bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuronal swelling and surface area regulation: elevated intracellular calcium is not a requirement
Authors: Herring T. L. , Slotin I. M. , Baltz J. M. , Morris C. E.
Published: December 2017

Neurons are mechanically robust. During prolonged swelling, molluscan neurons can triple their apparent membrane area. They gain surface area and capacitance independent of extracellular Ca concentration ([Ca]e), but it is unknown if an increase in intracellular Ca concentration ([Ca]i) is necessary. If Ca for stimulating exocytosis is unnecessary, it is possible that swelling-induced membrane tension changes directly trigger surface area readjustments. If, however, Ca-mediated but not tension-mediated membrane recruitment is responsible for surface area increases, swelling neurons should sustain elevated levels of [Ca]i. The purpose of this investigation is to determine if the [Ca]i in swelling neurons attains levels high enough to promote exocytosis and if any such increase is required. Lymnaeaneurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 μM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min) were done at either a standard [Ca]e or very low [Ca]e level (0.9 mM or 0.13 μM, respectively). In neither case did the [Ca]i increase to levels that drive exocytosis. We also monitored osmomechanically driven membrane dynamics [swelling, then formation and reversal of vacuole-like dilations (VLDs)] with the [Ca]i clamped below 40 nM via 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA). [Ca]idid not change with swelling, and VLD behavior was unaffected, consistent with tension-driven, [Ca]i-independent surface area adjustments. In addition, neurons with [Ca]i clamped at 0.1 μM via an ionophore could produce VLDs. We conclude that, under mechanical stress, neuronal membranes are compliant by virtue of surface area regulatory adjustments that operate independent of [Ca]i. The findings support the hypothesis that plasma membrane area is regulated in part by membrane tension.
State of actin in gastric parietal cells
Authors: Forte John G. , Ly Bernice , Rong Qinfen , Ogihara Shoji , Ramilo Marlon , Agnew Brian , Yao Xuebiao
Published: December 2017

Remodeling of the apical membrane-cytoskeleton has been suggested to occur when gastric parietal cells are stimulated to secrete HCl. The present experiments assayed the relative amounts of F-actin and G-actin in gastric glands and parietal cells, as well as the changes in the state of actin on stimulation. Glands and cells were treated with a Nonidet P-40 extraction buffer for separation into detergent-soluble (supernatant) and detergent-insoluble (pellet) pools. Two actin assays were used to quantitate actin: the deoxyribonuclease I binding assay to measure G-actin and F-actin content in the two pools and a simple Western blot assay to quantitate the relative amounts of actin in the pools. Functional secretory responsiveness was assayed by aminopyrine accumulation. About 5% of the total parietal cell protein is actin, with about 90% of the actin present as F-actin. Stimulation of acid secretion resulted in no measurable change in the relative amounts of G-actin and cytoskeletal F-actin. Treatment of gastric glands with cytochalasin D inhibited acid secretion and resulted in a decrease in F-actin and an increase in G-actin. No inhibition of parietal cell secretion was observed when phalloidin was used to stabilize actin filaments. These data are consistent with the hypothesis that microfilamentous actin is essential for membrane recruitment underlying parietal cell secretion. Although the experiments do not eliminate the importance of rapid exchange between G- and F-actin for the secretory process, the parietal cell maintains actin in a highly polymerized state, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.
Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells
Authors: Wendt Christine H. , Towle Howard , Sharma Renu , Duvick Sara , Kawakami Kiyoshi , Gick Gregory , Ingbar David H.
Published: December 2017

Na-K-ATPase plays a central role in a variety of physiological processes, including ion transport and regulation of cell volume. Our previous data showed that hyperoxia increased the expression of Na-K-ATPase α1 and β1 mRNA in lung type II cells. We similarly show that hyperoxia (≥95% O2 for 24–48 h) increased steady-state mRNA levels in both Na-K-ATPase subunits in Madin-Darby canine kidney (MDCK) cells. The mechanism of gene regulation by hyperoxia was assessed. Stability of the Na-K-ATPase mRNA levels of both subunits was unchanged in hyperoxia-exposed MDCK cells. To determine whether gene transcription was augmented by hyperoxia, MDCK cells were transfected with a β1-subunit promoter-reporter construct. Transfection with the wild-type promoter (β1-817) revealed a 1.9 ± 0.2-fold increase in promoter activity. Transfection with 5′ deletion constructs identified a 61-base pair (bp) region between −102 and −41 that was necessary for this increase in promoter activity by hyperoxia. Incorporation of this 61-bp region into a minimal promoter (mouse mammary tumor virus) similarly increased promoter activity 2.3-fold in the presence of hyperoxia. This increase in promoter activity was not seen when MDCK cells were incubated with various concentrations of hydrogen peroxide. In summary, hyperoxia increased Na-K-ATPase β1-subunit mRNA steady-state level due to increased transcription in MDCK cells. A region necessary for this hyperoxic effect on β1 transcription is located between base pairs −102 and −41 on the promoter.
Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells
Authors: Tyagi Suresh C.
Published: December 2017

Dynamic changes in the reduction-oxidation (redox) state of the tissue lead to the pathophysiological condition. Reduced homocysteine causes dysfunctions in endothelium. The proliferation of smooth muscle cells may lead to occlusive vascular disease, ischemia, and heart failure, but whether fibrosis and hypertension are a consequence of smooth muscle proliferation is unclear. Redox changes during hyperhomocyst(e)inemia may be one of the causes of premature atherosclerotic heart disease. To examine the effect of homocystine on human vascular smooth muscle cells (HVSMC), we isolated HVSMC from idiopathic dilated cardiomyopathic hearts. Coronaries in these hearts were apparently normal. HVSMC numbers in culture were measured by hemocytometer in the presence and absence of homocystine. Results show that homocystine induced cellular proliferation. This proliferation was reversed by the addition of the antioxidant N-acetylcysteine (NAC). Homocystine induces collagen expression in a dose- and time-dependent manner, as measured by Northern blot (mRNA) analysis. The 50% inhibitory concentration of 5 μM for collagen was estimated. The induction of collagen was reversed by the addition of NAC and reduced glutathione. To localize the receptor for homocystine on HVSMC, we synthesized fluorescamine-labeled homocystine conjugate. Incubation of labeled homocystine with HVSMC demonstrated membrane and cytosol localization of homocystine binding. The receptor-ligand binding was disrupted by NAC. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography, we observed a 40- to 25-kDa homocystine redox receptor in HVSMC. Our results suggested that the redox homocysteine induces HVSMC proliferation by binding to the redox receptor and may exacerbate atherosclerotic lesion formation by inducing collagen expression.
Metabolic fluctuation during a muscle contraction cycle
Authors: Chung Youngran , Sharman Robert , Carlsen Richard , Unger Steven W. , Larson Douglas , Jue Thomas
Published: December 2017

Gated31P-nuclear magnetic resonance followed the metabolic fluctuation in rat gastrocnemius muscle during a contraction cycle. Within 16 ms after stimulation, the phosphocreatine (PCr) level drops 11.3% from its reference state. The PCr minimum corresponds closely to the time of maximum force contraction. Pi increases stoichiometrically, while ATP remains constant. During a twitch, PCr hydrolysis produces 3.1 μmol ATP/g tissue, which is substantially higher than the reported 0.3 μmol ATP ⋅ twitch−1 ⋅ g tissue−1 derived from steady-state experiments. The results reveal that a substantial energy fluctuation accompanies a muscle twitch.
A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells
Authors: Lytle Christian
Published: December 2017

When Na-K-2Cl cotransport is activated in duck red blood cells by either osmotic cell shrinkage, norepinephrine, fluoride, or calyculin A, phosphorylation of the transporter occurs at a common set of serine/threonine sites. To examine the kinetics and regulation of the activating kinase, phosphatase activity was inhibited abruptly with calyculin A and the subsequent changes in transporter phosphorylation and activity were determined. Increases in fractional incorporation of 32P into the transporter and uptake of 86Rb by the cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in this manner, the activating kinase was 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, staurosporine, or N-ethylmaleimide, and 3) unaffected by norepinephrine or fluoride. The inhibitory effect of swelling on kinase activity was progressively relieved by calyculin A, suggesting that the kinase itself is switched on by phosphorylation. The kinetics of activation by calyculin A conformed to an autocatalytic model in which the volume-sensitive kinase is stimulated by a product of its own reaction (e.g., via autophosphorylation).
Time-dependent modulation of capacitative Ca2+ entry signals by plasma membrane Ca2+ pump in endothelium
Authors: Klishin Andrey , Sedova Marina , Blatter Lothar A.
Published: December 2017

In vascular endothelial cells, depletion of intracellular Ca2+ stores elicited capacitative Ca2+ entry (CCE) that resulted in biphasic changes of intracellular Ca2+ concentration ([Ca2+]i) with a rapid initial peak of [Ca2+]ifollowed by a gradual decrease to a sustained plateau level. We investigated the rates of Ca2+entry, removal, and sequestration during activation of CCE and their respective contributions to the biphasic changes of [Ca2+]i. Ca2+ buffering by mitochondria, removal by Na+/Ca2+exchange, and a fixed electrical driving force for Ca2+ (voltage-clamp experiments) had little effect on the CCE signal. The rates of entry of Mn2+ and Ba2+, used as unidirectional substitutes for Ca2+ entry through the CCE pathway, were constant and did not follow the concomitant changes of [Ca2+]i. Pharmacological inhibition of the plasma membrane Ca2+ pump, however, abolished the secondary decay phase of the CCE transient. The disparity between the biphasic changes of [Ca2+]iand the constant rate of Ca2+entry during CCE was the result of a delayed, Ca2+-dependent activation of the pump. These results suggest an important modulatory role of the plasma membrane Ca2+ pump in the net cellular gain of Ca2+ during CCE.
Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease
Authors: Lin King-Teh , Xue Ji-Yan , Lin Marie C. , Spokas Eric G. , Sun Frank F. , Wong Patrick Y.-K.
Published: December 2017

Apoptosis is an active process critical for the homeostasis of organisms. Enzymes of the caspase family are responsible for executing this process. We have previously shown that peroxynitrite (ONOO−), a biological product generated from the interaction of nitric oxide and superoxide, induces apoptosis of HL-60 cells. The aim of this study was to elucidate the mechanisms involved in the execution process of peroxynitrite-induced apoptosis. Proteolytic cleavage of poly(ADP-ribose) polymerase, an indication of caspase-3 family protease activation and an early biochemical event accompanying apoptosis, was observed in a time-dependent manner during peroxynitrite-induced apoptosis of HL-60 cells. Activation of caspase-3 during peroxynitrite-induced apoptosis was substantiated by monitoring proteolysis of the caspase-3 proenzyme and by measuring caspase-3 activity with a fluorogenic substrate. Furthermore, pretreatment of HL-60 cells with N-acetyl-Asp-Glu-Val-Asp-aldehyde, a specific inhibitor of caspase-3, but not N-acetyl-Tyr-Val-Ala-Asp-aldehyde, a specific inhibitor of caspase-1, decreased peroxynitrite-induced apoptosis. These results suggest that the activation of a caspase-3 family protease is essential for initiating the execution process of peroxynitrite-induced apoptosis of HL-60 cells.
Basal chloride currents in murine airway epithelial cells: modulation by CFTR
Authors: Tarran R. , Gray M. A. , Evans M. J. , Colledge W. H. , Ratcliff R. , Argent B. E.
Published: December 2017

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl− conductances and, under certain conditions, a small Na+conductance. Voltage-clamp experiments revealed three distinct Cl− conductances. I tv-indep was time and voltage independent with a linear current-voltage ( I- V) plot; I v-actexhibited activation at potentials greater than ±50 mV, giving an S-shaped I- Vplot; and I hyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I- Vplot. The current density sequence was I hyp-act = I v-act ≫ I tv-indep. These conductances had Cl−-to- N-methyl-d-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. I tv-indep and I v-act were present in wild-type and CF cells at equal density and frequency. However, I hyp-actwas detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).
Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata
Authors: Fong Peying , Rehfeldt Annett , Jentsch Thomas J.
Published: December 2017

Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel (ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of “ball” structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.
Neuropeptide Y inhibition of calcium channels in PC-12 pheochromocytoma cells
Authors: McCullough Laura A. , Egan Terrance M. , Westfall Thomas C.
Published: December 2017

We previously demonstrated, using rat PC-12 pheochromocytoma cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF), that neuropeptide Y (NPY) inhibits catecholamine synthesis as well as release. Inquiry into the mechanisms of these inhibitions implicated distinct pathways involving reduction of Ca2+ influx through voltage-activated Ca2+ channels. In the present investigation the effects of NPY on whole cell Ba2+ currents were examined to obtain direct evidence supporting the mechanisms suggested by those studies. NPY was found to inhibit the voltage-activated Ba2+ current in NGF-differentiated PC-12 cells in a reversible fashion with an EC50 of 13 nM. This inhibition was pertussis toxin sensitive and resulted from NPY modulation of L- and N-type Ca2+ channels. The inhibition of L-type channels was not seen with <1 nM free intracellular Ca2+ or when protein kinase C (PKC) was inhibited by chelerythrine or PKC-(19—31). Furthermore, the effect of NPY on L-type channels was mimicked by the PKC activator phorbol 12-myristate 13-acetate. These studies demonstrate that, in addition to inhibition of N-type Ca2+ channels, in NGF-differentiated PC-12 cells NPY inhibits L-type Ca2+ channels via an intracellular Ca2+- and PKC-dependent pathway.
Contractile activity-induced adaptations in the mitochondrial protein import system
Authors: Takahashi Mark , Chesley Alan , Freyssenet Damien , Hood David A.
Published: December 2017

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4- to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.
Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+exchange
Authors: Joiner C. H. , Jiang M. , Fathallah H. , Giraud F. , Franco R. S.
Published: December 2017

KCl cotransport activated by swelling of sickle red blood cells (SS RBC) is inhibited by deoxygenation. Yet recent studies found a Cl−-dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl−-dependent component of the deoxygenation-induced net K+efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl−-dependent K+ efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+ at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+ or Rb+ minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+exchange activity, known to be modulated by cytosolic Ca2+ elevation and cell shrinkage, remained silent under deoxygenation conditions.
MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells
Authors: Weiss Robert H. , Maga Elizabeth A. , Ramirez Al
Published: December 2017

Proteins comprising the mitogen-activated protein (MAP) kinase signaling cascade are activated by a variety of growth factors, but the precise role of this series of kinase reactions, especially Raf kinase and MAP kinase kinase (MEK), in vascular smooth muscle (VSM) cell mitogenesis is not known. In this study, a specific and selective inhibitor of MEK, PD-98059, was used to examine the role of MEK in DNA synthesis and Raf-1 activity in VSM cells stimulated with serum as well as with growth factors encompassing both tyrosine kinase and G protein-coupled receptor classes. Although significant increases in DNA synthesis are seen after stimulation of VSM cells with either 10% serum, platelet-derived growth factor (PDGF)-BB, or α-thrombin, preincubation of the cells with 50 μM PD-98059 for 1 h inhibits stimulation by PDGF and thrombin, but not by serum. There is a dose-dependent inhibition of the mitogenic effect by PD-98059 in all cases; these results are not affected when PD-98059 is added at times ranging from 4 h before to 2 h after growth factor addition (times at which PD-98059 exerts its inhibitory effect). In the presence of PD-98059, stimulated MAP kinase activity is attenuated when growth factors are added between 10 min and 4 h, times which correspond to both early and sustained phases of MAP kinase activity. In addition, Raf-1 activity is markedly increased by incubation of the cells with PD-98059, despite attenuation of hyperphosphorylation of this kinase. Thus growth factors coupled to both tyrosine kinase and G protein receptors require components of the MAP kinase cascade (MEK and/or MAP kinase) for VSM cell mitogenesis, whereas serum is capable of stimulatory effects in the absence of active MEK and MAP kinase. Furthermore, there exists a functional feedback stimulatory effect of inhibited MEK on its upstream activator Raf-1 in the case of serum as well as growth factors coupled to tyrosine kinase and G protein receptors.
Determinants of relaxation rate in skinned frog skeletal muscle fibers
Authors: Wahr Philip A. , Johnson J. David , Rall Jack. A.
Published: December 2017

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.
Glutamate transport and cellular glutamine metabolism: regulation in LLC-PK1 vs. LLC-PK1-F+cell lines
Authors: Meade Dale , Chess Catherine , Welbourne Tomas C.
Published: December 2017

The glutamate (Glu) transporter may modulate cellular glutamine (Gln) metabolism by regulating both the rates of hydrolysis and subsequent conversion of Glu to α-ketoglutarate and[Formula: see text]. By delivering Glu, a competitive inhibitor of Gln for the phosphate-dependent glutaminase (PDG) as well as an acid-load activator of glutamate dehydrogenase (GDH) flux, the transporter may effectively substitute extracellularly generated Glu from the γ-glutamyltransferase for that derived intracellularly from Gln. We tested this hypothesis in two closely related porcine kidney cell lines, LLC-PK1 and LLC-PK1-F+, the latter selected to grow in the absence of glucose, relying on Gln as their sole energy source. Both cell lines exhibited PDG suppression as the result of Glu uptake while disrupting the extracellularl-Glu uptake, withd-aspartate-accelerated intracellular Glu formation coupled primarily to the ammoniagenic pathway (GDH). Conversely, enhancing the extracellular Glu formation with p-aminohippurate and Glu uptake suppressed intracellular Gln hydrolysis while[Formula: see text] formation from Glu increased. Thus these results are consistent with the transporter’s dual role in modulating both PDG and GDH flux. Interestingly, PDG flux was actually higher in the Gln-adapted LLC-PK1-F+cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parental LLC-PK1 cells, revealing the importance of both Glu and Gln transport in the modulation of PDG flux. Nevertheless, when studied at physiological Gln concentration, PDG flux falls under tight Glu transporter control as Gln uptake decreases, suggesting that cellular Gln metabolism may indeed be under Glu transporter control in vivo.
Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes
Authors: Moroi Seiji , Saitou Mitinori , Fujimoto Kazushi , Sakakibara Akira , Furuse Mikio , Yoshida Osamu , Tsukita Shoichiro
Published: December 2017

Occludin is the only integral membrane protein identified to date as a component of tight junctions (TJs). Here, we examined the distribution and expression of occludin in murine testis bearing well-developed TJ. In the adult mouse testis, occludin was concentrated at TJ strands, which are located at the most basal regions of lateral membranes of Sertoli cells. In immunoblotting, occludin showed a characteristic multiple banding pattern, suggesting that occludin is highly phosphorylated in the testis. In 1-wk-old mouse testis, occludin was distributed diffusely at the lateral membranes of Sertoli cells, and even at this stage, highly phosphorylated occludin was detected. With development, occludin gradually became concentrated at the most basal regions of Sertoli cells. The same results were obtained in rat, but unexpectedly occludin was not detected in human or guinea pig Sertoli cells by immunofluorescence microscopy as well as by immunoblotting. Inasmuch as TJs are also well developed in Sertoli cells of these species, we concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of occludin or other TJ-associated integral membrane proteins that differ from occludin.
Hypertrophy-stimulated myogenic regulatory factor mRNA increases are attenuated in fast muscle of aged quails
Authors: Lowe Dawn A. , Lund Troy , Alway Stephen E.
Published: December 2017

Myogenic regulatory factors (MRFs) are a family of skeletal muscle-specific transcription factors that regulate the expression of several muscle genes. This study was designed to determine whether MRF transcripts were increased in hypertrophy-stimulated muscle of adult quails and whether equivalent increases occurred in muscles of older quails. Slow-tonic anterior latissimus dorsi and fast-twitch patagialis muscles of adult, middle-aged, aged, and senescent quails were stretch overloaded for 6, 24, or 72 h, with contralateral muscles serving as controls. RNase protection assays showed that MRF4 and MyoD transcript levels were increased and myogenin and Myf5 transcripts were induced in stretch-overloaded muscles. However, MRF4 and MyoD increases were significantly attenuated in patagialis muscles of older quails. RT-PCR analyses of three MRF-regulated genes showed that increases in the transcription of these genes occurred with stretch overload, but the increases were less in muscles of older quails. In summary, attenuated MRF responses in muscles from aged animals may partially explain why muscles from older animals do not hypertrophy to the same extent as muscles from younger animals.
Acid-induced responses in hamster chorda tympani and intracellular pH tracking by taste receptor cells
Authors: Stewart Robert E. , Lyall Vijay , Feldman George M. , Heck Gerard L. , DeSimone John A.
Published: December 2017

HCl- and NaCl-induced hamster chorda tympani nerve responses were recorded during voltage clamp of the lingual receptive field. Voltage perturbations did not influence responses to HCl. In contrast, responses to NaCl were decreased by submucosal-positive and increased by submucosal-negative voltage clamp. Responses to HCl were insensitive to the Na+ channel blockers, amiloride and benzamil, and to methylisobutylamiloride (MIA), an Na+/H+exchange blocker. Responses to NaCl were unaffected by MIA but were suppressed by benzamil. Microfluorometric and imaging techniques were used to monitor the relationship between external pH (pHo) and the intracellular pH (pHi) of fungiform papilla taste receptor cells (TRCs) following 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein loading. TRC pHi responded rapidly and monotonically to changes in pHo. This response was unaffected by Na+ removal or the presence of amiloride, benzamil, or MIA. The neural records and the data from isolated TRCs suggest that the principal transduction pathway for acid taste in hamster is similar to that in rat. This may involve the monitoring of changes in TRC pHimediated through amiloride-insensitive H+ transport across TRC membranes. This is an example of cell monitoring of environmental pH through pH tracking, i.e., a linear change in pHi in response to a change in pHo, as has been proposed for carotid bodies. In taste, the H+transport sites may be concentrated on the basolateral membranes of TRCs and, therefore, are responsive to an attenuated H+ concentration from diffusion of acids across the tight junctions.
Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells
Authors: Chen Lanlin , Glass J. Douglas , Walton Staci C. , Laurie Gordon W.
Published: December 2017

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 ( Cell Physiol. 39): C1743–C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the β1 and γ1 chains, but not α1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the β1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.
Theoretical insights into the mechanism of spiral Ca2+ wave initiation inXenopus oocytes
Authors: Dupont Geneviève
Published: December 2017

Spiral waves of intracellular Ca2+ have often been observed in Xenopus oocytes. Such waves can be accounted for by most realistic models for Ca2+ oscillations taking diffusion of cytosolic Ca2+ into account, but their initiation requires rather demanding and unphysiological initial conditions. Here, it is shown by means of numerical simulations that these spiral Ca2+ waves naturally arise if the cytoplasm is assumed to be heterogeneous both at the level of the synthesis and metabolism ofd- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] and at the level of the distribution of the Ins(1,4,5) P 3receptors. In such conditions, a spiral can be initiated in the simulations after an increase in Ins(1,4,5) P 3concentration, with the direction of rotation being determined by the position of the region of high receptor density with respect to the locus of Ins(1,4,5) P 3production.
Glutathione permeability of CFTR
Authors: Linsdell Paul , Hanrahan John W.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an ion channel that is permeable both to Cl− and to larger organic anions. Here we show, using macroscopic current recording from excised membrane patches, that the anionic antioxidant tripeptide glutathione is permeant in the CFTR channel. This permeability may account for the high concentrations of glutathione that have been measured in the surface fluid that coats airway epithelial cells. Furthermore, loss of this pathway for glutathione transport may contribute to the reduced levels of glutathione observed in airway surface fluid of cystic fibrosis patients, which has been suggested to contribute to the oxidative stress observed in the lung in cystic fibrosis. We suggest that release of glutathione into airway surface fluid may be a novel function of CFTR.
Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells
Authors: Danto Spencer I. , Borok Zea , Zhang Xiao-Ling , Lopez Melissa Z. , Patel Paryus , Crandall Edward D. , Lubman Richard L.
Published: December 2017

We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) α-, β-, and γ-subunits and Na+ pump α1- and β1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both α1 and β1Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump α1- and β1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump α1- and β1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+ pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+ resorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral membranes.
Myocardial gene reprogramming associated with a cardiac cross-resistant state induced by LPS preconditioning
Authors: Meng Xianzhong , Brown James M. , Ao Lihua , Rowland Robert T. , Nordeen Steven K. , Banerjee Anirban , Harken Alden H.
Published: December 2017

Lipopolysaccharide (LPS) preconditioning induces cardiac resistance to subsequent LPS or ischemia. This study tested the hypothesis that resistance to LPS and resistance to ischemia are two manifestations of cardiac cross-resistance which may involve reprogramming of cardiac gene expression. Rats were preconditioned with a single dose of LPS (0.5 mg/kg ip). Cardiac resistance to LPS was examined with a subsequent LPS challenge. Cardiac resistance to ischemia was determined by subjecting hearts to ischemia-reperfusion. Total RNA was extracted from myocardium for Northern analysis of mRNAs encoding protooncoproteins, antioxidant enzymes, and contractile protein isoforms. Rats preconditioned with LPS 1–7 days earlier acquired cardiac resistance to endotoxemic depression. This resistance temporally correlated with resistance to ischemia. Pretreatment with cycloheximide (0.5 mg/kg ip) abolished resistance to both LPS and ischemia. LPS preconditioning induced the expression of c- jun and c- fos mRNAs. LPS also transiently increased mRNAs encoding catalase and Mn-containing superoxide dismutase. The expression of both α- and β-myosin heavy chain mRNAs was upregulated, whereas the expression of cardiac α-actin mRNA was suppressed. We conclude that 1) LPS induces sustained cardiac resistance to both LPS and ischemia, 2) resistance to ischemia and resistance to LPS seem to be two mechanistically indistinct components of cardiac cross-resistance, and 3) the cardiac cross-resistance is associated with reprogramming of myocardial gene expression.
Functional expression of putative H+-K+-ATPase from guinea pig distal colon
Authors: Asano Shinji , Hoshina Satomi , Nakaie Yumi , Watanabe Toshiyuki , Sato Michihiko , Suzuki Yuichi , Takeguchi Noriaki
Published: December 2017

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase α-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854 ) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or TorpedoNa+-K+-ATPase β-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 μM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the β-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (α-subunit) of the colonic H+-K+-ATPase. Although the β-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase β-subunits were found to act as a surrogate for the colonic β-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC 50 = 0.4–1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).
Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise
Authors: Dupont-Versteegden Esther E. , Houlé John D. , Gurley Cathy M. , Peterson Charlotte A.
Published: December 2017

Muscles of spinal cord-transected rats exhibit severe atrophy and a shift toward a faster phenotype. Exercise can partially prevent these changes. The goal of this study was to investigate early events involved in regulating the muscle response to spinal transection and passive hindlimb exercise. Adult female Sprague-Dawley rats were anesthetized, and a complete spinal cord transection lesion (T10) was created in all rats except controls. Rats were killed 5 or 10 days after transection or they were exercised daily on motor-driven bicycles starting at 5 days after transection and were killed 0.5, 1, or 5 days after the first bout of exercise. Structural and biochemical features of soleus and extensor digitorum longus (EDL) muscles were studied. Atrophy was decreased in all fiber types of soleus and in type 2a and type 2x fibers of EDL after 5 days of exercise. However, exercise did not appear to affect fiber type that was altered within 5 days of spinal cord transection: fibers expressing myosin heavy chain 2x increased in soleus and EDL, and extensive coexpression of myosin heavy chain in soleus was apparent. Activation of satellite cells was observed in both muscles of transected rats regardless of exercise status, evidenced by increased accumulation of MyoD and myogenin. Increased expression was transient, except for MyoD, which remained elevated in soleus. MyoD and myogenin were detected both in myofiber and in satellite cell nuclei in both muscles, but in soleus, MyoD was preferentially expressed in satellite cell nuclei, and in EDL, MyoD was more readily detectable in myofiber nuclei, suggesting that MyoD and myogenin have different functions in different muscles. Exercise did not affect the level or localization of MyoD and myogenin expression. Similarly, Id-1 expression was transiently increased in soleus and EDL upon spinal cord transection, and no effect of exercise was observed. These results indicate that passive exercise can ameliorate muscle atrophy after spinal cord transection and that satellite cell activation may play a role in muscle plasticity in response to spinal cord transection and exercise. Finally, the mechanisms underlying maintenance of muscle mass are likely distinct from those controlling myosin heavy chain expression.
Monocyte/macrophages evoke epithelial dysfunction: indirect role of tumor necrosis factor-α
Authors: Zareie Mehri , McKay Derek M. , Kovarik Garrett G. , Perdue Mary H.
Published: December 2017

We examined the ability of monocytes (MΦ) activated by bacterial products to alter epithelial physiology. Confluent monolayers of the T84 colonic epithelial cell line were grown on filter supports and then cocultured in the presence of human MΦ with or without the activating agents bacterial lipopolysaccharide and the bacterial tripeptide formyl-methionyl-leucyl-phenylalanine. After 24 or 48 h, monolayers were mounted in Ussing chambers where parameters of epithelial function were measured. Exposure to activated MΦ resulted in a significant increase ( P < 0.05) in baseline short-circuit current (250% after 48 h) that was associated with enhanced secretion of Cl−. In addition, epithelial permeability was significantly increased as shown by reduced transepithelial resistance and increased flux of51Cr-EDTA. Activated MΦ produced substantial amounts (∼3 ng/ml at 48 h) of tumor necrosis factor-α (TNF-α). TNF-α was identified as a key mediator acting via an autocrine mechanism to induce epithelial pathophysiology. Our data show that MΦ, when activated by common bacterial components, are potent effector cells capable of initiating significant changes in the transport and barrier properties of a model epithelium.
Functional and molecular identification of a novel chloride conductance in canine colonic smooth muscle
Authors: Dick Gregory M. , Bradley Karri K. , Horowitz Burton , Hume Joseph R. , Sanders Kenton M.
Published: December 2017

Swelling-activated or volume-sensitive Cl− currents are found in numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for swelling-activated Cl−current in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrophysiological techniques and, if present, to characterize its functional and molecular properties. We hypothesized that ClC-3 is present in colonic smooth muscle and is regulated in a manner similar to the molecular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl− gradient. Current magnitude increased and reversed at more negative potentials when Cl− was replaced by I− or Br−. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene; 10 μM) and DIDS (100 μM) inhibited the current, whereas 25 μM niflumic acid, 10 μM nicardipine, and Ca2+ removal had no effect. Current was inhibited by 1 mM extracellular ATP in a voltage-dependent manner. Cl− current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl− current magnitude, while chelerythrine chloride (30 μM) activated it under isotonic conditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an important mechanism controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.
Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway
Authors: Chin Ting-Yu , Chueh Sheau-Huei
Published: December 2017

In cultured porcine aortic smooth muscle cells, sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced a rapid dose-dependent increase in the cytosolic Ca2+ concentration ([Ca2+]i) and also stimulated inositol 1,4,5-trisphosphate (IP3) generation. Pretreatment of cells with pertussis toxin blocked the SPC-induced IP3 generation and [Ca2+]iincrease but had no effect on the action of ATP or BK. In addition, SPC stimulated the mitogen-activated protein kinase (MAPK) and increased DNA synthesis, whereas neither ATP nor BK produced such effects. Both the SPC-induced MAPK activation and DNA synthesis were pertussis toxin sensitive. SPC-induced MAPK activation was blocked by treatment of cells with the phospholipase C inhibitor, U-73122, or the intracellular Ca2+-ATPase inhibitor, thapsigargin, but not by removal of extracellular Ca2+. Lysophosphatidic acid induced cellular responses similar to SPC in a pertussis toxin-sensitive manner in terms of [Ca2+]iincrease, IP3 generation, MAPK activation, and DNA synthesis. Platelet-derived growth factor (PDGF) also induced a [Ca2+]iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in [Ca2+]i. SPC-induced MAPK activation was inhibited by pretreatment of cells with staurosporine, W-7, or calmidazolium. Our results suggest that, in porcine aortic smooth muscle cells, MAPK is not activated by the increase in [Ca2+]iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role of Ca2+ in pertussis toxin-sensitive G protein-mediated MAPK activation.