bluuebunny/crossref_metadata_2025 huggingface dataset:
Aldosterone action: induction of p21 ras andfra-2 and transcription-independent decrease inmyc,jun, andfos
Authors: Spindler Benjamin , Verrey François
Published: December 2017

Adrenal steroids induce an increase in transcellular Na+ reabsorption across Xenopus laevis A6 cell epithelia that requires the action of transcriptionally regulated gene products. In a previous study we identified K- ras2 as an aldosterone-upregulated mRNA in A6 epithelia. Here, we show that in vivo injection of aldosterone in Xenopus (2.5 h) increases K- ras2 mRNA specifically in the kidney (2.5-fold) and that in A6 epithelia aldosterone (2.5 h) increases Ras protein synthesis (∼6-fold). Xl- ras, another ras mRNA expressed at a low level in A6 cells, was also induced (2-fold). Aldosterone was shown to regulate the mRNA levels of several transcription factors as well. After 2 h of aldosterone treatment, fra-2 mRNA was upregulated by 130%, whereas c- myc, c- jun, c- fos, and glucocorticoid receptor mRNAs were downregulated by 23–43%. After 16 h, c- fos and GR mRNAs were further decreased, whereas levels of fra-2, c- jun, and c- myc began to return to control levels. Interestingly, the downregulation of the protooncogene mRNAs was independent of transcription. These results support the view that aldosterone exerts complex pleiotropic transcriptional and nontranscriptional actions that involve the regulation of signaling cascade elements (i.e., K-Ras2) as well as that of transcription factors.
Tissue factor activity is increased in human endothelial cells cultured under elevated static pressure
Authors: Silverman M. D. , Waters C. R. , Hayman G. T. , Wigboldus J. , Samet M. M. , Lelkes P. I.
Published: December 2017

We tested the hypothesis that elevated blood pressure, a known stimulus for vascular remodeling and an independent risk factor for the development of atherosclerotic disease, can modulate basal and cytokine-induced tissue factor (TF; CD 142) expression in cultured human endothelial cells (EC). Using a chromogenic enzymatic assay, we measured basal and tumor necrosis factor-α (TNF-α; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC (HVCEC) cultured at atmospheric pressure and at 170 mmHg imposed pressure for up to 48 h. Basal TF activities were 22 ± 10 U/mg protein for HAEC and 14 ± 9 U/mg protein for HVCEC and were upregulated in both cell types >10-fold by TNF-α. Exposure to pressure for 5 h induced additional elevation of basal TF activity by 47 ± 16% ( P < 0.05, n = 6) for HAEC and 17 ± 5% ( P < 0.05, n = 3) for HVCEC. Pressurization also enhanced TF activity in TNF-α-treated cells from 240 ± 28 to 319 ± 32 U/mg protein in HAEC ( P < 0.05, n = 4) and from 148 ± 25 to 179 ± 0.8 U/mg protein ( P < 0.05, n = 3) in HVCEC. Cytokine stimulation caused an ∼100-fold increase in steady-state TF mRNA levels in HAEC, whereas pressurization did not alter either TF mRNA or cell surface antigen expression, as determined by quantitative RT-PCR methodology and ELISA. Elevated pressure, however, modulated the EC plasma membrane organization and/or permeability as inferred from the increased cellular uptake of the fluorescent amphipathic dye merocyanine 540 (33 ± 7%, P < 0.05). Our data suggest that elevated static pressure modulates the hemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.
Effects of osmolarity on taste receptor cell size and function
Authors: Lyall Vijay , Heck Gerard L. , DeSimone John A. , Feldman George M.
Published: December 2017

Osmotic effects on salt taste were studied by recording from the rat chorda tympani (CT) nerve and by measuring changes in cell volume of isolated rat fungiform taste receptor cells (TRCs). Mannitol, cellobiose, urea, or DMSO did not induce CT responses. However, the steady-state CT responses to 150 mM NaCl were significantly increased when the stimulus solutions also contained 300 mM mannitol or cellobiose, but not 600 mM urea or DMSO. The enhanced CT responses to NaCl were reversed when the saccharides were removed and were completely blocked by addition of 100 μM amiloride to the stimulus solution. Exposure of TRCs to hyperosmotic solutions of mannitol or cellobiose induced a rapid and sustained decrease in cell volume that was completely reversible, whereas exposure to hypertonic urea or DMSO did not induce sustained reductions in cell volume. These data suggest that the osmolyte-induced increase in the CT response to NaCl involves a sustained decrease in TRC volume and the activation of amiloride-sensitive apical Na+ channels.
PLA2 stimulation of Na+/H+antiport and proliferation in rat aortic smooth muscle cells
Authors: Rufini S. , De Vito P. , Balestro N. , Pescatori M. , Luly P. , Incerpi S.
Published: December 2017

The proliferative properties and the ability to stimulate the Na+/H+antiport activity of a secretory phospholipase A2 were studied in rat aortic smooth muscle cells in culture. The requirement of the enzymatic activity of phospholipase A2 to elicit mitogenesis was assessed by the use of ammodytin L, a Ser49 phospholipase A2 from the venom of Vipera ammodytes, devoid of hydrolytic activity. We propose that the proliferative effect is mediated by the same transduction pathway for both proteins. In particular, 1) both secretory phospholipase A2 and ammodytin L stimulated thymidine incorporation in a dose-dependent manner; 2) both proteins affected the cell cycle, as assessed by cell growth and fluorescence-activated cell sorting experiments; 3) both phospholipase A2 and ammodytin L increased intracellular pH, a permissive factor for cell proliferation, through activation of the Na+/H+antiport; 4) ammodytin L was able to displace the 125I-labeled phospholipase A2 from specific binding sites in a concentration range consistent with that capable of eliciting a cellular response; and 5) the inhibition by heparin was similar for both proteins, taking into account the ratio of heparin to protein. In conclusion, the enzymatic activity of phospholipase A2 is not required for the stimulation of mitogenesis. The inhibitory effect of heparin combined with its therapeutic potential could help to clarify the role of phospholipase A2 in the pathogenesis of several preinflammatory situations.
MHC class II molecules, cathepsins, and La/SSB proteins in lacrimal acinar cell endomembranes
Authors: Yang Tao , Zeng Hongtao , Zhang Jian , Okamoto Curtis T. , Warren Dwight W. , Wood Richard L. , Bachmann Michael , Mircheff Austin K.
Published: December 2017

Sjögren's syndrome is a chronic autoimmune disease affecting the lacrimal glands and other epithelia. It has been suggested that acinar cells of the lacrimal glands provoke local autoimmune responses, leading to Sjögren's syndrome when they begin expressing major histocompatibility complex (MHC) class II molecules. We used isopycnic centrifugation and phase partitioning to resolve compartments that participate in traffic between the basolateral membranes and the endomembrane system to test the hypothesis that MHC class II molecules enter compartments that contain potential autoantigens, i.e., La/SSB, and enzymes capable of proteolytically processing autoantigen, i.e., cathepsins B and D. A series of compartments identified as secretory vesicle membranes, prelysosomes, and microdomains of the trans-Golgi network involved in traffic to the basolateral membrane, to the secretory vesicles, and to the prelysosomes were all prominent loci of MHC class II molecules, La/SSB, and cathepsins B and D. These observations support the thesis that lacrimal gland acinar cells that have been induced to express MHC class II molecules function as autoantigen processing and presenting cells.
Role of HERG-like K+ currents in opossum esophageal circular smooth muscle
Authors: Akbarali Hamid I. , Thatte Hemant , He Xue Dao , Giles Wayne R. , Goyal Raj K.
Published: December 2017

An inwardly rectifying K+ conductance closely resembling the human ether-a-go-go-related gene (HERG) current was identified in single smooth muscle cells of opossum esophageal circular muscle. When cells were voltage clamped at 0 mV, in isotonic K+ solution (140 mM), step hyperpolarizations to −120 mV in 10-mV increments resulted in large inward currents that activated rapidly and then declined slowly (inactivated) during the test pulse in a time- and voltage- dependent fashion. The HERG K+ channel blockers E-4031 (1 μM), cisapride (1 μM), and La3+ (100 μM) strongly inhibited these currents as did millimolar concentrations of Ba2+. Immunoflourescence staining with anti-HERG antibody in single cells resulted in punctate staining at the sarcolemma. At membrane potentials near the resting membrane potential (−50 to −70 mV), this K+ conductance did not inactivate completely. In conventional microelectrode recordings, both E-4031 and cisapride depolarized tissue strips by 10 mV and also induced phasic contractions. In combination, these results provide direct experimental evidence for expression of HERG-like K+ currents in gastrointestinal smooth muscle cells and suggest that HERG plays an important role in modulating the resting membrane potential.
Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers
Authors: Duke Adrian M. , Steele Derek S.
Published: December 2017

The effects of Pi on sarcoplasmic reticulum (SR) Ca2+ regulation were studied in mechanically skinned rat skeletal muscle fibers. Brief application of caffeine was used to assess the SR Ca2+ content, and changes in concentration of Ca2+([Ca2+]) within the cytosol were detected with fura 2 fluorescence. Introduction of Pi (1–40 mM) induced a concentration-dependent Ca2+ efflux from the SR. In solutions lacking creatine phosphate (CP), the amplitude of the Pi-induced Ca2+ transient approximately doubled. A similar potentiation of Pi-induced Ca2+ release occurred after inhibition of creatine kinase (CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca2+ release was almost abolished, whereas Pi-induced Ca2+ release was unaffected. However, introduction of the SR Ca2+ ATPase inhibitor cyclopiazonic acid effectively abolished Pi-induced Ca2+ release. These data suggest that Pi induces Ca2+ release from the SR by reversal of the SR Ca2+ pump but not via the SR Ca2+ channel under these conditions. If this occurs in intact skeletal muscle during fatigue, activation of a Ca2+efflux pathway by Pi may contribute to the reported decrease in net Ca2+ uptake and increase in resting [Ca2+].
Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts
Authors: Panchenko Maria V. , Farber Harrison W. , Korn Joseph H.
Published: December 2017

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H2O2and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H2O2and CCCP on HO-1 mRNA. Addition of Fe2+, Fe3+, or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H2O2or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.
Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity
Authors: Woo Seung Kyoon , Maouyo Djikolngar , Handler Joseph S. , Kwon H. Moo
Published: December 2017

Tonicity-responsive enhancer binding protein (TonEBP) is the transcription factor that regulates tonicity-responsive expression of the genes for the sodium- myo-inositol cotransporter (SMIT) and the sodium-chloride-betaine cotransporter (BGT1). Hypertonicity stimulates the activity of TonEBP due to a combination of increased protein abundance and increased nuclear distribution (proportion of TonEBP that is in the nucleus). We found that inhibitors of proteasome activity markedly reduce the induction of SMIT and BGT1 mRNA in response to hypertonicity. These inhibitors also reduce hypertonicity-induced stimulation of expression of a reporter gene controlled by the tonicity-responsive enhancer. Western and immunohistochemical analyses revealed that the proteasome inhibitors reduce the hypertonicity-induced increase of TonEBP in the nucleus by inhibiting its nuclear redistribution without affecting its abundance. Although the nuclear distribution of TonEBP is sensitive to inhibition of proteasome activity as is that of nuclear factor (NF)-κB, the signaling pathways appear to be different in that hypertonicity does not affect the nuclear distribution of NF-κB. Conversely, treatment with tumor necrosis factor-α increases the nuclear distribution of NF-κB but not TonEBP.
cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes
Authors: Lader Alan S. , Wang Yihan , Jackson G. Robert , Borkan Steven C. , Cantiello Horacio F.
Published: December 2017

In this study, patch-clamp techniques were applied to cultured neonatal mouse cardiac myocytes (NMCM) to assess the contribution of cAMP stimulation to the anion permeability in this cell model. Addition of either isoproterenol or a cocktail to raise intracellular cAMP increased the whole cell currents of NMCM. The cAMP-dependent conductance was largely anionic, as determined under asymmetrical (low intracellular) Cl− conditions and symmetrical Cl−in the presence of various counterions, including Na+, Mg2+, Cs+, and N-methyl-d-glucamine. Furthermore, the cAMP-stimulated conductance was also permeable to ATP. The cAMP-activated currents were inhibited by diphenylamine-2-carboxylate, glibenclamide, and an anti-cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody. The anti-CFTR monoclonal antibody failed, however, to inhibit an osmotically activated anion conductance, indicating that CFTR is not linked to osmotically stimulated currents in this cell model. Immunodetection studies of both neonatal mouse heart tissue and cultured NMCM revealed that CFTR is expressed in these preparations. The implication of CFTR in the cAMP-stimulated Cl−- and ATP-permeable conductance was further verified with NMCM of CFTR knockout mice [ cftr(−/−)] in which cAMP stimulation was without effect on the whole cell currents. In addition, stimulation with protein kinase A and ATP induced Cl−-permeable single-channel activity in excised, inside-out patches from control, but not cftr(−/−) NMCM. The data in this report indicate that cAMP stimulation of NMCM activates an anion-permeable conductance with functional properties similar to those expected for CFTR, thus suggesting that CFTR may be responsible for the cAMP-activated conductance. CFTR may thus contribute to the permeation and/or regulation of Cl−- and ATP-permeable pathways in the developing heart.
Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro
Authors: Vicario I. , Rigual R. , Obeso A. , Gonzalez C.
Published: December 2017

The aim of this work was to determine contents and turnover rates for dopamine (DA) and norepinephrine (NE) and to identify the catecholamine (CA) released during stimulation of the rat carotid body (CB). Turnover rates and the release of CA were measured in an in vitro preparation using a combination of HPLC and radioisotopic methods. Mean rat CB levels of DA and NE were 209 and 45 pmol/mg tissue, respectively. With [3H]tyrosine as precursor, rat CB synthesized [3H]CA in a time- and concentration-dependent manner; calculated turnover times for DA and NE were 5.77 and 11.4 h, respectively. Hypoxia and dibutyryl adenosine 3′,5′-cyclic monophosphate significantly increased [3H]CA synthesis. In normoxia, rat CB released [3H]DA and [3H]NE in a ratio of 5:1, comparable to that of the endogenous tissue CA. Hypoxia and high K+ preferentially released [3H]DA, nicotine preferentially released [3H]NE, and acidic stimuli released both amines in proportion to tissue content. Release of [3H]CA induced by hypoxia and high K+ was nearly fully dependent on extracellular Ca2+, whereas basal normoxic release was not altered by removal of Ca2+ from the incubating solution. We conclude that the rat CB is an organ with higher levels of DA than NE that preferentially releases DA or NE in a stimulus-specific manner.
Gender-specific reduction in contractility and [Ca2+]i in vascular smooth muscle cells of female rat
Authors: Murphy Jason G. , Khalil Raouf A.
Published: December 2017

The hypothesis that vascular protection in females and its absence in males reflects gender differences in [Ca2+]i and Ca2+ mobilization mechanisms of vascular smooth muscle contraction was tested in fura 2-loaded aortic smooth muscle cells isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca2+), the resting length and [Ca2+]i were significantly different in intact males (64.5 ± 1.2 μm and 83 ± 3 nM) than in intact females (76.5 ± 1.5 μm and 64 ± 7 nM). In intact male WKY, phenylephrine (Phe, 10−5 M) caused transient increase in [Ca2+]i to 428 ± 13 nM followed by maintained increase to 201 ± 8 nM and 32% cell contraction. In intact female WKY, the Phe-induced [Ca2+]i transient was not significantly different, but the maintained [Ca2+]i (159 ± 7 nM) and cell contraction (26%) were significantly less than in intact male WKY. In Ca2+-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM) caused transient increases in [Ca2+]i and contraction that were not significantly different between males and females. Membrane depolarization by 51 mM KCl caused 31% cell contraction and increased [Ca2+]i to 259 ± 9 nM in intact male WKY, which were significantly greater than a 24% contraction and 214 ± 8 nM [Ca2+]i in intact female WKY. Maintained Phe- and KCl-stimulated cell contraction and [Ca2+]i were significantly greater in SHR than WKY in all groups of rats. Reduction in cell contraction and [Ca2+]i in intact females compared with intact males was significantly greater in SHR (∼30%) than WKY (∼20%). No significant differences in cell contraction or [Ca2+]i were observed between castrated males, ovariectomized (OVX) females, and intact males, or between OVX females with 17β-estradiol implants and intact females. Exogenous application of 17β-estradiol (10−8 M) to cells from OVX females caused greater reduction in Phe- and KCl-induced contraction and [Ca2+]i in SHR than WKY. Thus the basal, maintained Phe- and depolarization-induced [Ca2+]i and contraction of vascular smooth muscle triggered by Ca2+ entry from the extracellular space exhibit differences depending on gender and the presence or absence of female gonads. Cell contraction and [Ca2+]i due to Ca2+release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca2+]i in vascular smooth muscle of female rats is greater in SHR than WKY rats.
NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin
Authors: Gorodeski George I.
Published: December 2017

Human cervical epithelial cells express mRNA for the nitric oxide (NO) synthase (NOS) isoforms ecNOS, bNOS, and iNOS and release NO into the extracellular medium. N G-nitro-l-arginine methyl ester (l-NAME), an NOS inhibitor, and Hb, an NO scavenger, decreased paracellular permeability; in contrast, the NO donors sodium nitroprusside (SNP) and N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increased paracellular permeability across cultured human cervical epithelia on filters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect was blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but not by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583 and KT-5823 blocked the SNP-induced increase in permeability. Treatment with SNP increased cellular cGMP, and the effect was blocked by Hb and LY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulated cervical cation selectivity. In contrast, both agents increased fluorescence from fura 2-loaded cells in the Ca2+-insensitive wavelengths, indicating that SNP and 8-BrcGMP stimulate a decrease in cell size and in the resistance of the lateral intercellular space. Neither SNP nor 8-BrcGMP had an effect on total cellular actin, but both agents increased the fraction of G-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these results, it is suggested that NO acts on guanylate cyclase and stimulates an increase in cGMP; cGMP, acting via cGMP-dependent protein kinase, shifts actin steady-state toward G-actin; this fragments the cytoskeleton and renders cells more sensitive to decreases in cell size and resistance of the lateral intercellular space and, hence, to increases in permeability. These results may be important for understanding NO regulation of transcervical paracellular permeability and secretion of cervical mucus in the woman.
Probing the extracellular release site of the plasma membrane calcium pump
Authors: Xu Wanyan , Wilson Betty Jo , Huang Lin , Parkinson Emma L. , Hill Brent J. F. , Milanick Mark A.
Published: December 2017

The plasma membrane Ca2+ pump is known to mediate Ca2+/H+ exchange. Extracellular protons activated 45Ca2+ efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC50 for extracellular Ca2+ from ∼33 to ∼6 mM. Changing the membrane potential by >54 mV had no effect on the IC50 for extracellular Ca2+. This argues against Ca2+ release through a high-field access channel. Extracellular Ni2+ inhibited Ca2+ efflux with an IC50 of 11 mM. Extracellular Cd2+ inhibited with an IC50 of 1.5 mM, >10 times better than Ca2+. The Cd2+ IC50 also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca2+, Cd2+, and H+ competing for the same site. The higher affinity for inhibition by Ni2+and Cd2+ is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca2+ efflux. Our results are consistent with the notion that the release site contains a histidine.
Mechanical strain inhibits expression of osteoclast differentiation factor by murine stromal cells
Authors: Rubin Janet , Murphy Tamara , Nanes Mark S. , Fan Xian
Published: December 2017

Normal dynamic loading prevents bone resorption; however, the means whereby biophysical factors reduce osteoclast activity are not understood. We show here that mechanical strain (2% at 10 cycles per minute) applied to murine marrow cultures reduced 1,25(OH)2D3-stimulated osteoclast formation by 50%. This was preceded by decreased expression of osteoclast differentiation factor (ODF/TRANCE). RT-PCR for ODF/TRANCE revealed that ODF/TRANCE mRNA in strained cultures was 59 ± 3% of that seen in control cultures. No significant effects on total cell count, thymidine uptake, or alkaline phosphatase activity were induced by strain. To isolate the cell targeted by strain, primary stromal cells were cultured from marrow. Mechanical strain also reduced mRNA for ODF/TRANCE to 60% that of control in these cells. In contrast, mRNA for membrane-bound macrophage colony-stimulating factor was not significantly affected. Soluble ODF (∼2 ng/ml) was able to reverse the effect of strain, returning osteoclast numbers to control. Because osteoclast formation is dependent upon ODF/TRANCE expression, strain-induced reductions in this factor may contribute to the accompanying reduction in osteoclastogenesis.
Intracellular calcium events activated by ATP in murine colonic myocytes
Authors: Bayguinov Orline , Hagen Brian , Bonev Adrian D. , Nelson Mark T. , Sanders Kenton M.
Published: December 2017

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca2+-activated K+ (SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca2+ release. Isolated myocytes of the murine colon produced spontaneous, localized Ca2+ release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca2+ transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca2+ events in colonic myocytes “Ca2+ puffs.” The P2Y receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca2+ puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca2+ puffs. Pyridoxal-phospate-6-azophenyl-2′,4′-disculfonic acid tetrasodium, a P2 receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca2+ transients and the effects of 2-MeS-ATP on Ca2+ puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP3receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca2+. These data suggest that localized Ca2+ release modulates Ca2+-dependent ionic conductances in the plasma membrane. Localized Ca2+ release may contribute to the electrical responses resulting from purinergic stimulation.
Excitation, inhibition, and suppression by odors in isolated toad and rat olfactory receptor neurons
Authors: Sanhueza Magdalena , Schmachtenberg Oliver , Bacigalupo Juan
Published: December 2017

Vertebrate olfactory receptor neurons (ORNs) exhibit odor-induced increases in action potential firing rate due to an excitatory cAMP-dependent current. Fish and amphibian ORNs also give inhibitory odor responses, manifested as decreases in firing rate, but the underlying mechanism is poorly understood. In the toad, an odor-induced Ca2+-activated K+ current is responsible for the hyperpolarizing receptor potential that causes inhibition. In isolated ORNs, a third manner by which odors affect firing is suppression, a direct and nonspecific reduction of voltage-gated and transduction conductances. Here we show that in whole cell voltage-clamped toad ORNs, excitatory or inhibitory currents were not strictly associated to a particular odorant mixture. Occasionally, both odor effects, in addition to suppression, were concurrently observed in a cell. We report that rat ORNs also exhibit odor-induced inhibitory currents, due to the activation of a K+ conductance closely resembling that in the toad, suggesting that this conductance is widely distributed among vertebrates. We propose that ORNs operate as complex integrator units in the olfactory epithelium, where the first events in the process of odor discrimination take place.
CFTR induces the expression of DRA along with Cl−/HCO3 − exchange activity in tracheal epithelial cells
Authors: Wheat Valerie J. , Shumaker Holli , Burnham Charles , Shull Gary E. , Yankaskas James R. , Soleimani Manoocher
Published: December 2017

Thickening of airway mucus and lung dysfunction in cystic fibrosis (CF) results, at least in part, from abnormal secretion of Cl− and HCO3 −across the tracheal epithelium. The mechanism of the defect in HCO3 − secretion is ill defined; however, a lack of apical Cl−/HCO3 − exchange may exist in CF. To test this hypothesis, we examined the expression of Cl−/HCO3 − exchangers in tracheal epithelial cells exhibiting physiological features prototypical of cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1 cells transfected with functional wild-type CFTR, termed CFT-WT). Cells were grown on coverslips and were loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was monitored. Cl−/HCO3 −exchange activity increased by ∼300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1 cells to 0.11 in CFT-WT cells ( P < 0.001, n = 8). This activity was significantly inhibited by DIDS. The mRNA expression of the ubiquitous basolateral AE-2 Cl−/HCO3 − exchanger remained unchanged. However, mRNA encoding DRA, recently shown to be a Cl−/HCO3 − exchanger (Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274: 22855–22861, 1999.) was abundantly expressed in cells expressing functional CFTR but not in cells that lacked CFTR or that expressed mutant CFTR. In conclusion, CFTR induces the mRNA expression of “downregulated in adenoma” (DRA) and, as a result, upregulates the apical Cl−/HCO3 − exchanger activity in tracheal cells. We propose that the tracheal HCO3 −secretion defect in patients with CF is partly due to the downregulation of the apical Cl−/HCO3 −exchange activity mediated by DRA.
Differential Ca2+ sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin
Authors: Fruen Bradley R. , Bardy Jennifer M. , Byrem Todd M. , Strasburg Gale M. , Louis Charles F.
Published: December 2017

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) in the presence of nanomolar Ca2+ concentrations. However, the role of CaM activation in the mechanisms that control Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle and in the heart remains unclear. In media that contained 100 nM Ca2+, the rate of45Ca2+ release from porcine skeletal muscle SR vesicles was increased approximately threefold in the presence of CaM (1 μM). In contrast, cardiac SR vesicle45Ca2+ release was unaffected by CaM, suggesting that CaM activated the skeletal RyR1 but not the cardiac RyR2 channel isoform. The activation of RyR1 by CaM was associated with an approximately sixfold increase in the Ca2+ sensitivity of [3H]ryanodine binding to skeletal muscle SR, whereas the Ca2+ sensitivity of cardiac SR [3H]ryanodine binding was similar in the absence and presence of CaM. Cross-linking experiments identified both RyR1 and RyR2 as predominant CaM binding proteins in skeletal and cardiac SR, respectively, and [35S]CaM binding determinations further indicated comparable CaM binding to the two isoforms in the presence of micromolar Ca2+. In nanomolar Ca2+, however, the affinity and stoichiometry of RyR2 [35S]CaM binding was reduced compared with that of RyR1. Together, our results indicate that CaM activates RyR1 by increasing the Ca2+ sensitivity of the channel, and further suggest differences in CaM's functional interactions with the RyR1 and RyR2 isoforms that may potentially contribute to differences in the Ca2+ dependence of channel activation in skeletal and cardiac muscle.
Expression of the TGF-β receptor gene and sensitivity to growth inhibition following polyamine depletion
Authors: Rao Jaladanki N. , Li Li , Bass Barbara L. , Wang Jian-Ying
Published: December 2017

Our previous studies have shown that inhibition of polyamine biosynthesis increases the sensitivity of intestinal epithelial cells to growth inhibition induced by exogenous transforming growth factor-β (TGF-β). This study went further to determine whether expression of the TGF-β receptor genes is involved in this process. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, and spermine in IEC-6 cells. Polyamine depletion by DFMO increased levels of the TGF-β type I receptor (TGF-βRI) mRNA and protein but had no effect on the TGF-β type II receptor expression. The induced TGF-βRI expression after polyamine depletion was associated with an increased sensitivity to growth inhibition induced by exogenous TGF-β but not by somatostatin. Extracellular matrix laminin inhibited IEC-6 cell growth without affecting the TGF-β receptor expression. Laminin consistently failed to induce the sensitivity of TGF-β-mediated growth inhibition. In addition, decreasing TGF-βRI expression by treatment with retinoic acid not only decreased TGF-β-mediated growth inhibition in normal cells but also prevented the increased sensitivity to exogenous TGF-β in polyamine-deficient cells. These results indicate that 1) depletion of cellular polyamines by DFMO increases expression of the TGF-βRI gene and 2) increased TGF-βRI expression plays an important role in the process through which polyamine depletion sensitizes intestinal epithelial cells to growth inhibition induced by TGF-β.
Mechanism and regulation of GLUT-4 vesicle fusion in muscle and fat cells
Authors: Foster Leonard J. , Klip Amira
Published: December 2017

Twenty years ago it was shown that recruitment of glucose transporters from an internal membrane compartment to the plasma membrane led to increased glucose uptake into fat and muscle cells stimulated by insulin. The final step of this process is the fusion of glucose transporter 4 (GLUT-4)-containing vesicles with the plasma membrane. The identification of a neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex as a requirement for synaptic vesicle-plasma membrane fusion led to the search for homologous complexes outside the nervous system. Indeed, isoforms of the neuronal SNAREs were identified in muscle and fat cells and were shown to be required for GLUT-4 incorporation into the cell membrane. In addition, proteins that bind to nonneuronal SNAREs were cloned and proposed to regulate vesicle fusion. We have summarized the molecular mechanisms leading to membrane fusion in nonneuronal systems, focusing on the role of SNAREs and accessory proteins (Munc18c, synip, Rab4, and VAP-33) in incorporation of GLUT-4 into the plasma membrane. Potential modes of regulation of this process are discussed, including SNARE phosphorylation and interaction with the cytoskeleton.
Adaptation and survival of surface-deprived red blood cells in mice
Authors: Murdock Ryan C. , Reynolds Christopher , Sarelius Ingrid H. , Waugh Richard E.
Published: December 2017

The consequences of lost membrane area for long-term erythrocyte survival in the circulation were investigated. Mouse red blood cells were treated with lysophosphatidylcholine to reduce membrane area, labeled fluorescently, reinfused into recipient mice, and then sampled periodically for 35 days. The circulating fraction of the modified cells decreased on an approximately exponential time course, with time constants ranging from 2 to 14 days. The ratio of volume to surface area of the surviving cells, measured using micropipettes, decreased rapidly over the first 5 days after infusion to within 5% of normal. This occurred by both preferential removal of the most spherical cells and modification of others, possibly due to membrane stress developed during transient trapping of cells in the microvasculature. After 5 days, the cell area decreased with time in the circulation, but the ratio of volume to surface area remained essentially constant. These results demonstrate that the ratio of cell volume to surface area is a major determinant of the ability of erythrocytes to circulate properly.
Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells
Authors: Codina Juan , Cardwell Joseph , Gitomer Jeremy J. , Cui Yan , Kone Bruce C. , Dubose Thomas D.
Published: December 2017

Two H+-K+-ATPase isoforms are present in kidney: the gastric, highly sensitive to Sch-28080, and the colonic, partially sensitive to ouabain. Upregulation of Sch-28080-sensitive H+-K+-ATPase, or “gastric” H+-K+-ATPase, has been demonstrated in hypokalemic rat inner medullary collecting duct cells (IMCDs). Nevertheless, only colonic H+-K+-ATPase mRNA and protein abundance increase in this condition. This study was designed to determine whether Sch-28080 inhibits transporters other than the gastric H+-K+-ATPase. In the presence of bumetanide, Sch-28080 (200 μM) and ouabain (2 mM) inhibited86Rb+ uptake (>90%). That86Rb+ uptake was almost completely abolished by Sch-28080 indicates an effect of this agent on the Na+-K+-ATPase. ATPase assays in membranes, or lysed cells, demonstrated sensitivity to ouabain but not Sch-28080. Thus the inhibitory effect of Sch-28080 was dependent on cell integrity. 86Rb+-uptake studies without bumetanide demonstrated that ouabain inhibited activity by only 50%. Addition of Sch-28080 (200 μM) blocked all residual activity. Intracellular ATP declined after Sch-28080 (200 μM) but recovered after removal of this agent. In conclusion, high concentrations of Sch-28080 inhibit K+-ATPase activity in mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.
Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism
Authors: Gorodeski George I.
Published: December 2017

Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of l-[3H]arginine to l-[3H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.
MHC isoform composition and Ca2+- or Sr2+-activation properties of rat skeletal muscle fibers
Authors: Bortolotto Susan K. , Cellini Maria , Stephenson D. George , Stephenson Gabriela M. M.
Published: December 2017

Chemically skinned single fibers from adult rat skeletal muscles were used to test the hypothesis that, in mammalian muscle fibers, myosin heavy chain (MHC) isoform expression and Ca2+- or Sr2+-activation characteristics are only partly correlated. The fibers were first activated in Ca2+- or Sr2+-buffered solutions under near-physiological conditions, and then their MHC isoform composition was determined electrophoretically. Fibers expressing only the MHC I isoform could be appropriately identified on the basis of either the Ca2+- or Sr2+-activation characteristics or the MHC isoform composition. Fibers expressing one or a combination of fast MHC isoforms displayed no significant differences in their Ca2+- or Sr2+-activation properties; therefore, their MHC isoform composition could not be predicted from their Ca2+- or Sr2+-activation characteristics. A large proportion of fibers expressing both fast- and slow-twitch MHC isoforms displayed Ca2+- or Sr2+-activation properties that were not consistent with their MHC isoform composition; thus both fiber-typing methods were needed to fully characterize such fibers. These data show that, in rat skeletal muscles, the extent of correlation between MHC isoform expression and Ca2+- or Sr2+-activation characteristics is fiber-type dependent.
Cell cycle-related changes in transient K+ current density in the GH3 pituitary cell line
Authors: Czarnecki A. , Vaur S. , Dufy-Barbe L. , Dufy B. , Bresson-Bepoldin L.
Published: December 2017

Our aim was to determine whether the expression of K+ currents is related to the cell cycle in the excitable GH3 pituitary cell line. K+currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cell cycle. We show that the peak density of the transient outward K+ current ( I to) was 33% lower in cells in S phase (BrdU+) than in cells in other phases of the cell cycle (BrdU−). The voltage-dependence of I towas not modified. However, of the two kinetic components of I to inactivation, the characteristics of the fast component differed significantly between BrdU+ and BrdU− cells. Recovery from inactivation of I to showed biexponential and monoexponential function in BrdU− and BrdU+ cells, respectively. This suggests that the molecular basis of this current varies during the cell cycle. We further demonstrated that 4-aminopyridine, which blocks I to, inhibited GH3 cell proliferation without altering the membrane potential. These data suggest that I to may play a role in GH3 cell proliferation processes.
Regulation of the insulin and asialoglycoprotein receptors via cGMP-dependent protein kinase
Authors: De la Vega Luis A. , Stockert Richard J.
Published: December 2017

Biotin regulation of asialoglycoprotein receptor expression and insulin receptor activity has been established in two human hepatoblastoma cell lines, Hep G2 and HuH-7. Second messenger cGMP mimics the effect of biotin on asialoglycoprotein receptor expression at the translational level. Metabolic labeling and subsequent immunoprecipitation indicate that the loss of insulin receptor activity during biotin deprivation was due to suppression of receptor synthesis. Evidence for posttranscriptional regulation of insulin receptor synthesis was provided by rapid biotin induction of receptor synthesis without an increase in gene transcript number. Addition of a cGMP-dependent protein kinase (cGK) inhibitor prevented biotin induction of the insulin and asialoglycoprotein receptors, suggesting that protein phosphorylation propagates the cGMP signal transduction cascade. Coatomer protein COPI was recently identified as the trans-acting factor that regulates in vitro translation of the asialoglycoprotein receptor. Biotin repletion of the culture medium resulted in the hyperphosphorylation of α-COP, which was prevented by simultaneous addition of the cGK inhibitor. These findings suggest that the end point of this cGMP signal cascade is modulated by cGK and that a phosphorylation reaction governs the expression of both receptor proteins.
Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels
Authors: Ma Tonghui , Jayaraman Sujatha , Wang Kasper S. , Song Yuanlin , Yang Baoxue , Li Jiang , Bastidas J. Augusto , Verkman A. S.
Published: December 2017

Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3–3.5 wk of age, 10–12 g) gained 49 ± 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 ± 4%, AQP1 null mice gained only 4 ± 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceridemia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [14C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radioactivity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice.
Expression of the Na+/Ca2+exchanger in skeletal muscle
Authors: Fraysse Bodvaël , Rouaud Thierry , Millour Marie , Fontaine-Pérus Josiane , Gardahaut Marie-France , Levitsky Dmitri O.
Published: December 2017

The expression of the Na+/Ca2+ exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na+/Ca2+ exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.