bluuebunny/crossref_metadata_2025 huggingface dataset:
Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle
Authors: Herring B. Paul , Dixon Shelley , Gallagher Patricia J.
Published: December 2017

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or “nonmuscle” form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.
Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury
Authors: Dupont-Versteegden Esther E. , Murphy René J. L. , Houlé John D. , Gurley Cathy M. , Peterson Charlotte A.
Published: December 2017

We have shown that cycling exercise combined with fetal spinal cord transplantation restored muscle mass reduced as a result of complete transection of the spinal cord. In this study, mechanisms whereby this combined intervention increased the size of atrophied soleus and plantaris muscles were investigated. Rats were divided into five groups ( n = 4, per group): control, nontransected; spinal cord transected at T10 for 8 wk (Tx); spinal cord transected for 8 wk and exercised for the last 4 wk (TxEx); spinal cord transected for 8 wk with transplantation of fetal spinal cord tissue into the lesion site 4 wk prior to death (TxTp); and spinal cord transected for 8 wk, exercised for the last 4 wk combined with transplantation 4 wk prior to death (TxExTp). Tx soleus and plantaris muscles were decreased in size compared with control. Exercise and transplantation alone did not restore muscle size in soleus, but exercise alone minimized atrophy in plantaris. However, the combination of exercise and transplantation resulted in a significant increase in muscle size in soleus and plantaris compared with transection alone. Furthermore, myofiber nuclear number of soleus was decreased by 40% in Tx and was not affected in TxEx or TxTp but was restored in TxExTp. A strong correlation ( r = 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not in plantaris muscle, in which myonuclear number did not change with any of the experimental manipulations. 5′-Bromo-2′-deoxyuridine-positive nuclei inside the myofiber membrane were observed in TxExTp soleus muscles, indicating that satellite cells had divided and subsequently fused into myofibers, contributing to the increase in myonuclear number. The increase in satellite cell activity did not appear to be controlled by the insulin-like growth factors (IGF), as IGF-I and IGF-II mRNA abundance was decreased in Tx soleus and plantaris, and was not restored with the interventions. These results indicate that, following a relatively long postinjury interval, exercise and transplantation combined restore muscle size. Satellite cell fusion and restoration of myofiber nuclear number contributed to increased muscle size in the soleus, but not in plantaris, suggesting that cellular mechanisms regulating muscle size differ between muscles with different fiber type composition.
Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin
Authors: Illek Beate , Lizarzaburu Mike E. , Lee Vivien , Nantz Michael H. , Kurth Mark J. , Fischer Horst
Published: December 2017

Apigenin (4′,5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl−currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of Cl− currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation ( K s) was 9.1 ± 1.3 μM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4′-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and the 4′-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 ± 11 μM. Blocked Cl− currents were structurally determined by 7-hydroxyl = 4′-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, K s was 4.1 ± 0.9 μM, which was structurally determined by pyrone > all hydroxyls > single hydroxyls. In contrast, block of Cl− current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding.
Guanylyl cyclase stimulatory coupling to KCachannels
Authors: Nara M. , Dhulipala P. D. K. , Ji G. J. , Kamasani U. R. , Wang Y.-X. , Matalon S. , Kotlikoff M. I.
Published: December 2017

We coexpressed the human large-conductance, calcium-activated K (KCa) channel (α- and β-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell KCa currents by 21 ± 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased KCa currents (20 ± 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of KCacurrents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the α-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920–14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate KCa channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling.
Interleukin-1β induces apoptosis in GL15 glioblastoma-derived human cell line
Authors: Castigli Emilia , Arcuri Cataldo , Giovagnoli Luca , Luciani Roberto , Giovagnoli Leo , Secca Teresa , Gianfranceschi Gian Luigi , Bocchini Virginia
Published: December 2017

Interleukin 1-β (IL-1β) induces apoptosis in a glioblastoma-derived human cell line, exhibiting a poorly differentiated astrocytic phenotype. The apoptotic effect was demonstrated by analyzing nuclear morphology, in situ DNA fragmentation, and by ELISA detection of cytoplasmatic nucleosomes. We correlated the degree of differentiation of GL15 cells with the apoptotic response: 1) 4′,6-diamidino-2-phenylindole staining, combined with glial fibrillary acidic protein (GFAP) immunofluorescence, showed that the cells with apoptotic nuclei express low levels of GFAP; and 2) at 13 days of subculture, in a more differentiated state, GL15 cells did not respond with apoptosis to IL-1β. In this cell line, nonrandom chromosome changes and the expression of SV40 early region have been previously shown. The involvement of p42/p44 mitogen-activated protein kinase (MAPK) pathway in the induction of apoptosis by IL-1β was hypothesized. Previous studies have shown that SV40 small T antigen partially inhibits phosphatase 2A, leading to an enhancement of the steady-state activity of p42/p44 MAPK pathway. PD-098059, specific inhibitor of p42/p44 MAPK pathway, counteracts the apoptotic effect of IL-1β, whereas SB-203580, specific inhibitor of p38 stress-activated protein kinase (SAPK) pathway, is ineffective. The imbalance between MAPK and SAPK pathways has been proposed as a key factor in determination of cell fate. Our results demonstrate that a further stimulation of p42/p44 MAPK pathway can constitute a death signal in tumor cells in which genomic damage and MAPK pathway control alterations occur.
Into Ion Channel and Transporter Function. Caenorhabditis elegans ClC-type chloride channels: novel variants and functional expression
Authors: Nehrke Keith , Begenisich Ted , Pilato Jodi , Melvin James E.
Published: December 2017

Six ClC-type chloride channel genes have been identified in Caenorhabditis elegans, termed clh-1 through clh-6. cDNA sequences from these genes suggest that clh-2, clh-3, and clh-4 may code for multiple channel variants, bringing the total to at least nine channel types in this nematode. Promoter-driven green fluorescent protein (GFP) expression in transgenic animals indicates that the protein CLH-5 is expressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells, and the remaining isoforms vary from those restricted to a single cell to those expressed in over a dozen cells of the nematode. In an Sf9 cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did not form functional plasma membrane channels. In contrast, both CLH-1 and CLH-3b produced strong, inward-rectifying chloride currents similar to those arising from mammalian ClC2, but which operate over different voltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene family provides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.
Noninvasive measurement of hydrogen and potassium ion flux from single cells and epithelial structures
Authors: Smith P. J. S. , Trimarchi J.
Published: December 2017

This review introduces new developments in a technique for measuring the movement of ions across the plasma membrane. With the use of a self-referencing ion-selective (Seris) probe, transport mechanisms can be studied on a variety of preparations ranging from tissues to single cells. In this paper we illustrate this versatility with examples from the vas deferens and inner ear epithelium to large and small single cells represented by mouse single-cell embryos and rat microglia. Potassium and hydrogen ion fluxes are studied and pharmacological manipulation of the signals are reported. The strengths of the self-referencing technique are reviewed with regard to biological applications, and the expansion of self-referencing probes to include electrochemical and enzyme-based sensors is discussed.
Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts
Authors: Siwik Deborah A. , Pagano Patrick J. , Colucci Wilson S.
Published: December 2017

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H2O2 (0.05–5 μM) or the superoxide-generating system xanthine (500 μM) plus xanthine oxidase (0.001–0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H2O2 and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-13, MMP-2, and MMP-9. H2O2 and XXO decreased collagen synthesis (collagenase-sensitive [3H]proline incorporation) without affecting total protein synthesis ([3H]leucine incorporation). H2O2 and XXO decreased the expression of procollagen α1(I), α2(I), and α1(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H2O2, but not XXO, also decreased the expression of nonfibrillar procollagen α1(IV) and α2(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, 100 μM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; 10 μM) to increase intracellular H2O2. DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.
Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle
Authors: Sultan Karim R. , Dittrich Bernd T. , Leisner Elmi , Paul Nina , Pette Dirk
Published: December 2017

The present study investigates the role of two major proteolytic systems in transforming rabbit and rat muscles. The fast-to-slow transformation of rabbit muscle by chronic low-frequency stimulation (CLFS) induces fast-to-slow transitions of intact, mature fibers and replacement of degenerating fibers by newly formed slow fibers. Ubiquitination, an indicator of the ATP-dependent proteasome system, and calpain activity were measured in homogenates of control and stimulated extensor digitorum longus muscles. Calpain activity increased similarly (∼2-fold) in stimulated rat and rabbit muscles. CLFS had no effect on protein ubiquitination in rat muscle but led to elevations in ubiquitin protein conjugates in rabbit muscle. Immunohistochemistry was used to study the distribution of μ-calpain and m-calpain and of ubiquitinated proteins in myosin heavy chain-based fiber types. The findings suggest that both proteolytic systems are involved in fiber transformation and replacement. Transforming mature fibers displayed increases in μ-calpain and accumulation of ubiquitin protein conjugates. The majority of these fibers were identified as type IIA. Enhanced ubiquitination was also observed in degenerating and necrotic fibers. Such fibers additionally displayed elevated m-calpain levels. Conversely, p94, the skeletal muscle-specific calpain, decayed rapidly after stimulation onset and was hardly detectable after 4 days of CLFS.
Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages
Authors: Sjöholm Åke , Arkhammar Per , Berggren Per-Olof , Andersson Arne
Published: December 2017

To further evaluate the role of polyamines in insulin production and cell replication in diabetic pancreatic islets, we have studied hyperplastic islets of obese-hyperglycemic mice of different ages and normal islets of the same strain. The aims of the study were to investigate the impact of the diabetic state and aging on polyamine contents and requirements in these islets. Cultured islets from lean and obese animals contained significantly less polyamines than freshly isolated islets. Spermine-to-spermidine ratio was elevated in freshly isolated islets from young obese mice compared with those from lean mice. In islets from old obese animals, spermidine content was decreased, whereas the content of spermine was not different from that of young obese mice. The physiological significance of polyamines was investigated by exposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets but failed to affect polyamine content of cell nuclei. Insulin content was not affected in polyamine-deficient islets of obese mice, irrespective of age, in contrast to decreased islet insulin content in polyamine-depleted young lean animals. Polyamine depletion depressed DNA synthesis rate in obese mouse islets; in lean mice it actually stimulated DNA synthesis. We concluded that important qualitative and quantitative differences exist between islets from obese-hyperglycemic and normal mice with respect to polyamine content and requirements of polyamines for regulation of insulin content and cell proliferation. The results suggest that spermine may be involved in mediating the rapid islet cell proliferation noted early in obese-hyperglycemic syndrome, but changes in spermine concentration do not seem to account for the decline in islet cell DNA synthesis in aged normoglycemic animals.
cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl− conductance ( G Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G Cl is independent of protein kinase A. We activated G proteins and monitored CFTR G Cl in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5′- O-(3-thiotriphosphate) (10–100 μM) stimulated CFTR G Cl in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR G Cl required Mg2+ and ATP hydrolysis (5′-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR G Cl was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 μM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2′,5′-dideoxyadenosine (1 mM) and SQ-22536 (100 μM); and 3) independent of Ca2+, suggesting that Ca2+-dependent protein kinase C and Ca2+/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10−6 M forskolin plus 10−6 M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR G Cl endogenously via a cAMP-independent pathway in this native absorptive epithelium.
Differences in Ca2+ signaling underlie age-specific effects of secretagogues on colonic Cl−transport
Authors: Venkatasubramanian Jayashree , Selvaraj Nataraja , Carlos Maria , Skaluba Stanley , Rasenick Mark M. , Rao Mrinalini C.
Published: December 2017

Taurodeoxycholic acid (TDC) stimulates Cl− transport in adult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates that stimuli like neurotensin (NT) are also age specific and identifies the age-dependent signaling step. Bile acid actions are segment and bile acid specific. Thus although TDC and taurochenodeoxycholate stimulate Cl− transport in AD distal but not proximal colon, taurocholate has no effect in either segment. TDC increases intracellular Ca2+ concentration ([Ca2+]i) in AD, but not in WN and NB, colonocytes. In AD cells, TDC (5 min) action on Cl−transport needs intra- but not extracellular Ca2+. NT, histamine, and bethanechol increase Cl− transport and [Ca2+]i in AD, but not WN, distal colonocytes. However, A-23187 increased [Ca2+]i and Cl− transport in all age groups, suggesting that Ca2+-sensitive Cl−transport is present from birth. Study of the proximal steps in Ca2+ signaling revealed that NT, but not TDC, activates a GTP-binding protein, Gαq, in AD and WN cells. In addition, although WN and AD colonocytes had similar levels of phosphatidylinositol 4,5-bisphosphate, NT and TDC increased 1,4,5-inositol trisphosphate content only in AD cells. Nonresponsiveness of WN cells to Ca2+-dependent stimuli, therefore, is due to the absence of measurable phospholipase C activity. Thus delays in Ca2+ signaling afford a crucial protective mechanism to meet the changing demands of the developing colon.
G protein coupling to M1 and M3muscarinic receptors in sublingual glands
Authors: Luo W. , Latchney L. R. , Culp D. J.
Published: December 2017

Rat sublingual gland M1 and M3 muscarinic receptors each directly activate exocrine secretion. To investigate the functional role of coreceptor expression, we determined receptor-G protein coupling. Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both carbachol-stimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX. Carbachol enhances photoaffinity labeling ([α-32P]GTP-azidoaniline) of only 42-kDa proteins that are subsequently tractable to immunoprecipitation by antibodies specific for Gαq or Gα11 but not Gα12 or Gα13. Carbachol-stimulated photoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis is reduced 55% and 60%, respectively, by M1 receptor blockade with m1-toxin. Gαq/11-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar ratios of receptors to Gαq and Gα11. Although simultaneous activation of M1 and M3receptors is required for a maximal response, both receptor subtypes are coupled to Gαq and Gα11 to stimulate exocrine secretion via redundant mechanisms.
Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels
Authors: Li Jichang , Correa Ana M.
Published: December 2017

Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (∼17%) and maximum open probability (∼20%). Single-channel conductance was larger (∼20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.
A store-operated nonselective cation channel in lymphocytes is activated directly by Ca2+ influx factor and diacylglycerol
Authors: Su Zhengchang , Csutora Peter , Hunton Dacia , Shoemaker Richard L. , Marchase Richard B. , Blalock J. Edwin
Published: December 2017

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca2+ influx. While Ca2+ influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca2+ release-activated Ca2+ (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca2+release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca2+ influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca2+ store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.
Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons
Authors: Liu Liwang , Barrett Curtis F. , Rittenhouse Ann R.
Published: December 2017

We recently reported that arachidonic acid (AA) inhibits L- and N-type Ca2+ currents at positive test potentials in the presence of the dihydropyridine L-type Ca2+ channel agonist (+)-202-791 in dissociated neonatal rat superior cervical ganglion neurons [Liu L and Rittenhouse AR. J Physiol (Lond) 525: 291–404, 2000]. In this first of two companion papers, we characterized the mechanism of inhibition by AA at the whole cell level. In the presence of either ω-conotoxin GVIA or nimodipine, AA decreased current amplitude, confirming that L- and N-type currents, respectively, were inhibited. AA-induced inhibition was concentration dependent and reversible with an albumin-containing wash solution, but appears independent of AA metabolism and G protein activity. In characterizing inhibition, an AA-induced enhancement of current amplitude was revealed that occurred primarily at negative test potentials. Cell dialysis with albumin minimized inhibition but had little effect on enhancement, suggesting that AA has distinct sites of action. We examined AA's actions on current kinetics and found that AA increased holding potential-dependent inactivation. AA also enhanced the rate of N-type current activation. These findings indicate that AA causes multiple changes in sympathetic Ca2+ currents.
Amino acid depletion activates TonEBP and sodium-coupled inositol transport
Authors: Franchi-Gazzola Renata , Visigalli Rossana , Dall'Asta Valeria , Sala Roberto , Woo Seung Kyoon , Kwon H. Moo , Gazzola Gian C. , Bussolati Ovidio
Published: December 2017

The expression of the osmosensitive sodium/ myo-inositol cotransporter (SMIT) is regulated by multiple tonicity-responsive enhancers (TonEs) in the 5′-flanking region of the gene. In response to hypertonicity, the nuclear abundance of the transcription factor TonE-binding protein (TonEBP) is increased, and the transcription of the SMIT gene is induced. Transport system A for neutral amino acids, another osmosensitive mechanism, is progressively stimulated if amino acid substrates are not present in the extracellular compartment. Under this condition, as in hypertonicity, cells shrink and mitogen-activated protein kinases are activated. We demonstrate here that a clear-cut nuclear redistribution of TonEBP, followed by SMIT expression increase and inositol transport activation, is observed after incubation of cultured human fibroblasts in Earle's balanced salts (EBSS), an isotonic, amino acid-free saline. EBSS-induced SMIT stimulation is prevented by substrates of system A, although these compounds do not compete with inositol for transport through SMIT. We conclude that the incubation in isotonic, amino acid-free saline triggers an osmotic stimulus and elicits TonEBP-dependent responses like hypertonic treatment.
Perturbation of the pump-leak balance for Na+ and K+ in malaria-infected erythrocytes
Authors: Staines Henry M. , Ellory J. Clive , Kirk Kiaran
Published: December 2017

In human erythrocytes infected with the mature form of the malaria parasite Plasmodium falciparum, the cytosolic concentration of Na+ is increased and that of K+ is decreased. In this study, the membrane transport changes underlying this perturbation were investigated using a combination of86Rb+, 43K+, and22Na+ flux measurements and a semiquantitative hemolysis technique. From >15 h postinvasion, there appeared in the infected erythrocyte membrane new permeation pathways (NPP) that caused a significant increase in the basal ion permeability of the erythrocyte membrane and that were inhibited by furosemide (0.1 mM). The NPP showed the selectivity sequence Cs+ > Rb+ > K+ > Na+, with the K+-to-Na+permeability ratio estimated as 2.3. From 18 to 36 h postinvasion, the activity of the erythrocyte Na+/K+ pump increased in response to increased cytosolic Na+ (a consequence of the increased leakage of Na+ via the NPP) but underwent a progressive decrease in the latter 12 h of the parasite's occupancy of the erythrocyte (36–48 h postinvasion). Incorporation of the measured ion transport rates into a mathematical model of the human erythrocyte indicates that the induction of the NPP, together with the impairment of the Na+/K+pump, accounts for the altered Na+ and K+levels in the host cell cytosol, as well as predicting an initial decrease, followed by a lytic increase in the volume of the host erythrocyte.
Moderation of skeletal muscle reperfusion injury by a sLex-glycosylated complement inhibitory protein
Authors: Kyriakides Constantinos , Wang Yong , Austen William G. , Favuzza Joanne , Kobzik Lester , Moore Francis D. , Hechtman Herbert B.
Published: December 2017

The role of the sialyl Lewisx (sLex)-decorated version of soluble complement receptor type 1 (sCR1) in moderating skeletal muscle reperfusion injury, by antagonizing neutrophil endothelial selectin interaction and complement activation, is examined. Mice underwent 2 h of hindlimb ischemia and 3 h of reperfusion. Permeability index (PI) was assessed by extravasation of 125I-labeled albumin. Neutrophil depletion and complement inhibition with sCR1 reduced permeability by 72% (PI 0.81 ± 0.10) compared with a 42% decrease (PI 1.53 ± 0.08) observed in neutropenic mice, indicating that part of the complement-mediated injury is neutrophil independent. sCR1sLex treatment reduced PI by 70% (PI 0.86 ± 0.06), an additional 20% decrease compared with sCR1 treatment (PI 1.32 ± 0.08). Treatment with sCR1sLex 0.5 and 1 h after reperfusion reduced permeability by 63% (PI 0.09 ± 0.07) and 52% (PI 1.24 ± 0.09), respectively, compared with the respective decreases of 41% (PI 1.41 ± 0.10) and 32% (PI 1.61 ± 0.07) after sCR1 treatment. Muscle immunohistochemistry stained for sCR1 only on the vascular endothelium of sCR1sLex-treated mice. In conclusion, sCR1sLexis more effective than sCR1 in moderating skeletal muscle reperfusion injury.
Neutrophils injure cultured skeletal myotubes
Authors: Pizza Francis X. , McLoughlin Thomas J. , McGregor Stephen J. , Calomeni Edward P. , Gunning William T.
Published: December 2017

The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo.
Ionomycin causes activation of p38 and p42/44 mitogen-activated protein kinases in human neutrophils
Authors: Elzi David J. , Bjornsen A. Jason , MacKenzie Todd , Wyman Travis H. , Silliman Christopher C.
Published: December 2017

Many receptor-linked agents that prime or activate the NADPH oxidase in polymorphonuclear neutrophils (PMNs) elicit changes in cytosolic Ca2+ concentration and activate mitogen-activated protein (MAP) kinases. To investigate the role of Ca2+ in the activation of p38 and p42/44 MAP kinases, we examined the effects of the Ca2+-selective ionophore ionomycin on priming and activation of the PMN oxidase. Ionomycin caused a rapid rise in cytosolic Ca2+ that was due to both a release of cytosolic Ca2+ stores and Ca2+ influx. Ionomycin also activated (2 μM) and primed (20–200 nM) the PMN oxidase. Dual phosphorylation of p38 MAP kinase and phosphorylation of its substrate activating transcription factor-2 were detected at ionomycin concentrations that prime or activate the PMN oxidase, while dual phosphorylation of p42/44 MAP kinase and phosphorylation of its substrate Elk-1 were elicited at 0.2–2 μM. SB-203580, a p38 MAP kinase antagonist, inhibited ionomycin-induced activation of the oxidase (68 ± 8%, P < 0.05) and tyrosine phosphorylation of 105- and 72-kDa proteins; conversely, PD-98059, an inhibitor of MAP/extracellular signal-related kinase 1, had no effect. Treatment of PMNs with thapsigargin resulted in priming of the oxidase and activation of p38 MAP kinase. Chelation of cytosolic but not extracellular Ca2+ completely inhibited ionomycin activation of p38 MAP kinase, whereas chelation of extracellular Ca2+ abrogated activation of p42/44 MAP kinase. These results demonstrate the importance of changes in cytosolic Ca2+ for MAP kinase activation in PMNs.
Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance
Authors: Lange Klaus , Gartzke Joachim
Published: December 2017

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca2+ signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G0/G1 resting phase.
Resistance training improves single muscle fiber contractile function in older women
Authors: Trappe Scott , Godard Michael , Gallagher Philip , Carroll Chad , Rowden Greg , Porter David
Published: December 2017

The purpose of this study was to 1) examine single cell contractile mechanics of skeletal muscle before and after 12 wk of progressive resistance training (PRT) in older women ( n = 7; 74 ± 2 yr) and 2) to compare these results to our previously completed single cell PRT work with older men ( n = 7; 74 ± 2 yr) (Trappe S, Williamson D, Godard M, Porter D, Rowden G, and Costill D. J Applied Physiol 89:143–152, 2000). Knee extensor PRT was performed 3 days/wk at 80% of one-repetition maximum. Muscle biopsies were obtained from the vastus lateralis before and after the PRT. Chemically skinned single muscle fibers ( n = 313) were studied at 15°C for peak tension (Po), unloaded shortening velocity ( V o), and power. Due to the low number of hybrid fibers identified post-PRT, direct comparisons were limited to MHC I and IIa fibers. Muscle fiber diameter increased 24% (90 ± 2 to 112 ± 6 μm; P < 0.05) in MHC I fibers with no change in MHC IIa fibers. Po increased ( P< 0.05) 33% in MHC I (0.76 ± 0.04 to 1.01 ± 0.09 mN) and 14% in MHC IIa (0.73 ± 0.04 to 0.83 ± 0.05 mN) fibers. Muscle fiber V o was unaltered in both fiber types with PRT. MHC I and IIa fiber power increased ( P< 0.05) 50% [11 ± 2 to 17 ± 2 μN · fiber length (FL) · s−1] and 25% (40 ± 8 to 51 ± 6 μN · FL · s−1), respectively. However, when peak power was normalized to cell size, no pre- to postimprovements were observed. These data indicate that PRT in elderly women increases muscle cell size, strength, and peak power in both slow and fast muscle fibers, which was similar to the older men. However, in contrast to the older men, no change in fiber V o or normalized power was observed in the older women. These data suggest that older men and women respond differently at the muscle cell level to the same resistance-training stimulus.
Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells
Authors: Jaggar Jonathan H.
Published: December 2017

The regulation of intracellular Ca2+ signals in smooth muscle cells and arterial diameter by intravascular pressure was investigated in rat cerebral arteries (∼150 μm) using a laser scanning confocal microscope and the fluorescent Ca2+ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca2+spark frequency 2.6-fold, Ca2+ wave frequency 1.9-fold, and global intracellular Ca2+ concentration ([Ca2+]i) 1.4-fold in smooth muscle cells, and constricted arteries. Ryanodine (10 μM), an inhibitor of ryanodine-sensitive Ca2+ release channels, or thapsigargin (100 nM), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, abolished sparks and waves, elevated global [Ca2+]i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 μM), a voltage-dependent Ca2+ channel (VDCC) blocker, significantly reduced sparks, waves, and global [Ca2+]i, and dilated pressurized (60 mmHg) arteries. Steady membrane depolarization elevated Ca2+ signaling similar to pressure and increased transient Ca2+-sensitive K+ channel current frequency e-fold for ∼7 mV, and these effects were prevented by VDCC blockers. Data are consistent with the hypothesis that pressure induces a steady membrane depolarization that activates VDCCs, leading to an elevation of spark frequency, wave frequency, and global [Ca2+]i. In addition, pressure induces contraction via an elevation of global [Ca2+]i, whereas the net effect of sparks and waves, which do not significantly contribute to global [Ca2+]i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.
Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells
Authors: Schneider Eric , Schmid-Kotsas Alexandra , Zhao Jinshun , Weidenbach Hans , Schmid Roland M. , Menke Andre , Adler Guido , Waltenberger Johannes , Grünert Adolf , Bachem Max G.
Published: December 2017

The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle α-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-β1. Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) α- and PDGF β-receptors. TGF-β1and tumor necrosis factor (TNF)-α accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-β1 (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF-α (1.87 ± 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-β1 stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-β1 and TNF-α accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-β1, PDGF, and, to a lesser extent, TGF-α stimulate extracellular matrix synthesis of cultured rat PSC.
Peroxynitrite causes endothelial cell monolayer barrier dysfunction
Authors: Knepler James L. , Taher Loui N. , Gupta Mahesh P. , Patterson Carolyn , Pavalko Fred , Ober Michael D. , Hart C. Michael
Published: December 2017

Nitric oxide (·NO) attenuates hydrogen peroxide (H2O2)-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116–L126, 2001). However, ·NO rapidly combines with superoxide (O[Formula: see text]) to form the powerful oxidant peroxynitrite (ONOO−), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO− (500 μM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1–500 μM). SIN-1-mediated ONOO− formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO− and SIN-1 increased albumin clearance ( P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and β-catenin as detected by immunofluorescent confocal imaging. ONOO−-induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO− and SIN-1 nitrated tyrosine residues, including those on β-catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO− into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO− directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.
Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation
Authors: Teixeira Cristina C. , Mansfield Kyle , Hertkorn Caryn , Ischiropoulos Harry , Shapiro Irving M.
Published: December 2017

An elevation in inorganic phosphate (Pi) concentration activates epiphyseal chondrocyte apoptosis. To determine the mechanism of apoptosis, tibial chondrocytes were treated with Pi, and nitrate/nitrite (NO[Formula: see text]/NO[Formula: see text]) levels were determined. Pi induced a threefold increase in the NO[Formula: see text]/NO[Formula: see text] concentration; inhibitors of nitric oxide (NO) synthase activity and Pi transport significantly reduced NO[Formula: see text]/NO[Formula: see text]levels and prevented cell death. Furthermore, a dose-dependent increase in cell death was observed after exposure of chondrocytes to S-nitrosoglutathione. Pi increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protected chondrocytes from Pi-induced apoptosis. Pi caused a significant decrease in the mitochondrial membrane potential, while NO synthase inhibitors maintained mitochondrial function. While Pi caused thiol depletion, inhibition of Pi uptake or NO generation served to maintain glutathione levels. The results suggest that NO serves to mediate key metabolic events linked to Pi-dependent chondrocyte apoptosis.
High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism
Authors: Apostolova Margarita D. , Chen Shali , Chakrabarti Subrata , Cherian M. George
Published: December 2017

Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ETB antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ETA inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.
Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits
Authors: Gatto Craig , McLoud Scott M. , Kaplan Jack H.
Published: December 2017

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.