bluuebunny/crossref_metadata_2025 huggingface dataset:
Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress
Authors: Numazawa Satoshi , Ishikawa Makie , Yoshida Aya , Tanaka Sachiko , Yoshida Takemi
Published: May 2013

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of antioxidative proteins, including heme oxygenase-1 (HO-1). Nrf2 is sequestered in the cytoplasm by Keap1 under unstimulated conditions but translocates into the nucleus and transactivates the antioxidant responsive element (ARE) upon exposure to oxidative insults. It has recently been demonstrated that in vitro phosphorylation of Nrf2 on Ser40 by protein kinase C (PKC) facilitates the dissociation of Nrf2 from the Keap1 complex (Huang HC, Nguyen T, and Pickett CB. J Biol Chem 277: 42769–42774, 2002). The present study was designed to examine whether PKC is involved in oxidative stress-mediated nuclear translocation of Nrf2 in vivo and, if so, which PKC isoforms are involved. Induction of HO-1 gene expression by phorone, a glutathione depletor, and 4-hydroxy-2,3-nonenal (4-HNE), an end product of lipid peroxidation, was suppressed by a specific PKC inhibitor, Ro-31-8220, at concentrations that inhibit all isoforms in WI-38 cells. The induction of HO-1 was not affected by prolonged exposure of the cells to 12- O-tetradecanoylphorbol-13 acetate (TPA), suggesting that TPA-insensitive atypical PKC (aPKC) isoforms are involved. An immunocomplex kinase assay revealed that phorone and 4-HNE increased aPKCι activity. In COS-7 cells, 4-HNE induced nuclear translocation of the Nrf2-green fluorescent protein (GFP) fusion protein, but not the Nrf2(S40A)-GFP mutant. In the absence of oxidative insults, the Nrf2(S40E)-GFP mutant was distributed in the nucleus. The Nrf2-GFP accumulation in the nucleus was induced by coexpression of aPKCι, but not by a kinase inactive mutant aPKCι(K274W). The activity of an ARE-driven reporter was increased by coexpression of aPKCι, and this effect was eliminated by Ro-31-8220 in HepG2 cells. The reporter activity induced by 4-HNE was inhibited by coexpression of aPKCι(K274W). These results suggest that phosphorylation of Nrf2 Ser40 by aPKC(s) is involved in the nuclear translocation and ARE transactivation of Nrf2 by oxidative stress.
Caffeine-stimulated GTH-II release involves Ca2+stores with novel properties
Authors: Johnson James D. , Wong Calvin J. H. , Yunker Warren K. , Chang John P.
Published: May 2013

Modulation of Ca2+ stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca2+ store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca2+ imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K+ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca2+ signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid loading. Caffeine generated localized Ca2+ signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 μM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca2+ channels or sarco(endo)plasmic reticulum Ca2+-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca2+ stores found in endoplasmic reticulum.
Transcriptional regulation of IGF-I expression in skeletal muscle
Authors: McCall G. E. , Allen D. L. , Haddad F. , Baldwin K. M.
Published: May 2013

The present study investigated the role of transcription in the regulation of insulin-like growth factor (IGF)-I expression in skeletal muscle. RT-PCR was used to determine endogenous expression of IGF-I pre-mRNA and mRNA in control (Con) and functionally overloaded (FO) rat plantaris. The transcriptional activities of five different-length IGF-I promoter fragments controlling transcription of a firefly luciferase (FLuc) reporter gene were tested in vitro by transfection of myoblasts or in vivo during FO by direct gene transfer into the plantaris. Increased endogenous IGF-I gene transcription during 7 days of plantaris FO was evidenced by an ∼140-160% increase ( P < 0.0001) in IGF-I pre-mRNA (a transcriptional marker). IGF-I mRNA expression also increased by ∼90% ( P < 0.0001), and it was correlated ( R = 0.93; P < 0.0001) with the pre-mRNA increases. The three longest IGF-I exon 1 promoters induced reporter gene expression in proliferating C2C12 and L6E9 myoblasts. In differentiated L6E9 myotubes, promoter activity increased approximately two- to threefold over myoblasts. Overexpression of calcineurin and MyoD increased the activity of the -852/+192 promoter in C2C12 myotubes by ∼5- and ∼18-fold, respectively. However, FO did not induce these exogenous promoter fragments. Nevertheless, the present findings are consistent with the hypothesis that the IGF-I gene is transcriptionally regulated during muscle hypertrophy in vivo as evidenced by the induction of the endogenous IGF-I pre-mRNA during plantaris FO. The exon 1 promoter region of the IGF-I gene is sufficient to direct inducible expression in vitro; however, an in vivo response to FO may require elements outside the -852/+346 region of the exon 1 IGF-I promoter or features inherent to the endogenous IGF-I gene.
TSH signaling and cell survival in 3T3-L1 preadipocytes
Authors: Bell Andrea , Gagnon Annemarie , Dods Patti , Papineau Denise , Tiberi Mario , Sorisky Alexander
Published: May 2013

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29–76% in serum-deprived (6 h) preadipocytes treated with 1–20 μM TSH. In the presence of 20 μM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.
Effect of indomethacin on force responses and sarcoplasmic reticulum function in skinned skeletal muscle fibers and cytosolic [Ca2+] in myotubes
Authors: Han Renzhi , Suizu Takuya , Grounds Miranda D. , Bakker Anthony J.
Published: May 2013

In this study, the effects of phospholipase A2 (PLA2) inhibitors on excitation-contraction coupling (ECC) and sarcoplasmic reticulum (SR) function were examined in skinned extensor digitorum longus (EDL) muscle fibers of the rat. The nonspecific PLA2 inhibitor indomethacin (200 μM) significantly increased the peak (∼2-fold, P = 0.02) and the width (∼6-fold, P = 0.008) of depolarization-induced force responses (DIFRs) elicited in the fibers ( n = 4). Exposure of the skinned EDL fibers to indomethacin (200 μM) ( n = 7) and another PLA2 inhibitor quinacrine (200 μM) ( n = 5) resulted in the return of large DIFRs after use-dependent rundown. However, aristolochic acid (100 μM), an inhibitor of secretory PLA2, failed to return DIFRs after rundown. Indomethacin did not protect against the loss of DIFRs induced by exposure to elevated myofibrilar [Ca2+]. Indomethacin (200 μM) produced a small but significant increase in the Ca2+ sensitivity of the contractile apparatus of skinned EDL fibers and the maximum force production. Indomethacin (200 μM) also had significant effects on SR function, increasing SR Ca2+ loading in the skinned fibers (117.2 ± 3.0% of controls, P = 0.0008, n = 8) and inducing intracellular Ca2+ release in isolated intact flexor digitorum brevis (FDB) fibers ( n = 7) and C2C12 myotubes ( n = 6). These data suggest that intracellular PLA2 may be an important modulator of ECC in skeletal muscle.
Secretory modulation of basolateral membrane inwardly rectified K+ channel in guinea pig distal colonic crypts
Authors: Li Yingjun , Halm Dan R.
Published: May 2013

Cell-attached recordings revealed K+ channel activity in basolateral membranes of guinea pig distal colonic crypts. Inwardly rectified currents were apparent with a pipette solution containing 140 mM K+. Single-channel conductance (γ) was 9 pS at the resting membrane potential. Another inward rectifier with γ of 19 pS was observed occasionally. At a holding potential of −80 mV, γ was 21 and 41 pS, respectively. Identity as K+ channels was confirmed after patch excision by changing the bath ion composition. From reversal potentials, relative permeability of Na+ over K+ ( P Na/ P K) was 0.02 ± 0.02, with P Rb/ P K = 1.1 and P Cl/ P K < 0.03. Spontaneous open probability ( P o) of the 9-pS inward rectifier (gpKir) was voltage independent in cell-attached patches. Both a low ( P o = 0.09 ± 0.01) and a moderate ( P o = 0.41 ± 0.01) activity mode were observed. Excision moved gpKir to the medium activity mode; P o ofgpKir was independent of bath Ca2+activity and bath acidification. Addition of Cl− and K+ secretagogues altered P o ofgpKir. Forskolin or carbachol (10 μM) activated the small-conductance gpKir in quiescent patches and increased P o in low-activity patches. K+ secretagogues, either epinephrine (5 μM) or prostaglandin E2 (100 nM), decreased P o of gpKir in active patches. This gpKir may be involved in electrogenic secretion of Cl− and K+ across the colonic epithelium, which requires a large basolateral membrane K+ conductance during maximal Cl− secretion and, presumably, a lower K+ conductance during primary electrogenic K+ secretion.
Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally
Authors: Chun Lois G. , Ward Christopher W. , Schneider Martin F.
Published: May 2013

“Spontaneous” Ca2+ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca2+ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca2+ indicator dye fluo 4 to show that the frequency of spontaneous Ca2+ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca2+ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca2+ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl2 (5 mM) or nifedipine (30 μM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca2+ sparks may be triggered by locally elevated [Ca2+] due to Ca2+ influx via dihydropyridine receptor L-type Ca2+ channels in embryonic mammalian skeletal muscle.
Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line
Authors: Sautin Yuri Y. , Crawford James M. , Svetlov Stanislav I.
Published: May 2013

First published September 5, 2001; 10.1152/ajpcell.00077.2001.—Protective mechanisms for lysophosphatidic acid (LPA) against cell death caused by Clostridium difficile toxin, or tumor necrosis factor-α (TNF-α) plus d-galactosamine, were investigated in a murine hepatocyte cell line AML12 expressing Edg2 LPA receptor. In these models of hepatocellular injury, LPA prevented hepatocyte damage, suppressed apoptosis, and enhanced cell survival in a dose-dependent fashion. The protective effects of LPA were abolished by wortmannin and LY-294002, specific inhibitors of phosphatidylinositol 3-phosphate kinase (PI 3-kinase), and by PD-98059 and U-0126, inhibitors of MEK1/MEK2. In nontreated hepatocytes, LPA elicited a gradual and sustained increase in phosphorylation of Erk1/Erk2 over 180 min of stimulation and downstream phosphorylation of p90RSK and transcription factor Elk-1. In C. difficile toxin-treated cells, LPA-induced phosphorylation of Erk1/Erk2 was rapid but transient, while p90RSK and Elk-1 phosphorylation did not change significantly. LPA stimulated phosphorylation of Akt in a time-dependent manner in both intact and toxin-treated AML12 hepatocytes. Wortmannin and LY-294002 abolished phosphorylation of Akt, further supporting activation of PI 3-kinase/Akt as a signaling pathway, which mediates hepatocyte protection by LPA. Taken together, these results demonstrate that LPA prevents cell apoptosis induced by C. difficile toxin and TNF-α/d-galactosamine in the AML12 murine hepatocyte cell line. Cell protection by LPA involves activation of the mitogen-activated protein kinase Erk1/Erk2 cascade and PI 3-kinase-dependent phosphorylation of Akt.
Potential role for a novel AP180-related protein during endocytosis in MDCK cells
Authors: Kusner Linda , Carlin Cathleen
Published: May 2013

Clathrin assembly protein, AP180, was originally identified as a brain-specific protein localized to the presynaptic junction. AP180 acts to limit vesicle size and maintain a pool of releasable synaptic vesicles during rapid recycling. In this study, we show that polarized epithelial Madin-Darby canine kidney (MDCK) cells express two AP180-related proteins: the ubiquitously expressed 62-kDa clathrin assembly lymphoid myeloid leukemia (CALM, AP180-2) protein and a novel high-molecular-weight homolog that we have named AP180-3. Sequence analysis of AP180-3 expressed in MDCK cells shows high homology to AP180 from rat brain. AP180-3 contains conserved motifs found in brain-specific AP180, including the epsin NH2-terminal homology (ENTH) domain, the binding site for the α-subunit of AP-2, and DLL repeats. Our studies show that AP180-3 from MDCK cells forms complexes with AP-2 and clathrin and that membrane recruitment of these complexes is modulated by phosphorylation. We demonstrate by immunohistochemistry that AP180-3 is localized to cytoplasmic vesicles in MDCK cells and is also present in tubule epithelial cells from mouse kidney. We observed by immunodetection that a high-molecular-weight AP180-related protein is expressed in numerous cells in addition to MDCK cells.
Neurotrophic factors enhance the survival of muscle fibers in EDL, but not SOL, after neonatal nerve injury
Authors: Mousavi Kambiz , Miranda Wilson , Parry David J.
Published: May 2013

Neonatal sciatic nerve crush results in a sustained reduction of the mass of both extensor digitorum longus (EDL) and soleus (SOL) muscles in the rat. Type IIB fibers are selectively lost from EDL. We have investigated the effects of ciliary neurotrophic factor (CNTF) combined with neurotrophin (NT)-3 or NT-4 on muscle mass, as well as the number, cross-sectional area, and distribution of muscle fiber types and the number of motor neurons innervating EDL and SOL 3 mo after transient axotomy 5 days after birth. Both NT treatments prevented the axotomy-induced loss of muscle mass in both EDL and SOL and of total number of muscle fibers in EDL but not in SOL. Although IIB fiber loss was not prevented, both NT treatments resulted in altered fiber type distribution. Both NT combinations also reduced the loss of EDL motor neurons. These data suggest that a differential distribution of NT receptors on either motor neurons or muscle fibers may lead to different levels of susceptibility to neonatal axotomy.
Four novel splice variants of sulfonylurea receptor 1
Authors: Hambrock Annette , Preisig-Müller Regina , Russ Ulrich , Piehl Anke , Hanley Peter J. , Ray John , Daut Jürgen , Quast Ulrich , Derst Christian
Published: May 2013

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.
Circadian rhythms in surface molecules of rat blood lymphocytes
Authors: Pelegrı́ Carme , Vilaplana Jordi , Castellote Cristina , Rabanal Manel , Franch Àngels , Castell Margarida
Published: May 2013

The present article examines whether the expression of certain surface molecules that trigger immune responses shows a circadian rhythm. We also analyzed the rhythms in the number and percentage of lymphocyte subpopulations, in the leukocyte differential counts, and in the total red and white blood cell counts. Blood samples obtained from rats at 2-h intervals for 24 h were stained with several mouse monoclonal antibodies directed against lymphocyte surface molecules and processed by flow cytometry. The number of B, total T, Tγδ, Th, and Ts/c cells followed a 24-h rhythm with a peak in the first half of the resting period. The expression of CD45, CD5, CD3, and CD4 followed a circadian rhythm. Their acrophases suggested temporal association between CD45 and CD5 at the end of the active phase and between CD4 and CD3 at the beginning of this phase. This temporal organization could have an important role for immune cell function.
Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation
Authors: Zhou Rihong , Watson Charles , Fu Chuanhai , Yao Xuebiao , Forte John G.
Published: May 2013

Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1 H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation.
Cellular distributions of creatine kinase in branchia of euryhaline tilapia (Oreochromis mossambicus)
Authors: Lin Li-Yih , Chiang Chia-Chang , Gong Hong-Yi , Cheng Ching-Yi , Hwang Pung-Pung , Weng Ching-Feng
Published: May 2013

Although euryhaline teleosts can adapt to environmental fluctuation of salinity, their energy source for responding to changes in salinity and osmolarity remains unclear. This study examines the cellular localization of creatine kinase (CK) expression in branchia of tilapia ( Oreochromis mossambicus). Western blot analysis of muscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted to seawater did not change. With the use of immunocytochemistry, three CK isoforms (MM, MB, and BB) were localized in mitochondria-rich (MR) cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To our knowledge, this work presents the first evidence of CK expression in MR cells of tilapia gills, highlighting the potential role of CK in providing energy for ion transport.
Endothelial cells maintain a reduced redox environment even as mitochondrial function declines
Authors: Carlisle Ricarda , Rhoads Carol Ann , Aw Tak Yee , Harrison Lynn
Published: May 2013

Human umbilical vein endothelial cells (HUVECs) are an endothelial model of replicative senescence. Oxidative stress, possibly due to dysfunctional mitochondria, is believed to play a key role in replicative senescence and atherosclerosis, an age-related vascular disease. In this study, we determined the effect of cell division on genomic instability, mitochondrial function, and redox status in HUVECs that were able to replicate for ∼60 cumulative population doublings (CPD). After 20 CPD, the nuclear genome deteriorated and the protein content of the cell population increased. This indicated an increase in cell size, which was accompanied by an increase in oxygen consumption, ATP production, and mitochondrial genome copy number and ∼10% increase in mitochondrial mass. The antioxidant capacity increased, as seen by an increase in reduced glutathione, glutathione peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase. However, by CPD 52, the latter two enzymes decreased, as well as the ratio of mitochondrial-to-nuclear genome copies, the mitochondrial mass, and the oxygen consumption per milligram of protein. Our results signify that HUVECs maintain a highly reducing (GSH) environment as they replicate despite genomic instability and loss of mitochondrial function.
Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse
Authors: Wade James B. , Liu Jie , Coleman Richard A. , Cunningham Rochelle , Steplock Deborah A. , Lee-Kwon Whaseon , Pallone Thomas L. , Shenolikar Shirish , Weinman Edward J.
Published: May 2013

In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.
Structure-function correlation in airway smooth muscle adapted to different lengths
Authors: Kuo Kuo-Hsing , Herrera Ana M. , Wang Lu , Paré Peter D. , Ford Lincoln E. , Stephens Newman L. , Seow Chun Y.
Published: May 2013

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% ± 5.7 (SE) and 76.0% ± 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% ± 6.7, 34.6% ± 3.4, and 35.6% ± 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length.
USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells
Authors: Gupta Ashish K. , Kone Bruce C.
Published: May 2013

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.
A proteasome inhibitor reduces concurrent, sequential, and long-term IL-1β- and TNF-α-induced ECAM expression and adhesion
Authors: Dagia Nilesh M. , Goetz Douglas J.
Published: May 2013

A promising approach for reducing aberrant leukocyte-endothelial adhesion during pathological inflammation is to inhibit endothelial cell adhesion molecule (ECAM) expression at the transcription level. Several compounds have been shown to decrease cytokine-induced upregulation of ECAMs primarily by modulating the activity of transcription factors [e.g., nuclear factor-κB (NF-κB)]. The majority of the in vitro studies have focused on the effect of transcription inhibitors on endothelial cells exposed to a single cytokine [primarily tumor necrosis factor-α (TNF-α)] for a relatively short period of time (primarily 4-6 h). However, in the in vivo setting, multiple cytokines [e.g., interleukin-1β (IL-1β) and TNF-α] may be present for extended periods of time. Thus we studied the effects of a transcription inhibitor, the proteasome inhibitor lactacystin, on ECAM expression and myeloid (HL60) cell adhesion to human umbilical vein endothelial cells (HUVEC) activated by concurrent, sequential, and long-term (24 h) treatment with IL-1β and TNF-α. We show, for the first time, that lactacystin inhibits 1) 4-h concurrent IL-1β- and TNF-α-induced expression of E-selectin, VCAM-1, ICAM-1, and HL60 cell adhesion to HUVEC; 2) 4-h TNF-α-induced expression of E-selectin, VCAM-1, and HL60 cell adhesion to HUVEC that have become desensitized to IL-1β activation; 3) 24-h TNF-α-induced expression of E-selectin and VCAM-1 but not ICAM-1; and 4) 24-h TNF-α-induced HL60 cell adhesion to HUVEC. Combined, our results demonstrate that a proteasome inhibitor can reduce concurrent, sequential, and long-term IL-1β- and TNF-α-induced ECAM expression and myeloid cell adhesion.
Fluid shear- and time-dependent modulation of molecular interactions between PMNs and colon carcinomas
Authors: Jadhav Sameer , Konstantopoulos Konstantinos
Published: May 2013

This study compares the effects of fluid shear on the kinetics, adhesion efficiency, stability, and molecular requirements of polymorphonuclear leukocyte (PMN) binding to two colon adenocarcinoma cell-lines, the CD54-negative/sLex-bearing LS174T cells and the CD54-expressing/sLex-low HCT-8 cells. The efficiency of PMN-colon carcinoma heteroaggregation decreases with increasing shear, with PMNs binding HCT-8 more efficiently than LS174T cells at low shear (50–200 s−1). In the low shear regime, CD11b is sufficient to mediate PMN binding to LS174T cells. In contrast, both CD11a and CD11b contribute to PMN-HCT-8 heteroaggregation, with CD54 on HCT-8 cells acting as a CD11a ligand at early time points. At high shear, only PMN-LS174T heteroaggregation occurs, which is initiated by PMN L-selectin binding to a sialylated, O-linked, protease-sensitive ligand on LS174T cells. PMN-LS174T heteroaggregation is primarily dependent on the intercellular contact duration (or shear rate), whereas PMN-HCT-8 binding is a function of both the intercellular contact duration and the applied force (or shear stress). Cumulatively, these studies suggest that fluid shear modulates the kinetics and molecular mechanisms of PMN-colon carcinoma cell aggregation.
Reactive species mechanisms of cellular hypoxia-reoxygenation injury
Authors: Li Chuanyu , Jackson Robert M.
Published: May 2013

Exacerbation of hypoxic injury after restoration of oxygenation (reoxygenation) is an important mechanism of cellular injury in transplantation and in myocardial, hepatic, intestinal, cerebral, renal, and other ischemic syndromes. Cellular hypoxia and reoxygenation are two essential elements of ischemia-reperfusion injury. Activated neutrophils contribute to vascular reperfusion injury, yet posthypoxic cellular injury occurs in the absence of inflammatory cells through mechanisms involving reactive oxygen (ROS) or nitrogen species (RNS). Xanthine oxidase (XO) produces ROS in some reoxygenated cells, but other intracellular sources of ROS are abundant, and XO is not required for reoxygenation injury. Hypoxic or reoxygenated mitochondria may produce excess superoxide (O[Formula: see text]) and release H2O2, a diffusible long-lived oxidant that can activate signaling pathways or react vicinally with proteins and lipid membranes. This review focuses on the specific roles of ROS and RNS in the cellular response to hypoxia and subsequent cytolytic injury during reoxygenation.
Allosteric modulation of a neuronal K+ channel by 1-alkanols is linked to a key residue in the activation gate
Authors: Harris Thanawath , Graber Andrew R. , Covarrubias Manuel
Published: May 2013

The selective inhibition of neuronal Shaw2 K+ channels by 1-alkanols is conferred by the internal S4-S5 loop, a region that also contributes to the gating of voltage-gated K+ channels. Here, we applied alanine scanning mutagenesis to examine the contribution of the S5 and S6 segments to the allosteric modulation of Shaw2 K+ channels by 1-alkanols. The internal section of S6 is the main activation gate of K+ channels. While several mutations in S5 and S6 modulated the inhibition of the channels by 1-butanol and others had no effect, a single mutation at a key site in S6 (P410A) converted this inhibition into a dramatic dose-dependent potentiation (∼2-fold at 15 mM and ∼6-fold at 50 mM). P410 is the second proline in the highly conserved PVP motif that may cause a significant α-helix kink. The P410A currents in the presence of 1-butanol also exhibited novel kinetics (faster activation and slow inactivation). Internal application of 15 mM 1-butanol to inside-out patches expressing P410A did not significantly affect the mean unitary currents (∼2 pA at 0 mV) or the mean open time (5-6 ms) but clearly increased the opening frequency and open probability (∼2- to 4-fold). All effects displayed a fast onset and were fully reversible upon washout. The results suggest that the allosteric modulation of the Shaw2 K+ channel by 1-alkanols depends on a critical link between the PVP motif and activation gating. This study establishes the Shaw2 K+ channel as a robust model to investigate the mechanisms of alcohol intoxication and general anesthesia.
Gα13-mediated transformation and apoptosis are permissively dependent on basal ERK activity
Authors: Adarichev Vyacheslav A. , Vaiskunaite Rita , Niu Jiaxin , Balyasnikova Irina V. , Voyno-Yasenetskaya Tatyana A.
Published: May 2013

We previously reported that the α-subunit of heterotrimeric G13 protein induces either mitogenesis and neoplastic transformation or apoptosis in a cell-dependent manner. Here, we analyzed which signaling pathways are required for Gα13-induced mitogenesis or apoptosis using a novel mutant of Gα13. We have identified that in human cell line LoVo, the mutation encoding substitution of Arg260 to stop codon in mRNA of Gα13 subunit produced a mutant protein (Gα13-T) that lacks a COOH terminus and is endogenously expressed in LoVo cells as a polypeptide of 30 kDa. We found that Gα13-T lost its ability to promote proliferation and transformation but retained its ability to induce apoptosis. We found that full-length Gα13 could stimulate Elk1 transcription factor, whereas truncated Gα13 lost this ability. Gα13-dependent stimulation of Elk1 was inhibited by dominant-negative extracellular signal-regulated kinase (MEK) but not by dominant-negative MEKK1. Similarly, MEK inhibitor PD-98059 blocked Gα13-induced Elk1 stimulation, whereas JNK inhibitor SB-203580 was ineffective. In Rat-1 fibroblasts, Gα13-induced cell proliferation and foci formation were also inhibited by dominant-negative MEK and PD-98059 but not by dominant-negative MEKK1 and SB-203580. Whereas Gα13-T alone did not induce transformation, coexpression with constitutively active MEK partially restored its ability to transform Rat-1 cells. Importantly, full-length but not Gα13-T could stimulate Src kinase activity. Moreover, Gα13-dependent stimulation of Elk1, cell proliferation, and foci formation were inhibited by tyrosine kinase inhibitor, genistein, or by dominant-negative Src kinase, suggesting the involvement of a Src-dependent pathway in the Gα13-mediated cell proliferation and transformation. Importantly, truncated Gα13 retained its ability to stimulate apoptosis signal-regulated kinase ASK1 and c-Jun terminal kinase, JNK. Interestingly, the apoptosis induced by Gα13-T was inhibited by dominant-negative ASK1 or by SB-203580.
Intrahepatic bile ducts transport water in response to absorbed glucose
Authors: Masyuk Anatoly I. , Masyuk Tatyana V. , Tietz Pamela S. , Splinter Patrick L. , LaRusso Nicholas F.
Published: May 2013

The physiological relevance of the absorption of glucose from bile by cholangiocytes remains unclear. The aim of this study was to test the hypothesis that absorbed glucose drives aquaporin (AQP)-mediated water transport by biliary epithelia and is thus involved in ductal bile formation. Glucose absorption and water transport by biliary epithelia were studied in vitro by microperfusing intrahepatic bile duct units (IBDUs) isolated from rat liver. In a separate set of in vivo experiments, bile flow and absorption of biliary glucose were measured after intraportal infusion of d-glucose or phlorizin. IBDUs absorbedd-glucose in a dose- and phlorizin-dependent manner with an absorption maximum of 92.8 ± 6.2 pmol · min−1 · mm−1. Absorption of d-glucose by microperfused IBDUs resulted in an increase of water absorption ( J v = 3−10 nl · min−1 · mm−1, P f = 40 × 10−3 cm/sec). Glucose-driven water absorption by IBDUs was inhibited by HgCl2, suggesting that water passively follows absorbed d-glucose mainly transcellularly via mercury-sensitive AQPs. In vivo studies showed that as the amount of absorbed biliary glucose increased after intraportal infusion ofd-glucose, bile flow decreased. In contrast, as the absorption of biliary glucose decreased after phlorizin, bile flow increased. Results support the hypothesis that the physiological significance of the absorption of biliary glucose by cholangiocytes is likely related to regulation of ductal bile formation.
Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions
Authors: Zahradníková A. , Dura M. , Györke I. , Escobar A. L. , Zahradník I. , Györke S.
Published: May 2013

Mg2+, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca2+ release channels by competing with Ca2+ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca2+-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca2+ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca2+ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg2+ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca2+. Mg2+ dramatically slowed the rate of activation of RyRs in response to sustained (≥10-ms) elevations in Ca2+ concentration. Paradoxically, Mg2+ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca2+ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca2+ did not affect the peak open probability or the probability of responses to brief Ca2+ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg2+ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca2+ channels and RyRs in heart cells.
Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila
Authors: Broderick Kate E. , MacPherson Matthew R. , Regulski Michael , Tully Tim , Dow Julian A. T. , Davies Shireen A.
Published: May 2013

Signaling by nitric oxide (NO) and guanosine 3′,5′-cyclic monophosphate (cGMP) modulates fluid transport in Drosophila melanogaster. Expression of an inducible transgene encoding Drosophila NO synthase ( dNOS) increases both NOS activity in Malpighian (renal) tubules and DNOS protein in both type I (principal) and type II (stellate) cells. However, cGMP content is increased only in principal cells. DNOS overexpression results in elevated basal rates of fluid transport in the presence of the phosphodiesterase (PDE) inhibitor, Zaprinast. Direct assay of tubule cGMP-hydrolyzing phosphodiesterase (cG-PDE) activity in wild-type and dNOS transgenic lines shows that cG-PDE activity is Zaprinast sensitive and is elevated upon dNOS induction. Zaprinast treatment increases cGMP content in tubules, particularly at the apical regions of principal cells, suggesting localization of Zaprinast-sensitive cG-PDE to these areas. Potential cross talk between activated NO/cGMP and calcium signaling was assessed in vivo with a targeted aequorin transgene. Activated DNOS signaling alone does not modify either neuropeptide (CAP2b)- or cGMP-induced increases in cytosolic calcium levels. However, in the presence of Zaprinast, both CAP2b-and cGMP-stimulated calcium levels are potentiated upon DNOS overexpression. Use of the calcium channel blocker, verapamil, abolishes the Zaprinast-induced transport phenotype in dNOS-overexpressing tubules. Molecular genetic intervention in the NO/cGMP signaling pathway has uncovered a pivotal role for cell-specific cG-PDE in regulating the poise of the fluid transporting Malpighian tubule via direct effects on intracellular cGMP concentration and localization and via interactions with calcium signaling mechanisms.
PAR1-dependent and independent increases in COX-2 and PGE2 in human colonic myofibroblasts stimulated by thrombin
Authors: Seymour Michelle L. , Zaidi Nosheen F. , Hollenberg Morley D. , MacNaughton Wallace K.
Published: May 2013

Subepithelial myofibroblast-derived prostaglandin E2 (PGE2) regulates epithelial chloride secretion in the intestine. Thrombin is elevated in inflammatory conditions of the bowel. Therefore, we sought to determine a role for thrombin in regulating PGE2 synthesis by colonic myofibroblasts. Incubation of cultured CCD-18Co colonic myofibroblasts with thrombin, the proteinase-activated receptor 1 (PAR1)-activating peptide (Cit-NH2), and peptides corresponding to 2 noncatalytic regions of thrombin (TP367 and TP508) for 18 h increased both cyclooxygenase (COX)-2 expression (immunocytochemistry) and PGE2 synthesis (enzyme immunoassay). Inhibition of thrombin by D-Phe-Pro-Arg-chloromethylketone (PPACK) did not significantly reduce PGE2 synthesis, which remained elevated compared with control. We also investigated the basic fibroblast growth factor (bFGF) dependence of thrombin-induced PGE2 elevations. Recombinant human bFGF concentration dependently increased PGE2synthesis, and a bFGF neutralizing antibody inhibited PGE2synthesis induced by TP367 and TP508 (∼40%) and by thrombin (∼20%) (but not Cit-NH2). Thrombin, therefore, upregulates COX-2-derived PGE2 synthesis by both catalytic cleavage of PAR1 and bFGF-dependent noncatalytic activity. This presents a novel mechanism by which intestinal myofibroblasts might regulate epithelial chloride secretion.
Role of abnormal neutral endopeptidase-like activities inHyp mouse bone cells in renal phosphate transport
Authors: Dubois Stéphanie G. , Ruchon Andréa Frota , Delalandre Aline , Boileau Guy , Lajeunesse Daniel
Published: May 2013

We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) in the Hyp mouse affects the expression and activity of neprilysin (NEP) and of endothelin-converting enzyme-like endopeptidase (ECEL1/DINE) in bone marrow stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal. Conditioned media (CM) from Hyp BMSC and Ob inhibited inorganic phosphate (Pi) uptake by mouse proximal tubule cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal Pi uptake. A linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of Piuptake. NEP and ECEL1/DINE mRNA levels were higher in Hypcells than in normal cells, and in situ hybridization of ECEL1/DINE confirmed higher levels of expression in the Hyp mouse than in normal cells. In conclusion, we observed a correlation between the inhibition of Pi uptake by CM from Hyp cells and elevated NEP-like activities.
Lack of both oxygen and glucose contributes to I/R-induced changes in cardiac SR function
Authors: Temsah Rana M. , Netticadan Thomas , Kawabata Ken-Ichi , Dhalla Naranjan S.
Published: May 2013

Although ischemia-reperfusion (I/R) has been shown to depress cardiac performance and sarcoplasmic reticulum (SR) function, the mechanisms underlying these alterations are poorly understood. Because lack of oxygen and substrate deprivation are known to occur during the ischemic phase, we examined the effects of reperfusion on cardiac performance and SR function in hearts subjected to hypoxia and substrate lack. For this purpose, isolated rat hearts were perfused with hypoxic and/or glucose-free medium for 30 min and then reperfused with normal medium for 1 h; the SR vesicles were isolated for studying the Ca2+-transport activities. Reperfusion with normal medium of hearts deprived of oxygen or glucose showed no changes in cardiac performance and SR function. However, reperfusion of hearts perfused with hypoxic glucose-free medium showed ∼45% decrease in cardiac contractile activities as well as 23 and 64% reduction in SR Ca2+-uptake and Ca2+-release activities, respectively, without any change in the level of SR Ca2+-cycling proteins. Depressed SR function in these hearts was associated with a reduction in Ca2+/calmodulin-dependent protein kinase (CaMK) phosphorylation of the SR Ca2+-cycling proteins and 34% decrease in SR CaMK activity. These changes in cardiac performance, SR function, and SR CaMK activity in the hypoxic, glucose-deprived, reperfused hearts were similar to those observed in hearts subjected to 30 min of global ischemia and 60 min of reperfusion. The results therefore suggest that the lack of both oxygen and substrate during the ischemic phase may contribute to the I/R-induced alterations in cardiac performance and SR function. Furthermore, these abnormalities were associated with reduced SR CaMK activity.