bluuebunny/crossref_metadata_2025 huggingface dataset:
Mismatch between myosin heavy chain mRNA and protein distribution in human skeletal muscle fibers
Authors: Andersen J. L. , Schiaffino S.
Published: December 2017

The distribution of myosin heavy chain (MHC) isoforms was analyzed at the protein and mRNA levels in human skeletal muscle biopsies from young normal adult subjects. Using ATPase histochemical reactions, antibodies to fast- and slow-type MHCs, and in situ hybridization with probes specific for MHC-beta/slow, MHC-2A, and MHC-2X, we confirmed our previous results showing that most fibers contain either a single mRNA and isoprotein or a mixed 1/2A or 2A/2X phenotype with coexistence of two mRNAs and isoproteins. However, we also found a minor proportion of fibers showing a mismatch in the relative proportion of mRNA and protein, e.g., fibers containing MHC-2A mRNA but not the corresponding protein or fibers containing MHC-2A protein but not the corresponding transcript. These fibers were more frequent in biopsies obtained after a training or detraining period than before the training period. We propose that these fibers represent transitional fibers and that the relative content of each mRNA and isoprotein gives a clue as to the direction of change in MHC gene expression.
G protein-coupled receptors control vascular smooth muscle cell proliferation via pp60c-src and p21ras
Authors: Schieffer B. , Drexler H. , Ling B. N. , Marrero M. B.
Published: December 2017

The binding of vasoactive peptides to their respective G protein-coupled receptors has been implicated in the pathogenesis of vascular smooth muscle cell proliferation, leading to the development of hypertension, arteriosclerosis, and restenosis after vascular injury. We previously showed that the cytosolic tyrosine kinase pp60c-src is crucial for angiotensin II (ANG II)-induced activation of the protooncogene p21ras. Therefore, we investigated the role of pp60c-src and p21ras in rat aortic smooth muscle cell proliferation induced by several G protein-coupled receptors. ANG II, endothelin-1, or thrombin increased cell proliferation and DNA synthesis. Electroporation of anti-pp60c-src antibodies into cells abolished proliferation in response to these G protein-coupled receptor ligands but not in response to platelet-derived growth factor-BB (PDGF-BB). In contrast, electroporation of anti-p21ras antibody completely blocked DNA synthesis and cell proliferation in response to ANG II, endothelin-1, thrombin, and PDGF-BB. Our data indicate that the pp60c-src tyrosine kinase is necessary and specific for vascular smooth muscle cell proliferation and DNA synthesis in response to G protein-coupled receptors but not classic growth factor receptors.
Phosphocreatine content in single fibers of human muscle after sustained submaximal exercise
Authors: Sahlin K. , Soderlund K. , Tonkonogi M. , Hirakoba K.
Published: December 2017

The effect of sustained submaximal exercise on muscle energetics has been studied on the single-fiber level in human skeletal muscle. Seven subjects cycled to fatigue (mean 77 min) at a work rate corresponding to approximately 75% of maximal O2 uptake. Biopsies were taken from the vastus lateralis muscle at rest, at fatigue, and after 5 min of recovery. Muscle glycogen decreased from 444 +/- 40 (SE) mmol glucosyl units/kg dry wt at rest to 94 +/- 16. Postexercise glycogen was inversely correlated (P < 0.01) to muscle content of inosine monophosphate, a catabolite of ATP. Phosphocreatine (PCr) in mixed-fiber muscle decreased at fatigue to 37% but was restored above the initial value (106.5%, P < 0.025) after 5 min of recovery. The overshoot was localized to type I fibers. The rapid reversal of PCr is in contrast to the slow recovery in contraction force. Pi increased at fatigue but less than that expected from the changes in PCr and other phosphate compounds. Mean PCr at rest was approximately 20% higher in type II than in type I fibers (86.4 +/- 3.6 and 71.6 +/- 1.8 mmol/kg dry wt, respectively, P < 0.05), but at fatigue similar PCr contents were observed in the two fiber types. Reduction in PCr in all fibers at fatigue suggests that all fibers were recruited at the end of exercise. PCr content in single fibers showed a great variability in samples at rest, exercise, and recovery. The variability was more pronounced than for ATP, and the data suggest that it is due to interfiber physiological-biochemical differences. At fatigue ATP was maintained relatively high in all single fibers, but a pronounced depletion of PCr was observed in a large number of fibers, and this may contribute to fatigue through the associated increases in Pi or/and free ADP. It is noteworthy that the increase in calculated free ADP at fatigue was similar to that after high-intensity exercise.
Sapecin B, a novel fly toxin, blocks macroscopic K+ currents in the GH3 rat pituitary cell line
Authors: Suzuki N. , Hirono M. , Kawahara K. , Yoshioka T.
Published: December 2017

Sapecin B is structurally homologous to charybdotoxin (CTX), which is found in scorpion venom. This study investigated the effects of sapecin B on the Ca(2+)-activated K+ currents [IK(Ca)] and the rapidly inactivating K+ currents in clonal rat GH3 pituitary cells with whole cell voltage-clamp methods. Sapecin B (20 nM) reversibly blocked the CTX-sensitive Ix(Ca) (the BK current) in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 0.9 nM, comparable to that of 0.08-0.4 nM for CTX. The Ca2+ currents in GH3 cells, however, were not affected by sapecin B (40 nM), indicating that the blockade of IK(Ca) by sapecin B is not a secondary effect of Ca2+ current inhibition. The effect of sapecin B on IK(Ca) resembled that of CTX, as expected from the structural similarities shared by CTX and sapecin B. We also found that sapecin B largely inhibited the 4-aminopyridine-sensitive, rapidly inactivating K+ currents in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 40 nM, whereas CTX had little effect on this current in GH3 cells. Sapecin B may thus provide a useful tool, complementary to CTX, for probing the functional role of molecular domains in the BK channels and the structural similarities common to the BK and the rapidly inactivating A-type K+ channels.
Activation of glycolysis in human muscle in vivo
Authors: Conley K. E. , Blei M. L. , Richards T. L. , Kushmerick M. J. , Jubrias S. A.
Published: December 2017

We tested the cytoplasmic control mechanisms for glycolytic ATP synthesis in human wrist flexor muscles. The forearm was made ischemic and activated by maximal twitch stimulation of the median and ulnar nerves in 10 subjects. Kinetic changes in phosphocreatine, Pi, ADP, ATP, sugar phosphates, and pH were measured by 31P magnetic resonance spectroscopy at 7.1-s intervals. Proton production was determined from pH and tissue buffer capacity during stimulation. Glycolysis was activated between 30 and 50 stimulations, and the rate did not significantly change through the stimulation period. The independence of glycolytic rate on [Pi], [ADP], or [AMP] indicates that feedback regulation by these metabolites could not account for this activation of glycolysis. However, glycolytic H+ and ATP production increased sixfold from 0.5 to 3 Hz, indicating that glycolytic rate reflected muscle activation frequency. This dependence of glycolytic rate on muscle stimulation frequency and independence on metabolite levels is consistent with control of glycolysis by Ca2+.
Measurement of mitochondrial calcium in single living cardiomyocytes by selective removal of cytosolic indo 1
Authors: Griffiths E. J. , Stern M. D. , Silverman H. S.
Published: December 2017

The aim of the present study was to determine whether, in indo 1 acetoxymethyl ester (AM)-loaded rat cardiomyocytes, it was possible to remove cytosolic but not mitochondrial indo 1 by promoting loss of cytosolic indo 1 through plasma membrane anion pumps (which are blocked by probenecid). Isolated rat ventricular myocytes were loaded with indo 1-AM under conditions (15 min at 30 degrees C) in which about half of the dye is located within mitochondria. Cells were then maintained at 25 degrees C for 2.5 h followed by incubation at 37 degrees C for 1.5 h. After this "heat treatment," the myocyte fluorescence signal was 44% of the value of cells measured before heat treatment, and loss of fluorescence was prevented by 1 mM probenecid. The remaining fluorescence was shown to originate from mitochondria, since 1) Ca2+ uptake and efflux could be inhibited by ruthenium red and clonazepam, respectively, and 2) low concentrations of digitonin, which release only cytosolic marker enzymes, decreased fluorescence of untreated myocytes but had little effect on the fluorescence signal of heat-treated cells. We conclude that heat treatment selectively removes cytosolic indo 1, leaving a signal due to mitochondrial indo 1 only.
Myosin I
Authors: Coluccio L. M.
Published: December 2017

The class I myosins are single-headed, actin-binding, mechanochemical “motor” proteins with heavy chains in the molecular mass range of 110-130 kDa; they do not form filaments. Each myosin I heavy chain is associated with one to six light chains that bind to specific motifs known as IQ domains. In vertebrate myosin I isoforms, the light chain is calmodulin, which is thought to regulate motor activity. Proteins similar to calmodulin are associated with myosin I isoforms from lower eukaryotes. Some myosin I isoforms from lower eukaryotes are regulated by phosphorylation; however, the phosphorylation site is not present in vertebrate myosin I isoforms. Based on sequence analyses of the amino terminal “head” domains, myosin I can be subdivided into several subclasses. Analyses of the biochemical properties of the isolated molecules and localization studies support the proposal of roles for these molecules in intracellular trafficking and changes in membrane structure. Our present understanding of the properties of these molecules and their proposed roles is reviewed here.
Coordinated expression of myosin heavy chain isoforms and metabolic enzymes within overloaded rat muscle fibers
Authors: Dunn S. E. , Michel R. N.
Published: December 2017

We studied the coordinated regulation of myosin heavy chains (MHC) and metabolic enzymes within individual overloaded adult rat plantaris fibers. This was done using monoclonal antibodies raised against distinct developmental and adult MHCs, and quantitative microphotometric succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) enzyme assays. Overload shifted MHC expression in the order IIb-->IIx-->IIa-->alpha/I, with a tripling of cells coexpressing I and alpha-MHC, and a transient reexpression of two embryonic MHC and the neonatal isoform in preexisting myofibers. Overload caused a rapid, size-independent, 50% decrease in GPDH activity across all cell types, which recovered by 6 wk. Fiber SDH activities varied according to MHC composition, such that overloaded fibers coexpressing IIa MHC displayed control slow fiber SDH levels, whereas cells expressing IIx and IIb MHC displayed a transient 30% increase in SDH that recovered by 6 wk. Our results suggest that during overload, fibers adapt progressively to the new functional requirements and display more efficient cellular energy utilization and delivery characteristics. The time course of adaptations suggests a role for glycolytic enzymes in the initiation of these transformations.
Hormonal and neurogenic control of Na-K-ATPase and myosin isoforms in neonatal rat cardiac myocytes
Authors: Arystarkhova E. , Sweadner K. J.
Published: December 2017

In the rat heart there is a postnatal switch in the expression of isoforms of both Na-K-ATPase and myosin heavy chain (MHC). Here we investigated factors controlling isoform changes in cultures of neonatal cardiomyocytes. In serum-free medium, the compositions of either Na-K-ATPase or MHC isoforms resembled the neonatal phenotype. Thyroid hormone induced the MHC isoform switch but increased expression of all Na-K-ATPase isoforms to various extents. Dexamethasone failed to induce the MHC switch and inhibited Na-K-ATPase alpha 1 isoform expression without inducing the other isoforms. With both hormones, the adult phenotype for both MHC and Na-K-ATPase was seen but with low Na-K-ATPase alpha 2. The paucity of alpha 2 protein was not predicted by studies of mRNA levels. In serum, there was a gradual decline of Na-K-ATPase alpha 3 and the appearance of alpha 2, but again at a relatively low level. Expression of Na-K-ATPase alpha 2 was significantly upregulated when cardiomyocytes were cocultured with sympathetic neurons from superior cervical ganglia, without changes in the MHC isoforms. Thus innervation is postulated to play a specific role in modulating Na-K-ATPase gene expression.
Actin removal from cardiac myocytes shows that near Z line titin attaches to actin while under tension
Authors: Trombitas K. , Granzier H.
Published: December 2017

The I band of cardiac sarcomeres contains both actin and titin/connectin filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the approximately 100-nm-wide region adjoining the Z line, where titin has been found to be inelastic. To study the putative interaction between titin and actin, techniques for selective removal of actin from different regions of the I band are needed. Here we report studies with a gelsolin fragment (FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic: the majority of actin was extracted in approximately 10 min, whereas actin near the Z line (where titin is inelastic) required a approximately 10-fold longer extraction time. Thus, by controlling the extraction time, we could remove either the full actin filament outside the Z line or just the segment of the actin filament that extends beyond the inelastic region of titin that adjoins the Z line. The actin filament-free I band contained titin filaments, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the location of which in the sarcomere varied linearly with sarcomere length. The position in the sarcomere of the junction line coincided with the binding site of the anti-titin antibody 9D10. Actin removal significantly affected the slack sarcomere length. Slack sarcomere length was 1.85 +/- 0.04 microns in control cells and decreased to 1.71 +/- 0.05 microns after actin near the Z line was extracted. This length reduction may be caused by contraction of the titin segment that becomes exposed after actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.
Experimentally induced changes in the endocytic traffic of P-glycoprotein alter drug resistance of cancer cells
Authors: Kim H. , Barroso M. , Samanta R. , Greenberger L. , Sztul E.
Published: December 2017

The MDR-1 gene product, plasma membrane glycoprotein or P-glycoprotein (PGP), has been shown to confer drug resistance to cancer cells by acting as an energy-dependent drug-efflux pump. We have examined the endocytic traffic of PGP in human multidrug-resistant cells and tested whether the traffic and the steady-state intracellular localization of PGP can be experimentally modulated. Here we show that 1) under steady state approximately 70% of cellular PGP is on the surface whereas approximately 30% is intracellular, 2) surface PGP undergoes constitutive endocytosis and recycling, 3) endocytosis of PGP involves clathrin and adaptin complex 2-dependent mechanism, and 4) PGP cycles through a Rab5-responsive endosomal compartment. Biochemical (such as antibody crosslinking of PGP or treatment of cells with chloroquine) and molecular (such as overexpression of Rab5) treatments were used to modulate the endocytic/ recycling traffic of PGP. Such treatments resulted in the redistribution of PGP from the cell surface to intracellular compartments. Cells with such “mislocalized” PGP showed a decrease in multidrug resistance, suggesting that clinically relevant strategies can be attempted by modulating PGP's temporal and spatial distribution within cancer cells.
Normalization of ion transport in murine cystic fibrosis nasal epithelium using gene transfer
Authors: MacVinish L. J. , Goddard C. , Colledge W. H. , Higgins C. F. , Evans M. J. , Cuthbert A. W.
Published: December 2017

The murine nasal epithelium was investigated by the short-circuit current (SCC) technique. Electrogenic sodium absorption was revealed by addition of amiloride and calcium-dependent chloride secretion by the addition of amiloride and calcium-dependent chloride secretion by the addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ)/ionomycin. In the presence of these agents a further increase in SCC was obtained by addition of forskolin. Epithelia from both cystic fibrosis (CF) null (Cftrtm1Cam) and CF delta F508 (Cftrtm2Cam) mice had enhanced sodium absorption compared with controls, whereas only delta F508 epithelia had increased calcium-dependent chloride secretion. Both strains gave nasal epithelia that showed significantly reduced responses to forskolin, due to the absence of CF transmembrane conductance regulator (CFTR) chloride channels. In Cftrtm2Cam nasal epithelia the forskolin responses were not significantly different from zero. Transfection of these mice with the plasmid pTRIAL10-CFTR2 complexed with cationic liposomes normalized the transporting activity in the nasal epithelium. Basal SCC and calcium-dependent chloride secretion were significantly reduced, whereas CFTR-dependent chloride secretion was increased to normal values. Amiloride-sensitive SCC was reduced by transfection but failed to reach significance. The similarity of murine CF nasal epithelium to that in human CF airways makes the model valuable for gene therapy studies.
Effects of extracellular pH, PCO2, and HCO3- on intracellular pH in isolated rat taste buds
Authors: Lyall V. , Feldman G. M. , Heck G. L. , DeSimone J. A.
Published: December 2017

We studied the effects of changing external pH (pHo), external bicarbonate concentration ([HCO3-]o), and PCO2 on taste receptor cell (TRC) intracellular pH (pHi) in taste bud fragments (TBFs) isolated from rat circumvallate and fungiform papillae with the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using microfluorometric and imaging techniques. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions, TRC pHi responded rapidly and monotonically to changes in pHo between 6.5 and 8.0. The relationship between pHi and pHo was steep, with slopes varying between 0.8 and 1.2. Similarly, varying pHo by changing PCO2 at constant [HCO3-]o or changing [HCO3-]o at constant PCO2 led to rapid, monotonic changes in pHi. The relationship between pHi and pHo was once again steep, with slopes varying between 0.8 and 1.2. However, simultaneous changes in PCO2 and [HCO3-]o at constant pHo did not cause any significant changes in steady-state pHi. In imaging studies, single, isolated TRCs responded to changes in pHo, with parallel changes in pHi in the soma and apical process. In addition, changes in pHo induced parallel changes in pHi throughout TBFs. These data suggest that the steady-state TRC pHi is a function of pHo. Changes in TRC pHi may be involved in acid sensing, and salivary [HCO3-] may play a role in the maintainance of steady-state TRC pHi and in the neutralization of acid-induced changes in pHi.
Modulation of K+ and Ca2+ currents in cultured neurons by an angiotensin II type 1a receptor peptide
Authors: Zhu M. , Neubig R. R. , Wade S. M. , Posner P. , Gelband C. H. , Sumners C.
Published: December 2017

Angiotensin II (ANG II) inhibits delayed rectifier K+ current (IK) and stimulates total Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem, effects mediated via ANG II type 1 (AT1) receptors. Here, we identify potential G protein activator regions of the AT1 receptor responsible for initiating the intracellular changes that lead to alterations in these currents. Intracellular application into cultured neurons of a peptide corresponding to the third cytoplasmic loop of the AT1 receptor (AT1a/i3) mimicked the actions of ANG II on IK and ICa, whereas application of a peptide corresponding to the second cytoplasmic loop (AT1a/i2) did not alter these currents. This modulation of IK and ICa by AT1a/i3 involves intracellular messengers (G alpha q, protein kinase C, and intracellular Ca2+) that are identical to those involved in the modulation of IK and ICa following ANG II activation of AT1 receptors. These data provide functional evidence for a role of the third cytoplasmic loop of the AT1 receptor in G protein coupling and subsequent modulation of ion channel effectors.
A role for retrosomes in intracellular cholesterol transport from endosomes to the plasma membrane
Authors: Hornick C. A. , Hui D. Y. , DeLamatre J. G.
Published: December 2017

The recycling component (retrosome) of the endocytic pathway was evaluated as a potential vehicle for the recycling of lipoprotein-derived cholesterol and the maintenance of a high concentration of free cholesterol in plasma membranes. Receptor-to-ligand ratios were established in three distinct endosomal compartments using a recycling receptor (apolipoprotein B/E) to confirm isolated retrosomes as recycling vesicles. Compositional studies showed that retrosomes have twice the free cholesterol in their limiting membranes as do the endosomal compartments from which they derive. Furthermore, of the three isolated endosomal fractions, retrosomes showed the highest ratio of free to esterified cholesterol derived from injected very low density lipoprotein as well as the highest free-to-esterified cholesterol mass ratio overall, confirming endosomal cholesteryl ester hydrolysis and sorting. Endosomal neutral cholesterol esterase was identified by immunoblot, whereas electron microscopy employing membrane cholesterol-specific filipin revealed a high concentration of cholesterol in appendages that appear to be the formative stage of retrosomal biogenesis.
Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain
Authors: Owan I. , Burr D. B. , Turner C. H. , Qiu J. , Tu Y. , Onyia J. E. , Duncan R. L.
Published: December 2017

Mechanical force applied to bone produces two localized mechanical signals on the cell: deformation of the extracellular matrix (substrate strain) and extracellular fluid flow. To study the effects of these stimuli on osteoblasts, MC3T3-E1 cells were grown on type I collagen-coated plastic plates and subjected to four-point bending. This technique produces uniform levels of physiological strain and fluid forces on the cells. Each of these parameters can be varied independently. Osteopontin (OPN) mRNA expression was used to assess the anabolic response of MC3T3-E1 cells. When fluid forces were low, neither strain magnitude nor strain rate was correlated with OPN expression. However, higher-magnitude fluid forces significantly increased OPN message levels independently of the strain magnitude or rate. These data indicate that fluid forces, and not mechanical stretch, influence OPN expression in osteoblasts and suggest that fluid forces induced by extracellular fluid flow within the bone matrix may play an important role in bone formation in response to mechanical loading.
Sphingosine prevents diacylglycerol signaling to mitogen-activated protein kinase in airway smooth muscle
Authors: Tolan D. , Conway A. M. , Pyne N. J. , Pyne S.
Published: December 2017

Because many agonists utilize diacylglycerol (DAG) to initiate nuclear transcriptional activity via protein kinase C (PKC), we have investigated whether sphingosine might counter DAG. Sphingosine inhibited PKC activity in an isolated airway smooth muscle cell lysate and prevented the activation of mitogen-activated protein kinase (MAPK) by platelet-derived growth factor, bradykinin, and phorbol 12-myristate 13-acetate in intact cells. MAPK activation in response to all the agonists involves PKC. The stimulation of [3H]palmitate-labeled cells with sphingosine, in the presence of butan-1-ol (0.3%, vol/vol), induced an increase in [3H]phosphatidate (PtdOH) but was without effect on [3H]DAG. [3H]PtdOH synthesis was inhibited, whereas [3H]DAG levels were increased in the presence of the DAG kinase inhibitor R-59949, indicating that sphingosine stimulates phospholipase C/DAG kinase. Recycling of DAG from PtdOH was prevented by a sphingosine-dependent inhibition of PtdOH phosphohydrolase-2 activity. In conclusion, the sphingosine-induced conversion of DAG to PtdOH may serve to optimize the effect of sphingosine on MAPK. This may account for the antiproliferative action of sphingosine.
Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells
Authors: Stutzin A. , Eguiguren A. L. , Cid L. P. , Sepulveda F. V.
Published: December 2017

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.
Three-dimensional cell cultures: from molecular mechanisms to clinical applications
Authors: Mueller-Klieser Wolfgang
Published: December 2017

This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.
Glutamine utilization by rat neutrophils: presence of phosphate-dependent glutaminase
Authors: Curi Tania C. Pithon , De Melo Mariza P. , De Azevedo Ricardo B. , Zorn Telma M. T. , Curi Rui
Published: December 2017

The capacity of rat neutrophils to utilize glutamine was investigated by 1) determination of oxygen consumption in the presence of glucose or glutamine, 2) measurement of maximal activity of phosphate-dependent glutaminase, 3) Northern blot, Western blot, and immunocytochemical detection of glutaminase, and 4) measurement of glutamine utilization and also production of ammonia, glutamate, aspartate, alanine, and lactate and decarboxylation of [U-14C]glutamine in cells incubated for 1 h. The rate of respiration by isolated neutrophils in the absence of added substrate was 5.0 nmol ⋅ min−1 ⋅ 107cells−1. Maximal activity of phosphate-dependent glutaminase was 56 nmol ⋅ min−1 ⋅ mg protein−1 in freshly obtained neutrophils; the Michaelis-Menten constant was 3.5 mM for glutamine. This enzyme activity was inhibited by 2 mM glutamate, 2 mM oxoglutarate, and 2 mM NH4Cl. The presence of glutaminase protein (65 kDa) was confirmed by Western blot and immunocytochemical detection and the presence of the mRNA (6.0 kb) by Northern blot analysis. Glutamine was utilized by neutrophils incubated for 1 h at a rate of 12.8 nmol ⋅ min−1 ⋅ mg protein−1 when the amino acid was added to the medium at 2 mM, which is three to four times higher than the physiological concentration. In the presence of 0.5 mM glutamine, the amino acid was utilized at a rate of 2.9 nmol ⋅ min−1 ⋅ mg protein−1. The addition of 0.5 mM glutamate to the incubation medium caused a marked reduction (by 70%) in glutamine utilization by neutrophils. Glucose was utilized at 7.7 nmol ⋅ min−1 ⋅ mg protein−1 when cells were incubated in 5 mM glucose. The conversion of [U-14C]glutamine to14CO2was very low: <1% was totally oxidized. The formation of ammonia was ∼27% of glutamine utilization, and the conversion of glutamine to glutamate, aspartate, alanine, and lactate accounted for ∼84.6% of the total amino acid utilized by neutrophils. In this study, evidence is presented that, in addition to lymphocytes and macrophages, neutrophils also utilize glutamine.
Role of β1- and β3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes
Authors: Atgié Claude , D’Allaire François , Bukowiecki Ludwik J.
Published: December 2017

To evaluate the physiological functions of β1-, β2-, and β3-adrenoceptors (ARs) in brown adipose tissue, the lipolytic and respiratory effects of various adrenergic agonists and antagonists were studied in rat brown adipocytes. The β-agonists stimulated both lipolysis and respiration (8–10 times above basal levels), with the following order of potency (concentration eliciting 50% of maximum response): CL-316243 (β3) > BRL-37344 (β3) > isoproterenol (mainly β1/β2) > norepinephrine (NE; mainly β1/β2) > epinephrine (mainly β1/β2) ≫ dobutamine (β1) ≫ procaterol (β2). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (β1 antagonist) revealed that more than one type of receptor mediates NE action. It is concluded from our results that 1) NE, at low plasma levels (1–25 nM), stimulates lipolysis and respiration mainly through β1-ARs, 2) NE, at higher levels, stimulates lipolysis and respiration via both β1- and β3-ARs, 3) β2-ARs play only a minor role, and 4) β3-ARs may represent the physiological receptors for the high NE concentrations in the synaptic cleft, where the high-affinity β1-ARs are presumably desensitized. It is also suggested that lipolysis represents the flux-generating step regulating mitochondrial respiration.
Ca2+-sensing receptors in intestinal epithelium
Authors: Gama Lucio , Baxendale-Cox Lynn M. , Breitwieser Gerda E.
Published: December 2017

Expression of Ca2+-sensing receptors (CaR) was demonstrated in several human intestinal epithelial cell lines (T84, HT-29, and Caco-2) and in rat intestinal epithelium by both reverse transcriptase-polymerase chain reaction (PCR) and Northern blotting of RNA. Restriction patterns of the PCR products were of the sizes predicted by the human and rat sequences. CaR agonists (Ca2+, poly-l-arginine, protamine) mediated an increase in intracellular Ca2+ in HT-29–18-C1 cells (monitored by changes in fura 2 fluorescence), which was dependent on release from thapsigargin-sensitive stores. U-73122, an inhibitor of phosphatidylinositol-phospholipase C, eliminated the CaR agonist-mediated rise in intracellular Ca2+, whereas its inactive analog, U-73343, had no effect. Pertussis toxin pretreatment had no effect on CaR agonist-mediated modulation of intracellular Ca2+. Taken together, these studies demonstrate that CaR are expressed in intestinal epithelial cells and couple to mobilization of intracellular Ca2+. The presence of CaR in intestinal epithelial cells presents a new locus for investigations into the role(s) of extracellular Ca2+ in modulating intestinal epithelial cell differentiation and transepithelial Ca2+ transport.
Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells
Authors: Amlal Hassane , Wang Zhaohui , Soleimani Manoocher
Published: December 2017

The response of H+-ATPase to lethal acid stress is unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles of lethal acid stress. Cells were grown to confluence on coverslips, loaded with 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, and monitored for intracellular pH (pHi) recovery from an acid load. The rate of Na+-independent pHi recovery from an acid load in mutant cells was approximately fourfold higher than in parent cells ( P < 0.001). The Na+-independent H+ extrusion was ATP dependent and K+ independent and was completely inhibited in the presence of diethylstilbestrol, N, N′-dicyclohexylcarbodiimide, or N-ethylmaleimide. These results indicate that the Na+-independent H+ extrusion in cultured medullary cells is mediated via H+-ATPase and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained unchanged in mutant cells compared with parent cells. We propose that lethal acid stress results in increased H+-ATPase activity in inner medullary collecting duct cells. Upregulation of H+-ATPase could play a protective role against cell death in severe intracellular acidosis.
F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes
Authors: Zhang Jianping , Larsen Terje H. , Lieberman Melvyn
Published: December 2017

The integrity of F-actin and its association with the activation of a Cl− current ( I Cl) in cultured chick cardiac myocytes subjected to hyposmotic challenge were monitored by whole cell patch clamp and fluorescence confocal microscopy. Disruption of F-actin by 25 μM cytochalasin B augmented hyposmotic cell swelling by 51% (from a relative volume of 1.54 ± 0.10 in control to 2.33 ± 0.21), whereas stabilization of F-actin by 20 μM phalloidin attenuated swelling by 15% (relative volume of 1.31 ± 0.05). Trace fluorochrome-labeled (fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate) phalloidin revealed an intact F-actin conformation in control cells under hyposmotic conditions despite the considerable changes in cell volume. Sarcoplasmic F-actin was very disorganized and occurred only randomly beneath the sarcolemma in cells treated with cytochalasin B, whereas no changes in F-actin distribution occurred under either isosmotic or hyposmotic conditions in cells treated with phalloidin. Swelling-activated I Cl (68.0 ± 6.0 pA/pF at +60 mV) was suppressed by both cytochalasin B (22.7 ± 5.1 pA/pF) and phalloidin (22.5 ± 3.5 pA/pF). On the basis of these results, we suggest that swelling of cardiac myocytes initiates dynamic changes in the cytoarchitecture of F-actin, which may be involved in the volume transduction processes associated with activation of I Cl.
Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function
Authors: Grover Ashok K. , Samson Sue E.
Published: December 2017

We examined the effects of peroxide on the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump in pig coronary artery endothelium and smooth muscle at three organizational levels: Ca2+ transport in permeabilized cells, cytosolic Ca2+ concentration in intact cells, and contractile function of artery rings. We monitored the ATP-dependent, azide-insensitive, oxalate-stimulated45Ca2+uptake by saponin-permeabilized cultured cells. Low concentrations of peroxide inhibited the uptake less effectively in endothelium than in smooth muscle whether we added the peroxide directly to the Ca2+ uptake solution or treated intact cells with peroxide and washed them before the permeabilization. An acylphosphate formation assay confirmed the greater resistance of the SERCA pump in endothelial cells than in smooth muscle cells. Pretreating smooth muscle cells with 300 μM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic Ca2+ concentration in a Ca2+-free solution, but it did not affect the endothelial cells. Peroxide pretreatment inhibited the CPA-induced contraction in deendothelialized arteries with a 50% inhibitory concentration of 97 ± 13 μM, but up to 500 μM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 μM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced, endothelium-dependent relaxation by only 40 ± 13%. The greater resistance of the endothelium to reactive oxygen may be important during ischemia-reperfusion or in the postinfection immune response.
Cell cycle-dependent expression of a glioma-specific chloride current: proposed link to cytoskeletal changes
Authors: Ullrich Nicole , Sontheimer Harald
Published: December 2017

We recently demonstrated expression of a novel, glioma-specific Cl− current in glial-derived tumor cells (gliomas), including stable cell lines such as STTG1, derived from a human anaplastic astrocytoma. We used STTG1 cells to study whether glioma Cl−channel (GCC) activity is regulated during cell cycle progression. Cells were arrested in defined stages of cell cycle (G0, G1, G1/S, S, and M phases) using serum starvation, mevastatin, hydroxyurea, demecolcine, and cytosine β-d-arabinofuranoside. Cell cycle arrest was confirmed by measuring [3H]thymidine incorporation and by DNA flow cytometry. Using whole cell patch-clamp recordings, we demonstrate differential changes in GCC activity after cell proliferation and cell cycle progression was selectively altered; specifically, channel expression was low in serum-starved, G0-arrested cells, increased significantly in early G1, decreased during S phase, and increased after arrest in M phase. Although the link between the cell cycle and GCC activity is not yet clear, we speculate that GCCs are linked to the cytoskeleton and that cytoskeletal rearrangements associated with cell division lead to the observed changes in channel activity. Consistent with this hypothesis, we demonstrate the activation of GCC by disruption of F-actin using cytochalasin D or osmotic cell swelling.
ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells
Authors: Liakos Panagiotis , Bourmeyster Nicolas , Defaye Geneviève , Chambaz Edmond M. , Bottari Serge P.
Published: December 2017

Angiotensin II (ANG II) has long been known for its pressor and growth-promoting effects, which are both mediated by the AT1 receptor. By contrast, the AT2 receptor has recently been reported to mediate inhibition of proliferation through as yet undefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks the cells in G1 phase. Consistent with this, ANG II inhibits cyclin D1 expression and cyclin D1-associated kinase activity. The antimitogenic effect of ANG II is partly mimicked by the AT2-selective agonist CGP-42112. It is also blocked partly and in an additive fashion by the AT1- and AT2-selective antagonists losartan and PD-123319, indicating the contribution of both receptor subtypes to this response. AT1-dependent antiproliferation is selectively blocked by the cyclooxygenase inhibitor indomethacin and restored by prostaglandin E2, whereas AT2-receptor-mediated inhibition of growth is suppressed by the tyrosine phosphatase inhibitors orthovanadate and bpV(pic). Both pathways are, however, pertussis toxin sensitive. We hypothesize that, in fasciculata cells, the AT1 receptor inhibits bFGF-induced proliferation by stimulating prostaglandin synthesis, whereas the AT2 receptor mediates its effect through a pathway that requires protein tyrosine phosphatase activation.
Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation
Authors: Turner Jerrold R. , Rill Brian K. , Carlson Susan L. , Carnes Denise , Kerner Rachel , Mrsny Randall J. , Madara James L.
Published: December 2017

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.
Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37
Authors: Brink P. R. , Cronin K. , Banach K. , Peterson E. , Westphale E. M. , Seul K. H. , Ramanan S. V. , Beyer E. C.
Published: December 2017

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage ( G j/ V j) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The G j/ V jdata imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.
Letters to the Editor
Authors: Adorante Joseph S. , Edelman Jeffrey L.
Published: December 2017

The following is the abstract of the article discussed in the subsequent letter:

Mitchell, Claire H., Jin Jun Zhang, Liwei Wang, and Tim J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 ( Cell Physiol. 41): C212–C222, 1997.—The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob. J. Physiol. Lond. 491: 743–755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5′- O-(3-thiotriphosphate) (GTPγS) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTPγS-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C, an inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTPγS but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.