bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids
Authors: Kunz-Schughart L. A. , Habbersett R. C. , Freyer J. P.
Published: December 2017

Two mitochondrion-specific fluorochromes, 10- N-nonyl acridine orange (NAO) and rhodamine 123 (Rh123), were used to determine the mechanism responsible for alterations in energy metabolism of transformed rat embryo fibroblast cells isolated from different locations within multicellular spheroids. Accumulation of Rh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thus represents mitochondrial distribution only. A reproducible selective dissociation procedure was used to isolate cells from different locations within the spheroids. After isolation, cells were simultaneously stained with one mitochondrial stain and the DNA dye Hoechst 33342, and several parameters, including cell volume, were monitored via multilaser-multiparameter flow cytometry. Our data clearly show a decrease in the uptake of Rh123 in cells from the periphery to the inner regions of the tumor spheroids, reflecting a persistent alteration in mitochondrial function. However, NAO staining experiments showed no reduction in the total mitochondrial mass per unit cell volume. Because cells were exposed to stain under uniform conditions after isolation from the spheroid, these data indicate that downregulation of mitochondrial function is associated with cell quiescence rather than a transient effect of reduced nutrient availability. This result, which is in accordance with data from two other cell lines (EMT6 and 9L), might reflect a general phenomenon in multicellular spheroids, supporting the hypothesis that quiescent cells in the innermost viable spheroid layer stably reduce their mitochondrial function, presumably to compensate for lower nutrient supply and/or decreased energy demand.
Effect of 17 days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers
Authors: Widrick J. J. , Romatowski J. G. , Bain J. L. W. , Trappe S. W. , Trappe T. A. , Thompson J. L. , Costill D. L. , Riley D. A. , Fitts R. H.
Published: December 2017

The purpose of this study was to examine the effect of prolonged bed rest (BR) on the peak isometric force (Po) and unloaded shortening velocity ( V o) of single Ca2+-activated muscle fibers. Soleus muscle biopsies were obtained from eight adult males before and after 17 days of 6° head-down BR. Chemically permeabilized single fiber segments were mounted between a force transducer and position motor, activated with saturating levels of Ca2+, and subjected to slack length steps. V owas determined by plotting the time for force redevelopment vs. the slack step distance. Gel electrophoresis revealed that 96% of the pre- and 87% of the post-BR fibers studied expressed only the slow type I myosin heavy chain isoform. Fibers with diameter >100 μm made up only 14% of this post-BR type I population compared with 33% of the pre-BR type I population. Consequently, the post-BR type I fibers ( n = 147) were, on average, 5% smaller in diameter than the pre-BR type I fibers ( n = 218) and produced 13% less absolute Po. BR had no overall effect on Po per fiber cross-sectional area (Po/CSA), even though half of the subjects displayed a decline of 9–12% in Po/CSA after BR. Type I fiber V oincreased by an average of 34% with BR. Although the ratio of myosin light chain 3 to myosin light chain 2 also rose with BR, there was no correlation between this ratio and V o for either the pre- or post-BR fibers. In separate fibers obtained from the original biopsies, quantitative electron microscopy revealed a 20–24% decrease in thin filament density, with no change in thick filament density. These results raise the possibility that alterations in the geometric relationships between thin and thick filaments may be at least partially responsible for the elevated V o of the post-BR type I fibers.
Site-specific thrombin receptor antibodies inhibit Ca2+ signaling and increased endothelial permeability
Authors: Nguyen Lan T. , Lum Hazel , Tiruppathì Chinnaswamy , Malik Asrar B.
Published: December 2017

Thrombin receptor is activated by thrombin-mediated cleavage of the receptor’s NH2 terminus between Arg-41 and Ser-42, generating a new NH2terminus that functions as a “tethered ligand” by binding to sites on the receptor. We prepared antibodies (Abs) directed against specific receptor domains to study the tethered ligand-receptor interactions required for signaling the increase in endothelial permeability to albumin. We used polyclonal Abs directed against the peptide sequences corresponding to the extracellular NH2 terminus [residues 70–99 (AbDD) and 1–160 (AbEE)] and extracellular loops 1 and 2 [residues 161–178 (AbL1) and 244–265 (AbL2)] of the seven-transmembrane thrombin receptor. Receptor activation was determined by measuring changes in cytosolic Ca2+ concentration ([Ca2+]i) in human dermal microvascular endothelial cells (HMEC) loaded with Ca2+-sensitive fura 2-acetoxymethyl ester dye. The transendothelial125I-labeled albumin clearance rate (a measure of endothelial permeability) was determined across the confluent HMEC monolayers. AbEE (300 μg/ml), directed against the entire extracellular NH2-terminal extension, inhibited the thrombin-induced increases in [Ca2+]iand the endothelial 125I-albumin clearance rate (>90% reduction in both responses). AbDD (300 μg/ml), directed against a sequence within the NH2-terminal extension, inhibited 70% of the thrombin-induced increase in [Ca2+]iand 60% of the increased125I-albumin clearance rate. AbL2 (300 μg/ml) inhibited these responses by 70 and 80%, respectively. However, AbL1 (300 μg/ml) had no effect on either response. We conclude that NH2-terminal extension and loop 2 are critical sites for thrombin receptor activation in endothelial cells and thus lead to increased [Ca2+]iand transendothelial permeability to albumin.
Regulation of Ca2+ handling by phosphorylation status in mouse fast- and slow-twitch skeletal muscle fibers
Authors: Liu Yewei , Kranias Evangelia G. , Schneider Martin F.
Published: December 2017

The effects of phosphorylation status on Ca2+release and Ca2+ removal were studied in fast-twitch flexor digitorum brevis and slow-twitch soleus skeletal muscle fibers enzymatically isolated from wild-type and phospholamban knockout (PLBko) mice. In all fibers the adenosine 3′,5′-cyclic monophosphate-dependent protein kinase (PKA) inhibitor H-89 decreased the peak amplitude of the intracellular Ca2+ concentration ([Ca2+]) transient for a single action potential, and the PKA activator dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP) reversed this effect, indicating modulation of Ca2+release by phosphorylation status in all fibers. H-89 decreased the decay rate constant of the [Ca2+] transient and DBcAMP reversed this effect only in phospholamban-expressing fibers (wild-type soleus), indicating modulation of Ca2+ removal only in the presence of phospholamban. A high basal level of PKA phosphorylation in soleus fibers maintained under our control conditions was indicated by the lack of effect of direct application of DBcAMP on Ca2+ release or Ca2+ removal in wild-type or PLBko soleus fibers and was confirmed by analysis of phospholamban from wild-type soleus fibers.
Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of86Rb+uptake by external K+
Authors: Tepperman Katherine , Millette Lauren A. , Johnson Carl L. , Jewell-Motz Elizabeth A. , Lingrel Jerry B. , Wallick Earl T.
Published: December 2017

A competition assay of86Rb+uptake in HeLa cells transfected with ouabain-resistant Na+-K+-ATPase mutants revealed a stimulation of86Rb+uptake at low external concentrations (1 mM) of competitor (K+). Of the models that were tested, those that require that two K+ be bound before transport occurs gave the worst fits. Random and ordered binding schemes described the data equally well. General models in which both binding and transport were allowed to be cooperative yielded parameter errors larger than the parameters themselves and could not be utilized. Models that assumed noncooperative transport always showed positive cooperativity in binding. E327Q and E327L mutated forms of rat α2 had lower apparent affinities for the first K+ bound than did wild-type rat α2 modified to be ouabain resistant. The mutations did not affect the apparent affinity of the second K+ bound. Models that assumed noncooperativity in binding always showed positively cooperative transport, i.e., enzymes with two K+ bound had a higher flux than those with one K+ bound. Increases in external Na+ decreased the apparent affinity for K+ for all models and decreased the ratio of the apparent influx rate constants for E327L.
Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells
Authors: Inoue M. , Imanaga I.
Published: December 2017

The effects of cyanide (CN) on whole cell current measured with the perforated-patch method were studied in adrenal medullary cells. Application of CN produced initially inward and then outward currents at −52 mV or more negative. As the membrane potential was hyperpolarized, amplitude and latency of the outward current ( I o) by CN became small and long, respectively. A decrease in the external Na+ concentration did not affect the latency for CN-induced I o but enhanced the amplitude markedly. The CN I o reversed polarity at −85 mV, close to the Nernst potential for K+, and was suppressed by the K+ channel blockers curare and apamin but not by glibenclamide, suggesting that I o is due to the activation of Ca2+-dependent K+ channels. Consistent with this notion, the Ca2+-mobilizing agents, muscarine and caffeine, also produced I o. Exposure to CN in a Ca2+-deficient medium for 4 min abolished caffeine- or muscarine-induced I o without development of I o, and addition of Ca2+ to the CN-containing solution induced I o. We conclude that exposure to CN produces Ca2+-dependent K+ currents in an external Ca2+-dependent manner, probably via facilitation of Ca2+ influx.
Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel
Authors: Devor Daniel C. , Frizzell Raymond A.
Published: December 2017

We used single-channel recording techniques to identify and characterize a large-conductance, Ca2+-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability ( P o) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel P o was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3←→C2←→C1←→O. Arachidonic acid (AA) increased the P o of the channel, with an apparent stimulatory constant ( K s) of 1.39 μM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased P oalso, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect P o. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 μM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl− secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.
Na+ influx and Na+-K+pump activation during short-term exposure of cardiac myocytes to aldosterone
Authors: Mihailidou Anastasia S. , Buhagiar Kerrie A. , Rasmussen Helge H.
Published: December 2017

To examine the effect of aldosterone on sarcolemmal Na+ transport, we measured ouabain-sensitive electrogenic Na+-K+pump current ( I p) in voltage-clamped ventricular myocytes and intracellular Na+ activity ([Formula: see text]) in right ventricular papillary muscles. Aldosterone (10 nM) induced an increase in both I p and the rate of rise of [Formula: see text] during Na+-K+pump blockade with the fast-acting cardiac steroid dihydroouabain. The aldosterone-induced increase in I p and rate of rise of [Formula: see text] was eliminated by bumetanide, suggesting that aldosterone activates Na+ influx through the Na+-K+-2Cl−cotransporter. To obtain independent support for this, the Na+, K+, and Cl− concentrations in the superfusate and solution of pipettes used to voltage clamp myocytes were set at levels designed to abolish the inward electrochemical driving force for the Na+-K+-2Cl−cotransporter. This eliminated the aldosterone-induced increase in I p. We conclude that in vitro exposure of cardiac myocytes to aldosterone activates the Na+-K+-2Cl−cotransporter to enhance Na+influx and stimulate the Na+-K+pump.
Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy
Authors: Word R. Ann , Cornwell Trudy L.
Published: December 2017

Increases in guanosine 3′,5′-cyclic monophosphate (cGMP) induced by nitric oxide (NO), nitrovasodilators, and atrial peptides correlate with relaxation of vascular smooth muscle. Relaxation of myometrial smooth muscle by increases in cGMP, however, has required unusually high concentrations of the cyclic nucleotide. We tested the hypothesis that the sensitivity of myometrium to relaxation by cGMP is increased during pregnancy. Aortic smooth muscle was more sensitive to relaxation by cGMP than myometrial tissues, and, contrary to our hypothesis, myometrium from pregnant rats was least sensitive. Although levels of cGMP were elevated after treatment with the NO donor, S-nitroso- N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels of cGMP-dependent protein kinase (PKG) were significantly decreased in myometrium from pregnant rats compared with myometrium from nonpregnant cycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, and progesterone antagonized this response. We conclude that 1) myometrial tissues from pregnant rats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP is accompanied by progesterone-mediated decreases in the level of PKG expression.
Regulation of Na+/H+exchanger gene expression: mitogenic stimulation increases NHE1 promoter activity
Authors: Besson Pierre , Fernandez-Rachubinski Francoise , Yang Weidong , Fliegel Larry
Published: December 2017

We examined factors important in regulation of expression of the Na+/H+exchanger gene in NIH/3T3 cells. A stable fibroblast cell line was generated that contained a 1.1-kb proximal fragment of the mouse NHE1 promoter. The addition of serum to serum-starved cells resulted in an increase in activity of the NHE1 promoter. The mitogenic agonists insulin, thrombin, and epidermal growth factor also increased transcription from the NHE1 promoter. Phorbol esters also increased NHE1 promoter-directed transcription, whereas the serine/threonine protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine inhibited this stimulation. The protein kinase inhibitors GF-109203X, PD-98059, and genistein all stimulated promoter activity. Promoter deletion analysis and gel mobility shift assays showed that a region between 0.9 and 1.1 kb from the start site was involved in mediating the effect of mitogenic stimulation. The results show that a variety of mitogenic factors can activate the NHE1 promoter during cell growth and proliferation.
Letters to the Editor
Authors: Wagner Alexandra , Boos Joachim
Published: December 2017

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

Hutson, Richard G., Toshiyuki Kitoh, David A. Moraga Amador, Sanja Cosic, Sheldon M. Schuster, and Michael S. Kilberg.Amino acid control of asparagine synthetase: relation to asparaginase resistance in human leukemia cells. Am. J. Physiol. 272 ( Cell Physiol. 41): C1691–C1699, 1997.—Complete amino acid deprivation in mammalian cells causes a significant enhancement in gene expression for a number of important cellular activities; among these is asparagine synthetase (AS). The data presented demonstrate that, in both nonleukemic (rat Fao hepatoma cells) and human leukemia cells (MOLT-4, NALL-1, and BALL-1), AS mRNA levels, protein content, and enzymatic activity are induced after incubation in an otherwise complete tissue culture medium that is deficient in a single amino acid or in medium that has been depleted of the amino acid asparagine by the addition of asparaginase. Complete amino acid deprivation results in a concerted increase in AS mRNA, protein, and enzymatic activity, which, in conjunction with previously published research, suggests that the mechanism of this cellular response involves transcriptional control of the AS gene. Asparaginase treatment is a standard component of acute lymphoblastic leukemia therapy for which the effectiveness is related to the inability of these cells to upregulate AS activity to a sufficient level. With regard to the asparaginase sensitivity of the three human leukemia cell lines, there was a trend toward an inverse relation to the degree of AS expression. Selection for asparaginase-resistant MOLT-4 sublines resulted in enhanced AS mRNA and protein content regardless of whether the cells had been selected by asparaginase treatment directly or asparagine was removed from the culture medium. Collectively, the data illustrate that further advances in asparaginase therapy will require additional knowledge of amino acid-dependent regulation of AS gene expression and, conversely, that asparaginase resistance represents a model system for investigating metabolite control in a clinically relevant setting.
Actions of substance P on membrane potential and ionic currents in guinea pig stellate ganglion neurons
Authors: Gilbert Robert , Ryan Jennifer S. , Horackova Magda , Smith Frank M. , Kelly Melanie E. M.
Published: December 2017

Neuropeptides are known to modulate the excitability of mammalian sympathetic neurons by their actions on various types of K+ and Ca2+ channels. We used whole cell patch-clamp recording methods to study the actions of substance P (SP) on dissociated adult guinea pig stellate ganglion (SG) neurons. Under current-clamp conditions, SG neurons exhibited overshooting action potentials followed by afterhyperpolarizations (AHP). The K+ channel blocker tetraethylammonium (1 mM), the Ca2+ channel blocker Cd2+ (0.1–0.2 mM), and SP (500 nM) depolarized SG neurons, decreased the AHP amplitude, and increased the action potential duration. In the presence of Cd2+, the effect of SP on membrane potential and AHP was reduced. Under voltage-clamp conditions, several different K+ currents were observed, including a transient outward K+ conductance and a delayed rectifier outward K+ current ( I K) consisting of Ca2+-sensitive [ I K(Ca)] and Ca2+-insensitive components. SP (500 nM) inhibited I K. Pretreatment with Cd2+ (20–200 μM) or the high-voltage-activated Ca2+ channel blocker ω-conotoxin (10 μM) blocked SP’s inhibitory effects on I K. This suggests that SP reduces I K primarily through the inhibition of I K(Ca) and that this may occur, in part, via a reduction of Ca2+ influx through voltage-dependent Ca2+ channels. SP’s actions on I K were mediated by a pertussis toxin-insensitive G protein(s) coupled to NK1 tachykinin receptors. Furthermore, we have confirmed that 500 nM SP reduced an inward Cd2+- and ω-conotoxin-sensitive Ba2+ current in SG neurons. Thus the actions of SP on I K(Ca) may be due in part to a reduction in Ca2+influx occurring via N-type Ca2+channels. This study presents the first description of ionic currents in mammalian SG neurons and demonstrates that SP may modulate excitability in SG neurons via inhibitory actions on K+ and Ca2+ currents.
Nerve growth factor regulates HCO 3 − absorption in thick ascending limb: modifying effects of vasopressin
Authors: Good David W. ,   (With the Technical Assistance of Thampi George)
Published: December 2017

Growth factors stimulate Na+/H+exchange activity in many cell types but their effects on acid secretion via this mechanism in renal tubules are poorly understood. We examined the regulation of [Formula: see text]absorption by nerve growth factor (NGF) in the rat medullary thick ascending limb (MTAL), which absorbs [Formula: see text]via apical membrane Na+/H+exchange. MTAL were perfused in vitro with 25 mM[Formula: see text] solutions (pH 7.4; 290 mosmol/kgH2O). Addition of 0.7 nM NGF to the bath decreased [Formula: see text]absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol ⋅ min−1 ⋅ mm−1( P < 0.001). In contrast, with 10−10 M arginine vasopressin (AVP) in the bath, addition of NGF to the bath increased[Formula: see text] absorption from 8.0 ± 1.6 to 12.5 ± 1.3 pmol ⋅ min−1 ⋅ mm−1( P < 0.01). Both effects of NGF were blocked by genistein, consistent with the involvement of tyrosine kinase pathways. However, the AVP-dependent stimulation required activation of protein kinase C (PKC), whereas the inhibition was PKC independent, indicating that the NGF-induced signaling pathways leading to inhibition and stimulation of [Formula: see text]absorption are distinct. Hypertonicity blocked the inhibition but not the AVP-dependent stimulation, suggesting that hypertonicity and NGF may inhibit [Formula: see text] absorption via a common mechanism. These data demonstrate that NGF inhibits[Formula: see text] absorption in the MTAL under basal conditions but stimulates [Formula: see text]absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is a critical determinant of the response of the MTAL to growth factor stimulation and suggest that NGF can either inhibit or stimulate apical Na+/H+ exchange activity depending on its interactions with other regulatory factors. Locally produced growth factors such as NGF may play a role in regulating renal tubule [Formula: see text] absorption.
Inhibition of Ca2+-dependent Cl− secretion in T84 cells: membrane target(s) of inhibition is agonist specific
Authors: Barrett Kim E. , Smitham Jane , Traynor-Kaplan Alexis , Uribe Jorge M.
Published: December 2017

Previous studies have indicated that Ca2+-dependent Cl− secretion across monolayers of T84 epithelial cells is subject to a variety of negative influences that serve to limit the overall extent of secretion. However, the downstream membrane target(s) of these inhibitory influences had not been elucidated. In this study, nuclide efflux techniques were used to determine whether inhibition of Ca2+-dependent Cl− secretion induced by carbachol, inositol 3,4,5,6-tetrakisphosphate, epidermal growth factor, or insulin reflected actions at an apical Cl− conductance, a basolateral K+ conductance, or both. Pretreatment of T84 cell monolayers with carbachol or a cell-permeant analog of inositol 3,4,5,6-tetrakisphosphate reduced the ability of subsequently added thapsigargin to stimulate apical125I−, but not basolateral86Rb+, efflux. These data suggested an effect on an apical Cl− channel. Conversely, epidermal growth factor reduced Ca2+-stimulated86Rb+but not125I−efflux, suggesting an effect of the growth factor on a K+ channel. Finally, insulin inhibited125I−and86Rb+effluxes. Thus effects of agents that inhibit transepithelial Cl− secretion are also manifest at the level of transmembrane transport pathways. However, the precise nature of the membrane conductances targeted are agonist specific.
Hormonal regulation of ENaCs: insulin and aldosterone
Authors: Blazer-Yost Bonnie L. , Liu Xuehong , Helman Sandy I.
Published: December 2017

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current ( i Na), channel open probability ( P o), and functional channel density ( N T) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10–30 min the density of the pool of apical membrane ENaCs ( N T) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of i Na(10–20%) and decreases of P o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of i Na, P o, and N T were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.
Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases
Authors: Luo Jiexin , Pato Mary D. , Riordan John R. , Hanrahan John W.
Published: December 2017

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by >90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability ( P o). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating; P o declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.
Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle
Authors: Prakash Y. S. , Kannan Mathur S. , Walseth Timothy F. , Sieck Gary C.
Published: December 2017

The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with β-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM–10 μM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 μM) and by the RyR blockers ruthenium red (10 μM) and ryanodine (10 μM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]ioscillations induced by acetylcholine (ACh), addition of 100 nM and 1 μM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]ioscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]iresponse to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]ioscillations.
Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle
Authors: Suzuki Yosuke , Shen Tiansheng , Poyard Madeleine , Best-Belpomme Martin , Hanoune Jacques , Defer Nicole
Published: December 2017

Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.
Na+-K+-2Cl−cotransport in Ehrlich cells: regulation by protein phosphatases and kinases
Authors: Krarup Thomas , Jakobsen Lene D. , Jensen Bo S. , Hoffmann Else K.
Published: December 2017

To identify protein kinases (PK) and phosphatases (PP) involved in regulation of the Na+-K+-2Cl−cotransporter in Ehrlich cells, the effect of various PK and PP inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin A (Cal-A) was a potent activator of Na+-K+-2Cl−cotransport (EC50 = 35 nM). Activation by Cal-A was rapid (<1 min) but transient. Inactivation is probably due to a 10% cell swelling and/or the concurrent increase in intracellular Cl− concentration. Cell shrinkage also activates the Na+-K+-2Cl−cotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation with Cal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH and Ca2+/calmodulin dependent. Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA by H-89 and KT-5720 inhibited cotransport activity induced by Cal-A and by cell shrinkage, with IC50 values similar to reported inhibition constants of the respective kinases in vitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of the Na+-K+-2Cl−cotransport activity during regulatory volume increase.
Interferon-α and interferon-γ differentially affect pancreatic β-cell phenotype and function
Authors: Baldeón Manuel E. , Chun Taehoon , Gaskins H. Rex
Published: December 2017

To better clarify individual roles of interferon (IFN)-α and IFN-γ in β-cell pathology during the onset of type 1 diabetes mellitus, we compared the effects of these cytokines on insulin production and major histocompatibility complex (MHC) gene expression in pancreatic β-cell lines. IFN-γ but not IFN-α decreased secreted and intracellular insulin concentrations in βTC6-F7 and βTC3 cells. Likewise, IFN-γ but not IFN-α treatment of β-cells upregulated mRNA expression of MHC class IA antigen-processing genes and surface expression of class IA molecules. Alternatively, class IA MHC expression was upregulated by IFN-γ and IFN-α in the P388D1 macrophage cell line. The observation of constitutive Ifn-α6mRNA expression by a differentiated β-cell line substantiates previous indications that local expression of IFN-α in islets may trigger insulitis. Evidence that IFN-γ, a product of infiltrating leukocytes, directly decreases β-cell glucose sensitivity and increases MHC class IA cell surface expression supports the postulate that IFN-γ magnifies the insulitic process.
Overexpression of protein kinase C-δ increases tight junction permeability in LLC-PK1epithelia
Authors: Mullin James M. , Kampherstein Jennifer A. , Laughlin Kathleen V. , Clarkin Cheryl E. K. , Miller R. Daniel , Szallasi Zoltan , Kachar Bechara , Soler Alejandro Peralta , Rosson Dan
Published: December 2017

The Ca2+-independent δ-isoform of protein kinase C (PKC-δ) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-δ is expressed. Western immunoblots show that the overexpressed PKC-δ is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-δ produced subconfluent and confluent epithelial morphologies similar to that observed on exposure of wild-type cells to the phorbol ester 12- O-tetradecanoylphorbol-13-acetate. Transepithelial electrical resistance ( R T) in cell sheets overexpressing PKC-δ was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-δ and R Twere similar to those in LLC-PK1parental cell sheets. Overexpression of PKC-δ also elicited a significant increase in transepithelial flux ofd-[14C]mannitol and a radiolabeled 2 × 106-molecular-weight dextran, suggesting with the R T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-δ overexpression results in a multilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-δ results in a more disorganized arrangement of tight junctional strands. As with LLC-PK1 cell sheets treated with 12- O-tetradecanoylphorbol-13-acetate, the reduced R T, increasedd-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-δ overexpression suggest the involvement of PKC-δ in regulation of tight junctional permeability.
Conductive pathways for chloride and oxalate in rabbit ileal brush-border membrane vesicles
Authors: Freel Robert W. , Hatch Marguerite , Vaziri N. D.
Published: December 2017

To evaluate the possibility that an apical membrane conductive pathway for oxalate is present in the rabbit distal ileum, we studied oxalate ([14C]oxalate) and chloride (36Cl) uptake into brush-border membrane vesicles enriched 15- to 18-fold in sucrase activity. Voltage-sensitive pathways for oxalate and chloride were identified by the stimulation of uptake provided by an inwardly directed potassium diffusion potential in the presence of valinomycin. Additionally, outwardly directed oxalate (or chloride) gradients stimulated [14C]oxalate (or36Cl) uptake to a greater degree in the absence of valinomycin (when intracellular and extracellular potassium are equal) than in the presence of valinomycin. Voltage-dependent anion uptake was poorly saturable: apparent affinity constants were 141 ± 17 and 126 ± 8 mM for chloride and oxalate, respectively. Activation energies for the voltage-dependent uptake processes were low: 4.7 and 6.3 kcal/mol for chloride and oxalate, respectively. Sensitivity profiles of voltage-dependent chloride and oxalate uptake to anion transport inhibitors were similar. We conclude that an anion conductance is present in the apical membranes of ileal enterocytes and that this conductance is a candidate pathway for oxalate efflux from the enterocyte during transepithelial oxalate secretion.
Interstitial ATP level and degradation in control and postmyocardial infarcted rats
Authors: Kuzmin Alexander I. , Lakomkin Vladimir L. , Kapelko Valeri I. , Vassort Guy
Published: December 2017

With the aim of estimating interstitial levels and the breakdown process of ATP, cardiac microdialysis was performed in the left ventricular wall of in situ control and postinfarcted as well as of isolated, Langendorff-perfused rat hearts. With the use of a bioluminescence technique for dialysate ATP measurement, the baseline interstitial fluid ATP concentration in in situ hearts was estimated to be 38 ± 8 nM. Regional ischemia induced an early peak increase in interstitial fluid ATP to 373 ± 73 nM that correlates with the maximal incidence of ventricular arrhythmias. During continuous infusion of individual adenine nucleotides (50 μM ATP, ADP, or AMP), the dialysate samples were analyzed for adenine nucleotides, nucleosides, and bases using HPLC with ultraviolet detection. The patterns of catabolites were consistent with the major pathway of metabolism, that is, sequential dephosphorylation catalyzed by a chain of separate ecto-nucleotidases. In in situ postinfarcted hearts as well as in perfused hearts, a reduced catabolism rate of extracellular adenine nucleotides was observed. In conclusion, in in situ rat hearts, ATP can be released in substantial amounts in the interstitium where it readily undergoes enzymatic degradation. Dephosphorylation occurs sequentially and faster in in situ control hearts than in in situ postinfarcted or in perfused hearts.
Mechanism of depression in cardiac sarcolemmal Na+-K+-ATPase by hypochlorous acid
Authors: Kato Kiminori , Shao Qiming , Elimban Vijayan , Lukas Anton , Dhalla Naranjan S.
Published: December 2017

Oxidative stress during pathological conditions such as ischemia-reperfusion is known to promote the formation of hypochlorous acid (HOCl) in the heart and to result in depression of cardiac sarcolemmal (SL) Na+-K+-ATPase activity. In this study, we examined the direct effects of HOCl on SL Na+-K+-ATPase from porcine heart. HOCl decreased SL Na+-K+-ATPase activity in a concentration- and time-dependent manner. Characterization of Na+-K+-ATPase activity in the presence of different concentrations of MgATP revealed a decrease in the maximal velocity ( V max) value, without a change in affinity for MgATP on treatment of SL membranes with 0.1 mM HOCl. The V max value of Na+-K+-ATPase, when determined in the presence of different concentrations of Na+, was also decreased, but affinity for Na+ was increased when treated with HOCl. Formation of acylphosphate by SL Na+-K+-ATPase was not affected by HOCl. Scatchard plot analysis of [3H]ouabain binding data indicated no significant change in the affinity or maximum binding capacity value for ouabain binding following treatment of SL membranes with HOCl. Western blot analysis of Na+-K+-ATPase subunits in HOCl-treated SL membranes showed a decrease (34 ± 9% of control) in the β1-subunit without any change in the α1- or α2-subunits. These data suggest that the HOCl-induced decrease in SL Na+-K+-ATPase activity may be due to a depression in the β1-subunit of the enzyme.
Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel
Authors: Jensen Bo Skaaning , Strøbæk Dorte , Christophersen Palle , Jørgensen Tino Dyhring , Hansen Claus , Silahtaroglu Asli , Olesen Søren-Peter , Ahring Philip Kiær
Published: December 2017

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at −100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability ( P K/ P X) sequence K+ = Rb+ (1.0) > Cs+ (10.4) ≫ Na+, Li+, N-methyl-d-glucamine (>51).[Formula: see text] blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 μM), and cetiedil (IC50 79 μM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 μM.
Dichotomous development of the organic anion transport protein in liver and choroid plexus
Authors: Angeletti Ruth Hogue , Bergwerk Ari J. , Novikoff Phyllis M. , Wolkoff Allan W.
Published: December 2017

Both adult liver and choroid plexus express the organic anion transport protein (oatp1) and transport [35S]bromosulfophthalein (BSP). Studies of the developing rat liver reveal that oatp1 mRNA and protein do not begin to be expressed until 15 days postnatal and are at adult levels by 30 days. Uptake of [35S]BSP follows the same time course. In contrast, neonatal rat choroid plexus expresses oatp1 mRNA and protein. When quantified on a weight basis, the uptake of [35S]BSP in choroid plexus is lower in the adult than at earlier stages of development. Although fluorescence confocal microscopy of adult rat choroid plexus shows that oatp is localized to the apical surface, facing the cerebrospinal fluid, this method reveals an intracellular localization of oatp1 in the neonate. Approximately 12 wk are required for the appearance of the adult pattern of distribution. Changes in the localization and activity of oatp1 during development could play an important role in the pathobiology of maturation of the liver and the central nervous system.
Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH
Authors: French Stephanie A. , Territo Paul R. , Balaban Robert S.
Published: December 2017

Fluorescent determinations of NADH in porcine heart mitochondria were subject to significant errors caused by alterations in inner filter effects during numerous metabolic perturbations. These inner filter effects were primarily associated with changes in mitochondrial volume and accompanying light scattering. The observed effects were detected in a standard commercial fluorometer with emission orthogonal to the excitation light path and, to a lesser extent, in a light path geometry detecting only the surface fluorescence. A method was developed to detect and correct for inner filter effects on mitochondrial NADH fluorescence measurements that were independent of the optical path geometry using an internal fluorescent standard and linear least-squares spectral analysis. A simple linear correction with the inner fluorescence reference was found to adequately correct for inner filter effects. This approach may be useful for other fluorescence probes in isolated mitochondria or other light-scattering media.
Ascorbate and glutathione homeostasis in vascular smooth muscle cells: cooperation with endothelial cells
Authors: Voskoboinik Ilia , Söderholm Karin , Cotgreave Ian A.
Published: December 2017

Human umbilical vein smooth muscle cells (HUVSMCs) utilize extracellular cystine, glutathione (GSH), and N-acetylcysteine (NAC) to synthesize cellular GSH. Extracellular cystine was effective from 5 μM, whereas GSH and NAC were required at 100 μM for comparable effects. The efficacy of extracellular GSH was dependent on de novo GSH synthesis, indicating a dependence on cellular γ-glutamyltransferase (glutamyl transpeptidase). Coculture of syngenetic HUVSMCs and corresponding human umbilical vein endothelial cells (HUVECs) on porous supports restricted cystine- or GSH-stimulated synthesis of HUVSMC GSH when supplied on the “luminal” endothelial side. Thus HUVSMC GSH rapidly attained a steady-state level below that achieved in the absence of interposed HUVECs. HUVSMCs also readily utilize both reduced ascorbate (AA) and oxidized dehydroascorbate (DHAA) over the range 50–500 μM. Phloretin effectively blocked both AA- and DHAA-stimulated assimilation of intracellular AA, indicating a role for a glucose transporter in their transport. Uptake of extracellular AA was also sensitive to extracellular, but not intracellular, thiol depletion. When AA was applied to the endothelial side of the coculture model, assimilation of intracellular AA in HUVSMCs was restricted to a steady-state level below that achieved by free access.
Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver
Authors: Cefaratti Christie , Romani Andrea , Scarpa Antonio
Published: December 2017

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.