scholarly journals Biophysical Characterization of Epigallocatechin-3-Gallate Effect on the Cardiac Sodium Channel Nav1.5

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 902 ◽  
Author(s):  
Mohamed-Yassine Amarouch ◽  
Han Kurt ◽  
Lucie Delemotte ◽  
Hugues Abriel
Keyword(s):  
Md Simulations ◽  
Inactivation Kinetics ◽  
Free Energy Barrier ◽  
Dependent Manner ◽  
Biophysical Characterization ◽  
Inactivation Curve ◽  
Cardiac Sodium Channel ◽  
Dependent Inhibition ◽  
Steady State Inactivation ◽  
Dose Dependent

Epigallocatechin-3-Gallate (EGCG) has been extensively studied for its protective effect against cardiovascular disorders. This effect has been attributed to its action on multiple molecular pathways and transmembrane proteins, including the cardiac Nav1.5 channels, which are inhibited in a dose-dependent manner. However, the molecular mechanism underlying this effect remains to be unveiled. To this aim, we have characterized the EGCG effect on Nav1.5 using electrophysiology and molecular dynamics (MD) simulations. EGCG superfusion induced a dose-dependent inhibition of Nav1.5 expressed in tsA201 cells, negatively shifted the steady-state inactivation curve, slowed the inactivation kinetics, and delayed the recovery from fast inactivation. However, EGCG had no effect on the voltage-dependence of activation and showed little use-dependent block on Nav1.5. Finally, MD simulations suggested that EGCG does not preferentially stay in the center of the bilayer, but that it spontaneously relocates to the membrane headgroup region. Moreover, no sign of spontaneous crossing from one leaflet to the other was observed, indicating a relatively large free energy barrier associated with EGCG transport across the membrane. These results indicate that EGCG may exert its biophysical effect via access to its binding site through the cell membrane or via a bilayer-mediated mechanism.

scholarly journals Biophysical characterization of Epigallocatechin-3-Gallate effect on the cardiac sodium channel Nav1.5

2019 ◽  
Author(s):  
Mohamed Yassine AMAROUCH ◽  
Mehmet Hanifi Kurt ◽  
Lucie Delemotte ◽  
Hugues Abriel
Keyword(s):  
Md Simulations ◽  
Inactivation Kinetics ◽  
Free Energy Barrier ◽  
Dependent Manner ◽  
Biophysical Characterization ◽  
Inactivation Curve ◽  
Cardiac Sodium Channel ◽  
Dependent Inhibition ◽  
Steady State Inactivation ◽  
Dose Dependent

Epigallocatechin-3-Gallate (EGCG) has been extensively studied for its protective effect against cardiovascular disorders. This effect has been attributed to its action on multiple molecular pathways and transmembrane proteins, including the cardiac Nav1.5 channels, which are inhibited in a dose-dependent manner. However, the molecular mechanism underlying this effect remains to be unveiled. To this aim, we have characterized the EGCG effect on Nav1.5 using electrophysiology and molecular dynamics (MD) simulations. EGCG superfusion induced a dose-dependent inhibition of Nav1.5 expressed in tsA201 cells, negatively shifted the steady-state inactivation curve, slowed the inactivation kinetics, and delayed the recovery from fast inactivation. However, EGCG had no effect on the voltage-dependence of activation and showed little use-dependent block on Nav1.5. Finally, MD simulations suggested that EGCG does not preferentially stay in the center of the bilayer, but that it spontaneously relocates to the membrane headgroup region. Moreover, no sign of spontaneous crossing from one leaflet to the other was observed, indicating a relatively large free energy barrier associated with EGCG transport across the membrane. These results indicate that EGCG may exert its biophysical effect via access to its binding site through the cell membrane or via a bilayer-mediated mechanism.

scholarly journals Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

2011 ◽  
Vol 300 (3) ◽  
pp. C567-C575 ◽  
Author(s):  
Hee Jae Kim ◽  
Hye Sook Ahn ◽  
Bok Hee Choi ◽  
Sang June Hahn
Keyword(s):  
Steady State ◽  
Inactivation Kinetics ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Voltage Range ◽  
Closed State ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Steady State Inactivation

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K+ (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC50 of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The Ki for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.

Calcineurin-independent inhibition of KV1.3 by FK-506 (tacrolimus): a novel pharmacological property

2007 ◽  
Vol 292 (5) ◽  
pp. C1714-C1722 ◽  
Author(s):  
Hye Sook Ahn ◽  
Sung Eun Kim ◽  
Bok Hee Choi ◽  
Jin-Sung Choi ◽  
Myung-Jun Kim ◽  
...  
Keyword(s):  
Steady State ◽  
Chinese Hamster Ovary Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Fk 506 ◽  
Inactivation Curve ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Steady State Inactivation

The interaction of FK-506 with KV1.3, stably expressed in Chinese hamster ovary cells, was investigated with the whole cell patch-clamp technique. FK-506 inhibited KV1.3 in a reversible, concentration-dependent manner with an IC50 of 5.6 μM. Rapamycin, another immunosuppressant, produced effects that were similar to those of FK-506 (IC50 = 6.7 μM). Other calcineurin inhibitors (cypermethrin or calcineurin autoinhibitory peptide) alone had no effect on the amplitude or kinetics of KV1.3. In addition, the inhibitory action of FK-506 continued, even after the inhibition of calcineurin activity. The inhibition produced by FK-506 was voltage dependent, increasing in the voltage range for channel activation. At potentials positive to 0 mV (where maximal conductance is reached), however, no voltage-dependent inhibition was found. FK-506 exhibited a strong use-dependent inhibition of KV1.3. FK-506 shifted the steady-state inactivation curves of KV1.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant for FK-506 to inhibit KV1.3 in the inactivated state was estimated from the concentration-dependent shift in the steady-state inactivation curve and was calculated to be 0.37 μM. Moreover, the rate of recovery from inactivation of KV1.3 was decreased. In inside-out patches, FK-506 not only reduced the current amplitude but also accelerated the rate of inactivation during depolarization. FK-506 also inhibited KV1.5 and KV4.3 in a concentration-dependent manner with IC50 of 4.6 and 53.9 μM, respectively. The present results indicate that FK-506 inhibits KV1.3 directly and that this effect is not mediated via the inhibition of the phosphatase activity of calcineurin.

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability

1989 ◽  
Vol 66 (3) ◽  
pp. 1471-1476 ◽  
Author(s):  
H. Lum ◽  
P. J. Del Vecchio ◽  
A. S. Schneider ◽  
M. S. Goligorsky ◽  
A. B. Malik
Keyword(s):  
Endothelial Cells ◽  
Endothelial Permeability ◽  
Base Line ◽  
Dependent Manner ◽  
Extracellular Medium ◽  
Influx Rate ◽  
Before And After ◽  
Dependent Inhibition ◽  
Permeability Increase ◽  
Dose Dependent

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.

ACTIVATION DEPENDENT ALTERATIONS IN THE CHEMICAL CROSSLINKING OF ARGINYL-GLYCYL-ASPARTIC ACID (RGD) PEPTIDES WITH PLATELET GLYCOPROTEIN (GP) GPIIb-IIIa

1987 ◽  
Author(s):  
S E D’Souza ◽  
M H Ginaberg ◽  
S Lam ◽  
E A Plow
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Amino Acid Sequences ◽  
Platelet Glycoprotein ◽  
Chemical Crosslinking ◽  
Dependent Manner ◽  
Rgd Peptides ◽  
Dependent Inhibition ◽  
Adhesive Proteins ◽  
Dose Dependent

The platelet adhesive proteins, fibrinogen, fibronectin and von WillebrandFactor, contain RGD amino acid sequences; RGD-containing peptides inhibit the binding of these adhesive proteins to platelets; and a membrane receptor for these adhesive proteins binds to Arg-Gly-Asp and contains GPIIb-IIIa. The present study was undertaken to characterize the interaction of RGDpeptides with GPIIb-IIIa using a chemical crosslinking approach. A radioiodinated RGD-containing heptapeptide was bound to washed human platelets under conditions at which ≥ 85% of theinteraction was inhibited by excess nonlabeled peptide. After binding of the peptide to platelets for 45 min at22°, a homobifunctional crosslinking reagent was added, and the platelets were extracted and analyzed on polyacrylamide gels. With resting platelets,autoradiography of the gels revealedthat the peptide crosslinked tobothGPIIb and GPIIIa. This interaction wasinhibited by excess nonlabeled peptide but not by certain conservatively substituted RGD peptides. Stimulation of the platelets caused a dramatic increase in crosslinking of the peptide to only one of the two subunitsof GPIIb-IIIa. The stimulus dependentincrease in the crosslinking reactionwas specific and saturable as it was inhibited by RGD peptides in a dose dependent manner. In addition, peptides corresponding in structure to the carboxy terminus of the γ chain of fibrinogen also produced concentration dependent inhibition of the interaction. The increase in crosslinking induced by platelet stimulation was divalent ion dependent. Similar results werealso obtained with a second, larger RGD-containing peptide and with asecond chemical crosslinking reagent.Theseresults indicate that platelet stimulation in the presence of divalent ions causes a change which permitsmoreefficient crosslinking of RGD-containing peptides to only one of the two subunits of GPIIb-IIIa. The results are also compatible with a proximalrelationship of both subunits tothe RGD binding sites on the plateletmembrane.

scholarly journals Hirudin interruption of heparin-resistant arterial thrombus formation in baboons

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1006-1012 ◽  
Author(s):  
AB Kelly ◽  
UM Marzec ◽  
W Krupski ◽  
A Bass ◽  
Y Cadroy ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Fibrin Deposition ◽  
Recombinant Hirudin ◽  
Arterial Thrombus ◽  
Dependent Inhibition ◽  
Antithrombotic Effects ◽  
Dose Dependent

Abstract To determine the role of thrombin in high blood flow, platelet- dependent thrombotic and hemostatic processes we measured the relative antithrombotic and antihemostatic effects in baboons of hirudin, a highly potent and specific antithrombin, and compared the effects of heparin, an antithrombin III-dependent inhibitor of thrombin. Thrombus formation was determined in vivo using three relevant models (homologous endarterectomized aorta, collagen-coated tubing, and Dacron vascular graft) by measuring: (1) platelet deposition, using gamma camera imaging of 111In-platelets; (2) fibrin deposition, as assessed by the incorporation of circulating 125I-fibrinogen; and (3) occlusion. The continuous intravenous infusion of 1, 5, and 20 nmol/kg per minute of recombinant hirudin (desulfatohirudin) maintained constant plasma levels of 0.16 +/- 0.03, 0.79 +/- 0.44, and 3.3 +/- 0.77 mumol/mL, respectively. Hirudin interrupted platelet and fibrin deposition in a dose-dependent manner that was profound at the highest dose for all three thrombogenic surfaces and significant at the lowest dose for thrombus formation on endarterectomized aorta. Thrombotic occlusion was prevented by all doses studied. In contrast, heparin did not inhibit either platelet or fibrin deposition when administered at a dose that maximally prolonged clotting times (100 U/kg) (P greater than .1), and only intermediate effects were produced at 10-fold that dose (1,000 U/kg). Moreover, heparin did not prevent occlusion of the test segments. Hirudin inhibited platelet hemostatic function in concert with its antithrombotic effects (bleeding times were prolonged by the intermediate and higher doses). By comparison, intravenous heparin failed to affect the bleeding time at the 100 U/kg dose (P greater than .5), and only minimally prolonged the bleeding time at the 1,000 U/kg dose (P less than .05). We conclude that platelet-dependent thrombotic and hemostatic processes are thrombin-mediated and that the biologic antithrombin hirudin produces a potent, dose-dependent inhibition of arterial thrombus formation that greatly exceeds the minimal antithrombotic effects produced by heparin.

Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro

1999 ◽  
Vol 277 (3) ◽  
pp. L543-L549 ◽  
Author(s):  
Etsuro Sato ◽  
Keith L. Simpson ◽  
Matthew B. Grisham ◽  
Sekiya Koyama ◽  
Richard A. Robbins
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Chemotactic Activity ◽  
Dependent Manner ◽  
No Donor ◽  
Monocyte Chemoattractant Protein 1 ◽  
Dependent Inhibition ◽  
Monocyte Chemotaxis ◽  
Dose Dependent

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), can nitrate tyrosine residues, resulting in compromised protein function. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that attracts monocytes and has a tyrosine residue critical for function. We hypothesized that peroxynitrite would alter MCP-1 activity. Peroxynitrite attenuated MCP-1-induced monocyte chemotactic activity (MCA) in a dose-dependent manner ( P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum MCA. The reducing agents dithionite, deferoxamine, and dithiothreitol reversed the MCA inhibition by peroxynitrite, and exogenous l-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, an NO donor, or superoxide generated by xanthine and xanthine oxidase did not show an inhibitory effect on MCA induced by MCP-1. The peroxynitrite generator 3-morpholinosydnonimine caused a concentration-dependent inhibition of MCA by MCP-1. Peroxynitrite reduced MCP-1 binding to monocytes and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite, with subsequent inhibition of MCP-1 binding to monocytes, and suggest that peroxynitrite may play a role in regulation of MCP-1-induced monocyte chemotaxis.

scholarly journals A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1446-1454 ◽  
Author(s):  
N Katayama ◽  
M Nishikawa ◽  
F Komada ◽  
N Minami ◽  
S Shirakawa
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Gm Csf ◽  
Erythroid Progenitor Cells ◽  
Dependent Inhibition ◽  
Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

scholarly journals Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog.

1989 ◽  
Vol 94 (5) ◽  
pp. 937-951 ◽  
Author(s):  
G Cota ◽  
E Stefani
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Voltage Dependence ◽  
Muscle Fibers ◽  
Dependent Manner ◽  
Inactivation Curve ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Intact Muscle ◽  
Ca2 Channels

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.

scholarly journals Effect of L. usitatissimum (Flaxseed/Linseed) Fixed Oil against Distinct Phases of Inflammation

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Gaurav Kaithwas ◽  
Dipak K. Majumdar
Keyword(s):  
Vascular Permeability ◽  
Inflammatory Reaction ◽  
Linum Usitatissimum ◽  
Acid Metabolism ◽  
Capillary Permeability ◽  
Leukocyte Migration ◽  
Dependent Manner ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

The present investigation summarizes the effect of Linum usitatissimum fixed oil against different phases of acute inflammatory reaction, namely, protein exudation, peritoneal capillary permeability, and leukocyte migration. The fixed oil exhibited dose-dependent inhibition of protein exudation vascular permeability, comparable to standard aspirin. The oil also inhibited the leukocyte migration in pleural exudates in a dose-dependent manner. Production of less vasodilatory (PGE3) and chemotactic (LTB5) eicosanoids through EPA (derived from linolenic acid) metabolism could account for the above observations.

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