Increase of 5-HT levels is induced both in mouse brain and HEK-293 cells following their exposure to a non-viral tryptophan hydroxylase construct

AbstractTryptophan hydroxylase type 2 (Tph2) is the rate-limiting enzyme for serotonin (5-HT) biosynthesis in the brain. Dysfunctional Tph2 alters 5-HT biosynthesis, leading to a deficiency of 5-HT, which could have repercussions on human behavior. In the last decade, several studies have associated polymorphisms of the TPH2 gene with suicidal behavior. Additionally, a 5-HT deficiency has been implicated in various psychiatric pathologies, including alcoholism, impulsive behavior, anxiety, and depression. Therefore, the TPH2 gene could be an ideal target for analyzing the effects of a 5-HT deficiency on brain function. The aim of this study was to use the construct pIRES-hrGFP-1a-Tph2-FLAG to treat CD1-male mice and to transfect HEK-293-cells and then to evaluate whether this treatment increases 5-HT production. 5-HT levels were enhanced 48 h post-transfection, in HEK-293 cells. Three days after the ocular administration of pIRES-hrGFP-1a-Tph2-FLAG to mice, putative 5-HT production was significantly higher than in the control in both hypothalamus and amygdala, but not in the brainstem. Further research will be needed on the possible application of this treatment for psychiatric diseases involving a Tph2 dysfunction or serotonin deficiency.

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Type 2 IP3 receptors are involved in uranyl acetate induced apoptosis in HEK 293 cells

Toxicology ◽

10.1016/j.tox.2009.05.006 ◽

2009 ◽

Vol 262 (1) ◽

pp. 73-79 ◽

Cited By ~ 10

Author(s):

Juraj Kopacek ◽

Karol Ondrias ◽

Barbora Sedlakova ◽

Jana Tomaskova ◽

Lucia Zahradnikova ◽

...

Keyword(s):

Uranyl Acetate ◽

Ip3 Receptors ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Induced Apoptosis

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Ablation of small conductance calcium-activated potassium type-2 channel (SK2) delays occurrence of bupivacaine-induced cardiotoxicity in isolated mouse hearts

Human & Experimental Toxicology ◽

10.1177/0960327120958102 ◽

2020 ◽

pp. 096032712095810

Author(s):

H Chen ◽

F Xia ◽

X Chen ◽

Y Cai ◽

Z Jin

Keyword(s):

Rate Pressure Product ◽

Hek 293 ◽

Hek 293 Cells ◽

Severe Arrhythmia ◽

293 Cells ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Dose Dependent ◽

Small Conductance

Bupivacaine is frequently used for conducting regional anesthesia. When accidentally injected or excessively absorbed into circulation, bupivacaine can induce severe arrhythmia and potentially lead to cardiac arrest. The specific mechanisms underlying this cardiotoxicity, however, remain to be clarified. We transfected HEK-293 cells to express the small conductance calcium-activated potassium type-2 channel (SK2), and used a whole-cell patch clamp method in order to explore how bupivacaine affected these channels. We subsequently used SK2 knockout mice to explore the relevance of SK2 channels in bupivacaine-induced cardiotoxicity in isolating mouse hearts, mounting them on a Langendorff apparatus, and perfusing them with bupivacaine. Using this system, arrhythmia, asystole, and cardiac functions were monitored. We observed dose-dependent inhibition of SK2 channels by bupivacaine: half-maximal inhibitory concentration (IC50) value = 18.6 μM (95% CI 10.8–32.1). When SK2 knockout (SK2 −/−) or wild-type (WT) mice were perfused with Krebs-Henseleit buffer (KHB), we did not observe any instances of arrhythmia. When SK2 −/− mice or WT were perfused with KHB containing bupivacaine (40 μM), the time to arrhythmia (Tarrhythmia) and time to asystole (Tasystole) were both significantly longer in SK2 −/− mice relative to WT mice ( P < 0.001). Similarly, SK2 −/− mice exhibited a significantly longer time to 25%, 50%, and 75% reductions in heart rate (HR) and rate-pressure product (RPP) relative to WT mice following bupivacaine perfusion ( P < 0.001). These results reveal that bupivacaine was able to mediate a dose-dependent inhibition of SK2 channels in HEK-293 cells, and deletion of SK2 channels can delay bupivacaine-induced cardiotoxicity in isolated mouse hearts.

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Bupivacaine inhibits a small conductance calcium-activated potassium type 2 channel (SK2) in HEK 293 cells

10.21203/rs.2.338/v1 ◽

2019 ◽

Author(s):

Hongfei Chen ◽

Fangfang Xia ◽

Zhousheng Jin ◽

Yuting He ◽

Zhengjie Chen ◽

...

Keyword(s):

Inhibitory Effect ◽

Heart Cells ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Ic50 Value ◽

Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause severe cardiotoxicity. Small conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unknown. This study investigated the effect of bupivacaine on SK2 channels. Methods: The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch clamp technique was used to study the effect of bupivacaine on SK2 channels. The inhibitory effect of various concentrations of bupivacaine on SK2 currents exhibited a non-linear relation, and the half-maximal inhibitory concentration (IC50) value was determined. Results: Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine and lidocaine on the SK2 current was 133.7, 189.3, and 885.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium. Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity. Keywords: Bupivacaine, SK2 channel, inhibition, cardiotoxicity, HEK 293.

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Statins and Downstream Inhibitors of the Isoprenylation Pathway Increase Type 2 Iodothyronine Deiodinase Activity

Endocrinology ◽

10.1210/en.2012-1117 ◽

2012 ◽

Vol 153 (8) ◽

pp. 4039-4048 ◽

Cited By ~ 1

Author(s):

B. T. Miller ◽

C. B. Ueta ◽

V. Lau ◽

K. G. Jacomino ◽

L. M. Wasserman ◽

...

Keyword(s):

Active Form ◽

Critical Determinant ◽

Iodothyronine Deiodinase ◽

Hek 293 ◽

Hek 293 Cells ◽

Reductase Inhibitors ◽

293 Cells ◽

Brown Adipose ◽

Prenyl Transferase

The type 2 iodothyronine selenodeiodinase (D2) is a critical determinant of local thyroid signaling, converting T4 to the active form T3 at the cytoplasmic face of the endoplasmic reticulum, thus supplying the nucleus with T3 without immediately affecting circulating thyroid hormone levels. Although inhibitors of the cholesterol synthesis/isoprenylation pathway, such as hydroxy-methyl-glutaryl-coenzyme A reductase inhibitors (statins) have been to shown to down-regulate selenoproteins via interruption of normal selenocysteine incorporation, little is known about the effect of statins on D2. Here, we report that statins and prenyl transferase inhibitors actually increase D2 activity in cells with endogenous D2 expression. Although we confirmed that lovastatin (LVS) decreases the activity of transiently expressed D2 in HEK-293 cells, the prenyl transferase inhibitors increase activity in this system as well. LVS treatment increases endogenous Dio2 mRNA in MSTO-211H cells but does not alter transiently expressed Dio2 mRNA in HEK-293 cells. The prenyl transferase inhibitors do not increase Dio2 mRNA in either system, indicating that a posttranscriptional mechanism must exist. Cotreatment with LVS or the prenyl transferase inhibitors with the proteasome inhibitor MG-132 did not lead to additive increases in D2 activity, indirectly implicating the ubiquitin-proteasomal system in the mechanism. Finally, C57BL/6J mice treated with LVS or farnesyl transferase inhibitor-277 for 24 h exhibited increased D2 activity in their brown adipose tissue. These data indicate that statins and downstream inhibitors of the isoprenylation pathway may increase thyroid signaling via stimulation of D2 activity.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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