline (pH 9.0, 4°C), was administered as a bolus through a portal vein catheter at 0, 60, and 120 min after reperfusion. Results showed that superoxide generation in the ischemic lobes of the liver and plasma alanine aminotransferase (ALT) activity of group C were decreased by 43% and 45%, respectively, compared with group D. Leukocyte accumulations in the ischemic lobes of liver and circulating leukocytes were decreased by 40% and 27%, respectively, in group C vs. D. The ratios of mRNA of P-selectin and intercellular adhesion molecule-1 (ICAM-1) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA extracted from the ischemic lobes of the liver of group C were decreased compared with group D. There were no differences between the groups A and B in terms of plasma ALT activity, circulating leukocytes, superoxide generation, and leukocyte infiltration in the ischemic lobes of the liver. Moreover, hemodynamic parameters (i.e., mean arterial blood pressure, cardiac index, stroke index, and systemic vascular resistance) were not significantly different among groups B, C, and D. These results suggest that administration of peroxynitrite via the portal vein only has a local effect. Exogenous peroxynitrite at physiological concentrations attenuates leukocyte-endothelial interaction and reduces leukocyte infiltration. The mechanism of the reduction of leukocyte infiltration into ischemic lobes of the liver appears because of decreased expression of mRNA of P-selectin and ICAM-1. The net effect of administration of peroxynitrite may be to reduce adhesion molecule-mediated, leukocyte-dependent reperfusion injury.
Protein kinase C contributes to desensitization of ANG II signaling in adult rat cardiac fibroblasts
Authors: Meszaros J. Gary , Raphael Robert , Lio Francisco M. , Brunton Laurence L.
Published: December 2017

We have studied Gq-linked ANG II signaling [inositol phosphate (IP) accumulation, Ca2+ mobilization] in primary cultures of rat cardiac fibroblasts (CFs) and have found that ANG II initiates a protein kinase C (PKC)-mediated negative feedback loop that rapidly terminates the ANG II response. Pharmacological inhibition of PKC by staurosporine and GF-109203X doubled IP production over that achieved in response to ANG II alone. Inhibition of PKC also led to larger Ca2+ transients in response to ANG II, suggesting that Ca2+ mobilization was proportional to Gq-phospholipase C-IP3 activity under the conditions studied. Depletion of cellular PKC by overnight treatment with phorbol 12-myristate 13-acetate (PMA) similarly augmented ANG II-induced IP production. Acute activation of PKC by PMA halved IP formation, with an EC50≈1 nM; 4α-PMA was inactive. Time course data demonstrated that ANG II-mediated IP production fully desensitized within 30 s; PKC inhibition reduced the rate and extent of this desensitization. In cells desensitized to ANG II, a purinergic agonist still mobilized intracellular Ca2+, indicating that desensitization was homologous. The ANG II-induced Ca2+ signal was fully resensitized within 30 min. The data demonstrate that a large portion of the IP-Ca2+ responses of rat CFs to ANG II are short-lived because of rapid, PKC-mediated desensitization.
Activation of pancreatic acinar cells on isolation from tissue: cytokine upregulation via p38 MAP kinase
Authors: Blinman Thane A. , Gukovsky Ilya , Mouria Michelle , Zaninovic Vjekoslav , Livingston Edward , Pandol Stephen J. , Gukovskaya Anna S.
Published: December 2017

Cytokines produced by pancreatic acinar cells may mediate cell death and recruitment of inflammatory cells into pancreas in pancreatitis and other disorders. Here, we demonstrate mRNA expression for a number of cytokines in acini isolated from rat pancreas. Using RNA from microscopically selected individual cells, we confirmed the acinar cell as a source for cytokine expression. Competitive RT-PCR, Western blot analysis, and immunocytochemistry showed large amounts of monocyte chemotactic protein-1 and interleukin-6 compared with other cytokines. Cytokine expression was inhibited by either inhibitors of p38 mitogen-activated protein kinase (MAPK), SB-202190 and SB-203580, or (less strongly) by the transcription factor nuclear factor (NF)-κB inhibitor MG-132. A combination of SB-203580 and MG-132 inhibited mRNA expression of all cytokines by >90%. The results suggest a major role for p38 MAPK and involvement of NF-κB in cytokine expression in pancreatic acinar cells. In contrast to isolated acini, we detected no or very low cytokine expression in normal rat pancreas. Our results indicate that activation of p38 MAPK, transcription factors, and cytokines occurs during removal of the pancreas from the animal and isolation of acini.
Requirement of cortical actin organization for bombesin, endothelin, and EGF receptor internalization
Authors: Lunn J. Adrian , Wong Helen , Rozengurt Enrique , Walsh John H.
Published: December 2017

The role of actin organization in occupancy-induced receptor internalization remains poorly defined. Here we report that treatment of mouse Swiss 3T3 cells with latrunculin A, a potent inhibitor of actin polymerization (including cortical actin), inhibited the internalization of the endogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judged by uptake of 125I-labeled GRP or fluorescent Cy3-labeled bombesin. In contrast, cells pretreated with cytochalasin D showed minimal inhibition of bombesin/GRP receptor internalization. Similarly, pretreatment of Swiss 3T3 cells with the potent Rho-kinase inhibitor HA-1077, at concentrations (10–20 μM) that abrogated bombesin-mediated stress fiber formation, did not significantly alter receptor-mediated internalization of125I-GRP. These results indicate that bombesin/GRP receptor internalization depends on latrunculin A-sensitive cortical actin rather than on rapidly turning over actin stress fibers that are disrupted by either cytochalasin D or HA-1077. The rates and total levels of internalization of the endogenously expressed endothelin A receptor and epidermal growth factor receptor were also markedly reduced by latrunculin A in Swiss 3T3 cells. The potency of latrunculin A for inhibiting G protein-coupled receptor endocytosis was comparable to that for reducing internalization of the epidermal growth factor tyrosine kinase receptor. We conclude that cortical actin structures, disrupted by latrunculin A, are necessary for occupancy-induced receptor internalization in animal cells.
Activation of MT2 melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock
Authors: Hunt Amanda E. , Al-Ghoul Walid M. , Gillette Martha U. , Dubocovich Margarita L.
Published: December 2017

The aim of this study was to identify the melatonin receptor type(s) (MT1 or MT2) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[125I]iodomelatonin and in situ hybridization histochemistry, with either 33P- or digoxigenin-labeled antisense MT1 and MT2 melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT1/MT2nonselective luzindole and the MT2-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 μM) did not affect circadian phase on their own; however, they blocked both the phase advances (∼4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT2 melatonin receptor signaling.
Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells
Authors: Yingst Douglas R. , Davis Joanne , Schiebinger Rick
Published: December 2017

Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (Ko) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive 86Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing Ko from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Cao) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing Ko from 4 to 10 mM in the absence of Cao stimulated the sodium pump ∼30% and did not increase intracellular free calcium concentration ([Ca2+]i). In some experiments, addition of 1.8 mM Cao in the presence of 4 mM Ko increased [Ca2+]i above the levels observed in the absence of Cao and stimulated the sodium pump up to 100%. Ca-dependent stimulation of the sodium pump by Ko and Cao was inhibited by isradipine (10 μM), a blocker of L- and T-type calcium channels, by compound 48/80 (40 μg/ml) and calmidizolium (10 μM), which inhibits calmodulin (CaM), and by KN-62 (10 μM), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 μM), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Cao increased [Ca2+]i to the level observed in the presence of 10 mM Ko and 1.8 mM Cao and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Cao was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that Ko stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Cao. The resulting increase in [Ca2+]i may stimulate the sodium pump by activating CaM and/or CaMKII.
A proposed mechanism for the potentiation of cAMP- mediated acid secretion by carbachol
Authors: Muto Yuko , Nagao Taku , Yamada Maki , Mikoshiba Katsuhiko , Urushidani Tetsuro
Published: December 2017

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [14C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca2+ and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 μM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca2+ concentration ([Ca2+]i) increase due to release from the Ca2+ store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca2+ release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca2+store and the receptor is maintained by actin microfilaments.
The ΔF508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells
Authors: Heda Ghanshyam D. , Tanwani Mridul , Marino Christopher R.
Published: December 2017

Although the biosynthetic arrest of the ΔF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of ΔF508 and wild-type CFTR at the cell surface of transfected LLC-PK1 cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane ΔF508 CFTR to be ∼4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the ΔF508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the ΔF508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.
Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2Kb-tsA58transgene
Authors: Takacs-Jarrett Marcia , Sweeney William E. , Avner Ellis D. , Cotton Calvin U.
Published: December 2017

Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) −/−] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33°C with interferon-γ). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl− permeability and cAMP-stimulated Cl− secretion. In contrast, the four CFTR −/− cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl− secretory responses. Ca2+-activated Cl− secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.
Chromaffin-adrenocortical cell interactions: effects of chromaffin cell activation in adrenal cell cocultures
Authors: Shepherd S. P. , Holzwarth M. A.
Published: December 2017

Although the adrenal cortex and medulla are both involved in the maintenance of homeostasis and stress response, the functional importance of intra-adrenal interactions remains unclear. When primary cocultures of frog ( Rana pipiens) adrenocortical and chromaffin cells were used, selective chromaffin cell activation dramatically affected both chromaffin and adrenocortical cells. Depolarization with 50 μm veratridine enhanced chromaffin cell neuronal phenotype, contacts with adrenocortical cells, and secretion of norepinephrine, epinephrine, and serotonin. Time-lapse video microscopy recorded the rapid establishment of growth cones on the activated chromaffin cell neurites, neurite branching, and outgrowth toward adrenocortical cells. Simultaneously, adrenocortical cells migrated toward chromaffin cells. Following chromaffin cell activation, adrenocortical cell Fos protein expression and corticosteroid secretion were increased, indicating that chromaffin cell modulation of adrenocortical cells is at the transcriptional level. These results provide evidence that intra-adrenal interactions affect cellular differentiation and modulate steroidogenesis. Furthermore, this suggests that the activity-related plasticity of chromaffin and adrenocortical cells is developmentally and physiologically important.
Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment
Authors: Spector Jason A. , Mehrara Babak J. , Greenwald Joshua A. , Saadeh Pierre B. , Steinbrech Douglas S. , Bouletreau Pierre J. , Smith Lee P. , Longaker Michael T.
Published: December 2017

Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions ( P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH ( P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive.
Modulation of P2Z/P2X7 receptor activity in macrophages infected with Chlamydia psittaci
Authors: Coutinho-Silva Robson , Perfettini Jean-Luc , Persechini Pedro M. , Dautry-Varsat Alice , Ojcius David M.
Published: December 2017

Given the role that extracellular ATP (ATPo)-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATPo has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATPo-induced killing via P2Z/P2X7 purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATPo, because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X7 purinergic receptors. Incubation with ATPo but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATPo effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATPo-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATPo to permeabilize macrophages to small molecules and by abrogating a sustained Ca2+ influx previously associated with ATPo-induced apoptosis.
Effect of culture Po 2 on macrophage (RAW 264.7) nitric oxide production
Authors: Otto Cynthia M. , Baumgardner James E.
Published: December 2017

Macrophages are commonly cultured at a Po 2 of 149 Torr, but tissue macrophages in vivo live in an environment of much lower oxygen tension. Despite the many potential mechanisms for changes in oxygen tension to influence nitric oxide (NO) synthesis, there have been few reports investigating the effect of Po 2 on macrophage NO production. With the use of a culture chamber designed to rigorously control oxygen tension, we investigated the effects of culture Po 2 on macrophage NO production, inducible nitric oxide synthase (iNOS) activity, iNOS protein, and tumor necrosis factor production. NO production and iNOS activity were linearly related in the range of 39.4 to 677 Torr, but not in the range of 1.03 to 39.4 Torr. Therefore, results obtained in vitro for the high oxygen tensions commonly used in cell culture were quantitatively and qualitatively different from results obtained in cells cultured at the lower oxygen tensions that more accurately reflect the in vivo environment. The influence of oxygen tension on NO production has implications for cell culture methodology and for the relationship between microcirculatory dysfunction and inflammatory responses in rodent models of sepsis.
Cryptdin-3 induces novel apical conductance(s) in Cl− secretory, including cystic fibrosis, epithelia
Authors: Merlin Didier , Yue Gang , Lencer Wayne I. , Selsted Michael E. , Madara James L.
Published: December 2017

Opening of anion-conductive pathways in apical membranes of secretory cells lining mucosal surfaces is a critical step in salt and water secretion and, thus, hydration of sites including airway and intestine. In intestine, Paneth cells are positioned at the base of the secretory gland (crypt) and release defensin peptide, in mice termed cryptdins, into the crypt lumen. Because at least some defensins have been shown to form anion-conductive channels in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes. To directly evaluate the possibility of cryptdin-3-mediated apical anion conductance ( G ap), we have utilized amphotericin B to selectively permeabilize basolateral membranes of electrically tight monolayers of polarized human intestinal secretory epithelia (T84 cells), thus isolating the apical membrane for study. Cryptdin-3 induces G ap that is voltage independent (Δ G ap = 1.90 ± 0.60 mS/cm2) and exhibits ion selectivity contrasting to that elicited by forskolin or thapsigargin (for cryptdin-3, Cl− = gluconate; for forskolin and thapsigargin, Cl− ≫ gluconate). We cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of cation and Cl− currents. Cryptdin-3 induces a current in basolaterally permeabilized epithelial monolayers derived from airway cells harboring the ΔF508 mutation of cystic fibrosis (CF; Δ G ap = 0.80 ± 0.06 mS/cm2), demonstrating that cryptdin-3 restores anion secretion in CF cells; this occurs independently of the CF transmembrane conductance regulator channel. These results support the idea that cryptdin-3 may associate with apical membranes of Cl−-secreting epithelia and self-assemble into conducting channels capable of mediating a physiological response.
Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A
Authors: Westfall Margaret V. , Turner Immanuel I. , Albayya Faris P. , Metzger Joseph M.
Published: December 2017

Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI. PKA-mediated phosphorylation of a second TnI chimera, N-card/slow-C TnI, which has the amino terminal region of cTnI, caused a decrease in the Ca2+ sensitivity of tension comparable in magnitude to control myocytes. Based on these results, we propose the amino terminal region shared by cTnI and N-card/slow-C TnI plays a central role in determining the magnitude of the PKA-mediated shift in myofilament Ca2+ sensitivity, independent of the isoform-specific functional domains previously defined within the carboxyl terminal backbone of TnI. Interestingly, exposure of permeabilized myocytes to acidic pH after PKA-mediated phosphorylation of cTnI resulted in an additive decrease in myofilament Ca2+ sensitivity. The isoform-specific, pH-sensitive region within TnI lies in the carboxyl terminus of TnI, and the additive response provides further evidence for the presence of a separate domain that directly transduces the PKA phosphorylation signal.
Essential role for IL-6 in postresuscitation inflammation in hemorrhagic shock
Authors: Meng Zhi Hong , Dyer Kevin , Billiar Timothy R. , Tweardy David J.
Published: December 2017

Interleukin-6 (IL-6) is produced within multiple tissues and can be readily detected in the circulation in resuscitated hemorrhagic shock (HS). Instillation of IL-6 into lungs of normal rats induces polymorphonuclear neutrophilic granulocyte (PMN) infiltration and lung damage, while infusion of IL-6 into the systemic circulation of rats during resuscitation from HS reduces PMN recruitment and lung injury. The current study was designed to determine whether or not IL-6 makes an essential contribution to postresuscitation inflammation and which of the two effects of IL-6, its local proinflammatory effect or its systemic anti-inflammatory effect, is dominant in HS. Wild-type and IL-6-deficient mice were subjected to HS followed by resuscitation and death 4 h later. IL-6-deficient mice subjected to HS did not demonstrate any features of postresuscitation inflammation observed in wild-type mice, including increased PMN infiltration into the lungs, increased alveolar cross-sectional surface area, increased PMN infiltration into the liver, increased liver necrosis, increased signal transducer and activator of transcription 3 activation, and increased nuclear factor-κB activity. These findings indicate that IL-6 is an essential component of the postresuscitation inflammatory cascade in HS and that the local proinflammatory effects of IL-6 on PMN infiltration and organ damage in HS dominate over the anti-inflammatory effects of systemic IL-6.
Differential expression of KvLQT1 and its regulator IsK in mouse epithelia
Authors: Demolombe Sophie , Franco Diego , de Boer Piet , Kuperschmidt Sabina , Roden Dan , Pereon Yann , Jarry Anne , Moorman Antoon F. M. , Escande Denis
Published: December 2017

KCNQ1 is the human gene responsible in most cases for the long QT syndrome, a genetic disorder characterized by anomalies in cardiac repolarization leading to arrhythmias and sudden death. KCNQ1 encodes a pore-forming K+channel subunit termed KvLQT1 which, in association with its regulatory β-subunit IsK (also called minK), produces the slow component of the delayed-rectifier cardiac K+ current. We used in situ hybridization to localize KvLQT1 and IsK mRNAs in various tissues from adult mice. We showed that KvLQT1 mRNA expression is widely distributed in epithelial tissues, in the absence (small intestine, lung, liver, thymus) or presence (kidney, stomach, exocrine pancreas) of its regulator IsK. In the kidney and the stomach, however, the expression patterns of KvLQT1 and IsK do not coincide. In many tissues, in situ data obtained with the IsK probe coincide with β-galactosidase expression in IsK-deficient mice in which the bacterial lacZgene has been substituted for the IsK coding region. Because expression of KvLQT1 in the presence or absence of its regulator generates a K+ current with different biophysical characteristics, the role of KvLQT1 in epithelial cells may vary depending on the expression of its regulator IsK. The high level of KvLQT1 expression in epithelial tissues is consistent with its potential role in K+secretion and recycling, in maintaining the resting potential, and in regulating Cl− secretion and/or Na+absorption.
Negative regulation of epithelium-neutrophil interactions via activation of CD44
Authors: Si-Tahar Mustapha , Sitaraman Shanthi , Shibahara Takeshi , Madara James L.
Published: December 2017

Polymorphonuclear neutrophil (PMN) migration across epithelia is a common feature of active inflammation. Given the suggested role of carbohydrates in this process, we examined the receptor CD44. The standard CD44 isoform was expressed at the cell surface of PMN. PMN migration across model polarized intestinal epithelia was reduced (by 60%) if the CD44 receptor was activated by either a specific antibody (clone IM7) or the natural soluble ligand, hyaluronic acid. This inhibitory effect following receptor activation occurred with both basolateral-to-apical- and apical-to-basolateral-directed migration. The anti-CD44 antibody similarly reduced PMN migration through filters in the absence of epithelia, while preincubation of the antibody with the epithelium did not alter subsequent PMN transepithelial migration. These data suggest that PMN, rather than epithelial, CD44 is responsible for these effects. A similar inhibitory effect of anti-CD44 antibody was also observed on migration of intraepithelial lymphocytes. The molecular mechanism involved in such negative signaling following CD44 activation may include modulation of outside-in cell signaling. While neither the anti-CD44 antibody nor CD44 ligand affected PMN mobilization of intracellular Ca2+, both led to increased adenylate cyclase activity, an inhibitory signal for PMN migration. Together, these results suggest that CD44 of PMN may potentially serve as a negative regulator of leukocyte migration across biological surfaces such as columnar epithelia.
Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-κB in malignant melanoma cells
Authors: Brar Sukhdev S. , Kennedy Thomas P. , Whorton A. Richard , Sturrock Anne B. , Huecksteadt Thomas P. , Ghio Andrew J. , Hoidal John R.
Published: December 2017

The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in malignancies from enhanced activity of inhibitor of NF-κB (IκB) kinase, with accelerated IκBα degradation. We studied whether redox signaling might stimulate these events. Cultured melanoma cells generated superoxide anions (O[Formula: see text]) without serum stimulation. O[Formula: see text]generation was reduced by the NAD(P)H:quinone oxidoreductase (NQO) inhibitor dicumarol and the quinone analog capsaicin, suggesting that electron transfer from NQO through a quinone-mediated pathway may be an important source of endogenous reactive oxygen species (ROS) in tumor cells. Treatment of malignant melanoma cells with the H2O2 scavenger catalase, the sulfhydryl donor N-acetylcysteine, the glutathione peroxidase mimetic ebselen, or dicumarol decreased NF-κB activation. Catalase, N-acetylcysteine, ebselen, dicumarol, and capsaicin also inhibited growth of melanoma and other malignant cell lines. These results raise the possibility that ROS produced endogenously by mechanisms involving NQO can constitutively activate NF-κB in an autocrine fashion and suggest the potential for new antioxidant strategies for interruption of oxidant signaling of melanoma cell growth.
Prostaglandin endoperoxide H synthase expression in human thyroid epithelial cells
Authors: Gianoukakis Andrew G. , Cao H. James , Jennings Timothy A. , Smith Terry J.
Published: December 2017

KAT-50, an established human thyrocyte cell line, expresses constitutively high levels of prostaglandin endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Here, we examine primary human thyrocytes. We find that they, too, express PGHS-2 mRNA and protein under control culture conditions. A substantial fraction of the basal prostaglandin E2(PGE2) produced by these cells can be inhibited by SC-58125 (5 μM), a PGHS-2-selective inhibitor. Interleukin (IL)-1β (10 ng/ml) induces PGHS-2 expression and PGE2 production in primary thyrocytes. The induction of PGHS-2 and PGE2synthesis by IL-1β could be blocked by glucocorticoid treatment. Unlike KAT-50, most of the culture strains also express PGHS-1 protein. Our observations suggest that both cyclooxygenase isoforms may have functional roles in primary human thyroid epithelial cells, and PGHS-2 might predominate under basal and cytokine-activated culture conditions.
Ontogenetic, gravity-dependent development of rat soleus muscle
Authors: Ohira Yoshinobu , Tanaka Takato , Yoshinaga Tomoo , Kawano Fuminori , Nomura Takeshi , Nonaka Ikuya , Allen David L. , Roy Roland R. , Edgerton V. Reggie
Published: December 2017

We tested the hypothesis that rat soleus muscle fiber growth and changes in myosin phenotype during the postnatal, preweaning period would be largely independent of weight bearing. The hindlimbs of one group of pups were unloaded intermittently from postnatal day 4 to day 21: the pups were isolated from the dam for 5 h during unloading and returned for nursing for 1 h. Control pups were either maintained with the dam as normal or put on an alternating feeding schedule as described above. The enlargement of mass (∼3 times), increase in myonuclear number (∼1.6 times) and myonuclear domain (∼2.6 times), and transformation toward a slow fiber phenotype (from 56 to 70% fibers expressing type I myosin heavy chain) observed in controls were inhibited by hindlimb unloading. These properties were normalized to control levels or higher within 1 mo of reambulation beginning immediately after the unloading period. Therefore, chronic unloading essentially stopped the ontogenetic developmental processes of 1) net increase in DNA available for transcription, 2) increase in amount of cytoplasm sustained by that DNA pool, and 3) normal transition of myosin isoforms that occur in some fibers from birth to weaning. It is concluded that normal ontogenetic development of a postural muscle is highly dependent on the gravitational environment even during the early postnatal period, when full weight-bearing activity is not routine.
Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-ATPase
Authors: Sweadner Kathleen J. , Feschenko Marina S.
Published: December 2017

Regulation of Na-K-ATPase by cAMP-dependent protein kinase occurs in a variety of tissues. Phosphorylation of the enzyme's catalytic subunit at a classical phosphorylation consensus motif has been observed with purified enzyme. Demonstration of phosphorylation at the same site in normal living cells or tissues has been more difficult, however, making it uncertain that the Na-K-ATPase is a direct physiological substrate of the kinase. Recently, the structure of the homologous sarco(endo)plasmic reticulum Ca-ATPase (SERCA1a) has been determined at 2.6 Å resolution (Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Nature 405: 647–655, 2000.), and the Na-K- ATPase should have the same fold. Here, the Na-K-ATPase sequence has been aligned with the Ca-ATPase structure to examine the predicted disposition of the phosphorylation site. The location is close to the membrane and partially buried by adjacent loops, and the site is unlikely to be accessible to the kinase in this conformation. Conditions that may expose the site or further bury it are discussed to highlight the issues facing future research on regulation of Na-K-ATPase by cAMP-dependent pathways.
Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs
Authors: Dumaswala U. J. , Zhuo L. , Mahajan S. , Nair P. N. M. , Shertzer H. G. , Dibello P. , Jacobsen D. W.
Published: December 2017

Oxidant stress, in vivo or in vitro, is known to induce oxidative changes in human red blood cells (RBCs). Our objective was to examine the effect of augmenting RBC glutathione (GSH) synthesis on 1) degenerative protein loss and 2) RBC chemokine- and free radical-scavenging functions in the oxidatively stressed human RBCs by using banked RBCs as a model. Packed RBCs were stored up to 84 days at 1–6°C in Adsol or in the experimental additive solution (Adsol fortified with glutamine, glycine, and N-acetyl-l-cysteine). Supplementing the conventional additive with GSH precursor amino acids improved RBC GSH synthesis and maintenance. The rise in RBC γ-glutamylcysteine ligase activity was directly proportional to the GSH content and inversely proportional to extracellular homocysteine concentration, methemoglobin formation, and losses of the RBC proteins band 3, band 4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffy antigen ( P < 0.01). Reduced loss of Duffy antigen correlated well with a decrease in chemokine RANTES (regulated upon activation, normal T-cell expressed, and secreted) concentration. We conclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSH synthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, in which RBCs are subjected to chronic/acute oxidant stresses.
Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD
Authors: Wei Yuan , Lu Ming , Wang WenHui
Published: December 2017

We investigated the effect of inhibiting Na+-K+-ATPase on the basolateral 18-pS K+ channel in the cortical collecting duct (CCD) of the rat kidney. Inhibiting Na+-K+-ATPase with strophanthidin decreased the activity of the 18-pS K+channel and increased the intracellular Ca2+ to 420 nM. Removal of extracellular Ca2+ abolished the effect of strophanthidin. When intracellular Ca2+ was raised with 5 μM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the 18-pS K+ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of Ca2+-induced inhibition, the effect of 400 nM Ca2+ on channel activity was studied in the presence of calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62, inhibitors of calmodulin-dependent kinase II. Addition of calphostin C or KN-93 or KN-62 failed to block the inhibitory effect of high concentrations of Ca2+. This suggested that the inhibitory effect of high concentrations of Ca2+ was not mediated by protein kinase C or calmodulin-dependent kinase II pathways. To examine the possibility that the inhibitory effect of high concentrations of Ca2+ was mediated by the interaction of nitric oxide with superoxide, we investigated the effect of 400 nM Ca2+ on channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or N ω-nitro-l-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or N ω-nitro-l-arginine methyl ester completely abolished the inhibitory effect of 400 nM Ca2+ on channel activity. Moreover, application of 4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect of strophanthidin. We conclude that the effect of inhibiting Na+-K+-ATPase is mediated by intracellular Ca2+ and the inhibitory effect of high concentrations of Ca2+ is the result of interaction of nitric oxide with superoxide.
Ca2+-induced contraction of cat esophageal circular smooth muscle cells
Authors: Cao W. , Chen Q. , Sohn U. D. , Kim N. , Kirber M. T. , Harnett K. M. , Behar J. , Biancani P.
Published: December 2017

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+ influx and activation of protein kinase Cε (PKCε). PKCε, however, is known to be Ca2+ independent. To determine where Ca2+ is needed in this PKCε-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+ (0.2–1.0 μM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCε (but not by PKCβII or PKCγ antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPβs. Addition of 1 μM Ca2+ to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF3 (cytosolic phospholipase A2inhibitor). These data suggest that Ca2+ may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCε, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPγS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+ and G protein activation), producing DAG and AA, and activating PKCε-dependent mechanisms to cause contraction.
Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion
Authors: Takami Mimi , Herrera Roman , Petruzzelli Lilli
Published: December 2017

Activated neutrophils display an array of physiological responses, including initiation of the oxidative burst, phagocytosis, and cell migration, that are associated with cellular adhesion. Under conditions that lead to cellular adhesion, we observed rapid tyrosine phosphorylation of an intracellular protein with an approximate relative molecular mass of 92 kDa (p92). Phosphorylation of p92 was inducible when Mac-1 was activated by phorbol 12-myristate 13-acetate, the β2-specific activating antibody CBR LFA-1/2, or interleukin-8 (77 amino acids). In addition, tyrosine phosphorylation of p92 was dependent on engagement of Mac-1 with ligand. Several observations suggest that this event may be an important step in the signaling pathway initiated by Mac-1 binding. p92 phosphorylation was specifically blocked with antibodies to CD11b, the α-subunit of Mac-1, and was rapidly reversible on disengagement of the integrin ligand interaction. Integrin-stimulated phosphorylation of p92 created binding sites that were recognized in vitro by the SH2 domains of c-CrkII and Src. Our observations suggest that neutrophil adhesion mediated through the binding of the β2-integrin Mac-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interactions via SH2 domains, a key process shared with growth factor signaling pathways.
Increased contractility and altered Ca2+ transients of mouse heart myocytes conditionally expressing PKCβ
Authors: Huang Lin , Wolska Beata M. , Montgomery David E. , Burkart Eileen M. , Buttrick Peter M. , Solaro R. John
Published: December 2017

Activation of protein kinase C (PKC) in heart muscle signals hypertrophy and may also directly affect contractile function. We tested this idea using a transgenic (TG) mouse model in which conditionally expressed PKCβ was turned on at 10 wk of age and remained on for either 6 or 10 mo. Compared with controls, TG cardiac myocytes demonstrated an increase in the peak amplitude of the Ca2+ transient, an increase in the extent and rate of shortening, and an increase in the rate of relengthening at both 6 and 10 mo of age. Phospholamban phosphorylation and Ca2+-uptake rates of sarcoplasmic reticulum vesicles were the same in TG and control heart preparations. At 10 mo, TG skinned fiber bundles demonstrated the same sensitivity to Ca2+ as controls, but maximum tension was depressed and there was increased myofilament protein phosphorylation. Our results differ from studies in which PKCβ was constitutively overexpressed in the heart and in studies that reported a depression of myocyte contraction with no change in the Ca2+ transient.
Coupling of L-type voltage-sensitive calcium channels to P2X2 purinoceptors in PC-12 cells
Authors: Hur Eun-Mi , Park Tae-Ju , Kim Kyong-Tai
Published: December 2017

Extracellular ATP elevates cytosolic Ca2+ by activating P2X and P2Y purinoceptors and voltage-sensitive Ca2+ channels (VCCCs) in PC-12 cells, thereby facilitating catecholamine secretion. We investigated the mechanism by which ATP activates VSCCs. 2-Methylthioadenosine 5′-triphosphate (2-MeS-ATP) and UTP were used as preferential activators of P2X and P2Y, respectively. Nifedipine inhibited the ATP- and 2-MeS-ATP-evoked cytosolic Ca2+concentration increase and [3H]norepinephrine secretion, but not the UTP-evoked responses. Studies with Ca2+ channel blockers indicated that L-type VSCCs were activated after the P2X activation. Mn2+ entry profiles and studies with thapsigargin revealed that Ca2+ entry, rather than Ca2+ release, was sensitive to nifedipine. Although P2X2 and P2X4 receptor mRNAs were detected, studies with pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid revealed that P2X2 was mainly coupled to the L-type VSCCs. The inhibitory effect of nifedipine did not occur in the absence of extracellular Na+, suggesting that Na+ influx, which induces depolarization, was essential for the P2X2-mediated activation of VSCCs. We report that depolarization induced by Na+ entry through the P2X2 purinoceptors effectively activates L-type VSCCs in PC-12 cells.
In vivo regulation of the β-myosin heavy chain gene in hypertensive rodent heart
Authors: Wright Carola E. , Bodell P. W. , Haddad F. , Qin A. X. , Baldwin K. M.
Published: December 2017

The main goal of this study was to examine the transcriptional activity of different-length β-myosin heavy chain (β-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by ∼45% relative to control. Results show that β-MHC promoter activity of all tested wild-type constructs, i.e., −3500, −408, −299, −215, −171, and −71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the −71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting βe1, βe2, GATA, and βe3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the β-MHC gene. Collectively, these results indicate that three separate regions on the β-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between −408 and −3500 bp, 2) a proximal region between −299 and −215 bp, and 3) a basal region within −71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate β-MHC transcriptional regulation in response to pressure overload.
Mouse MCT3 gene is expressed preferentially in retinal pigment and choroid plexus epithelia
Authors: Philp Nancy J. , Yoon Heeyong , Lombardi Lorraine
Published: December 2017

Monocarboxylate transporters (MCTs) are a family of highly homologous membrane proteins that mediate the 1:1 transport of a proton and a lactate ion. In chicken, MCT3 is preferentially expressed in the retinal pigment epithelium (RPE). We have isolated the mouse MCT3 cDNA and gene and characterized the pattern of tissue expression. MCT3 is a single copy gene with a 1.8-kb transcript that encodes a protein with a predicted molecular mass of 51.5 kDa. Based on Northern hybridization analysis, MCT3 transcript was expressed in only two tissues: RPE and choroid plexus epithelium (CPE). The choroid plexus forms a barrier between the cerebrospinal fluid and fenestrated capillaries, similar to the organization of the RPE and choroidal vessels. Immunohistochemical staining demonstrated that MCT3 was restricted to the basolateral membranes of both epithelia but was more abundant in RPE than CPE. Differences in the level of protein expression were confirmed by Western blot analysis. The cloning of MCT3 identifies a specific transporter that could regulate lactate levels in fluid-bathing neuronal tissues.