scholarly journals Lucifer Yellow Slows Voltage‐Gated Na + Current Inactivation in a Light‐Dependent Manner in Mice

2003 ◽  
Vol 550 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Yoko Higure ◽  
Yoshimi Katayama ◽  
Keita Takeuchi ◽  
Yoshitaka Ohtubo ◽  
Kiyonori Yoshii
Keyword(s):  
Lucifer Yellow ◽  
Dependent Manner ◽  
Na Current ◽  
Voltage Gated

Ochratoxin A challenges the intestinal epithelial cell integrity: results obtained in model experiments with Caco-2 cells

2019 ◽  
Vol 12 (4) ◽  
pp. 399-407 ◽  
Author(s):  
A. Alizadeh ◽  
P. Akbari ◽  
S. Varasteh ◽  
S. Braber ◽  
H. Malekinejad ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ochratoxin A ◽  
Lucifer Yellow ◽  
Intestinal Barrier ◽  
Clinical Importance ◽  
Hazardous Substances ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Cell Integrity ◽  
Barrier Integrity

Contamination of human and animal diets with different mycotoxins have gained significant attention over the past decade. The intestinal barrier is the first site of exposure and a primary target for nutritional contaminants and hazardous substances including mycotoxins. In this study, the potential impact of ochratoxin A (OTA) on intestinal barrier integrity was highlighted using a human intestinal Caco-2 cell line. Cell viability following OTA exposure was determined by lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, markers of barrier integrity, such as transepithelial electrical resistance (TEER) as well as the permeability of Lucifer Yellow (LY) and fluorescein isothiocyanate (FITC)-dextran, were assessed. Furthermore, the protein expression of different tight junction (TJ) proteins, as main constituents of barrier integrity, was evaluated by Western blot. Results show that OTA reduces TEER values in a concentration- and time-dependent manner and increase the permeability of LY through the intestinal epithelial layer, while the cell viability did not change significantly. However, the damage was not severe enough to change the permeability to larger molecules, such as FITC-dextran. OTA exposure down-regulated the expression of TJ proteins claudin-1, -3 and -4 and up-regulated the expression of zona occludens 1. The observation that OTA can disrupt the epithelial barrier is of clinical importance as it may lead to an increased passage of luminal antigens into the systemic circulation.

scholarly journals Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803

2006 ◽  
Vol 11 (3) ◽  
Author(s):  
Hui Ling ◽  
Liang-Yun Zhang ◽  
Qi Su ◽  
Ying Song ◽  
Zhao-Yang Luo ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Specific Activity ◽  
Lucifer Yellow ◽  
Gastric Cancer Cell Line ◽  
Major Constituent ◽  
Diallyl Disulfide ◽  
Human Gastric Cancer ◽  
Dependent Manner ◽  
Gastric Cancer Cells ◽  
Induced Differentiation

AbstractDiallyl disulfide (DADS) is a major constituent of garlic. Previously, we found that DADS both inhibited proliferation in human gastric cancer cells in vitro and in vivo, and induced G2/M arrest. In this study, we investigated whether this differentiation effect was induced by DADS in human gastric cancer MGC803 cells, and whether it was related to an alteration in ERK activity. The results showed that the growth of MGC803 cells was inhibited by DADS. Cells treated with DADS displayed a lower nucleocytoplasmic ratio and tended to form gland and intercellular conjunction structures. The ConA-mediated cell agglutination ratio and cells’ ALP specific activity decreased. In MGC803 cells, dye transfer was limited to a few cells neighbouring the dye-injected cell and to a depth of 1–2 layers beneath the scrape site. However, after treatment with DADS, the LY (Lucifer Yellow) was transferred to several cells immediately neighbouring the microinjected cell and to a depth of 2–4 cell layers from the scrape site. This indicated that DADS induced differentiation in MGC803 cells. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls. At 30 mg·L−1, DADS inhibited the activation of ERK1/2 in 15–30 min. These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway.

Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Piet van Vliet ◽  
Teun P de Boer ◽  
Marcel A van der Heyden ◽  
Joost P Sluijter ◽  
Pieter A Doevendans ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Troponin I ◽  
Lucifer Yellow ◽  
Myogenic Differentiation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Cardiomyogenic Differentiation ◽  
Low Potassium ◽  
Calcineurin Signaling

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation. Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway. Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.

scholarly journals Dopamine Receptor Activation Can Reduce Voltage-Gated Na+ Current by Modulating Both Entry Into and Recovery From Inactivation

2004 ◽  
Vol 92 (5) ◽  
pp. 3134-3141 ◽  
Author(s):  
Yuki Hayashida ◽  
Andrew T. Ishida
Keyword(s):  
Dopamine Receptor ◽  
Ganglion Cells ◽  
Receptor Activation ◽  
Resting Potential ◽  
Retinal Ganglion ◽  
Na Current ◽  
Voltage Gated ◽  
Recovery From Inactivation ◽  
Rate Of Recovery ◽  
Type Receptor

We tested whether dopamine receptor activation modulates the voltage-gated Na+ current of goldfish retinal ganglion cells, using a fast voltage-clamp amplifier, perforated-patch whole cell mode, and a physiological extracellular Na+ concentration. As found in other cells, activators of D1-type dopamine receptors and of protein kinase A reduced the amplitude of current activated by depolarizations from resting potential without altering the current kinetics or activation range. However, D1-type dopamine receptor activation also accelerated the rate of entry into inactivation during subthreshold depolarizations and slowed the rate of recovery from inactivation after single, brief depolarizations. Our results provide the first evidence in any preparation that D1-type receptor activation can produce both of these latter effects.

scholarly journals Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

1985 ◽  
Vol 86 (5) ◽  
pp. 739-762 ◽  
Author(s):  
G K Wang ◽  
G Strichartz
Keyword(s):  
Steady State ◽  
Ionic Currents ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Current ◽  
Toxin Concentration ◽  
Toxin Molecule ◽  
Channel Inactivation ◽  
Na Currents ◽  
Voltage Dependent

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

Low [Mg2+]o induces contraction and [Ca2+]i rises in cerebral arteries: roles of Ca2+, PKC, and PI3

2000 ◽  
Vol 279 (6) ◽  
pp. H2898-H2907 ◽  
Author(s):  
Zhi-Wei Yang ◽  
Jun Wang ◽  
Tao Zheng ◽  
Bella T. Altura ◽  
Burton M. Altura
Keyword(s):  
Cerebral Arteries ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Voltage Gated ◽  
Selective Antagonist ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Intracellular Ca ◽  
Basilar Arteries ◽  
Specific Antagonist

Removal of extracellular Ca2+ concentration ([Ca2+]o) and pretreatment of canine basilar arterial rings with either an antagonist of voltage-gated Ca2+ channels (verapamil), a selective antagonist of the sarcoplasmic reticulum Ca2+ pump [thapsigargin (TSG)], caffeine plus a specific antagonist of ryanodine-sensitive Ca2+ release (ryanodine), or ad- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]- mediated Ca2+ release antagonist (heparin) markedly attenuates low extracellular Mg2+ concentration ([Mg2+]o)-induced contractions. Low [Mg2+]o-induced contractions are significantly inhibited by pretreatment of the vessels with Gö-6976 [a protein kinase C-α (PKC-α)- and PKC-βI-selective antagonist], bisindolylmaleimide I (Bis, a specific antagonist of PKC), and wortmannin or LY-294002 [selective antagonists of phosphatidylinositol-3 kinases (PI3Ks)]. These antagonists were also found to relax arterial contractions induced by low [Mg2+]o in a concentration-dependent manner. The absence of [Ca2+]o and preincubation of the cells with verapamil, TSG, heparin, or caffeine plus ryanodine markedly attenuates the transient and sustained elevations in the intracellular Ca2+ concentration ([Ca2+]i) induced by low-[Mg2+]o medium. Low [Mg2+]o-produced increases in [Ca2+]i are also suppressed markedly in the presence of Gö-6976, Bis, wortmannin, or LY-294002. The present study suggests that both Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from intracellular stores [both Ins(1,4,5)P3sensitive and ryanodine sensitive] play important roles in low-[Mg2+]o medium-induced contractions of isolated canine basilar arteries. Such contractions are clearly associated with activation of PKC isoforms and PI3Ks.

Relationship between Na+ current expression and cell-cell coupling in astrocytes cultured from rat hippocampus

1991 ◽  
Vol 65 (4) ◽  
pp. 989-1002 ◽  
Author(s):  
H. Sontheimer ◽  
S. G. Waxman ◽  
B. R. Ransom
Keyword(s):  
Lucifer Yellow ◽  
Na Channel ◽  
Dye Coupling ◽  
Cell Coupling ◽  
Na Current ◽  
Na Currents ◽  
Coupled Cells ◽  
Hippocampal Astrocytes ◽  
Cell Cell

1. Cell-cell coupling between hippocampal astrocytes in culture was studied by following the intracellular spread of the low molecular weight fluorescent dye Lucifer yellow (LY). Dye coupling appeared as early as 24 h after plating, at which time approximately 20% of all astrocytes that physically contacted neighboring cells showed dye coupling. 2. The percentage of coupled cells increased with time in culture and peaked after 10 days in vitro (DIV) when approximately 50% of astrocytes showed coupling. Further time in culture, up to 20 DIV, did not increase the percentage of coupled cells. Thus, coupled and noncoupled astrocytes coexist in hippocampal cultures in approximately equal numbers. 3. Na+ currents were expressed in a subpopulation of hippocampal astrocytes and changed characteristics during in vitro development. A "neuronal type" of Na+ current, so called because of an h alpha curve that had a midpoint near -60 mV, was observed within the first 5 days post-plating. A "glial type" of Na+ current, characterized by a -25 mV shift in its h alpha curve, was only expressed after 6 days in culture. 4. Na+ current expression was restricted to hippocampal astrocytes that did not exhibit dye coupling; astrocytes that exhibited dye coupling (n = 39) did not show measurable Na+ currents. 5. The failure to see Na+ currents in coupled astrocytes cannot be explained by insufficient space-clamp since astrocytes acutely uncoupled with octanol (10 microM) did not reveal Na+ current expression. Control experiments showed that low concentrations of octanol (i.e., 10-100 microM) did not block Na+ currents; blockage of Na+ currents by octanol was only observed at high concentrations (e.g., 50-fold the concentration used for uncoupling). These observations support the idea that Na(+)-channel expression was restricted to noncoupled astrocytes. 6. The time courses for the development of cell coupling and Na+ current expression appeared to be inversely correlated and suggested a gradual increase in cell coupling in concert with a loss in Na+ current expression with time in culture.

scholarly journals Evidence That the Product of the Human X-Linked Cgd Gene, Gp91-phox, Is a Voltage-Gated H+ Pathway

1999 ◽  
Vol 114 (6) ◽  
pp. 771-786 ◽  
Author(s):  
Lydia M. Henderson ◽  
Robert W. Meech
Keyword(s):  
Cell Membrane ◽  
Time Course ◽  
Lucifer Yellow ◽  
Chinese Hamster ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Histidine Residues ◽  
Voltage Gated

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H+ conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O2·2). Suspensions of CHO91 cells exhibit arachidonate-activatable H+ fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909–5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H+. As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199–216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590–1598), a lowered external pH (pHo) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 μM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10–20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2′7′ bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pHi) was ∼6.9, as though pHi was largely determined by endogenous cellular regulation. Arachidonate (20 μM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl− concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH2-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H+ permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H+ selectivity. Mechanisms of H+ permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319–1327) and the transfer of protons across an “H-X-X-X-H-X-X-X-H” motif lining a conducting pore.

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