scholarly journals Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells

1995 ◽  
Vol 311 (2) ◽  
pp. 385-392 ◽  
Author(s):  
L Anderson ◽  
C L Alexander ◽  
E Faccenda ◽  
K A Eidne
Keyword(s):  
Hormone Receptor ◽  
Cyclopiazonic Acid ◽  
Thyrotropin Releasing Hormone ◽  
Receptor Desensitization ◽  
Hek 293 ◽  
Releasing Hormone ◽  
Hek 293 Cells ◽  
C Terminus ◽  
293 Cells ◽  
Dose Dependent

This study uses fluorescence microscopy combined with dynamic video imaging to examine the events associated with the rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R). In single non-pituitary human embryonic kidney 293 (HEK-293) cells, expressing either the rat or human TRH-Rs, TRH produced a rapid dose-dependent monophasic rise in [Ca2+]i. This Ca2+ transient was completely abolished by pretreatment of cells with the intracellular Ca2+ antagonists thapsigargin or cyclopiazonic acid, but not EGTA, the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine or the second-messenger-operated Ca2+ channel antagonist SK&F 96365. These results suggest that TRH causes the mobilization of Ca2+ from thapsigargin/cyclopiazonic acid-sensitive intracellular Ca2+ stores but not the influx of extracellular Ca2+. HEK-293 cells also failed to respond to KCl or the slow Ca(2+)-channel activator BAY K 8644, suggesting that they lack L-type VOCCs. Rat and human TRH-Rs are highly conserved except at the C-terminus where the sequence differs. The C-terminus is believed to be important in receptor desensitization. Despite differences in this region, rat and human TRH-Rs expressed in HEK-293 cells underwent rapid (within 1 min) desensitization. This desensitization was dose-dependent and did not involve receptor loss. Similarly the bradykinin receptor endogenous to HEK-293 cells also displays a rapid desensitization. We conclude that in TRH-R-expressing non-pituitary HEK-293 cells, TRH mobilizes intracellular Ca2+ resulting in a monophasic Ca2+ transient. The rat and human TRH-Rs as well as the endogenous bradykinin receptor also displayed rapid receptor desensitization.

scholarly journals Agonist-dependent up-regulation of thyrotrophin-releasing hormone receptor protein

2004 ◽  
Vol 380 (3) ◽  
pp. 815-821 ◽  
Author(s):  
Laurie B. COOK ◽  
Patricia M. HINKLE
Keyword(s):  
Phorbol Ester ◽  
Hormone Receptor ◽  
Fluorescent Protein ◽  
Receptor Protein ◽  
Thyrotrophin Releasing Hormone ◽  
Hek 293 ◽  
Releasing Hormone ◽  
293 Cells ◽  
Receptor Concentration ◽  
Trh Receptor

To study the effect of agonist on the TRH (thyrotrophin-releasing hormone) receptor protein, an epitope-tagged receptor was stably expressed in HEK-293 cells (human embryonic kidney 293 cells) and receptor levels were measured by immunoblotting. TRH caused a 5–25-fold increase in receptor protein during 48 h, which was half-maximal at 1 nM and was slowly reversible after hormone withdrawal. Chlordiazepoxide, an inverse agonist, had no effect. TRH up-regulation was mimicked by phorbol ester and blocked by the protein kinase C inhibitor GF109203X in combination with thapsigargin, which prevents a calcium response. TRH and phorbol ester increased the density of immunoreactive receptors localized at the cell surface and [3H]MeTRH (where MeTRH stands for [N3-methyl-His]TRH) binding. TRH also increased the concentration of a truncated, internalization-defective receptor. Analysis of cell lines stably expressing TRH receptors fused to the green fluorescent protein on a fluorescence-activated cell sorter showed that TRH and phorbol ester caused 2.7- and 6.8-fold increases in fusion protein expression respectively. TRH receptor up-regulation was only partially accounted for by changes in receptor mRNA, which increased 1.7-fold. TRH caused a small increase in receptor concentration in the presence of cycloheximide, actinomycin D or MG132. In contrast with the results obtained with the TRH receptor, agonist decreased the concentration of stably expressed β2-adrenergic receptors. These results show that TRH increases receptor concentration by a complex mechanism that requires signal transduction but not receptor endocytosis.

scholarly journals Tightly regulated and inducible expression of a yoked hormone-receptor complex in HEK 293 cells

2004 ◽  
Vol 32 (1) ◽  
pp. 247-255
Author(s):  
TP Meehan ◽  
D Puett ◽  
P Narayan
Keyword(s):  
Hormone Receptor ◽  
Receptor Activation ◽  
Receptor Expression ◽  
Luteinizing Hormone Receptor ◽  
Receptor Complex ◽  
Hek 293 ◽  
Expression Levels ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Clonal Lines

We have previously reported the construction of a constitutively active luteinizing hormone receptor by covalently linking a fused heterodimeric hormone to the extracellular domain of the G protein-coupled receptor. This yoked hormone-receptor complex (YHR) was found to produce high levels of cAMP in the absence of exogenous hormone. Stable lines expressing YHR were generated in HEK 293 cells to obtain lines with different expression levels; however, in a relatively short time of continued passage, it was found that YHR expression was greatly reduced. Herein, we describe the development of clonal lines of HEK 293 cells in which the expression of YHR is under the control of a tetracycline-regulated system. Characterization of clonal lines revealed tight control of YHR expression both by dose and time of incubation with doxycycline. These experiments demonstrated a good correlation between expression levels of the receptor and basal cAMP production. Moreover, the reduction in receptor expression following doxycycline removal revealed that YHR mRNA and protein decayed at similar rates, again suggesting a strong linkage between mRNA and protein levels. The controlled expression of YHR in this cell system will allow for a more detailed analysis of the signaling properties associated with constitutive receptor activation and may prove to be advantageous in developmental studies with transgenic animals.

Abstract 16460: Enhanced Trpc Channel-Mediated Ca2+ Influx in the Cells With Phospholipase C-δ1 Overexpression

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kazuo Murakami ◽  
Tomohiro Osanai ◽  
Makoto Tanaka ◽  
Kimitaka Nishizaki ◽  
Ikuyo Narita ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Coronary Spasm ◽  
Dependent Manner ◽  
Hek 293 ◽  
Trpc Channel ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dose Dependent

Background: We recently showed that ergometrine induces coronary spasm in the mice with enhanced phospholipase C (PLC)-δ1 activity, which was detected in patients with coronary spasm (Circulation 2012), and PLC-mediated Ca2+ entry is involved in the genesis of coronary spams. We examined the role of enhanced PLC-δ1 in extracellular Ca2+ entry and its underlying mechanisms in human embryonic kidney (HEK)-293 cells and human coronary arteries smooth muscle cells (CASMC). Methods and Results: The cells were stimulated with acetylcholine (ACh) in a extracellular Ca2+-free condition, and the increase in intracellular free Ca2+ concentration ([Ca2+]i) after addition of extracellular Ca2+ was defined as Ca2+ influx. [Ca2+]i was measured by fura-2. In HEK-293 cells, ACh-induced Ca2+ influx (nM ) was 21±2 in the control and 52±6 in the cells with PLC-δ1 overexpression (p<0.05). ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by nifedipine in a dose-dependent manner but was partially by 36±13% at 10-5M (p<0.05), thereby even after treatment with nifedipine at 10-5M, ACh-induced Ca2+ influx was increased by 2.9±0.1 times by enhanced PLC-δ1 compared with the control (p<0.05). To clarify the role of diacylglycerol (DAG) in Ca2+ influx, the effect of blockers for DAG-activated transient receptor potential (TRPC) channel was examined. ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by 2-APB, an inhibitor of non-selective cation channel TRPC, in a dose-dependent manner, and was almost completely blocked by 89±12% at 10-4M (p<0.05). While TRPC3 siRNA did not affect ACh-induced Ca2+ influx (59±27 vs 75±23 nM, p=ns), TRPC6 siRNA suppressed Ca2+ influx to 37±28 nM (p<0.05). In human CASMC, ACh-induced Ca2+ influx was 41±11 in the control and 64±15 in the cells with PLC-δ1 overexpression (p<0.05). Like HEK-293 cells, pretreatment with nifedipine partially suppressed Ca2+ influx, whereas either 2-APB or TRPC6 siRNA almost completely blocked it. Conclusion: ACh-induced Ca2+ influx, mediated by both voltage-gated Ca2+ channels and non-selective cation channels TRPC6, is enhanced by PLC-δ1 overexpression. Inhibition of TRPC may be effective in enhanced PLC-δ1-mediated coronary spasm.

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

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.

scholarly journals Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1

2000 ◽  
Vol 346 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Tomas DRMOTA ◽  
Graeme MILLIGAN
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Thyrotropin Releasing Hormone ◽  
Peptide Ligands ◽  
Full Length ◽  
Hek 293 ◽  
Releasing Hormone ◽  
293 Cells ◽  
Kinetics Of ◽  
Trh Receptor

The C-terminal tail of the long splice variant of the rat thyrotropin-releasing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in response to [3H]TRH was dependent upon the degree of truncation. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the re-ceptor, which altered both the association and dissociation rates of [3H]TRH, the kinetics of ligand binding were unaffected by C-terminal truncation. Equally, the rate of recycling to the plasma membrane of internalized receptors was unaffected by C-terminal truncation. Although the extent of internalization of the full-length receptor was impaired by pre-exposure of cells to TRH, this was not true of C-terminal truncation mutants, which displayed limited steady-state internalization ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length receptor.

scholarly journals Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells

2000 ◽  
Vol 352 (1) ◽  
pp. 203-210 ◽  
Author(s):  
Amy L. TUCKER ◽  
LiGuo JIA ◽  
Diane HOLETON ◽  
Allen J. TAYLOR ◽  
Joel LINDEN
Keyword(s):  
Adenylate Cyclase ◽  
G Proteins ◽  
Adenosine Receptors ◽  
Wild Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
A2a Receptors ◽  
Recombinant Receptors ◽  
C Terminus ◽  
293 Cells

A1 adenosine receptors inhibit adenylate cyclase by activating Gi/Go, whereas A2A receptors activate Gs. We examined how regions of A1 and A2A receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to Gi/Go and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A1/A2A 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A1 3ICL sequence (A2A/A1L or A2A/A1LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to Gi/Go. These chimaeras have basal cAMP levels lower than those of wild-type A2A receptors, similar to wild-type A1 receptors. The A1 C-terminus modulates the coupling of receptors with A1 3ICL such that A2A/A1LT is better coupled to Gi/Go than A2A/A1L. The C-terminus has little impact on coupling to receptors containing A2A 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to Gi/Go mediated by A1 3ICL and not by other intracellular domains that favour Gi coupling. The C-terminus sequence has little or no effect on coupling to Gs. For doubly Gs/Gi-coupled adenosine receptors in HEK-293 cells, Gs-mediated stimulation predominates over Gi/Go-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

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

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
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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