scholarly journals Oligomycin inhibits store-operated channels by a mechanism independent of its effects on mitochondrial ATP

1997 ◽  
Vol 324 (3) ◽  
pp. 971-980 ◽  
Author(s):  
Jwa Hwa CHO ◽  
M. BALASUBRAMANYAM ◽  
Galina CHERNAYA ◽  
Jeffrey P. GARDNER ◽  
Abraham AVIV ◽  
...  
Keyword(s):  
T Cells ◽  
Rank Order ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Jurkat T Cells ◽  
Ovary Cells ◽  
Atp Production ◽  
Patch Pipette ◽  
Pump Activity

Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration ∼2 μM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.

Costimulation of  T cells by B7-H2, a B7-like molecule that binds ICOS

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2808-2813 ◽  
Author(s):  
Shengdian Wang ◽  
Gefeng Zhu ◽  
Andrei I. Chapoval ◽  
Haidong Dong ◽  
Koji Tamada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Ovary Cells ◽  
Inducible Costimulator ◽  
Immature Dendritic Cells ◽  
Dose Dependent

Abstract This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

Retigabine Stimulates Human KCNQ2/Q3 Channels in the Presence of Bupivacaine

2004 ◽  
Vol 101 (2) ◽  
pp. 430-438 ◽  
Author(s):  
Mark A. Punke ◽  
Patrick Friederich
Keyword(s):  
Membrane Potential ◽  
Local Anesthetic ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Ovary Cells ◽  
Novel Approach ◽  
Concentration Response Curve

Background Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic. Methods KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique. Results Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 microm, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 microm) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential. Conclusions The anticonvulsant retigabine at nanomolar concentrations reverses the inhibitory effect of micromolar concentrations of bupivacaine. Our results allow the hypothesis that activation of KCNQ2/Q3 channels by retigabine may offer a novel therapeutic approach for the treatment of bupivacaine-induced seizures.

Costimulation of  T cells by B7-H2, a B7-like molecule that binds ICOS

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2808-2813 ◽  
Author(s):  
Shengdian Wang ◽  
Gefeng Zhu ◽  
Andrei I. Chapoval ◽  
Haidong Dong ◽  
Koji Tamada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Ovary Cells ◽  
Inducible Costimulator ◽  
Immature Dendritic Cells ◽  
Dose Dependent

This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

scholarly journals Antiarrhythmic Mechanisms of Chinese Herbal Medicine Dingji Fumai Decoction

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Bo Liang ◽  
Yan Zhou ◽  
Ling Fu ◽  
Hui-Ling Liao
Keyword(s):  
Ventricular Arrhythmia ◽  
Connexin 43 ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Barium Chloride ◽  
Inhibitory Effect ◽  
Ovary Cells ◽  
Whole Cell Patch Clamp ◽  
And Oxidative Stress ◽  
Electrophysiological Mechanism

Background. Dingji Fumai decoction (DFD) is used to treat ventricular arrhythmia, and it has provided a very good curative effect. However, its cellular electrophysiological mechanism is unknown. Methods. Electrocardiogram was recorded, and oxidative stress response and ion-channel-related molecules were detected in rats with barium chloride- and aconitine-induced ventricular arrhythmia. Moreover, whole-cell patch-clamp assay was used to investigate the inhibitory effect of DFD on Nav1.5 in Chinese hamster ovary cells. Results. DFD prolonged the occurrence time and shortened the duration of ventricular arrhythmia, decreased the malondialdehyde and increased the superoxide dismutase, and alleviated the activation of Na+-K+-ATPase and connexin-43. DFD suppressed Nav1.5dose-dependently with an IC50 of 24.0 ± 2.4 mg/mL. Conclusions. The clinical antiarrhythmic mechanisms of DFD are based on its antioxidant potential, alleviation of Na+-K+-ATPase and connexin-43, and class I antiarrhythmic properties by suppressing Nav1.5dose-dependently with an IC50 of 24.0 ± 2.4 mg/mL.

Linoleic acid both enhances activation and blocks Kv1.5 and Kv2.1 channels by two separate mechanisms

2001 ◽  
Vol 281 (4) ◽  
pp. C1277-C1284 ◽  
Author(s):  
M. Craig McKay ◽  
Jennings F. Worley
Keyword(s):  
Linoleic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Activation Process ◽  
Activation Kinetics ◽  
Pipette Solution ◽  
Ovary Cells ◽  
Cooperative Interactions ◽  
Patch Pipette ◽  
Current Activation

Linoleic acid (LA) had two effects on human Kv1.5 and Kv2.1 channels expressed in Chinese hamster ovary cells: an increase in the speed of current activation process (EC50 = 2.4 and 2.7 μM for Kv1.5 and Kv2.1, respectively) and current inhibition (IC50 = 6.6 and 7.4 for Kv1.5 and Kv2.1, respectively). LA affected the activation kinetics via two processes: a leftward shift in the instantaneous activation curves and an increase in the rate of current rise. Current inhibition by LA was time dependent but voltage independent. Hill slopes for plots of current inhibition (3.5 and 3.9 for Kv1.5 and Kv2.1, respectively) vs. dose of LA suggested that cooperativity was involved in the mechanism of current inhibition. A similar analysis of the effects of LA on current activation did not reveal cooperative interactions. The effects of LA were mediated from the external side of the channels, since addition of 10 μM LA to the patch pipette solution was without effect. Additionally, the methyl ester of LA was effective at enhancing peak current and promoting channel activation for Kv1.5 and Kv2.1 without inducing significant current inhibition.

Interaction of Ropivacaine with Cloned Cardiac Kv4.3/KChIP2.2 Complexes

2004 ◽  
Vol 101 (6) ◽  
pp. 1347-1356 ◽  
Author(s):  
Patrick Friederich ◽  
Anna Solth
Keyword(s):  
Local Anesthetic ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Outward Current ◽  
Ventricular Myocardium ◽  
Inhibitory Effect ◽  
Transient Outward Current ◽  
Qtc Interval ◽  
Ovary Cells ◽  
Ic50 Value

Background Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels. Methods Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method. Results Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 microm (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 microm (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels. Conclusions : The results are consistent with the idea that ropivacaine, by blocking Kv4.3/KChIP2.2 from the open state, interferes with the gating modifying effects of KChIP2.2 on Kv4.3 channels. Inhibition of Kv4.3/KChIP2.2 channels by the local anesthetic may contribute to the deterioration of cardiac function during events of intoxication.

scholarly journals CFTR Gating I

2005 ◽  
Vol 125 (4) ◽  
pp. 361-375 ◽  
Author(s):  
Silvia G. Bompadre ◽  
Tomohiko Ai ◽  
Jeong Han Cho ◽  
Xiaohui Wang ◽  
Yoshiro Sohma ◽  
...  
Keyword(s):  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Kinetic Mechanism ◽  
Inhibitory Effect ◽  
Unexpected Result ◽  
Ovary Cells ◽  
Gating Mechanisms ◽  
Inside Out ◽  
R Domain

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

scholarly journals Effect of Colcemid on the Centriole Cycle in Chinese Hamster Ovary Cells

1982 ◽  
Vol 53 (1) ◽  
pp. 155-171 ◽  
Author(s):  
RYOKO KURIYAMA
Keyword(s):  
Chinese Hamster Ovary ◽  
Structural Changes ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Ultrastructural Changes ◽  
Prolonged Treatment ◽  
Ovary Cells ◽  
Almost All ◽  
Daughter Centriole

The structural changes in the centrioles in Chinese hamster ovary cells were monitored by electron microscopy of whole mount preparations to investigate the effects of colcemid on the events in the centriole cycle. The population of mitotic cells increased with time of incubation with colcemid, but the arrest at mitosis by this drug was soon overcome, resulting in the formation of nuclei and a change in the shape of the cells again spreading over the substrate. The maximal mitotic index was reached every 25 h in the presence of either 0.10 or 0.91 μg/ml of colcemid. During this time, cells became multinucleated, increased greatly in size, and accumulated 8 to 10-nm filamentous bundles in the cytoplasm instead of microtubules, almost all of which had been depolymerized after exposure to colcemid. In the cells that were continuously treated with colcemid, a pair of centrioles became disoriented and each subsequently produced a daughter centriole. However, these daughter centrioles elongated to only half their full length; many unusual figures in the centriolar pairs resulted from their proceeding normally to the phases for disorientation and nucleation for centrioles in the next cycle. Although the rate of centriole elongation and the frequency of formation of the daughter centrioles were decreased by increasing the concentration of colcemid, the disorientation of the centrioles was not disturbed by this drug. The inhibitory effect of colcemid on centriolar nucleation and elongation was found to be totally reveisible; the formation and elongation of new daughter centrioles occurred again just after removal of the drug. Prolonged treatment of cells with colcemid caused ultrastructural changes in the centrioles, such as the outgrowth of microtubules from the wall of centriolar triplets or the formation of unusual bundles of microtubules around the centrioles.

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