bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelin-B receptors activate Gα13
Authors: Kitamura Kenichiro , Shiraishi Naoki , Singer William D. , Handlogten Mary E. , Tomita Kimio , Miller R. Tyler
Published: December 2017

Endothelin (ET) receptors activate heterotrimeric G proteins that are members of the Gi, Gq, and Gs families but may also activate members of other families such as Gα12/13. Gα13 has multiple complex cellular effects that are similar to those of ET. We studied the ability of ET receptors to activate Gα13 using an assay for G protein α-chain activation that is based on the fact that an activated (GTP-bound) α-chain is resistant to trypsinization compared with an inactive (GDP-bound) α-chain. Nonhydrolyzable guanine nucleotides and AlMgF protected Gα13 from degradation by trypsin. In membranes from human embryonic kidney 293 cells that coexpress ETB receptors and α13, ET-3 and 5′-guanylylimidodiphosphate [Gpp(NH)p] increased the protection of α13 compared with Gpp(NH)p alone. The specificity of ETBreceptor-α13 coupling was documented by showing that β2receptors and isoproterenol or ETAreceptors and ET-1 did not activate α13 and that a specific antagonist for ETB receptors blocked ET-3-dependent activation of α13.
Cloning and expression of the Na+/H+exchanger from Amphiuma RBCs: resemblance to mammalian NHE1
Authors: McLean Lee Anne , Zia Shaheen , Gorin Fredric A. , Cala Peter M.
Published: December 2017

The cDNA encoding the Na+/H+exchanger (NHE) from Amphiumaerythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89% similarity at the amino acid level. Sequence comparisons with other NHEs indicate that the Amphiuma tridactylum NHE isoform 1 (atNHE1) is likely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1 protein, when stably transfected into the NHE-deficient AP-1 cell line (37), demonstrates robust Na+-dependent proton transport that is sensitive to amiloride but not to the potent NHE1 inhibitor HOE-694. Interestingly, chimeric NHE proteins constructed by exchanging the amino and carboxy termini between atNHE1 and hNHE1 exhibited drug sensitivities similar to atNHE1. Based on kinetic, sequence, and functional similarities between atNHE1 and mammalian NHE1, we propose that the Amphiuma exchanger should prove to be a valuable model for studying the control of pH and volume regulation of mammalian NHE1. However, low sensitivity of atNHE1 to the NHE inhibitor HOE-694 in both native Amphiuma red blood cells (RBCs) and in transfected mammalian cells distinguishes this transporter from its mammalian homologue.
Effect of β-adrenoceptor activation on [Ca2+]i regulation in murine skeletal myotubes
Authors: Prakash Y. S. , van der Heijden H. F. M. , Gallant E. M. , Sieck G. C.
Published: December 2017

The present study used real-time confocal microscopy to examine the effects of the β2-adrenoceptor agonist salbutamol on regulation of intracellular Ca2+ concentration ([Ca2+]i) in myotubes derived from neonatal mouse limb muscles. Immunocytochemical staining for ryanodine receptors and skeletal muscle myosin confirmed the presence of sarcomeres. The myotubes displayed both spontaneous and ACh-induced rapid (<2-ms rise time) [Ca2+]itransients. The [Ca2+]itransients were frequency modulated by both low and high concentrations of salbutamol. Exposure to α-bungarotoxin and tetrodotoxin inhibited ACh-induced [Ca2+]itransients and the response to low concentrations of salbutamol but not the response to higher concentrations. Preexposure to caffeine inhibited the subsequent [Ca2+]iresponse to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the [Ca2+]iresponse to caffeine. Inhibition of dihydropyridine-sensitive Ca2+ channels with nifedipine or PN-200-110 did not prevent [Ca2+]ielevations induced by higher concentrations of salbutamol. The effects of salbutamol were mimicked by the membrane-permeant analog dibutyryl adenosine 3′,5′-cyclic monophosphate. These data indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulum Ca2+ release.
PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells
Authors: Vayro Steven , Silverman Mel
Published: December 2017

We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose transporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), caused a significant decrease (38.03 ± 0.05%) in methyl α-d-glucopyranoside transport activity that could not be emulated by 4α-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC inhibitor bisindolylmaleimide I. The maximal rate of Na+-glucose cotransport activity ( V max) was decreased from 1.29 ± 0.09 to 0.85 ± 0.04 nmol ⋅ min−1 ⋅ mg protein−1 after PMA exposure. However, measurement of high-affinity Na+-dependent phloridzin binding revealed that there was no difference in the number of cell surface transporters after PMA treatment; maximal binding capacities were 1.54 ± 0.34 and 1.64 ± 0.21 pmol/mg protein for untreated and treated cells, respectively. The apparent sugar binding affinity (Michaelis-Menten constant) and phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced V max without affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrier, resulting in a lowering of the transporter turnover rate by a factor of two.
An E-box within the MHC IIB gene is bound by MyoD and is required for gene expression in fast muscle
Authors: Wheeler Matthew T. , Snyder Emily C. , Patterson Melissa N. , Swoap Steven J.
Published: December 2017

The myosin heavy chain (MHC) IIB gene is selectively expressed in skeletal muscles, imparting fast contractile kinetics. Why the MHC IIB gene product is expressed in muscles like the tibialis anterior (TA) and not expressed in muscles like the soleus is currently unclear. It is shown here that the mutation of an E-box within the MHC IIB promoter decreased reporter gene activity in the fast-twitch TA muscle 90-fold as compared with the wild-type promoter. Reporter gene expression within the TA required this E-box for activation of a heterologous construct containing upstream regulatory regions of the MHC IIB promoter linked to the basal 70-kDa heat shock protein TATA promoter. Electrophoretic mobility shift assays demonstrated that mutation of the E-box prevented the binding of both MyoD and myogenin to this element. In cotransfected C2C12myotubes and Hep G2 cells, MyoD preferentially activated the MHC IIB promoter in an E-box-dependent manner, whereas myogenin activated the MHC IIB promoter to a lesser extent, and in an E-box-independent manner. A time course analysis of hindlimb suspension demonstrated that the unweighted soleus muscle activated expression of MyoD mRNA before the de novo expression of MHC IIB mRNA. These data suggest a possible causative role for MyoD in the observed upregulation of MHC IIB in the unweighted soleus muscle.
Importance of glucose-6-phosphate dehydrogenase activity in cell death
Authors: Tian Wang-Ni , Braunstein Leigh D. , Apse Kira , Pang Jiongdong , Rose Mark , Tian Xiaoni , Stanton Robert C.
Published: December 2017

The intracellular redox potential plays an important role in cell survival. The principal intracellular reductant NADPH is mainly produced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by 6-phosphogluconate dehydrogenase. Considering the importance of NADPH, we hypothesized that G6PDH plays a critical role in cell death. Our results show that 1) G6PDH inhibitors potentiated H2O2-induced cell death; 2) overexpression of G6PDH increased resistance to H2O2-induced cell death; 3) serum deprivation, a stimulator of cell death, was associated with decreased G6PDH activity and resulted in elevated reactive oxygen species (ROS); 4) additions of substrates for G6PDH to serum-deprived cells almost completely abrogated the serum deprivation-induced rise in ROS; 5) consequences of G6PDH inhibition included a significant increase in apoptosis, loss of protein thiols, and degradation of G6PDH; and 6) G6PDH inhibition caused changes in mitogen-activated protein kinase phosphorylation that were similar to the changes seen with H2O2. We conclude that G6PDH plays a critical role in cell death by affecting the redox potential.
Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation
Authors: Tian Jean Q. , Quaroni Andrea
Published: December 2017

Using the conditionally immortalized human cell line tsFHI, we have investigated the role of cyclin-dependent kinase inhibitors (CKIs) in intestinal epithelial cell differentiation. Expression of cyclins, cyclin-dependent kinases (Cdk), and CKIs was examined under conditions promoting growth, growth arrest, or expression of differentiated traits. Formation of complexes among cell cycle regulatory proteins and their kinase activities were also investigated. The tsFHI cells express three CKIs: p16, p21, and p27. With differentiation, p21 and p27 were strongly induced, but with different kinetics: the p21 increase was rapid but transient and the p27 increase was delayed but sustained. Our results suggest that the function of p16 is primarily to inhibit cyclin D-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 for pRb phosphorylation and G1/S progression. Furthermore, they indicate that p21 is the main CKI involved in irreversible growth arrest during the early stages of cell differentiation in association with D-type cyclins, cyclin E, and Cdk2, whereas p27 may induce or stabilize expression of differentiated traits acting independently of cyclin-Cdk function.
Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland
Authors: Gruber Achim D. , Schreur Kevin D. , Ji Hong-Long , Fuller Catherine M. , Pauli Bendicht U.
Published: December 2017

The CLCA family of Ca2+-activated Cl− channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.
Nitric oxide modulation of focal adhesions in endothelial cells
Authors: Goligorsky Michael S. , Abedi Husna , Noiri Eisei , Takhtajan Alice , Lense Sheri , Romanov Victor , Zachary Ian
Published: December 2017

A permissive role of nitric oxide (NO) in endothelial cell migration and angiogenesis promoted by vascular endothelial growth factor (VEGF), endothelin, and substance P has previously been established. The present studies were designed to examine the mechanism(s) involved in the NO effect on focal adhesions. Time-lapse videomicroscopy of human umbilical vein endothelial cells (HUVECs) plated on the silicone rubber substrate revealed that unstimulated cells were constantly remodeling the wrinkling pattern, indicative of changing tractional forces. Application of NO donors reversibly decreased the degree of wrinkling, consistent with the release of tractional forces exerted by focal adhesions and stress fibers. Morphometric and immunocytochemical analyses showed that NO inhibited adhesion and spreading of HUVECs and attenuated recruitment of paxillin to focal adhesions. NO also had a profound dose-dependent effect on the formation of stress fibers by HUVECs. De novo formation of focal adhesions in HUVECs was significantly diminished in the presence of NO donors. Migration of HUVECs showed an absolute requirement for the functional NO synthase. NO donors did not interfere with focal adhesion kinase recruitment to focal adhesions but affected the state of its tyrosine phosphorylation, as judged from the results of immunoprecipitation and immunoblotting experiments. Videomicroscopy of HUVECs presented with VEGF in a micropipette showed that the rate of cell migration was slowed down by NO synthase inhibition as well as by inhibition of tyrosine phosphorylation. Collectively, these data indicate that NO reversibly releases tractional forces exerted by spreading endothelial cells via interference with the de novo formation of focal adhesions, tyrosine phosphorylation of components of focal adhesion complexes, and assembly of stress fibers.
Choline uptake across the ventricular membrane of neonate rat choroid plexus
Authors: Villalobos Alice R. , Parmelee Judith T. , Renfro J. Larry
Published: December 2017

The uptake of [3H]choline from the cerebrospinal fluid (CSF) side of the rat neonatal choroid plexus was characterized in primary cultures of the choroidal epithelium grown on solid supports. Cell-to-medium concentration ratios were ∼5 at 1 min and as high as 70 at 30 min. Apical choline uptake was facilitated; the K m was ∼50 μM. Several organic cations (e.g., hemicholinium-3 and N 1-methylnicotinamide) inhibited uptake. The reduction or removal of external Na+ or the addition of 5 mM LiCl had no effect on uptake. However, increasing external K+ concentration from 3 to 30 mM depolarized ventricular membrane potential (−70 to −15 mV) and reduced uptake to 45% of that for the control. Treatment with 1 mM ouabain or 2 mM BaCl2 reduced uptake 45%, and intracellular acidification reduced uptake to ∼90% of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled to Na+ influx but is sensitive to plasma membrane electrical potential.
Effect of subunit composition and Liddle’s syndrome mutations on biosynthesis of ENaC
Authors: Prince Lawrence S. , Welsh Michael J.
Published: December 2017

The epithelial Na+ channel (ENaC) is comprised of three homologous subunits: α, β, and γ, all of which are required for formation of the fully functional channel. This channel is responsible for salt reabsorption in the kidney, the airway, and the large bowel. Mutations in ENaC can cause human disease by increasing channel function in Liddle’s syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We previously showed that ENaC is expressed on the cell surface as a minimally glycosylated, Triton-insoluble protein. In the present study we found that ENaC existed initially as a Triton-soluble protein that contained high-mannose glycosylation, presumably in the endoplasmic reticulum. This form of the protein disappeared as the Triton-insoluble, minimally glycosylated form became the more prevalent species. In pulse-chase studies of individually expressed subunits, we found that the Triton-soluble form of β-ENaC accumulated initially, whereas the Triton-soluble form of α-ENaC decreased throughout the time course. However, when all three subunits were coexpressed, the α- and β-subunits showed a similar pattern. The complex became Triton insoluble at some point after the endoplasmic reticulum, as incubation at 15°C blocked the conversion to the insoluble form. Deletion of the carboxy-terminal tail of β-ENaC causes Liddle’s syndrome. This mutation increased the amount of newly synthesized Triton-insoluble ENaC heteromultimers but did not affect the half-life of insoluble protein. Therefore, subunit composition and mutations in individual subunits can influence biosynthesis of the ENaC complex.
Molecular mechanism underlying a Cx50-linked congenital cataract
Authors: Pal J. D. , Berthoud V. M. , Beyer E. C. , Mackay D. , Shiels A. , Ebihara L.
Published: December 2017

Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474–1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526–532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of ∼5 μS 4–7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels when coinjected with wild-type connexin 50 cRNA. Cells injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA exhibited gap junctional coupling of ∼8% and 39% of wild-type coupling, respectively. Based on these findings, we conclude that only one P88S mutant subunit is necessary per gap junctional channel to abolish channel function.
Norepinephrine-induced Ca2+waves depend on InsP3 and ryanodine receptor activation in vascular myocytes
Authors: Boittin François-Xavier , Macrez Nathalie , Halet Guillaume , Mironneau Jean
Published: December 2017

In rat portal vein myocytes, Ca2+ signals can be generated by inositol 1,4,5-trisphosphate (InsP3)- and ryanodine-sensitive Ca2+ release channels, which are located on the same intracellular store. Using a laser scanning confocal microscope associated with the patch-clamp technique, we showed that propagated Ca2+ waves evoked by norepinephrine (in the continuous presence of oxodipine) were completely blocked after internal application of an anti-InsP3 receptor antibody. These propagated Ca2+ waves were also reduced by ∼50% and transformed in homogenous Ca2+ responses after application of an anti-ryanodine receptor antibody or ryanodine. All-or-none Ca2+ waves obtained with increasing concentrations of norepinephrine were transformed in a dose-response relationship with a Hill coefficient close to unity after ryanodine receptor inhibition. Similar effects of the ryanodine receptor inhibition were observed on the norepinephrine- and ACh-induced Ca2+ responses in non-voltage-clamped portal vein and duodenal myocytes and on the norepinephrine-induced contraction. Taken together, these results show that ryanodine-sensitive Ca2+release channels are responsible for the fast propagation of Ca2+ responses evoked by various neurotransmitters producing InsP3in vascular and visceral myocytes.
Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts
Authors: Carballo Ester , Pitterle Diana M. , Stumpo Deborah J. , Sperling Robert T. , Blackshear Perry J.
Published: December 2017

Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright’s stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45–60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.
Na+-K+pump and metabolic activities of trout erythrocytes during anoxia
Authors: Pesquero Jesús , Roig Teresa , Sánchez Josep , Bermúdez Jordi
Published: December 2017

Metabolic activity in the red blood cells of brown trout was monitored under conditions of oxygen depletion and chemically induced anoxia. Although metabolic activity was reduced during anoxia to one-third of the normoxic value, these cells maintained their ATP contents stable and were viable for hours in the absence of oxygen. In addition, Na+-K+pump activity was not down-regulated when metabolic activity was reduced during anoxia. The compatibility of this finding with energy equilibrium and ion homeostasis was investigated.
Membrane phospholipid composition affects function of potassium channels from rabbit colon epithelium
Authors: Turnheim Klaus , Gruber Johannes , Wachter Christoph , Ruiz-Gutiérrez Valentina
Published: December 2017

We tested the effects of membrane phospholipids on the function of high-conductance, Ca2+-activated K+ channels from the basolateral cell membrane of rabbit distal colon epithelium by reconstituting these channels into planar bilayers consisting of different 1:1 mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambient K+ concentrations single-channel conductance is higher in PE/PS and PE/PI bilayers than in PE/PC bilayers. At high K+concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayers increases channel conductance, whereas the positively charged dodecyltrimethylammonium has the opposite effect. All these findings are consistent with modulation of channel current by the charge of the lipid membrane surrounding the channel. But the K+ that permeates the channel senses only a small fraction of the full membrane surface potential of the charged phospholipid bilayers, equivalent to separation of the conduction pathway from the charged phospholipid head groups by 20Å. This distance appears to insulate the channel entrance from the bilayer surface potential, suggesting large dimensions of the channel-forming protein. In addition, in PE/PC and PE/PI bilayers, but not in PE/PS bilayers, the open-state probability of the channel decreases with time (“channel rundown”), indicating that phospholipid properties other than surface charge are required to maintain channel fluctuations.
Myofibroblasts. II. Intestinal subepithelial myofibroblasts
Authors: Powell D. W. , Mifflin R. C. , Valentich J. D. , Crowe S. E. , Saada J. I. , West A. B.
Published: December 2017

Intestinal subepithelial myofibroblasts (ISEMF) and the interstitial cells of Cajal are the two types of myofibroblasts identified in the intestine. Intestinal myofibroblasts are activated and proliferate in response to various growth factors, particularly the platelet-derived growth factor (PDGF) family, which includes PDGF-BB and stem cell factor (SCF), through expression of PDGF receptors and the SCF receptor c- kit. ISEMF have been shown to play important roles in the organogenesis of the intestine, and growth factors and cytokines secreted by these cells promote epithelial restitution and proliferation, i.e., wound repair. Their role in the fibrosis of Crohn’s disease and collagenous colitis is being investigated. Through cyclooxygenase (COX)-1 and COX-2 activation, ISEMF augment intestinal ion secretion in response to certain secretagogues. By forming a subepithelial barrier to Na+ diffusion, they create a hypertonic compartment that may account for the ability of the gut to transport fluid against an adverse osmotic gradient. Through the paracrine secretion of prostaglandins and growth factors (e.g., transforming growth factor-β), ISEMF may play a role in colonic tumorigenesis and metastasis. COX-2 in polyp ISEMF may be a target for nonsteroidal anti-inflammatory drugs (NSAIDs), which would account for the regression of the neoplasms in familial adenomatous polyposis and the preventive effect of NSAIDs in the development of sporadic colon neoplasms. More investigation is needed to clarify the functions of these pleiotropic cells.
PGE2-mediated inhibition of T cell p59fyn is independent of cAMP
Authors: Choudhry Mashkoor A. , Ahmed Zulfiqar , Sayeed Mohammed M.
Published: December 2017

We recently observed that prostaglandin E2(PGE2)-mediated suppression of T cell functions could result from an attenuation of p59fyn protein tyrosine kinase activity. The present study evaluated the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), as well as those of cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cell p59fyn kinase activity. The study allowed us to assess whether PGE2-mediated activation of adenylate cyclase by itself or the elevation in intracellular cAMP levels is an integral event in the modulation of anti-CD3-linked p59fyn activation in T cells. The experiments were carried out with splenic T cells from male Sprague-Dawley rats. A 30–50% suppression in the autophosphorylation and the kinase activity of p59fyn in T cells incubated with PGE2 or forskolin was observed. Pretreatment of T cells with SQ-22536 prevented significant PGE2-mediated inhibition of T cell p59fyn kinase activity. In contrast, no change in p59fynautophosphorylation and kinase activity in T cells treated with cAMP analogues was observed. These data suggest that PGE2-mediated suppression of p59fyn autophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.
Systemic administration of the NF-κB inhibitor curcumin stimulates muscle regeneration after traumatic injury
Authors: Thaloor Deepa , Miller Kristy J. , Gephart Jonathan , Mitchell Patrick O. , Pavlath Grace K.
Published: December 2017

Skeletal muscle is often the site of tissue injury due to trauma, disease, developmental defects or surgery. Yet, to date, no effective treatment is available to stimulate the repair of skeletal muscle. We show that the kinetics and extent of muscle regeneration in vivo after trauma are greatly enhanced following systemic administration of curcumin, a pharmacological inhibitor of the transcription factor NF-κB. Biochemical and histological analyses indicate an effect of curcumin after only 4 days of daily intraperitoneal injection compared with controls that require >2 wk to restore normal tissue architecture. Curcumin can act directly on cultured muscle precursor cells to stimulate both cell proliferation and differentiation under appropriate conditions. Other pharmacological and genetic inhibitors of NF-κB also stimulate muscle differentiation in vitro. Inhibition of NF-κB-mediated transcription was confirmed using reporter gene assays. We conclude that NF-κB exerts a role in regulating myogenesis and that modulation of NF-κB activity within muscle tissue is beneficial for muscle repair. The striking effects of curcumin on myogenesis suggest therapeutic applications for treating muscle injuries.
Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion
Authors: Netticadan Thomas , Temsah Rana , Osada Mitsuru , Dhalla Naranjan S.
Published: December 2017

Although the sarcoplasmic reticulum (SR) is known to regulate the intracellular concentration of Ca2+ and the SR function has been shown to become abnormal during ischemia-reperfusion in the heart, the mechanisms for this defect are not fully understood. Because phosphorylation of SR proteins plays a crucial role in the regulation of SR function, we investigated the status of endogenous Ca2+/calmodulin-dependent protein kinase (CaMK) and exogenous cAMP-dependent protein kinase (PKA) phosphorylation of the SR proteins in control, ischemic (I), and ischemia-reperfused (I/R) hearts treated or not treated with superoxide dismutase (SOD) plus catalase (CAT). SR and cytosolic fractions were isolated from control, I, and I/R hearts treated or not treated with SOD plus CAT, and the SR protein phosphorylation by CaMK and PKA, the CaMK- and PKA-stimulated Ca2+ uptake, and the CaMK, PKA, and phosphatase activities were studied. The SR CaMK and CaMK-stimulated Ca2+ uptake activities, as well as CaMK phosphorylation of Ca2+ pump ATPase (SERCA2a) and phospholamban (PLB), were significantly decreased in both I and I/R hearts. The PKA phosphorylation of PLB and PKA-stimulated Ca2+ uptake were reduced significantly in the I/R hearts only. Cytosolic CaMK and PKA activities were unaltered, whereas SR phosphatase activity in the I and I/R hearts was depressed. SOD plus CAT treatment prevented the observed alterations in SR CaMK and phosphatase activities, CaMK and PKA phosphorylations, and CaMK- and PKA-stimulated Ca2+ uptake. These results indicate that depressed CaMK phosphorylation and CaMK-stimulated Ca2+ uptake in I/R hearts may be due to a depression in the SR CaMK activity. Furthermore, prevention of the I/R-induced alterations in SR protein phosphorylation by SOD plus CAT treatment is consistent with the role of oxidative stress during ischemia-reperfusion injury in the heart.
Separate taurine and chloride efflux pathways activated during regulatory volume decrease
Authors: Stutzin Andrés , Torres Rubén , Oporto Macarena , Pacheco Patricio , Eguiguren Ana Luisa , Cid L. Pablo , Sepúlveda Francisco V.
Published: December 2017

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl− by anions that are more permeant through the volume-activated Cl− channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the125I, used as a tracer for Cl−, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl−-rich medium the increase in I− efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl− conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl− conductance-dependent cell shrinkage but only ∼4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl− are separate.
Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+
Authors: Buhagiar Kerrie A. , Hansen Peter S. , Gray David F. , Mihailidou Anastasia S. , Rasmussen Helge H.
Published: December 2017

Treatment of rabbits with angiotensin-converting enzyme (ACE) inhibitors increases the apparent affinity of the Na+-K+pump for Na+. To explore the mechanism, we voltage clamped myocytes from control rabbits and rabbits treated with captopril with patch pipettes containing 10 mM Na+. When pipette solutions were K+ free, pump current ( I p) for myocytes from captopril-treated rabbits was nearly identical to that for myocytes from controls. However, treatment caused a significant increase in I pmeasured with pipettes containing K+. A similar difference was observed when myocytes from rabbits treated with the ANG II receptor antagonist losartan and myocytes from controls were compared. Treatment-induced differences in I p were eliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed of amino acids 530–558 in pipette solutions. Treatment with captopril had no effect on the voltage dependence of I p. We conclude that ANG II regulates the pump’s selectivity for intracellular Na+ at sites near the cytoplasmic surface. Protein kinase C is implicated in the messenger cascade.
Apolipoprotein A-I gene expression is regulated by cellular zinc status in Hep G2 cells
Authors: Wu John Y. J. , Wu Yan , Reaves Scott K. , Wang Yi Ran , Lei Polin P. , Lei Kai Y.
Published: December 2017

The influence of Zn on the expression of the apolipoprotein A-I (apoA-I) gene in Hep G2 cells was examined. Zn depletion was achieved with a low-Zn (ZD) medium prepared from Zn-free growth medium (Opti), a ZD medium containing Chelex 100-extracted fetal bovine serum (CHE), and a medium containing chelator 1,10-phenanthroline (OP). Compared with those for their respective controls, cellular Zn levels were reduced by 55, 48, and 46% and apoA-I mRNA abundances were reduced by 20, 29, and 28% in Opti, CHE, and OP systems, respectively, after one passage in ZD media or 24 h in OP medium. To establish the specificity of Zn treatment, groups of ZD cells were treated with their respective control media for the last 24 h (ZDA) or normal cells were cultured with OP medium supplemented with Zn (OP-Zn). ZDA treatments partially normalized cellular Zn levels in the Opti system and restored or elevated apoA-I mRNA levels in the Opti or CHE system, respectively. Similarly, the OP-Zn treatment restored the cellular Zn and apoA-I mRNA levels. Furthermore, one passage of culture with Zn-supplemented media in both the Opti and CHE systems resulted in higher cellular Zn and apoA-I mRNA levels than those for controls. Most significantly, short-term high-Zn induction to normal cells markedly elevated the cellular Zn (3-fold) and apoA-I mRNA (5-fold) levels. Data derived from this study strongly suggest that the expression of apoA-I is regulated by cellular Zn status.
PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase
Authors: Turner J. R. , Angle J. M. , Black E. D. , Joyal J. L. , Sacks D. B. , Madara J. L.
Published: December 2017

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 ( Gastrointest. Liver Physiol. 29): G214–G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 ( Cell Physiol. 42): C1378–C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.
Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate
Authors: Scheving Lawrence A. , Thomas Jiji R. , Zhang Linda
Published: December 2017

Cell suspensions of ileal mucosa undergo a rapid and synchronized form of programmed cell death when cultured in a simple medium at 37°C. Because tyrosine phosphorylation of proteins plays a crucial role in the signal transduction of many cellular processes, we examined its role in intestinal programmed cell death by use of immunoblot and immunohistochemical methods. We observed a 50–70% reduction in tyrosine phosphorylation during the initial 10 min of intestinal epithelial cell culture. We hypothesized that the inhibition of protein tyrosine phosphatases would increase protein tyrosine phosphorylation in these suspensions and decrease programmed cell death. A strong inhibitor of these phosphatases (peroxovanadate) but not a weaker one (sodium orthovanadate) abolished the DNA fragmentation/laddering normally seen in dying enterocytes. Peroxovanadate enhanced protein tyrosine phosphorylation of many intestinal proteins, dramatically increasing the dually phosphorylated and active form of mitogen-activated protein kinase. Immunohistochemistry revealed a particularly high level of increased tyrosine phosphorylation in the intestinal crypts in peroxovanadate-treated mucosa. Kinetic studies indicated that the pivotal time for protein tyrosine phosphatase inhibition occurred within 5 min of ex vivo culture, precisely when protein tyrosine phosphorylation declined. Our data suggest that tyrosine kinase inactivation or tyrosine phosphatase activation may initiate intestinal epithelial cell death.
Osteocyte hypoxia: a novel mechanotransduction pathway
Authors: Dodd J. S. , Raleigh J. A. , Gross T. S.
Published: December 2017

Bone is a unique tissue in which to examine mechanotransduction due to its essential role in weight bearing. Within bone, the osteocyte is an ideal cellular mechanotransducer candidate. Because osteocytes reside distant from the blood supply, their metabolic needs are met by a combination of passive diffusion and enhanced diffusion, arising when the tissue is loaded during functional activity. Therefore, we hypothesized that depriving a bone of mechanical loading (and thus eliminating diffusion enhanced by loading) would rapidly induce osteocyte hypoxia. Using the avian ulna model of disuse osteopenia, we found that 24 h of unloading results in significant osteocyte hypoxia (8.4 ± 1.8%) compared with control levels (1.1 ± 0.5%; P = 0.03). Additionally, we present preliminary data suggesting that a brief loading regimen is sufficient to rescue osteocytes from this fate. The rapid onset of the observed osteocyte hypoxia, the inhibition of hypoxia by brief loading, and the cellular consequences of oxygen deprivation are suggestive of a novel mechanotransduction pathway with implications across organ systems.
Expression of an inwardly rectifying K+ channel, Kir4.1, in satellite cells of rat cochlear ganglia
Authors: Hibino Hiroshi , Horio Yoshiyuki , Fujita Akikazu , Inanobe Atsushi , Doi Katsumi , Gotow Takahiro , Uchiyama Yasuo , Kubo Takeshi , Kurachi Yoshihisa
Published: December 2017

Satellite cells are glial cells wrapped around somata of sensory and autonomic ganglion neurons. Neither their functional roles nor electrical properties have been fully clarified so far. Using immunohistochemistry, we found that inwardly rectifying K+ channel subunit Kir4.1 (also called Kir1.2 or KAB-2) was expressed prominently in the satellite cells of cochlear ganglia. The Kir4.1 immunoreactivity was localized specifically at the myelin sheaths of satellite cells wrapping the somata of the ganglion neurons. Developmental expression of Kir4.1 in satellite cells paralleled development of the action potential in the auditory nerve. These results suggest that this channel in satellite cells may be responsible for the regulation of K+ extruded from the ganglion neurons during excitation.
Cloning, expression, and characterization of the trout cardiac Na+/Ca2+exchanger
Authors: Xue Xiao-Hua , Hryshko Larry V. , Nicoll Debora A. , Philipson Kenneth D. , Tibbits Glen F.
Published: December 2017

Isoform 1 of the cardiac Na+/Ca2+exchanger (NCX1) is an important regulator of cytosolic Ca2+ concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7°C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout ( Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca2+ binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3–4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at ∼23°C demonstrated Na+-dependent inactivation and Ca2+-dependent activation in a manner qualitatively similar to that for NCX1 currents.
Maitotoxin and P2Z/P2X7purinergic receptor stimulation activate a common cytolytic pore
Authors: Schilling William P. , Wasylyna Tanya , Dubyak George R. , Humphreys Benjamin D. , Sinkins William G.
Published: December 2017

The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X7ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X7 receptors or 2) MTX and P2Z/P2X7 receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-γ, a maneuver known to upregulate P2Z/P2X7receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X7 receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X7 channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca2+ concentration ([Ca2+]i); the initial increase reflects MTX-induced Ca2+ influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca2+]iand ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X7 receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X7 receptor, MTX-induced increases in [Ca2+]iand ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca2+]ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22°C, a characteristic shared by the P2Z/P2X7-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X7 receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.
The mechanism of histamine secretion from gastric enterochromaffin-like cells
Authors: Prinz Christian , Zanner Robert , Gerhard Markus , Mahr Sabine , Neumayer Nina , Höhne-Zell Barbara , Gratzl Manfred
Published: December 2017

Enterochromaffin-like (ECL) cells play a pivotal role in the peripheral regulation of gastric acid secretion as they respond to the functionally important gastrointestinal hormones gastrin and somatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the key stimulus of histamine release from ECL cells in vivo and in vitro. Voltage-gated K+ and Ca2+ channels have been detected on isolated ECL cells. Exocytosis of histamine following gastrin stimulation and Ca2+ entry across the plasma membrane is catalyzed by synaptobrevin and synaptosomal-associated protein of 25 kDa, both characterized as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein. Histamine release occurs from different cellular pools: preexisting vacuolar histamine immediately released by Ca2+ entry or newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized by cytoplasmic HDC and accumulated in secretory vesicles by proton-histamine countertransport via the vesicular monoamine transporter subtype 2 (VMAT-2). The promoter region of HDC contains Ca2+-, cAMP-, and protein kinase C-responsive elements. The gene promoter for VMAT-2, however, lacks TATA boxes but contains regulatory elements for the hormones glucagon and somatostatin. Histamine secretion from ECL cells is thereby under a complex regulation of hormonal signals and can be targeted at several steps during the process of exocytosis.
Apoptosis and atrophy in rat slow skeletal muscles in chronic heart failure
Authors: dalla Libera Luciano , Zennaro Roberta , Sandri Marco , Ambrosio Giovanni Battista , Vescovo Giorgio
Published: December 2017

Congestive heart failure is characterized by a skeletal muscle myopathy with muscle bulk loss. The mechanisms responsible for these changes are not clear at present. We have investigated the role of apoptosis in the rat “slow” soleus muscle during the development of heart failure, which was induced by injection of monocrotaline (30 mg/kg). We looked at the time course of apoptosis by studying six animals at each of the following time points: 0, 17, 24, and 30 days. We found a decreased expression of the antiapoptotic protein Bcl-2, which was accompanied by a rise of proapoptotic caspase-3. Ubiquitin levels did not change. DNA nick-end labeling showed an increased number of apoptotic nuclei both in myofibers and interstitial cells when heart failure occurred. At variance with previous observations in the fast-twitch tibialis anterior muscle in the same animals, in which tumor necrosis factor-α (TNF-α) increased at the time that apoptosis occurred, the magnitude of apoptosis is lower in soleus muscle and there is no appearance of muscle atrophy. In soleus muscle, apoptosis is accompanied by activation of the caspase-3 system. There is no activation of the TNF-α- and ubiquitin-dependent protein waste. In conclusion, slow muscles are less prone to develop apoptosis than fast muscles. Muscle atrophy appears earlier in these latter ones.