scholarly journals Evidence for the Capability of Roxadustat (FG-4592), an Oral HIF Prolyl-Hydroxylase Inhibitor, to Perturb Membrane Ionic Currents: An Unidentified yet Important Action

2019 ◽  
Vol 20 (23) ◽  
pp. 6027 ◽  
Author(s):  
Wei-Ting Chang ◽  
Yi-Ching Lo ◽  
Zi-Han Gao ◽  
Sheng-Nan Wu
Keyword(s):  
Time Course ◽  
Hypoxia Inducible Factor ◽  
Ionic Currents ◽  
Membrane Depolarization ◽  
Prolyl Hydroxylase ◽  
Gh3 Cells ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Hif Prolyl Hydroxylase

Roxadustat (FG-4592), an analog of 2-oxoglutarate, is an orally-administered, heterocyclic small molecule known to be an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase. However, none of the studies have thus far thoroughly investigated its possible perturbations on membrane ion currents in endocrine or heart cells. In our studies, the whole-cell current recordings of the patch-clamp technique showed that the presence of roxadustat effectively and differentially suppressed the peak and late components of IK(DR) amplitude in response to membrane depolarization in pituitary tumor (GH3) cells with an IC50 value of 5.71 and 1.32 μM, respectively. The current inactivation of IK(DR) elicited by 10-sec membrane depolarization became raised in the presence of roxadustatt. When cells were exposed to either CoCl2 or deferoxamine (DFO), the IK(DR) elicited by membrane depolarization was not modified; however, nonactin, a K+-selective ionophore, in continued presence of roxadustat, attenuated roxadustat-mediated inhibition of the amplitude. The steady-state inactivation of IK(DR) could be constructed in the presence of roxadustat. Recovery of IK(DR) block by roxadustat (3 and 10 μM) could be fitted by a single exponential with 382 and 523 msec, respectively. The roxadustat addition slightly suppressed erg-mediated K+ or hyperpolarization-activated cation currents. This drug also decreased the peak amplitude of voltage-gated Na+ current with a slowing in inactivation rate of the current. Likewise, in H9c2 heart-derived cells, the addition of roxadustat suppressed IK(DR) amplitude in combination with the shortening in inactivation time course of the current. In high glucose-treated H9c2 cells, roxadustat-mediated inhibition of IK(DR) remained unchanged. Collectively, despite its suppression of HIF prolyl hydroxylase, inhibitory actions of roxadustat on different types of ionic currents possibly in a non-genomic fashion might provide another yet unidentified mechanism through which cellular functions are seriously perturbed, if similar findings occur in vivo.

scholarly journals Effective Activation by Kynurenic Acid and Its Aminoalkylated Derivatives on M-Type K+ Current

2021 ◽  
Vol 22 (3) ◽  
pp. 1300
Author(s):  
Yi-Ching Lo ◽  
Chih-Lung Lin ◽  
Wei-Yu Fang ◽  
Bálint Lőrinczi ◽  
István Szatmári ◽  
...  
Keyword(s):  
Kynurenic Acid ◽  
Time Course ◽  
Ionic Currents ◽  
Gh3 Cells ◽  
Excitable Cells ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Type K ◽  
K Current

Kynurenic acid (KYNA, 4-oxoquinoline-2-carboxylic acid), an intermediate of the tryptophan metabolism, has been recognized to exert different neuroactive actions; however, the need of how it or its aminoalkylated amide derivative N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-oxo-1,4-dihydroquinoline-2-carboxamide (KYNA-A4) exerts any effects on ion currents in excitable cells remains largely unmet. In this study, the investigations of how KYNA and other structurally similar KYNA derivatives have any adjustments on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were performed by patch-clamp technique. KYNA or KYNA-A4 increased the amplitude of M-type K+ current (IK(M)) and concomitantly enhanced the activation time course of the current. The EC50 value required for KYNA- or KYNA-A4 -stimulated IK(M) was yielded to be 18.1 or 6.4 μM, respectively. The presence of KYNA or KYNA-A4 shifted the relationship of normalized IK(M)-conductance versus membrane potential to more depolarized potential with no change in the gating charge of the current. The voltage-dependent hysteretic area of IK(M) elicited by long-lasting triangular ramp pulse was observed in GH3 cells and that was increased during exposure to KYNA or KYNA-A4. In cell-attached current recordings, addition of KYNA raised the open probability of M-type K+ channels, along with increased mean open time of the channel. Cell exposure to KYNA or KYNA-A4 mildly inhibited delayed-rectifying K+ current; however, neither erg-mediated K+ current, hyperpolarization-activated cation current, nor voltage-gated Na+ current in GH3 cells was changed by KYNA or KYNA-A4. Under whole-cell, current-clamp recordings, exposure to KYNA or KYNA-A4 diminished the frequency of spontaneous action potentials; moreover, their reduction in firing frequency was attenuated by linopirdine, yet not by iberiotoxin or apamin. In hippocampal mHippoE-14 neurons, the addition of KYNA also increased the IK(M) amplitude effectively. Taken together, the actions presented herein would be one of the noticeable mechanisms through which they modulate functional activities of excitable cells occurring in vivo.

scholarly journals The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2

2018 ◽  
Author(s):  
Nina Kozlova ◽  
Daniela Mennerich ◽  
Anatoly Samoylenko ◽  
Elitsa Y. Dimova ◽  
Peppi Koivunen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Survival Function ◽  
Hypoxia Inducible Factor ◽  
Adaptor Protein ◽  
Prolyl Hydroxylase ◽  
Binding Partner ◽  
And Migration ◽  
Hif Prolyl Hydroxylase ◽  
Promote Breast Cancer

SummaryThe EGFR-adaptor protein CIN85 has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill-defined. Here, we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SH3 domains of CIN85 interact with the proline-arginine rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF-degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9 edited cells affected growth and migration properties as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2, and uncovered an essential survival function in tumor cells linking growth factor adaptors with hypoxia signaling.

scholarly journals Effectiveness in Block by Dexmedetomidine of Hyperpolarization-Activated Cation Current, Independent of Its Agonistic Effect on α2-Adrenergic Receptors

2020 ◽  
Vol 21 (23) ◽  
pp. 9110
Author(s):  
Te-Ling Lu ◽  
Te-Jung Lu ◽  
Sheng-Nan Wu
Keyword(s):  
Adrenergic Receptors ◽  
Time Course ◽  
Ionic Currents ◽  
Inhibitory Effect ◽  
Firing Frequency ◽  
Neuroendocrine Cells ◽  
Gh3 Cells ◽  
Membrane Excitability ◽  
Cation Current

Dexmedetomidine (DEX), a highly selective agonist of α2-adrenergic receptors, has been tailored for sedation without risk of respiratory depression. Our hypothesis is that DEX produces any direct perturbations on ionic currents (e.g., hyperpolarization-activated cation current, Ih). In this study, addition of DEX to pituitary GH3 cells caused a time- and concentration-dependent reduction in the amplitude of Ih with an IC50 value of 1.21 μM and a KD value of 1.97 μM. A hyperpolarizing shift in the activation curve of Ih by 10 mV was observed in the presence of DEX. The voltage-dependent hysteresis of Ih elicited by long-lasting triangular ramp pulse was also dose-dependently reduced during its presence. In continued presence of DEX (1 μM), further addition of OXAL (10 μM) or replacement with high K+ could reverse DEX-mediated inhibition of Ih, while subsequent addition of yohimbine (10 μM) did not attenuate the inhibitory effect on Ih amplitude. The addition of 3 μM DEX mildly suppressed the amplitude of erg-mediated K+ current. Under current-clamp potential recordings, the exposure to DEX could diminish the firing frequency of spontaneous action potentials. In pheochromocytoma PC12 cells, DEX was effective at suppressing Ih together with a slowing in activation time course of the current. Taken together, findings from this study strongly suggest that during cell exposure to DEX used at clinically relevant concentrations, the DEX-mediated block of Ih appears to be direct and would particularly be one of the ionic mechanisms underlying reduced membrane excitability in the in vivo endocrine or neuroendocrine cells.

scholarly journals Evidence for Effective Inhibitory Actions on Hyperpolarization-Activated Cation Current Caused by Ganoderma Triterpenoids, the Main Active Constitutents of Ganoderma Spores

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4256 ◽  
Author(s):  
Wei-Ting Chang ◽  
Zi-Han Gao ◽  
Yi-Ching Lo ◽  
Sheng-Nan Wu
Keyword(s):  
Ionic Currents ◽  
Inhibitory Effect ◽  
Firing Frequency ◽  
Gh3 Cells ◽  
Heart Cells ◽  
Excitable Cells ◽  
Cell Current ◽  
Spontaneous Action

The triterpenoid fraction of Ganoderma (Ganoderma triterpenoids, GTs) has been increasingly demonstrated to provide effective antioxidant, neuroprotective or cardioprotective activities. However, whether GTs is capable of perturbing the transmembrane ionic currents existing in electrically excitable cells is not thoroughly investigated. In this study, an attempt was made to study whether GTs could modify hyperpolarization-activated cation currents (Ih) in pituitary tumor (GH3) cells and in HL-1 atrial cardiomyocytes. In whole-cell current recordings, the addition of GTs produced a dose-dependent reduction in the amplitude of Ih in GH3 cells with an IC50 value of 11.7 µg/mL, in combination with a lengthening in activation time constant of the current. GTs (10 µg/mL) also caused a conceivable shift in the steady-state activation curve of Ih along the voltage axis to a more negative potential by approximately 11 mV. Subsequent addition of neither 8-cyclopentyl-1,3-dipropylxanthine nor 8-(p-sulfophenyl)theophylline, still in the presence of GTs, could attenuate GTs-mediated inhibition of Ih. In current-clamp voltage recordings, GTs diminished the firing frequency of spontaneous action potentials in GH3 cells, and it also decreased the amplitude of sag potential in response to hyperpolarizing current stimuli. In murine HL-1 cardiomyocytes, the GTs addition also suppressed the amplitude of Ih effectively. In DPCPX (1 µM)-treated HL-1 cells, the inhibitory effect of GTs on Ih remained efficacious. Collectively, the inhibition of Ih caused by GTs is independent of its possible binding to adenosine receptors and it might have profound influence in electrical behaviors of different types of electrically excitable cells (e.g., pituitary and heart cells) if similar in vitro or in vivo findings occur.

scholarly journals Methadone block of K+ current in squid giant fiber lobe neurons.

1996 ◽  
Vol 107 (2) ◽  
pp. 243-260 ◽  
Author(s):  
F T Horrigan ◽  
W F Gilly
Keyword(s):  
Time Course ◽  
Strong Dependence ◽  
Ionic Currents ◽  
Gh3 Cells ◽  
K Channel ◽  
Bathing Solution ◽  
Dependent Manner ◽  
Giant Fiber ◽  
Patch Clamp Technique ◽  
Pipette Solution

Voltage-dependent ionic currents were recorded from squid giant fiber lobe neurons using the whole-cell patch-clamp technique. When applied to the bathing solution, methadone was found to block IK, I Na and I Ca. Both I Na and I Ca were reduced without apparent change in kinetics and exhibited IC(50)'s of 50-100 and 250-500 mu M, respectively, at +10 mV. In contrast, IK was reduced in a time-dependent manner that is well fit by a simple model of open channel block (K(D)= 32+/- or 2 mu M, +60 mV, 10 degrees Celsius). The mechanism of I(K) block was examined in detail and involves a direct action of methadone, a tertiary amine, on K channels rather than an opioid receptor-mediated pathway. The kinetics of I(K) block resemble those reported for internally applied long chain quaternary ammonium (QA) compounds; and recovery from I(K) block is QA-like in its slow time course and strong dependence on holding potential. A quaternary derivative of methadone (N-methyl-methadone) only reproduced the effects of methadone on I(K) when included in the pipette solution; this compound was without effect when applied externally. I(K) block thus appears to involve diffusion of methadone into the cytoplasm and occlusion of the open K channel at the internal QA blocking site by the protonated form of the drug. This proposed mode of action is supported by the pH and voltage dependence of block as well as by the observation that high external K+ speeds the rate of drug dissociation. In addition, the effect of methadone on I(K) evoked during prolonged (300 ms) depolarizations suggests that methadone block may interfere with endogenous K+ channel inactivation. The effects of temperature, methadone stereoisomers, and the methadone-like drugs propoxyphene and nor-propoxyphene on IK block were examined. Methadone was also found to block I(K) in GH3 cells and in chick myoblasts.

scholarly journals Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

2002 ◽  
Vol 22 (19) ◽  
pp. 6842-6853 ◽  
Author(s):  
Sofía Lavista-Llanos ◽  
Lázaro Centanin ◽  
Maximiliano Irisarri ◽  
Daniela M. Russo ◽  
Jonathan M. Gleadle ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Time Course ◽  
Hypoxia Inducible Factor ◽  
Ectopic Expression ◽  
Prolyl Hydroxylase ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Oxygen Homeostasis ◽  
Multicellular Organisms

ABSTRACT In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-α and HIF-β homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

scholarly journals Effective Perturbations on the Amplitude and Hysteresis of Erg-Mediated Potassium Current Caused by 1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoate (SM-102), a Cationic Lipid

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1367
Author(s):  
Hsin-Yen Cho ◽  
Tzu-Hsien Chuang ◽  
Sheng-Nan Wu
Keyword(s):  
Endocrine Cells ◽  
Time Course ◽  
Cationic Lipid ◽  
Ionic Currents ◽  
Gh3 Cells ◽  
Bath Application ◽  
Bv2 Cells ◽  
Rat Pituitary ◽  
K Current

SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate) is an amino cationic lipid that has been tailored for the formation of lipid nanoparticles and it is one of the essential ingredients present in the ModernaTM COVID-19 vaccine. However, to what extent it may modify varying types of plasmalemmal ionic currents remains largely uncertain. In this study, we investigate the effects of SM-102 on ionic currents either in two types of endocrine cells (e.g., rat pituitary tumor (GH3) cells and mouse Leydig tumor (MA-10) cells) or in microglial (BV2) cells. Hyperpolarization-activated K+ currents in these cells bathed in high-K+, Ca2+-free extracellular solution were examined to assess the effects of SM-102 on the amplitude and hysteresis of the erg-mediated K+ current (IK(erg)). The SM-102 addition was effective at blocking IK(erg) in a concentration-dependent fashion with a half-maximal concentration (IC50) of 108 μM, a value which is similar to the KD value (i.e., 134 μM) required for its accentuation of deactivation time constant of the current. The hysteretic strength of IK(erg) in response to the long-lasting isosceles-triangular ramp pulse was effectively decreased in the presence of SM-102. Cell exposure to TurboFectinTM 8.0 (0.1%, v/v), a transfection reagent, was able to inhibit hyperpolarization-activated IK(erg) effectively with an increase in the deactivation time course of the current. Additionally, in GH3 cells dialyzed with spermine (30 μM), the IK(erg) amplitude progressively decreased; moreover, a further bath application of SM-102 (100 μM) or TurboFectin (0.1%) diminished the current magnitude further. In MA-10 Leydig cells, the IK(erg) was also blocked by the presence of SM-102 or TurboFectin. The IC50 value for SM-102-induced inhibition of IK(erg) in MA-10 cells was 98 μM. In BV2 microglial cells, the amplitude of the inwardly rectifying K+ current was inhibited by SM-102. Taken together, the presence of SM-102 concentration-dependently inhibited IK(erg) in endocrine cells (e.g., GH3 or MA-10 cells), and such action may contribute to their functional activities, assuming that similar in vivo findings exist.

Voltage-dependent kinetics of Na-Ca exchange current in Ca(2+)-loaded guinea pig heart cells

1993 ◽  
Vol 265 (5) ◽  
pp. C1258-C1265 ◽  
Author(s):  
K. Benndorf ◽  
C. Biskup ◽  
M. Friedrich
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Exchange Current ◽  
Heart Cells ◽  
Current Component ◽  
Patch Clamp Technique ◽  
Initial Current ◽  
Current Increase ◽  
Voltage Dependent ◽  
Kinetics Of

Voltage-dependent properties of Na-Ca exchange current were revealed with the patch-clamp technique in Ca(2+)-overloaded guinea pig ventricular myocytes in the whole cell configuration. With the assumption that the transient inward current (Iti) is mediated by the Na-Ca exchanger, oscillations of internal Ca2+ concentration ([Ca2+]i) were used to investigate voltage-dependent kinetics of exchange current differences at two [Ca2+]i values. After Iti was elicited by clamping from -45 mV to basic pulses of +10 mV, pairs of equipotential short test pulses were applied during the basic pulse at both the phase of low [Ca2+]i (between two neighboring Iti values) and the phase of high [Ca2+]i (at the peak of Iti). The test pulses were short enough to leave the time course of Iti during the basic pulse approximately unchanged, which allowed study of the voltage dependence of the respective current differences without disturbing the underlying oscillation of [Ca2+]i. The current differences were inward at all potentials between -140 and +70 mV, started from an equal initial value, and obeyed characteristic voltage-dependent time courses: hyperpolarization to potentials negative to -70 mV caused an initial current increase, which was followed by a decay to very small amplitudes or zero with a decay time constant decreasing toward hyperpolarization e-fold per 45.6 mV. Depolarizing pulses caused a decay of the current differences to smaller levels. Respective current differences formed during a slowly decaying current component, following the Ca current spike, showed equal voltage-dependent properties. This indicates that the slowly decaying current component is preferentially also carried by the Na-Ca exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Carcinogenicity Assessment of Daprodustat (GSK1278863), a Hypoxia-Inducible Factor (HIF)-Prolyl Hydroxylase Inhibitor

2019 ◽  
Vol 48 (2) ◽  
pp. 362-378 ◽  
Author(s):  
David F. Adams ◽  
Mark S. Watkins ◽  
Luc Durette ◽  
Josée Laliberté ◽  
Félix Goulet ◽  
...  
Keyword(s):  
Cell Mass ◽  
Hypoxia Inducible Factor ◽  
Carcinogenic Risk ◽  
Prolyl Hydroxylase ◽  
High Dose ◽  
Sprague Dawley ◽  
Time Curve ◽  
Clinical Exposure ◽  
High Doses ◽  
Hif Prolyl Hydroxylase

Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat’s biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat’s 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration–time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.

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