bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport
Authors: Armsby C. C. , Stuart-Tilley A. K. , Alper S. L. , Brugnara C.
Published: December 2017

The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis.
NH4Cl-induced hypertrophy is mediated by weak base effects and is independent of cell cycle processes
Authors: Franch H. A. , Preisig P. A.
Published: December 2017

Renal hypertrophy occurs in a number of clinical conditions, some of which are associated with increases in ambient ammonia concentrations. NH4Cl induces hypertrophy in cultured renal epithelial cells. The present studies examined the mechanism of NH4Cl-induced hypertrophy in NRK-52E cells. Hypertrophy was also induced by methylammonium chloride, a related weak base, but not by tetramethylammonium chloride, a weak base analogue that can neither accept nor donate protons. Bafilo-mycin A1, an inhibitor of vacuolar proton pumps, also induced hypertrophy. Together, these studies suggest that NH4Cl-induced hypertrophy is mediated by its weak base property, allowing it to enter and alkalinize acid vesicular compartments. Additional studies demonstrated that NH4Cl-induced hypertrophy is not mediated by modulation of cell cycle processes. NH4Cl addition had no effect on the following: c-fos mRNA abundance, typically associated with entrance into the cell cycle; cyclin E protein abundance, which increases as cells progress through G1; or protein synthesis, which also increases during G1. In addition, inactivation of pRB by overexpression of human papilloma virus-16 carrying the E7 gene, which inhibits cell cycle-dependent hypertrophy, had no effect on the ability of NH4Cl to induce hypertrophy. Based on these data, we postulate that, in hypertrophic conditions associated with increased ammoniagenesis, hypertrophy is mediated by vesicular alkalinization and occurs independently of processes that govern progression through the cell cycle.
Fractal analysis and imaging of the proximal nephron cell
Authors: Welling D. , Urani J. , Welling L. , Wagner E.
Published: December 2017

Cells of the S1 proximal renal tubule were examined to determine whether their peculiar shapes are a result of certain constructs of fractal mathematics. Morphometric measurements of the cell perimeter were made at several levels of cell height by measuring the intercellular boundaries that appear on electron micrographs of tubule cross sections. When the measurements were made over a range of scale lengths, the fractal dimension, D, of the cell perimeter was found to increase from 1.3 near the cell apex to 1.78 near the cell base. The length of scale was found to range between 8 and 0.4 micron and to represent the approximate dimensions of actual cell processes. Fractal patterns that conformed to the measured parameters were then constructed from a fractal generator composed of budlike formations that originated near the cell apex and that increased in number and decreased in size with cell depth according to a fractal scaling. It was found that the fractal rule of keeping a constant relative scale could be maintained between budding processes but, to obtain patterns that resemble biological structure, the processes must be positioned randomly on the cell periphery. It is shown that when the relative sizes of the buds decrease exponentially and their numbers increase geometrically, the perimeter can grow to the correct length without overlap. This suggests that patterns of the cell periphery corresponding to different levels of cell height obey a law of scale but occur randomly in a way that increases to high fractal dimension or near plane-filling values at the cell base. The fractal patterns that correspond to the measured fractal dimensions can be assembled into a three-dimensional model that closely resembles the known shape of the proximal tubule cell.
Relationship between replication and differentiation in cultured human adipocyte precursor cells
Authors: Entenmann G. , Hauner H.
Published: December 2017

The aim of this study was to investigate the role of cell replication for the differentiation of human adipocyte precursor cells in primary culture. When cells were seeded in a medium supplemented with 10% fetal bovine serum, they started to proliferate within 48 h after exposure, as assessed by cell counting and [3H]thymidine autoradiography. When cells were inoculated in the absence of serum, a significant degree of cell proliferation was not detectable. Histochemical investigations using bromodeoxyuridine incorporation demonstrated that cells replicating their DNA did not accumulate lipid droplets. Inoculating adipocyte precursor cells under completely serum-free conditions resulted in a 30-50% higher expression of lipogenic enzymes such as glycerol-3-phosphate dehydrogenase and lipoprotein lipase than keeping cells in serum-supplemented medium for the initial 16 h. Addition of cytosine arabinoside at concentrations that effectively block mitosis did not interfere with adipocyte development. In conclusion, adipocyte precursor cells from human adipose tissue do not require cell division to enter the differentiation process in vitro. These cells may have already undergone possibly critical cell divisions in vivo and may be in a late stage of adipocyte development.
Lactate production by white adipocytes in relation to insulin sensitivity
Authors: Faintrenie G. , Geloen A.
Published: December 2017

Besides its lipogenic and lipolytic functions, adipose tissue has been recognized as an important site of lactate production by epididymal adipocytes under experimental conditions in which insulin sensitivity was increased (cold exposure) or reduced (glucose perfusion). Insulin sensitivity of adipocytes was assessed by its antilipolytic effect and its stimulation of glucose uptake. We showed that, in adipocytes isolated from control and cold-exposed animals, glycerol and lactate production rose simultaneously, in response to norepinephrine. On the other hand, in adipocytes from glucose-perfused animals, we observed a dissociation between lipolytic and glycolytic responses. Indeed, lactate production in response to norepinephrine reached 60% of maximal response (10(-7)M) while the lipolytic response was still basal. This phenomenon was the result of a desensitization of beta-adrenergic receptors. However, the sensitivity of adipocytes to stimulation of lactate production by catecholamines was not different between control and glucose-perfused rats. Because lactate production in response to norepinephrine was not affected by desensitization, it suggests that lactate production is under alpha 1-adrenergic control. We confirmed this hypothesis.
Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice
Authors: Wiedenman J. L. , Rivera-Rivera I. , Vyas D. , Tsika G. , Gao L. , Sheriff-Carter K. , Wang X. , Kwan L. Y. , Tsika R. W.
Published: December 2017

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.
Isoform-independent heart rate-related variation in cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase activity
Authors: Rouslin W. , Broge C. W.
Published: December 2017

In the present study, we compared the activities of the cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase and the content of cardiac muscle mitochondrial ATPase inhibitor protein (IF1) of several mammalian species covering broad ranges of body mass and heart rate, i.e., from beef cattle to mouse. The cardiac myofibrillar ATPase from each species was assayed over a range of pCa values at pH 7.4. While the cardiac myofibrillar ATPase from all species examined showed essentially identical Ca2+ concentration dependencies with the ATPase in each species activating steeply between pCa 6.5 and 5.5, the maximal ATPase specific activity reached varied considerably from species to species, and this variation was largely independent of the predominant cardiac myosin ATPase isoform present. Thus, while adult beef cattle, pig, dog, and rabbit all contain predominantly the slow cardiac myosin ATPase isoform the cardiac myofibrillar ATPase specific activities of these four species varied over approximately a fourfold range. Moreover, there was a fairly smooth curvilinear relationship between maximum Ca(2+)-activated myofibrillar ATPase activity and median conscious heart rate for the slow cardiac myosin ATPase-possessing species examined. This smooth continuum also extended to include two species possessing the fast cardiac myosin ATPase isoform, rat and mouse. This relationship between myofibrillar ATPase activity and heart rate that appears to be applicable to a broad range of species suggests that the myofibrillar ATPase is specifically modeled or fine-tuned to the kinetic (heart rate) demand of each species and, within slow and fast heart rate ranges, is essentially independent of myosin ATPase isoform per se. Only hearts containing predominantly the slow myosin ATPase isoform contained functional levels of IF1. Finally, while it has been reported that the ratio of myosin Ca(2+)-ATPase to actomyosin Mg(2+)-ATPase activity is a good index of the percent of the fast myosin ATPase in rabbit myofibrillar preparations, we found that this relationship may be applicable to only some species.
MDR1/P-glycoprotein function. I. Effect of hypotonicity and inhibitors on rhodamine 123 exclusion
Authors: Weaver J. L. , McKinney L. , Schoenlein P. V. , Goldenberg S. , Gottesman M. M. , Aszalos A.
Published: December 2017

The MDR1 protein (P-glycoprotein) is a membrane ATPase whose expression results in resistance to several anti-tumor drugs. It has been proposed that the MDR1 protein, in addition to its pumplike properties, can function as (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54:618-622, 1994) or mediate the activity of (Hardy et al. EMBO J. 14: 68-75, 1995) a hypotonic stress-induced Cl- current. In addition, one study found that drug transport and Cl- channel-associated functions of MRD1 were separable and mutually exclusive and that, when cells were swelled, the MDR1 protein could not transport substrate. This hypothesis was tested in four pairs of isogenic cell lines with MDR1 transfectants expression 8,000-55,000 MDR1 antibody binding sites per cell. Cytoplasmic exclusion of rhodamine 123 was used as an indicator of MDR1 function to measure the effect of hypotonic stress, MDR1 inhibitors, and Cl- channel blockers on MRD1 transport function. It was found that MDR1 activity and its inhibition by cyclosporine A or flufenamic acid were unaffected by hypotonicity alone or in combination with Cl- channel blockers.
Adaptation of the skeletal muscle calcium-release mechanism to weight-bearing condition
Authors: Kandarian S. C. , Peters D. G. , Favero T. G. , Ward C. W. , Williams J. H.
Published: December 2017

In the present study, we examined whether weight-bearing condition can regulate the sarcoplasmic reticulum (SR) Ca(2+)-release mechanism. Measurements of alpha 1-subunit dihydropyridine (alpha 1-DHP) and ryanodine receptor levels were made in hypertrophied fast-twitch plantaris muscles 5 wk after surgical removal of synergist muscles (increased weight bearing) and in atrophied slowtwitch soleus muscles (14 days of non-weight bearing) of the rat. Rates of AgNO3-induced SR Ca2+ release were measured with fura 2 as the Ca2+ indicator and pyrophosphate as the precipitating ion during vesicular Ca2+ loading. Ca(2+)-release rates were 38, 49, and 58% lower in vesicles from hypertrophied vs. control muscles at AgNO3 concentrations of 0.05, 0.5, and 5 microM, respectively (control = 18.2 +/- 1.4 microM.mg-1. min-1). Western blots showed no differences in the relative expression of alpha 1-DHP or ryanodine receptor when IIID5 (monoclonal) or GP3 (polyclonal) antibodies were used. There was also no difference in ryanodine (10 nM) binding in Ca(2+)-incubated SR vesicles from hypertrophied muscles, suggesting no difference in the number of channels. In contrast, expression of alpha 1-DHP and ryanodine receptors was increased by 144 and 157% in non-weight-bearing soleus muscles, respectively. Scatchard analysis of DHP binding showed a 40% increase in maximum binding capacity and no change in the dissociation constant with non-weight-bearing muscles. The direction of modification of the SR Ca(2+)-release mechanism is opposite with increased and decreased weight bearing, but the mechanism by which this is achieved appears to be different.
SRF and TEF-1 control of chicken skeletal alpha-actin gene during slow-muscle hypertrophy
Authors: Carson J. A. , Schwartz R. J. , Booth F. W.
Published: December 2017

The purpose of this study was to delineate the alpha-actin regulatory elements and transcription factors that are responsible for conferring stretch-overload responsiveness during hypertrophy of the anterior latissimus dorsi (ALD) muscle of young chickens by weighting one wing. Minimal promoter constructs were evaluated by direct injection into the ALD, which demonstrated that both serum response element 1 (SRE1) and the transcriptional enhancer factor 1 (TEF-1) elements were sufficient for increased expression during stretch overload. A mutated SRE1 prevented expression in both basal and stretched ALD muscles, whereas a mutated TEF-1 element reduced actin promoter function in both control and stretched muscles. The serum response factor (SRF)-SRE1 binding complex demonstrated faster migration in mobility shift assays from day 3-and day 6-stretched ALD nuclear extracts relative to their control. TEF-1 binding was qualitatively increased in stretched extracts at day 3 but not day 6 of stretch overload. Skeletal alpha-actin mRNA accumulated from day 3 to day 6 of stretch overload. These data demonstrate that SRE1 is necessary and sufficient for stretch-overload responsiveness from the skeletal alpha-actin promoter and that the SRF-SRE1 binding complex migrates faster in stretched nuclear extracts of hypertrophied relative to control extracts from intact ALD muscles of chickens.
N tau-methylhistidine turnover in skeletal muscle cells measured by GC-MS
Authors: Thompson M. G. , Palmer R. M. , Thom A. , Garden K. , Lobley G. E. , Calder G.
Published: December 2017

A method that employs gas chromatography-mass spectrometry has been developed to measure N tau-methylhistidine (3-methylhistidine; 3-MH) synthesis and release from skeletal muscle myotubes in vitro. It shows excellent linearity (0.9999) over the range studied (0-4 nmol), high recovery (92.6%), and low coefficient of variation (1.6%). 3-MH release from myotubes was essentially linear over a 96-h incubation, whereas the loss of 3-MH from cell protein accelerated with increasing time, an effect due, at lest in part, to decreasing rates of total protein synthesis. When incubated in either glutamine-free or methionine-free medium for 48 h, 3-MH in cell protein and appearing in the medium were greatly reduced compared with the 48-h controls, suggesting that hypertrophy was greatly reduced. Similar but lesser trends were observed with adenosine 3',5' -cyclic monophosphate. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) appeared to both stimulate 3-MII synthesis and inhibit its release during a 48-h incubation. The development of this method facilitates detailed investigation into the mechanisms through which agents such as TPA regulate myofibrillar protein degradation.
Expression and action of parathyroid hormone-related peptide in human cervical epithelial cells
Authors: Kremer R. , Woodworth C. D. , Goltzman D.
Published: December 2017

Parathyroid hormone-related peptide (PTHRP) expression and activity were analyzed in normal human ectocervical keratinocytes (HCX) and keratinocytes immortalized by transfection with human papillomavirus (HPV) types 16 and 18 DNAs. In normal cells, trans-retinoic acid (RA) and 2.0 mM Ca2+ significantly stimulated PTHRP mRNA expression and secretion and led to a significant reduction in the rate of proliferation. In contrast, the basal level of PTHRP production decreased sharply in confluent HCX, and induction by Ca2+ or exogenous growth factors was reduced or lost. After stable transfection with HPV16 and HPV18 DNAs, we observed a sharp decrease of PTHRP production in high-passage poorly differentiated HCX. Finally, addition of exogenous PTHRP-(1-141) inhibited proliferation of both normal cells and low-passage well-differentiated HPV16 immortalized cells. High-passage poorly differentiated cells were refractory to PTHRP. These results demonstrate that PTHRP production varies greatly with the degree of cell proliferation and differentiation and suggest that this peptide acts as an autocrine negative growth regulator for cervical keratinocytes.
cAMP- but not Ca(2+)-regulated Cl- conductance is lacking in cystic fibrosis mice epididymides and seminal vesicles
Authors: Leung A. Y. , Wong P. Y. , Yankaskas J. R. , Boucher R. C.
Published: December 2017

Cystic fibrosis (CF) reflects the loss of adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- secretion consequent to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In humans, but not mice, with CF, the disease is associated with male infertility. The present study investigated the relative magnitudes of the cAMP pathways and an alternative Ca(2+)-regulated Cl- secretory pathway in primary cultures of the epididymides and the seminal vesicles of normal and CF mice. The basal equivalent short-circuit currents (Ieq) of cultures derived from the epididymides and the seminal vesicles from the CF mice were lower (6.0 +/- 0.6 and 4.0 +/- 1.0 muA/cm2, respectively) than those from normal mice (11.1 +/- 1.0 and 6.6 +/- 0.6 muA/cm2, respectively). Forskolin induced significant Ieq responses in both the epididymis (8.0 +/- 0.7 muA/cm2) and seminal vesicles (4.0 +/- 0.5 muA/cm2) from normal mice, whereas forskolin-induced changes in Ieq in CF mouse epididymis and seminal vesicles were absent, consistent with defective cAMP-CFTR-mediated Cl- secretion in CF mice. Ieq responses to agonists (ionomycin, ATP) that raise intracellular Ca2+ (Ca2+i) were larger than forskolin responses in normal animals (6.6 +/- 0.9 and 13.4 +/- 1.8 muA/cm2, respectively) and were preserved in CF (6.5 +/- 0.9 and 17.1 +/- 1.0 muA/cm2, respectively). We speculate that the fertility of male CF mice is maintained by persistent expression of the predominant alternative Ca(2+)-mediated Cl- transport system in the epididymides and seminal vesicles.
Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport
Authors: Flatman P. W. , Adragna N. C. , Lauf P. K.
Published: December 2017

K-Cl cotransport in sheep erythrocytes can be activated by treatment either with A-23187 and EDTA to reduce concentration of internal ionized Mg [Mg]i) to submicromolar levels, with staurosporine, a potent kinase inhibitor, or with N-ethylmaleimide (NEM). Activation by these maneuvers is prevented and reversed by genistein [inhibition constant (Ki) of 15 microM], which inhibits tyrosine kinases (TK). The related glycosidated compound genistin, which does not inhibit TK, does not inhibit transport, whereas another TK inhibitor, tyrphostin B46, inhibits both basal and stimulated transport (Ki of 28 microM). Cotransport activation by NEM is prevented and reversed by the phosphatase inhibitor, calyculin A, and activation by staurosporine occurs only if cells contain ATP. Increasing [Mg]i inhibits cotransport in the presence of calyculin A whether or not staurosporine is present as well. Our work suggests that genistein inhibits cotransport through a TK and that staurosporine and NEM activate cotransport, probably through inhibition of other kinases, causing stimulation through dephosphorylation of a protein (possibly the transporter itself) be a serine/threonine phosphatase. [Mg]i inhibits cotransport by activating a kinase (concentration for half-maximal activation of 10 microM) that phosphorylates this protein.
Cellular mechanisms underlying adenosine actions on cholinergic transmission in enteric neurons
Authors: Barajas-Lopez C. , Peres A. L. , Espinosa-Luna R.
Published: December 2017

Whole cell recordings were used to investigate the effects of adenosine and several of its analogues on voltage-activated calcium currents (VACC) of myenteric and submucosal neurons. Electrophysiological and pharmacological properties of the soma VACC recorded in myenteric neurons indicate that they are carried through N-type calcium channels, similar to those of the submucosal neurons and to those of the calcium conductance that mediates acetylcholine release at the submucosal ganglia. Adenosinergic compounds inhibited, in a concentration-response and in a voltage-dependent manner, VACC in neurons from both enteric plexuses. The pharmacological profile of the receptors that mediate this effect was similar to that of the receptors involved in presynaptic inhibition in enteric neurons and likely of the A1 subtype. The effects of 2-chloroadenosine (CADO) on VACC were prevented by pretreatment with pertussis toxin (PTX), became irreversible with guanosine 5'-O-(3-thiotriphosphate) (inside the pipette), and were abolished with N-ethylmaleimide (NEM; known to uncouple receptors from G protein complexes). Intracellular recordings were used to further evaluate presynaptic effects of adenosine at the submucosal plexus. Adenosinergic compounds reduced the amplitude of fast excitatory postsynaptic potentials (EPSPs) by acting at nerve terminals. This effect was insensitive to PTX and staurosporine (a protein kinase inhibitor) but was abolished by NEM. CADO effects on EPSPs were not reversed by increasing the extracellular calcium concentration. In conclusion, activation of A1 adenosine receptors inhibits VACC via PTX-sensitive G proteins in myenteric and submucosal neurons. Reduction of cholinergic transmission also involves A1 adenosine receptors and appears to involve the activation of PTX-insensitive G proteins.
IFN-gamma modulates CD1d surface expression on intestinal epithelia
Authors: Colgan S. P. , Morales V. M. , Madara J. L. , Polischuk J. E. , Balk S. P. , Blumberg R. S.
Published: December 2017

In vivo, epithelial cells that line the intestine are intimately associated with lymphocytes, termed intestinal intraepithelial lymphocytes (iIEL). A putative ligand for iIEL on intestinal epithelial cells is CD1d, and recent studies demonstrate a surface form of this molecule exists on intestinal epithelia. At present, it is not known whether CD1d expression is regulated by cytokines in the intestinal microenvironment. Thus we examined the impact of relevant cytokines on CD1d at the level of mRNA and cell surface expression. Using a sensitive whole cell enzyme-linked immunosorbent assay, we assessed the impact of relevant cytokines on CD1d expression on intestinal epithelial cell lines. We were readily able to detect CD1d on the surface of T84 cells, a cryptlike intestinal epithelial cell line. Epithelial cell exposure to human recombinant interferon-gamma (IFN-gamma) resulted in increased CD1d expression in a dose- and time-dependent manner. Polymerase chain reaction amplification of CD1d cDNA revealed a time-dependent induction after exposure to IFN-gamma. This IFN-gamma effect on CD1d expression was cytokine specific and was evident with epithelial cell lines other than T84, including Caco-2 and HT-29 cells. Finally, we were not able to detect significant surface expression of CD1a, CD1b, or CD1c on intestinal epithelial cell lines in the presence or absence of relevant cytokines. These results indicate that CD1d cell surface protein and cellular mRNA, like other major histocompatibility complex-related molecules, is cytokine regulated in intestinal epithelial cell lines.
Role for protein phosphatase in the regulation of Ca2+ influx in parotid gland acinar cells
Authors: Sakai T. , Ambudkar I. S.
Published: December 2017

Stimulation of Ca2+ (and Mn2+) entry in salivary epithelial cells by carbachol, or thapsigargin, is mediated by an, as yet, unknown mechanism that is dependent on the depletion of Ca2+ from intracellular Ca2+ stores. This study assesses the possible role of protein phosphorylation in the regulation of Ca2+ entry in rat parotid gland acinar cells. Treatment of cells with the protein phosphatase inhibitors okadaic acid, calyculin A, and pervanadate induced a dose-dependent inhibition of carbachol and thapsigargin stimulation of Ca2+ and Mn2+ entry. All three inhibitors decreased carbachol stimulation of internal Ca2+ release, which likely accounts for the inhibition of carbachol-stimulated Ca2+ entry. Thapsigargin-induced internal Ca2+ release was not affected by the treatments. Additionally, all three phosphatase inhibitors decreased Mn2+ entry into cells with depleted internal Ca2+ store(s) (achieved by incubation with either carbachol or thapsigargin in Ca2+-free medium). Treatment of cells with phorbol 12-myristate 13-acetate, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, or staurosporine did not affect divalent cation entry into unstimulated cells or thapsigargin treated cells. Importantly, when cells with depleted internal Ca2+ store(s) were pretreated with staurosporine, or K-252a, the inhibition of Ca2+ entry by calyculin A and okadaic acid, but not by pervanadate, was attenuated. Although the effect of pervanadate remains to be clarified, these results demonstrate a role for protein phosphorylation in the regulation of divalent cation influx in rat parotid acinar cells.
Ca(2+)-dependent nitric oxide release in endothelial but not R3230Ac rat mammary adenocarcinoma cells
Authors: Lindberg R. A. , Dewhirst M. W. , Buckley B. J. , Hughes C. S. , Whorton A. R.
Published: December 2017

We have characterized the ability of several cell types associated with the microvasculature of solid tumors to release nitric oxide (NO.) in response to increases in cytosolic Ca2+ concentration ([Ca2+]c). EA.hy926 immortalized human umbilical vein endothelial cells (EC), rat fibroblasts (RFL), and tumorigenic cells isolated from R3230Ac rat mammary adenocarcinoma (MaC) were treated with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase. NO. output was measured via a chemiluminescence detection system. Baseline NO. output was detectable only for EC. TG caused a significant increase in EC NO. output that could be blocked with NG-monomethyl-L-arginine and restored with L-arginine. TG did not stimulate NO. release from RFL or MaC cells, despite elevating [Ca2+]c in all cells. A Ca(2+)-dependent isoform of NO synthase (eNOS) was detected by immunoblot only in EC. These data indicate that EC, but not RFL or MaC, are capable of Ca(2+)-dependent NO. release and suggest that any Ca(2+)-dependent NO. release within this tumor is primarily of endothelial (and not tumorigenic cell) origin.
Enhanced NO production during Mg deficiency and its role in mediating red blood cell glutathione loss
Authors: Mak I. T. , Komarov A. M. , Wagner T. L. , Stafford R. E. , Dickens B. F. , Weglicki W. B.
Published: December 2017

The effect of dietary Mg deficiency on nitric oxide (NO) production and its role in mediating oxidative depletion of red blood cell (RBC) glutathione in rats were investigated. Male Sprague-Dawley rats were placed on Mg-deficient or Mg-sufficient diets for up to 3 wk. Plasma nitrate plus nitrite levels, determined by the Escherichia coli reductase/Griess reagent procedures, increased 1.7-fold during the 1st wk and increased 2- to 2.4-fold during the 2nd and 3rd wk on the Mg-deficient diet. In association, substantial losses (approximately 50%) of RBC glutathione occurred during the 2nd and 3rd wk. Administration of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in drinking water (0.5 mg/ml) effectively blunted the increases in plasma nitrate/nitrite during Mg deficiency. Concomitantly, losses of RBC glutathione exhibited by Mg-deficient rats were significantly attenuated. Packed RBCs, obtained from Mg-deficient but not from Mg-sufficient animals, displayed a prominent nitrosyl hemoglobin signal detected by electron spin resonance spectroscopy; the signals of the samples from the L-NAME-treated Mg-deficient rats were greatly reduced. With isolated RBCs, losses of the glutathione could be induced directly by peroxynitrite or 3-morpholinosydnonimine, which generates NO + .O2-, but not by NO (from sodium nitroprusside) alone, in a concentration-dependent manner. The results clearly indicate that NO overproduction occurs and participates in RBC glutathione loss during Mg deficiency. Because neutrophil activation also occurs, we suggest that NO might interact with superoxide anions to form peroxynitrite, which then directly oxidizes RBC glutathione.
Na(+)-K+(NH4+)-2Cl- cotransport in medullary thick ascending limb: control by PKA, PKC, and 20-HETE
Authors: Amlal H. , Legoff C. , Vernimmen C. , Paillard M. , Bichara M.
Published: December 2017

Cell pH was monitored in suspensions of medullary thick ascending limbs (MTALs) of rat kidney to determine possible effects of various transduction pathways on apical Na(+)-K+ (NH4+)-2Cl- cotransport, the activity of which was measured as the bumetanide-sensitive component of cell acidification caused by abrupt exposure to 4 mM NH4Cl. 8-Bromoadenosine 3',5'-cyclic monophosphate stimulated cotransport activity through activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), since the cAMP effect was abolished by N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89); stimulation by cAMP (P < 0.02) was observed even when other Na+, Cl-, and K+ carriers were blocked by ouabain, diphenylamine-2-carboxylate, and barium, which indicates that cotransport was directly affected by PKA. Phorbol 12,13-dibutyrate also stimulated cotransport activity (P < 0.03), which was abolished by protein kinase C (PKC) blockade by staurosporine. In contrast, cotransport activity was reduced (P < 0.001) by arachidonic acid or 20-hydroxyeicosatetraenoic acid (20-HETE), as well as by an ionomycin-induced rise in cytosolic Ca2+ ([Ca2+]i). Inhibition by arachidonic acid or ionomycin was abolished by econazole and SKF-525A that inhibit cytochrome P-450-dependent monoxygenase, which produces 20-HETE from arachidonic acid in the MTAL, and the ionomycin effect was prevented when phospholipase A2 (PLA2) was blocked by 4-bromophenacyl bromide or oleyloxyethyl phosphorylcholine. The results demonstrate that MTAL apical Na(+)-K+(NH4+)-2Cl- cotransport is stimulated by PKA and PKC and inhibited by 20-HETE that may be produced after a rise in [Ca2+]i through PLA2 activation.
Bovine adrenal endothelial cells express nucleoside transporters nonregulated by protein kinases A and C
Authors: Sen R. P. , Sobrevia L. , Delicado E. G. , Yudilevich D. , Miras-Portugal M. T.
Published: December 2017

The present investigation characterizes the nucleoside transporters in bovine adrenomedullary endothelial cells and their possible regulation by the action of protein kinases A and C to establish comparisons with the nucleoside transport system in chromaffin cells. The nucleoside transport proved to be a nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with high affinity for adenosine. These endothelial cells had a high density of nucleoside transporters (660,000 +/- 130,000 transporters/ cell), measured by NBTI binding, and the efficiency was close to 2 adenosine molecules internalized transporter-1.s-1. The stimulation of the cells with bradykinin and P1,P4-di(adenosine-5')tetraphosphate, which raise the intracellular Ca2+ concentration, did not modulate the adenosine transport. When the cells were stimulated with signals coupled to adenosine 3',5'-cyclic monophosphate intracellular production, such as norepinephrine and isoproterenol, the adenosine transport was not modified. Furthermore, the treatment of the cells with direct activators of both protein kinases A and C had no effect on adenosine transport, in contrast to that reported in chromaffin cells.
Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane
Authors: Merlin D. , Guo X. , Martin K. , Laboisse C. , Landis D. , Dubyak G. , Hopfer U.
Published: December 2017

HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.
Force-velocity and force-power properties of single muscle fibers from elite master runners and sedentary men
Authors: Widrick J. J. , Trappe S. W. , Costill D. L. , Fitts R. H.
Published: December 2017

Gastrocnemius muscle fiber bundles were obtained by needle biopsy from five middle-aged sedentary men (SED group) and six age-matched endurance-trained master runners (RUN group). A single chemically permeabilized fiber segment was mounted between a force transducer and a position motor, subjected to a series of isotonic contractions at maximal Ca2+ activation (15 degrees C), and subsequently run on a 5% polyacrylamide gel to determine myosin heavy chain composition. The Hill equation was fit to the data obtained for each individual fiber (r2 > or = 0.98). For the SED group, fiber force-velocity parameters varied (P < 0.05) with fiber myosin heavy chain expression as follows: peak force, no differences: peak tension (force/fiber cross-sectional area), type IIx > type IIa > type I; maximal shortening velocity (Vmax, defined as y-intercept of force-velocity relationship), type IIx = type IIa > type I; a/Pzero (where a is a constant with dimensions of force and Pzero is peak isometric force), type IIx > type IIa > type I. Consequently, type IIx fibers produced twice as much peak power as type IIa fibers, whereas type IIa fibers produced about five times more peak power than type I fibers. RUN type I and IIa fibers were smaller in diameter and produced less peak force than SED type I and IIa fibers. The absolute peak power output of RUN type I and IIa fibers was 13 and 27% less, respectively, than peak power of similarly typed SED fibers. However, type I and IIa Vmax and a/Pzero were not different between the SED and RUN groups, and RUN type I and IIa power deficits disappeared after power was normalized for differences in fiber diameter. Thus the reduced absolute peak power output of the type I and IIa fibers from the master runners was a result of the smaller diameter of these fibers and a corresponding reduction in their peak isometric force production. This impairment in absolute peak power production at the single fiber level may be in part responsible for the reduced in vivo power output previously observed for endurance-trained athletes.
Induction of beta-MHC transgene in overloaded skeletal muscle is not eliminated by mutation of conserved elements
Authors: Tsika G. L. , Wiedenman J. L. , Gao L. , McCarthy J. J. , Sheriff-Carter K. , Rivera-Rivera I. D. , Tsika R. W.
Published: December 2017

Mechanical overload leads to hypertrophy, increased type I fiber composition, and beta-myosin heavy chain (beta-MHC) induction in the fast-twitch plantaris muscle. To better understand the mechanism(s) involved in beta-MHC induction, we have examined inducible expression of transgenes carrying the simultaneous mutation of three DNA regulatory subregions [muscle CAT (MCAT), C-rich, and beta e3] in the context of either 5,600-base pair (bp; beta 5.6mut3) or 600-bp (beta 0.6mut3) beta-MHC promoter in overloaded plantaris muscles of transgenic mice. Protein extract from mechanically overloaded plantaris muscle of mice, harboring either mutant transgene beta 5.6mut3 or beta 0.6mut3, showed an unexpected 2.8- to 4.5-fold increase in chloramphenicol acetyltransferase (CAT) specific activity relative to their respective controls. Similar results were obtained with wild-type (wt) beta-MHC transgenes (beta 5.6wt, beta 0.6wt). Histochemical staining for both myofibrillar ATPase and CAT activity and CAT immunohistochemistry revealed a striking increase in type I fibers and that CAT expression was restricted to these fibers in overloaded plantaris muscle of beta 5.6mut3 transgenic mice. Our transgenic data suggest that beta-MHC transgenes, and perhaps the endogenous beta-MHC gene, are induced by mechanical overload via a mechanism(s) that does not exclusively require the MCAT, C-rich, or beta e3 subregions.
Volume regulatory taurine release in human tracheal 9HTEo- and multidrug resistant 9HTEo-/Dx cells
Authors: Galietta L. J. , Romeo G. , Zegarra-Moran O.
Published: December 2017

The intracellular taurine release evoked by hypotonic shock is accomplished by volume-activated Cl- channels whose activity has been related to the expression of the multidrug resistance protein (MDR-1). We studied taurine transport in 9HTEo- cells and in the derived cell line 9HTEo-/Dx expressing MDR-1. [3H]taurine release from preloaded cells increased upon reduction of extracellular osmolality. This process was not inhibited by preincubation with phorbol 12-myristate 13-acetate but was reduced by inhibitors of volume-sensitive Cl- channels such as 1,9-dideoxiforskolin, La3+, and arachidonate. Verapamil, a substrate of MDR-1, increased the osmotically evoked taurine efflux. Replacement of extracellular Cl- with I- or gluconate or of extracellular Na+ with Li+ significantly reduced the taurine efflux, whereas substitution of N-methyl-D-glucamine for Na+ increased it. Application of ATP and 2-chloroadenosine stimulated the efflux in isotonic medium. No differences were seen between 9HTEo- and 9HTEo-/Dx cells with respect to hypotonically induced taurine efflux and the response to phorbol ester, channel blockers, ion replacement, and purinergic agents. Our results reveal novel properties of the osmotically induced taurine release and demonstrate its independence from MDR-1 gene expression.
Cellular localization and regulation of CHIF in kidney and colon
Authors: Capurro C. , Coutry N. , Bonvalet J. P. , Escoubet B. , Garty H. , Farman N.
Published: December 2017

Channel inducing factor (CHIF) is a novel cDNA recently cloned from a rat distal colon cDNA library of dexamethasone-treated animals. While its expression in Xenopus oocytes evokes a potassium channel activity similar to that induced by Isk (minK), its cellular role is not clear. CHIF exhibits significant homologies with proteins that are putatively regulatory (phospholemman, gamma-subunit of Na(+)-K(+)-ATPase, Mat-8) while it differs from the small-conductance potassium channel Isk. We have studied the tissue specificity of CHIF expression in rat by in situ hybridization. CHIF is selectively present in the distal parts of the nephron (medullary and papillary collecting ducts and end portions of cortical collecting tubule) and in the epithelial cells of the distal colon. No expression of CHIF was found in renal proximal tubule, loop of Henle and distal tubule, proximal colon, small intestine, lung, choroid plexus, salivary glands, or brain. To gain some insight into CHIF function, we have investigated, using in situ hybridization and ribonuclease protection assay, whether CHIF mRNA expression could be altered in some situations. In the distal colon, corticosteroid hormones, sodium restriction, low-potassium diet, and metabolic acidosis significantly increased CHIF mRNA expression. In the kidney, metabolic acidosis was the only condition that showed an increase in CHIF mRNA expression. Some of these treatments also altered the expression of the colonic H(+)-K(+)-ATPase mRNA. In summary, CHIF mRNA is selectively expressed in the medullary collecting duct of the kidney and in the epithelium of the distal colon; its expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion, and metabolic acidosis. The precise cell-specific functions of CHIF remain to be established.
Effect of intracellular pH on force development depends on temperature in intact skeletal muscle from mouse
Authors: Wiseman R. W. , Beck T. W. , Chase P. B.
Published: December 2017

The cellular mechanism of muscle fatigue is still in debate. Opposite conclusions regarding the role of intracellular pH (pHi) in fatigue have been drawn from skinned fiber vs. isolated perfused muscle studies. Because these experiments are typically performed at different temperatures, we tested the hypothesis that temperature alters the effects of pH on force. Tetanic force of isolated mouse extensor digitorum longus was measured at temperatures between 13 and 25 degrees C in either normocapnia (5% CO2) or hypercapnia (25% CO2). Hypercapnia decreased pHi (monitored by 31P nuclear magnetic resonance spectroscopy) by the same amount at both 15 and 25 degrees C. However, inhibition of force by hypercapnia was greater at the lower temperature. A similar pattern of temperature-dependent inhibition of force by pH was observed in glycerinated fibers from rabbit psoas at maximum Ca2+ activation. We conclude that temperature differences are responsible for disparate conclusions on the role of pHi in muscle fatigue. Based on our results, we suggest that changes in pHi may have little or no role in the loss in force production associated with muscular fatigue at physiological temperatures.
pH dependence of neutrophil-endothelial cell adhesion and adhesion molecule expression
Authors: Serrano C. V. , Fraticelli A. , Paniccia R. , Teti A. , Noble B. , Corda S. , Faraggiana T. , Ziegelstein R. C. , Zweier J. L. , Capogrossi M. C.
Published: December 2017

Neutrophil adhesion to the vascular endothelium is enhanced during tissue ischemia and/or inflammation, conditions that are associated with tissue acidosis. This study examined the effects of hypercarbic acidosis (10 or 20% CO2) and of hypocarbic alkalosis (0% CO2) on human neutrophil CD18 and human aortic endothelial cell intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin expression quantified by flow cytometry. Acidosis with 20% CO2 for 4 h decreased ICAM-1 to 60.6 +/- 9.7% of control. In contrast, alkalosis with 0% CO2 for 4 h enhanced ICAM-1 expression to 143.8 +/- 10.1% of control. There was no pH dependence of VCAM-1 or E-selectin expression. Tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) increased endothelial ICAM-1, E-selectin, and VCAM-1; under these conditions, acidosis with 20% CO2 blunted both ICAM-1 and E-selectin surface expression compared with 5% CO2-, TNF-alpha-treated cells. Hypercarbic acidosis with 20% CO2 increased neutrophil CD18 expression and enhanced neutrophil adhesion. This latter effect was inhibited by neutrophil pretreatment with an anti-CD18 monoclonal antibody. In contrast, when only endothelial cells were preincubated with the hypercarbic buffer, neutrophil adhesion diminished to 55.6 +/- 7.8% of control. The results suggest that acidosis generated during tissue ischemia/inflammation may induce CD18-mediated neutrophil adhesion despite a decrease in ICAM-1 expression.
K(86Rb) transport heterogeneity in the low-density fraction of sickle cell anemia red blood cells
Authors: Etzion Z. , Lew V. L. , Bookchin R. M.
Published: December 2017

Previous studies have suggested ion transport heterogeneity among sickle cell anemia (SS) reticulocytes that could influence their dehydration susceptibility. We examined Ca2(+)-independent K transport in the lowest density (F1), reticulocyte-rich SS cells, measuring the effects of acidification, ouabain, and bumetanide on their unidirectional K(86Rb) fluxes. Unlike those of normal red blood cells and SS discocytes, the SS-F1 K(86Rb) fluxes were highly nonlinear, with large 5-min flux components (previously unobserved) and a more gradual decline over 60 min. Analysis revealed two distinct K pools: a rapid-turnover pool in a small fraction of cells, whose major ouabain-resistant K(86Rb) transport path showed distinctive properties including inhibition by high concentrations of bumetanide (> or = 1 mM) and stimulation at pH 7.0, and another heterogeneous, relatively slow-turnover pool, in most of the F1 cells, whose main ouabain-resistant K(86Rb) path was insensitive to bumetanide but was stimulated at pH 7.0, which is consistent with heterogeneous expression of the acid-sensitive K-Cl cotransport and with both rapid and slower generation of dehydrated SS cells.