Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor

2002 ◽  
Vol 282 (6) ◽  
pp. C1461-C1468 ◽  
Author(s):  
Bok Hee Choi ◽  
Jin-Sung Choi ◽  
Duck-Joo Rhie ◽  
Shin Hee Yoon ◽  
Do Sik Min ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Time Course ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Voltage Range

The action of tyrphostin AG-1478, a potent protein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels (Kv1.5) stably expressed in Chinese hamster ovary cells was investigated using the whole cell patch-clamp technique. AG-1478 rapidly and reversibly inhibited Kv1.5 currents at 50 mV in a concentration-dependent manner with an IC50 of 9.82 μM. AG-1478 accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. Pretreatment with the structurally dissimilar PTK inhibitors (genistein and lavendustin A) had no effect on the AG-1478-induced inhibition of Kv1.5 and did not modify the AG-1478-induced current kinetics. The rate constants for binding and unbinding of AG-1478 were 1.46 μM−1 · s−1 and 10.19 s−1, respectively. The AG-1478-induced inhibition of Kv1.5 channels was voltage dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG-1478 produced no significant effect on the steady-state activation or inactivation curves. AG-1478 slowed the deactivation time course, resulting in a tail crossover phenomenon. Inhibition of Kv1.5 by AG-1478 was use dependent. The present results suggest that AG-1478 acts directly on Kv1.5 currents as an open-channel blocker and independently of the effects of AG-1478 on PTK activity.

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

2005 ◽  
Vol 289 (2) ◽  
pp. C425-C436 ◽  
Author(s):  
Bok Hee Choi ◽  
Jung-Ah Park ◽  
Kyung-Ryoul Kim ◽  
Ggot-Im Lee ◽  
Yong-Tae Lee ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cytochalasin B ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk− cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC50 of 4.2 μM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC50 of 1.4 μM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 μM/s and 7.5 s−1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 μM) also inhibited an ultrarapid delayed rectifier K+ current ( IK,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous IK,ur in a cytoskeleton-independent manner as an open-channel blocker.

Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity

2007 ◽  
Vol 292 (5) ◽  
pp. F1583-F1591 ◽  
Author(s):  
Ryan M. Pelis ◽  
Yodying Dangprapai ◽  
Theresa M. Wunz ◽  
Stephen H. Wright
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Inorganic Mercury ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Tubular Secretion ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Transport Activity ◽  
Concentration Dependent Manner ◽  
Quadruple Mutant

Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO2-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg2+(as HgCl2) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [3H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl2, with an IC50of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl2caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg2+, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl2than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl2effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg2+with these residues causes reduced TEA transport activity.

scholarly journals RF9 Acts as a KISS1R Agonist In Vivo and In Vitro

Endocrinology ◽  
2015 ◽  
Vol 156 (12) ◽  
pp. 4639-4648 ◽  
Author(s):  
Le Min ◽  
Silvia Leon ◽  
Huan Li ◽  
Leonor Pinilla ◽  
Rona S. Carroll ◽  
...  
Keyword(s):  
Time Course ◽  
Inositol Phosphate ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Gonadotropin Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Lh Secretion

RF9, a reported antagonist of the mammalian gonadotropin-inhibitory hormone receptor, stimulates gonadotropin secretion in mammals. Recent studies have suggested that the stimulatory effect of RF9 on gonadotropin secretion relies on intact kisspeptin receptor (KISS1R) signaling, but the underlying mechanisms remain to be elucidated. Using Chinese Hamster Ovary cells stably transfected with KISS1R, we show that RF9 binds specifically to KISS1R, with a Kd of 1.6 × 10−5M, and stimulates an increase in intracellular calcium and inositol phosphate accumulation in a KISS1R-dependent manner, with EC50 values of 3.0 × 10−6M and 1.6 × 10−7M, respectively. RF9 also stimulated ERK phosphorylation, with a time course similar to that of kisspeptin-10. RFRP-3, the putative endogenous ligand for NPFFR1, did not stimulate inositol phosphate accumulation or pERK, nor did it alter responses to of kisspeptin-10 or RF9. In agreement with these in vitro data, we found that RF9 stimulated a robust LH increase in Npffr1−/− mice, similar to that in wild-type littermates, whereas the stimulatory effect of RF9 was markedly reduced in Kiss1r−/− and double Kiss1r−/−/Npfrr1−/− mice. The stimulatory effect of RF9 on LH secretion was restored by the selective rescue of Kiss1r expression in GnRH neurons, in Kiss1r−/−T mice. Taken together, our study demonstrates that RF9 acts primarily as a KISS1R agonist, but not as an allosteric modulator, to stimulate LH secretion. Our findings raise questions regarding the utility of RF9 for assessing NPFF1R function and de-emphasize a predominant role of this signaling system in central regulation of reproduction.

scholarly journals IL-1α Stimulates Cathepsin K Expression in Osteoclasts via the Tyrosine Kinase-NF-κB Pathway

2004 ◽  
Vol 83 (10) ◽  
pp. 791-796 ◽  
Author(s):  
S. Kamolmatyakul ◽  
W. Chen ◽  
S. Yang ◽  
Y. Abe ◽  
R. Moroi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Protein A ◽  
Cathepsin K ◽  
Underlying Mechanism ◽  
Transcriptional Level ◽  
Pyrrolidine Dithiocarbamate ◽  
Dependent Manner ◽  
Powerful Activator

Interleukin-1α (IL-1α) is a powerful activator of osteoclast cells. However, the underlying mechanism for this activation is unknown. In this study, we reveal that IL-1α up-regulates the expression of cathepsin K protein, a key protease in bone resorption, by five-fold. Northern blot analysis and promoter analysis show that this induction occurs at the transcriptional level, in a dose-responsive and time-dependent manner. No increase in expression occurs in the presence of either pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-κB, or Genistein, a protein tyrosine kinase inhibitor, suggesting that IL-1α up-regulation may be via the tyrosine kinase-NF-κB pathway to regulate cathepsin K expression. Antisense oligonucleotides to p65, but not the p50 subunit of NF-κB, suppress the IL-1α-induced expression of cathepsin K. We therefore conclude that IL-1α up-regulates cathepsin K gene expression at the transcription level, and this regulation may be via the tyrosine-kinase-NF-κB pathway.

scholarly journals Antithrombotic effect of recombinant human thrombomodulin on thrombin- induced thromboembolism in mice

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1396-1399 ◽  
Author(s):  
K Gomi ◽  
M Zushi ◽  
G Honda ◽  
S Kawahara ◽  
O Matsuzaki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Recombinant Thrombomodulin

Abstract Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

scholarly journals Progesterone and the zona pellucida activate different transducing pathways in the sequence of events leading to diacylglycerol generation during mouse sperm acrosomal exocytosis

1996 ◽  
Vol 320 (3) ◽  
pp. 1017-1023 ◽  
Author(s):  
Tetsuma MURASE ◽  
Eduardo R. S. ROLDAN
Keyword(s):  
Tyrosine Kinase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Zona Pellucida ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Sequence Of Events ◽  
Acrosomal Exocytosis

We tested the involvement of protein tyrosine kinase and G-protein transducing pathways in the formation of diacylglycerol (DAG) during exocytosis in mouse spermatozoa. In capacitated spermatozoa, stimulation with solubilized zona pellucida (ZP) or progesterone led to the formation of DAG and to exocytosis of the acrosomal granule. Stimulation of DAG formation and exocytosis by ZP were inhibited in a concentration-dependent fashion by pre-exposure to tyrphostin A48, a protein tyrosine kinase inhibitor. These ZP-induced responses were also reduced in a concentration-dependent manner by prior incubation with pertussis toxin, a G-protein (Gi class) inhibitor. On the other hand, generation of DAG and exocytosis triggered by progesterone were inhibited if spermatozoa were preincubated with different concentrations of tyrphostin A48, but were not affected by pre-exposure to pertussis toxin. Progesterone acts on at least two novel surface receptors, one being a γ-aminobutyric acid (GABA) type A (GABAA)-like receptor. Transducing mechanisms coupled to this receptor were tested directly by stimulating spermatozoa with GABA. Treatment of capacitated spermatozoa with GABA resulted in DAG formation and exocytosis. These responses were not seen when cells were preincubated with tyrphostin A48. Pertussis toxin, however, did not affect the generation of DAG and exocytosis triggered by GABA, in agreement with results obtained using progesterone. Taken together, these results indicate that DAG formation during acrosomal exocytosis is differentially regulated by transducing pathways activated by oocyte-associated agonists.

Endothelin receptor in osteoblastic cells is coupled to multiple messenger signals

1994 ◽  
Vol 267 (5) ◽  
pp. C1329-C1337 ◽  
Author(s):  
J. Green ◽  
O. Foellmer ◽  
C. R. Kleeman ◽  
M. M. Basic
Keyword(s):  
Tyrosine Kinase ◽  
Phorbol Esters ◽  
Kinase Inhibitor ◽  
Rank Order ◽  
Receptor Subtype ◽  
Dependent Manner ◽  
Response Curves ◽  
Concentration Dependent Manner ◽  
Single Class ◽  
Umr 106

We analyzed the functional characteristics of endothelin (ET) peptides in the osteoblastic UMR-106 cells by studying receptor binding as well as dose-response curves for ET-1 and ET-3 on two biological responses: 1) induction of Ca2+ transients and 2) activation of the Na(+)-H+ exchanger. ET specifically binds to a single class of receptor with a rank order of affinity ET-1 >> ET-3. ET-1 and ET-3 dose dependently stimulated a rise in intracellular Ca2+ ([Ca2+]i), with ET-1 being two orders of magnitude more potent than ET-3 [50% effective concentration (EC50) = 8 x 10(-10) and 9 x 10(-8) M for ET-1 and ET-3, respectively; P < 0.01]. The effect of ET-1 on [Ca2+]i was 90% inhibitable by the ETA antagonist BQ-123. The activity of Na(+)-H+ exchange was studied by using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein as well as by 22Na+ fluxes. ET-1 and ET-3 activated the exchange in a concentration-dependent manner and with similar potencies (EC50 approximately 10(-10) M). The action of ETs on Na(+)-H+ exchange was mimicked neither by phorbol esters nor by Ca2+ ionophores. It was, however, blocked by BQ-123 as well as by the protein tyrosine kinase inhibitor genistein. We conclude that in UMR-106 cells, a single ET receptor subtype is coupled to multiple effectors, a Ca2+ message system and a tyrosine-kinase system which, in turn, activates the Na(+)-H+ exchanger.

scholarly journals Differential Effects of Tyrosine Kinase Inhibitors on Volume-sensitive Chloride Current in Human Atrial Myocytes

2004 ◽  
Vol 123 (4) ◽  
pp. 427-439 ◽  
Author(s):  
Xin-Ling Du ◽  
Zhan Gao ◽  
Chu-Pak Lau ◽  
Shui-Wah Chiu ◽  
Hung-Fat Tse ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Broad Spectrum ◽  
Kinase Inhibitor ◽  
Chloride Current ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Atrial Myocytes ◽  
Concentration Dependent Manner ◽  
Human Atrial Myocytes ◽  
Protein Tyrosine

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (ICl.vol) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO4−3). ICl.vol evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC50 = 22.4 μM); 100 μM genistein stimulated ICl.vol by 122.4 ± 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 150 μM) and tamoxifen (20 μM), blockers of ICl.vol. Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl− current in 1T. In contrast to the stimulatory effects of genistein, 100 μM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited ICl.vol by 38.2 ± 4.9% and 40.9 ± 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter ICl.vol. In addition, the PTP inhibitor VO4−3 (1 mM) reduced ICl.vol by 53.5 ± 4.5% (IC50 = 249.6 μM). Pretreatment with VO4−3 antagonized genistein-induced augmentation and A23- or A25-induced suppression of ICl.vol. Furthermore, the selective Src-family PTK inhibitor PP2 (5 μM) stimulated ICl.vol, mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 μM) reduced ICl.vol, mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO4−3. The results suggest that ICl.vol is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on ICl.vol, and multiple target proteins are likely to be involved.

scholarly journals Kinetic mechanism and characterization of human β-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells

1994 ◽  
Vol 304 (1) ◽  
pp. 281-288 ◽  
Author(s):  
S Zhang ◽  
J D McCarter ◽  
Y Okamura-Oho ◽  
F Yaghi ◽  
A Hinek ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Time Course ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Dependent Manner ◽  
Ph Optimum ◽  
Apparent Homogeneity ◽  
Arginine Residues ◽  
Beta Galactosidase

Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.

Lysophosphatidylcholine transduces Ca2+ signaling via the platelet-activating factor receptor in macrophages

1997 ◽  
Vol 272 (1) ◽  
pp. H17-H24 ◽  
Author(s):  
T. Ogita ◽  
Y. Tanaka ◽  
T. Nakaoka ◽  
R. Matsuoka ◽  
Y. Kira ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Peritoneal Macrophages ◽  
Free Calcium ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Paf Receptor ◽  
Web 2086

To clarify the molecular mechanism underlying the lysophosphatidylcholine (LPC) signaling, we studied the effect of LPC on the intracellular free calcium concentration ([Ca2+]i) in murine peritoneal macrophages. LPC when added alone induced biphasic elevation of [Ca2+]i, which consisted of a rapid increase followed by sustained elevation. LPC, when added with equimolar cholesterol, induced only the rapid increase in [Ca2+]i, which was blocked by WEB-2086, a selective platelet-activating factor (PAF) receptor antagonist. These results suggest LPC exerts a specific Ca2+ signaling. The sustained elevation reflected the cell lysis. Furthermore, we confirmed its pathway in a more specific manner using cloned PAF receptors expressed in Chinese hamster ovary cells. LPC induced an elevation of [Ca2+]i in a concentration-dependent manner only when the PAF receptor had been expressed, and the elevation of [Ca2+]i was blocked by WEB-2086. Taken together, LPC transduces Ca2+ signaling via the PAF receptor. Activation of the PAF receptor by LPC may indicate its novel important role in the pathogenesis of atherosclerosis.

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