scholarly journals Expression of tachykinin receptors (tacr1a and tacr1b) in zebrafish: influence of cocaine and opioid receptors

2012 ◽  
Vol 50 (2) ◽  
pp. 115-129 ◽  
Author(s):  
Roger López-Bellido ◽  
Katherine Barreto-Valer ◽  
Raquel E Rodríguez
Keyword(s):  
Embryonic Development ◽  
Opioid Receptors ◽  
Phylogenetic Analyses ◽  
Critical Role ◽  
Cocaine Exposure ◽  
Peripheral Tissues ◽  
Tachykinin Receptors ◽  
Hek 293 ◽  
293 Cells ◽  
Gene Study

Opioid and tachykinin receptors (TACRs) are closely related in addiction and pain processes. In zebrafish, opioid receptors have been cloned and characterized both biochemically and pharmacologically. However, thetacr1gene has not yet been described in zebrafish. The aim of this research was to identify thetacr1gene, study the effects of cocaine ontacr1, and analyze the interaction betweentacr1and opioid receptors. We have identified a duplicate oftacr1gene in zebrafish, designated astacr1aandtacr1b. Phylogenetic analyses revealed an alignment of these receptors in the Tacr1 fish cluster, with a clear distinction from other TACR1s of amphibians, birds, and mammals. Our qPCR results showed thattacr1aandtacr1bmRNAs are expressed during embryonic development. Whole-mountin situhybridization showedtacr1expression in the CNS and in the peripheral tissues. Cocaine (1.5 μM) induced an upregulation oftacr1aandtacr1bat 24 and 48 h post-fertilization (hpf; except fortacr1aat 48 hpf, which was downregulated). By contrast, HEK-293 cells transfected withtacr1aandtacr1band exposed to cocaine showed a downregulation oftacr1s. The knockdown of ZfDOR2 and ZfMOR, opioid receptors, induced a down- and upregulation oftacr1aandtacr1brespectively. In conclusion,tacr1aandtacr1bin zebrafish are widely expressed throughout the CNS and peripherally, suggesting a critical role of thesetacr1sduring embryogenesis.tacr1aandtacr1bmRNA expression is altered by cocaine exposure and by the knockdown of opioid receptors. Thus, zebrafish can provide clues for a better understanding of the relationship between tachykinin and opioid receptors in pain and addiction during embryonic development.

An Extrasynaptic GABAA Receptor Mediates Tonic Inhibition in Thalamic VB Neurons

2005 ◽  
Vol 94 (6) ◽  
pp. 4491-4501 ◽  
Author(s):  
Fan Jia ◽  
Leonardo Pignataro ◽  
Claude M. Schofield ◽  
Minerva Yue ◽  
Neil L. Harrison ◽  
...  
Keyword(s):  
Critical Role ◽  
Brain Slices ◽  
Oscillatory Activity ◽  
Thalamic Neurons ◽  
Hek 293 ◽  
Subunit Protein ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Hypnotic Agent ◽  
Tonic Current

Whole cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) and reticular (RTN) neurons in mouse brain slices. A bicuculline-sensitive tonic current was observed in VB, but not in RTN, neurons; this current was increased by the GABAA receptor agonist 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol (THIP; 0.1 μM) and decreased by Zn2+ (50 μM) but was unaffected by zolpidem (0.3 μM) or midazolam (0.2 μM). The pharmacological profile of the tonic current is consistent with its generation by activation of GABAA receptors that do not contain the α1 or γ2 subunits. GABAA receptors expressed in HEK 293 cells that contained α4β2δ subunits showed higher sensitivity to THIP (gaboxadol) and GABA than did receptors made up from α1β2δ, α4β2γ2s, or α1β2γ2s subunits. Western blot analysis revealed that there is little, if any, α3 or α5 subunit protein in VB. In addition, co-immunoprecipitation studies showed that antibodies to the δ subunit could precipitate α4, but not α1 subunit protein. Confocal microscopy of thalamic neurons grown in culture confirmed that α4 and δ subunits are extensively co-localized with one another and are found predominantly, but not exclusively, at extrasynaptic sites. We conclude that thalamic VB neurons express extrasynaptic GABAA receptors that are highly sensitive to GABA and THIP and that these receptors are most likely made up of α4β2δ subunits. In view of the critical role of thalamic neurons in the generation of oscillatory activity associated with sleep, these receptors may represent a principal site of action for the novel hypnotic agent gaboxadol.

scholarly journals Proteasome Involvement in Agonist-induced Down-regulation of μ and δ Opioid Receptors

2001 ◽  
Vol 276 (15) ◽  
pp. 12345-12355 ◽  
Author(s):  
Kirti Chaturvedi ◽  
Persis Bandari ◽  
Norihiro Chinen ◽  
Richard D. Howells
Keyword(s):  
Opioid Receptor ◽  
Opioid Receptors ◽  
Proteasome Inhibitors ◽  
Metabolic Labeling ◽  
Down Regulation ◽  
Hek 293 ◽  
293 Cells ◽  
Ubiquitin Proteasome ◽  
Μ Opioid Receptor ◽  
Δ Opioid Receptor

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged δ and μ receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [35S]methionine metabolic labeling indicated that the turnover rate of δ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional Giand Goproteins by pertussis toxin-attenuated down-regulation of the μ opioid receptor, while down-regulation of the δ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on μ and δ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced μ and δ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state μ and δ opioid receptor levels. Immunoprecipitation of μ and δ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

2008 ◽  
Vol 410 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Ingeborg Winge ◽  
Jeffrey A. Mckinney ◽  
Ming Ying ◽  
Clive S. D'Santos ◽  
Rune Kleppe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tryptophan Hydroxylase ◽  
Enzyme Stability ◽  
Enzyme Activation ◽  
Site Directed Mutagenesis ◽  
Peripheral Tissues ◽  
Hek 293 ◽  
Rate Limiting Step ◽  
293 Cells ◽  
Dependent Protein

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca2+/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser19, but PKA also phosphorylated Ser104, as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser19, Ser99, Ser104 and Ser306. On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser19. This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins γ, ϵ and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.

Abstract 355: Modulation of Cardiac L-type Calcium Channels by Phospholemman

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Xianming Wang ◽  
Guofeng Gao ◽  
Blaise Z Peterson
Keyword(s):  
Plasma Membranes ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Ion Homeostasis ◽  
Functional Coupling ◽  
Mouse Heart ◽  
Channel Activation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Ca 2+ entry through L-type Ca 2+ channels plays a critical role in shaping the action potential and is the initial trigger for EC-coupling. The gating, expression and targeting Ca 2+ channels are tightly regulated by auxiliary subunits and second messenger signaling mechanisms. Here, we report that cardiac Ca 2+ channels are directly modulated by phospholemman (PLM), a single transmembrane protein that is important for regulating ion homeostasis in the heart through its interactions with the Na,K-ATPase and Na/Ca Exchanger (NCX). Experiments using confocal immunofluorescence microscopy indiate that PLM and the Ca 2+ channel alpha-1 subunit, Ca V 1.2, co-localize to the plasma membranes of HEK 293 and COS-7 cells. Recipricol co-immunoprecipitation studies demonstrate that PLM and Ca V 1.2 are specifically associated in the mouse heart (see Figure ) and HEK 293 cells expressing the two proteins (not shown). Whole-cell patch-clamp was used to assess the functional consequences of the interaction between PLM and the Ca V 1.2 subunit using HEK 293 cells transfected with PLM ((+)PLM) or empty PLM vector ((−)PLM). These studies demonstrate that PLM substantially slows the activation kinetics of Ca V 1.2 channels (see Figure ), but has no effect on neuronal Ca V 2.1 Ca 2+ channels (not shown). As a result, the level of Ca 2+ entry during the first 50 msec of channel activation is decreased by up to 32%. Since PLM is upregulated in post-ischemic rat hearts and due to the tight functional coupling between the cardiac Ca 2+ channel and NCX, we propose that PLM-induced slowing channel activation and PLM-dependent inhibition of NCX combine synergistically to reduce peak [Ca 2+ ]i in infacted myocytes.

Hetero-oligomers of α2A-adrenergic and μ-opioid receptors do not lead to transactivation of G-proteins or altered endocytosis profiles

2004 ◽  
Vol 32 (5) ◽  
pp. 856-860 ◽  
Author(s):  
Y.Q. Zhang ◽  
L.E. Limbird
Keyword(s):  
Opioid Receptors ◽  
Pertussis Toxin ◽  
Adrenergic Receptors ◽  
Functional Communication ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
293 Cells ◽  
Μ Opioid Receptors ◽  
Transmembrane Span ◽  
In Cells

Complexes of α2A-ARs (α2A-adrenergic receptors) and MORs (μ-opioid receptors), probably hetero-oligomers, were detected by co-immunoisolation after extraction from HEK-293 cells (human embryonic kidney 293 cells). Functional communication between these receptors is revealed by α2A-AR activation of a pertussis toxin-insensitive Giα subunit (termed as Gi1) when fused with the MOR and evaluated in membranes from pertussis toxin-treated cells. However, the α2A-AR does not require transactivation through MOR, since quantitatively indistinguishable results were observed in cells co-expressing α2A-AR and a fusion protein of Gi1 with the first transmembrane span of MOR (myc–MOR-TM1). Functional cross-talk among these α2A-AR–MOR complexes does not occur for internalization profiles; incubation with adrenaline (epinephrine) leads to endocytosis of α2A-AR but not MOR, while incubation with DAMGO ([D-Ala,NMe-Phe,Gly-ol]enkephalin) leads to endocytosis of MOR but not α2A-AR in cells co-expressing both the receptors. Hence, α2A-AR and MOR hetero-oligomers, although they occur, do not have an obligatory functional influence on one another in the paradigms studied.

Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms

2006 ◽  
Vol 291 (4) ◽  
pp. F796-F805 ◽  
Author(s):  
Balasubramaniem Ashokkumar ◽  
Nosratola D. Vaziri ◽  
Hamid M. Said
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Mrna Levels ◽  
Cellular Functions ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Glomerular Filtrate ◽  
Intracellular Ca ◽  
Ph Dependent

Thiamin (vitamin B1) is essential for normal cellular functions. The kidneys play a critical role in regulating body thiamin homeostasis, by salvaging the vitamin via reabsorption from the glomerular filtrate, but little is known about the mechanism(s) and regulation of thiamin transport in the human renal epithelia at cellular and molecular levels. Using the human-derived renal epithelial HEK-293 cells as a model, we have addressed these issues. Our results showed [3H]thiamin uptake to be 1) temperature and energy dependent but Na+ independent, 2) pH dependent with higher uptake at alkaline/neutral buffer pH compared with acidic pH, 3) saturable as a function of concentration over the nanomolar (apparent Km = 70.0 ± 18.4 nM) and micromolar (apparent Km = 2.66 ± 0.18 μM) ranges, 4) cis-inhibited by unlabeled thiamin and its structural analogs but not by unrelated organic cations, 5) trans-stimulated by unlabeled thiamin, and 6) competitively inhibited by amiloride with an apparent Ki of 0.6 mM. Using a gene-specific small-interference RNAs (siRNAs) approach, human thiamin transporters 1 and 2 (hTHTR-1 and hTHTR-2) were both found to be expressed and contributed toward total carrier-mediated thiamin uptake. Maintaining the cells in thiamin-deficient medium led to a significant ( P < 0.01) and specific upregulation in [3H]thiamin uptake, which was associated with an increase in hTHTR-1 and hTHTR-2 protein and mRNA levels as well as promoter activities. Uptake of thiamin by HEK-293 cells also appeared to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. These studies demonstrate for the first time that thiamin uptake by HEK-293 cells is mediated via a specific pH-dependent process, which involves both the hTHTR-1 and hTHTR-2. In addition, the uptake process appears to be under the regulation of an intracellular Ca2+/CaM-mediated pathway and also adaptively upregulated in thiamin deficiency via transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.

scholarly journals MICU1 modulates MCU ion selectivity and tolerance to manganese stress

2018 ◽  
Author(s):  
Jennifer Wettmarshausen ◽  
Valerie Goh ◽  
Utkarsh Tripathi ◽  
Anja Leimpek ◽  
Yiming Cheng ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Critical Role ◽  
Yeast Cells ◽  
Mitochondrial Calcium ◽  
Hek 293 ◽  
Regulatory Subunits ◽  
Calcium Uniporter ◽  
293 Cells ◽  
Phylogenetic Profiling

SUMMARYThe mitochondrial calcium uniporter is a highly selective ion channel composed of species-and tissue-specific structural and regulatory subunits. However, the contribution of each component to uniporter-mediated activity still remains unclear. Here, we employ an evolutionary and synthetic biology approach to investigate the functional inter-dependence between the pore-forming subunit MCU and the EF-hand protein MICU1. Using phylogenetic profiling and genetic complementation analyses, we show that MCU and MICU1 constitute the minimal eukaryotic unit of the uniporter, pointing towards a strong selective pressure behind their co-occurrence. Heterologous reconstitution of MCU-mediated and MICU1-gated mitochondrial calcium entry in vivo in yeast cells demonstrates that MICU1 per se is essential to protect yeast from MCU-dependent manganese cytotoxicity. Accordingly, MICU1 deletion significantly sensitizes human HEK-293 cells to manganese-induced stress. Our study identifies a critical role of MICU1 in the regulation of MCU ion selectivity, with potential implications for patients with MICU1 deficiency.

scholarly journals Single-Cell Analyses Reveal That KISS1R-Expressing Cells Undergo Sustained Kisspeptin-Induced Signaling That Is Dependent upon An Influx of Extracellular Ca2+

Endocrinology ◽  
2012 ◽  
Vol 153 (12) ◽  
pp. 5875-5887 ◽  
Author(s):  
Andy V. Babwah ◽  
Macarena Pampillo ◽  
Le Min ◽  
Ursula B. Kaiser ◽  
Moshmi Bhattacharya
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Single Cell ◽  
Critical Role ◽  
Ovary Cell ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Kinase C ◽  
293 Cells

Abstract The kisspeptin receptor (KISS1R) is a Gαq/11-coupled seven-transmembrane receptor activated by a group of peptides referred to as kisspeptins (Kps). The Kp/KISS1R signaling system is a powerful regulator of GnRH secretion, and inactivating mutations in this system are associated with hypogonadotropic hypogonadism. A recent study revealed that Kp triggers prolonged signaling; not from the inability of the receptor to undergo rapid desensitization, but instead from the maintenance of a dynamic and active pool of KISS1R at the cell surface. To investigate this further, we hypothesized that if a dynamic pool of receptor is maintained at the cell surface for a protracted period, chronic Kp-10 treatment would trigger the sustained activation of Gαq/11 as evidenced through the prolonged activation of phospholipase C, protein kinase C, and prolonged mobilization of intracellular Ca2+. Through single-cell analyses, we tested our hypothesis in human embryonic kidney (HEK) 293 cells and found that was indeed the case. We subsequently determined that prolonged KISS1R signaling was not a phenomenon specific to HEK 293 cells but is likely a conserved property of KISS1R-expressing cells because evidence of sustained KISS1R signaling was also observed in the GT1–7 GnRH neuronal and Chinese hamster ovary cell lines. While exploring the regulation of prolonged KISS1R signaling, we identified a critical role for extracellular Ca2+. We found that although free intracellular Ca2+, primarily derived from intracellular stores, was sufficient to trigger the acute activation of a major KISS1R secondary effector, protein kinase C, it was insufficient to sustain chronic KISS1R signaling; instead extracellular Ca2+ was absolutely required for this.

scholarly journals DNA methylome signatures of prenatal exposure to synthetic glucocorticoids in hippocampus and peripheral whole blood of female guinea pigs in early life

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aya Sasaki ◽  
Margaret E. Eng ◽  
Abigail H. Lee ◽  
Alisa Kostaki ◽  
Stephen G. Matthews
Keyword(s):  
Dna Methylation ◽  
Whole Blood ◽  
Guinea Pigs ◽  
Critical Role ◽  
Methylation Status ◽  
Peripheral Tissues ◽  
Epigenetic Biomarkers ◽  
Differentially Methylated Genes ◽  
Increased Risk ◽  
Synthetic Glucocorticoids

AbstractSynthetic glucocorticoids (sGC) are administered to women at risk of preterm delivery, approximately 10% of all pregnancies. In animal models, offspring exposed to elevated glucocorticoids, either by administration of sGC or endogenous glucocorticoids as a result of maternal stress, show increased risk of developing behavioral, endocrine, and metabolic dysregulation. DNA methylation may play a critical role in long-lasting programming of gene regulation underlying these phenotypes. However, peripheral tissues such as blood are often the only accessible source of DNA for epigenetic analyses in humans. Here, we examined the hypothesis that prenatal sGC administration alters DNA methylation signatures in guinea pig offspring hippocampus and whole blood. We compared these signatures across the two tissue types to assess epigenetic biomarkers of common molecular pathways affected by sGC exposure. Guinea pigs were treated with sGC or saline in late gestation. Genome-wide modifications of DNA methylation were analyzed at single nucleotide resolution using reduced representation bisulfite sequencing in juvenile female offspring. Results indicate that there are tissue-specific as well as common methylation signatures of prenatal sGC exposure. Over 90% of the common methylation signatures associated with sGC exposure showed the same directionality of change in methylation. Among differentially methylated genes, 134 were modified in both hippocampus and blood, of which 61 showed methylation changes at identical CpG sites. Gene pathway analyses indicated that prenatal sGC exposure alters the methylation status of gene clusters involved in brain development. These data indicate concordance across tissues of epigenetic programming in response to alterations in glucocorticoid signaling.

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