scholarly journals Prolonged Amphetamine Exposures Increase the Endogenous Human Dopamine Receptors 2 at the Cellular Membrane in Cells Lacking the Dopamine Transporter

2021 ◽  
Vol 15 ◽  
Author(s):  
Vindhya Nawaratne ◽  
Sean P. McLaughlin ◽  
Felix P. Mayer ◽  
Zayna Gichi ◽  
Alyssa Mastriano ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Specific Inhibitor ◽  
Cellular Membrane ◽  
Hek293 Cells ◽  
Imaging Data ◽  
Western Blots ◽  
Synaptic Terminals ◽  
And Function ◽  
In Cells

The dopamine 2 receptors (D2R) are G-protein coupled receptors expressed both in pre- and post-synaptic terminals that play an important role in mediating the physiological and behavioral effects of amphetamine (Amph). Previous studies have indicated that the effects of Amph at the D2R mainly rely on the ability of Amph to robustly increase extracellular dopamine through the dopamine transporter (DAT). This implies that the effects of Amph on D2R require the neurotransmitter dopamine. However, because of its lipophilic nature, Amph can cross the cellular membrane and thus potentially affect D2R expression independently of dopamine and DAT, e.g., in post-synaptic terminals. Here we used an in vitro system to study whether Amph affects total expression, cellular distribution, and function of the human D2R (hD2R), endogenously expressed in HEK293 cells. By performing Western blot experiments, we found that prolonged treatments with 1 or 50 μM Amph cause a significant decrease of the endogenous hD2R in cells transfected with human DAT (hDAT). On the other hand, in cells lacking expression of DAT, quantification of the hD2R-mediated changes in cAMP, biotinylation assays, Western blots and imaging experiments demonstrated an increase of hD2R at the cellular membrane after 15-h treatments with Amph. Moreover, imaging data suggested that barbadin, a specific inhibitor of the βarrestin-βadaptin interaction, blocked the Amph-induced increase of hD2R. Taken together our data suggest that prolonged exposures to Amph decrease or increase the endogenous hD2R at the cellular membrane in HEK293 cells expressing or lacking hDAT, respectively. Considering that this drug is often consumed for prolonged periods, during which tolerance develops, our data suggest that even in absence of DAT or dopamine, Amph can still alter D2R distribution and function.

scholarly journals Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

2006 ◽  
Vol 128 (5) ◽  
pp. 509-522 ◽  
Author(s):  
Alexander T. Stein ◽  
Carmen A. Ufret-Vincenty ◽  
Li Hua ◽  
Luis F. Santana ◽  
Sharona E. Gordon
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Specific Inhibitor ◽  
Thermal Hyperalgesia ◽  
Hek293 Cells ◽  
Drg Neurons ◽  
Excised Patches ◽  
Phosphoinositide 3 Kinase ◽  
Inside Out

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

scholarly journals Positive and Negative TAFII Functions That Suggest a Dynamic TFIID Structure and Elicit Synergy with TRAPs in Activator-Induced Transcription

2001 ◽  
Vol 21 (20) ◽  
pp. 6882-6894 ◽  
Author(s):  
Mohamed Guermah ◽  
Yong Tao ◽  
Robert G. Roeder
Keyword(s):  
Basal Transcription ◽  
Absolute Level ◽  
Western Blots ◽  
Core Promoters ◽  
Activated State ◽  
Cloned Cdna ◽  
Sequence Relationships ◽  
And Function ◽  
Human Transcription Factor

ABSTRACT Human transcription factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFIIs). To elucidate the structural organization and function of TFIID, we expressed and characterized the product of a cloned cDNA encoding human TAFII135 (hTAFII135). Comparative far Western blots have shown that hTAFII135 interacts strongly with hTAFII20, moderately with hTAFII150, and weakly with hTAFII43 and hTAFII250. Consistent with these observations and with sequence relationships of hTAFII20 and hTAFII135 to histones H2B and H2A, respectively, TFIID preparations that contain higher levels of hTAFII135 also contain higher levels of hTAFII20, and the interaction between hTAFII20 and hTAFII135 is critical for human TFIID assembly in vitro. From a functional standpoint, hTAFII135 has been found to interact strongly and directly with hTFIIA and (within a complex that also contains hTBP and hTAFII250) to specifically cooperate with TFIIA to relieve TAFII250-mediated repression of TBP binding and function on core promoters. Finally, we report a functional synergism between TAFIIs and the TRAP/Mediator complex in activated transcription, manifested as hTAFII-mediated inhibition of basal transcription and a consequent TRAP requirement for both a high absolute level of activated transcription and a high and more physiological activated/basal transcription ratio. These results suggest a dynamic TFIID structure in which the switch from a basal hTAFII-enhanced repression state to an activator-mediated activated state on a promoter may be mediated in part through activator or coactivator interactions with hTAFII135.

scholarly journals Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

1996 ◽  
Vol 109 (6) ◽  
pp. 1479-1495 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Rodriguez-Paris ◽  
J.M. Bush ◽  
L. Zhang ◽  
J.A. Cardelli
Keyword(s):  
Morphological Changes ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Contractile Vacuole ◽  
Dependent Manner ◽  
Concanamycin A ◽  
Latex Beads ◽  
And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum

1994 ◽  
Vol 107 (3) ◽  
pp. 645-659 ◽  
Author(s):  
G.G. Gundersen ◽  
I. Kim ◽  
C.J. Chapin
Keyword(s):  
Growth Factors ◽  
Calf Serum ◽  
Tgf Beta ◽  
Wound Edge ◽  
Western Blots ◽  
Polypeptide Growth Factors ◽  
3T3 Fibroblasts ◽  
Mild Acid ◽  
In Cells

Previous studies have shown that fibroblasts induced to migrate into an in vitro wound rapidly generate an array of stable, post-translationally detyrosinated microtubules (Glu MTs) oriented toward the direction of migration. To understand how cells generate a stable array of MTs at a specific location, we have analyzed the contribution of media components to the formation of oriented Glu MTs in wounded monolayers of 3T3 fibroblasts. When confluent monolayers were placed in serum-free medium (SFM) for 2 days before wounding, the cells contained virtually no Glu MTs or nocodazole-resistant MTs and were incapable of generating Glu MTs in response to wounding. Such SFM-treated monolayers were capable of generating oriented Glu MTs within 1 hour of wounding, if calf serum (CS) was added back to the medium. The Glu MTs in the CS refed cells were oriented toward the wound in cells at the wound edge, and were juxtanuclear in cells within the monolayer, demonstrating that CS restored the Glu MT array characteristic of each cell type. To determine the nature of the ‘Glu MT-inducing’ factor in CS, we subjected CS to different treatments and found that the CS factor was nondialyzable, resistant to heat, mild acid and trypsin, but inactivated by treatment with dithiothreitol. The factor was not absorbed by charcoal and was present in lipoprotein-deficient serum. These properties are consistent with the properties of a number of polypeptide growth factors, so we screened purified growth factors for their ability to induce Glu MTs in wounded SFM-treated monolayers. Of all the growth factors tested, only TGF-beta 1 and TGF-beta 2 induced a significant level (> or = 70% of the CS response) of oriented Glu MTs. The SFM-treated cells were exquisitely sensitive to TGF-beta 1, with significant induction of Glu MTs observed at 0.01 ng/ml TGF-beta 1. Induction of Glu MTs observed by immunofluorescence after CS or TGF-beta treatments were paralleled by increases in Glu tubulin detected on western blots. The Glu MTs formed after either CS or TGF-beta 1 treatment showed enhanced resistance to nocodazole, confirming that both treatments increased the level of stable MTs in cells. The TGF-beta 1 induction of stable MTs was slower than that of CS (2-4 hours onset versus 1 hour onset), but by 24 hours the level of MT stabilization in TGF-beta 1 was even greater than that in CS.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Effects of epigallocatechin gallate on regulatory T cell number and function in obese v. lean volunteers

2010 ◽  
Vol 103 (12) ◽  
pp. 1771-1777 ◽  
Author(s):  
Jung-Mi Yun ◽  
Ishwarlal Jialal ◽  
Sridevi Devaraj
Keyword(s):  
Epigallocatechin Gallate ◽  
Cell Number ◽  
Normal Weight ◽  
Western Blots ◽  
Egcg Treatment ◽  
Hdac Activity ◽  
Obese Subjects ◽  
Specific Regulation ◽  
And Function

Obesity predisposes to an increased incidence of diabetes and CVD. Also, obesity is a pro-inflammatory state. Regulatory T cells (Tregs) are essential negative regulators of inflammation and are down-regulated in pro-inflammatory states. Animal models of obesity are associated with decreased Tregs. The dietary modulation of Tregs could be used as a therapeutic strategy to control inflammation. Epigallocatechin gallate (EGCG) is a potent anti-inflammatory agent and an active ingredient of green tea and is suggested to have a role as a preventive agent in obesity, diabetes and CVD. The role of EGCG in the modulation of Tregs has, however, not been studied. Thus, the aim of the present study was to determine the effect of EGCG on the number and function of Tregs in obese and lean human subjects in vitro, and to delineate its specific regulation mechanisms. Tregs were isolated from normal-weight and obese subjects. Tregs were cultured in the absence or presence of EGCG (20 μm) for 24 h. Foxp3-expressing Tregs were enumerated using flow cytometry. Histone deacetylase (HDAC) activity and nuclear NF-κBp65 level were measured by ELISA and Western blots. Obese subjects had lower Tregs and IL-10 production than lean subjects. EGCG treatment significantly enhanced the number of Foxp3-expressing Tregs and IL-10 production in vitro (P < 0·05) in both groups. Also, EGCG decreased NF-κB activity and increased HDAC activity and HDAC-2 expression in Tregs (P < 0·05) in both groups. Thus, in part, EGCG enhances the functionality of Tregs, i.e. IL-10 production and number by suppressing the NF-κB signalling pathway via inducing epigenetic changes.

scholarly journals The actin nucleation factor JMY is a negative regulator of neuritogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4563-4574 ◽  
Author(s):  
Elif Nur Firat-Karalar ◽  
Peter P. Hsiue ◽  
Matthew D. Welch
Keyword(s):  
Cell Lines ◽  
Regulatory Protein ◽  
Neuronal Cell ◽  
Full Length ◽  
Negative Regulator ◽  
Neurite Formation ◽  
A Minor ◽  
And Function ◽  
In Cells

Junction-mediating and regulatory protein (JMY) is a p53 cofactor that was recently shown to nucleate actin assembly by a hybrid mechanism involving tandem actin monomer binding and Arp2/3 complex activation. However, the regulation and function of JMY remain largely uncharacterized. We examined the activity of JMY in vitro and in cells, its subcellular distribution, and its function in fibroblast and neuronal cell lines. We demonstrated that recombinant full-length JMY and its isolated WASP homology 2 domain, connector, and acidic region (WWWCA) have potent actin-nucleating and Arp2/3-activating abilities in vitro. In contrast, the activity of full-length JMY, but not the isolated WWWCA domain, is suppressed in cells. The WWWCA domain is sufficient to promote actin-based bead motility in cytoplasmic extracts, and this activity depends on its ability to activate the Arp2/3 complex. JMY is expressed at high levels in brain tissue, and in various cell lines JMY is predominantly cytoplasmic, with a minor fraction in the nucleus. Of interest, silencing JMY expression in neuronal cells results in a significant enhancement of the ability of these cells to form neurites, suggesting that JMY functions to suppress neurite formation. This function of JMY requires its actin-nucleating activity. These findings highlight a previously unrecognized function for JMY as a modulator of neuritogenesis.

scholarly journals Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 185
Author(s):  
Poyil Pratheeshkumar ◽  
Abdul K. Siraj ◽  
Sasidharan Padmaja Divya ◽  
Sandeep Kumar Parvathareddy ◽  
Sarah Siraj ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Inhibitor ◽  
Papillary Thyroid ◽  
Self Renewal ◽  
Functional Correlation ◽  
Xenograft Growth ◽  
Molecular Therapeutic ◽  
And Function

The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

scholarly journals Cardiomyocyte-Specific RIP2 Overexpression Exacerbated Pathologic Remodeling and Contributed to Spontaneous Cardiac Hypertrophy

2021 ◽  
Vol 9 ◽  
Author(s):  
Jing-jing Yang ◽  
Nan Zhang ◽  
Zi-ying Zhou ◽  
Jian Ni ◽  
Hong Feng ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cardiac Remodeling ◽  
Specific Inhibitor ◽  
Pressure Overload ◽  
Neonatal Rat ◽  
Hek293 Cells ◽  
Cardiomyocyte Hypertrophy ◽  
Rat Cardiomyocytes

This study aimed to investigate the role and mechanisms of Receptor interacting protein kinase 2 (RIP2) in pressure overload-induced cardiac remodeling. Human failing or healthy donor hearts were collected for detecting RIP2 expression. RIP2 cardiomyocyte-specific overexpression, RIP2 global knockout, or wild-type mice were subjected to sham or aortic banding (AB) surgery to establish pressure overload-induced cardiac remodeling in vivo. Phenylephrine (PE)-treated neonatal rat cardiomyocytes (NRCMs) were used for further investigation in vitro. The expression of RIP2 was significantly upregulated in failing human heart, mouse remodeling heart, and Ang II-treated NRCMs. RIP2 overexpression obviously aggravated pressure overload-induced cardiac remodeling. Mechanistically, RIP2 overexpression significantly increased the phosphorylation of TAK1, P38, and JNK1/2 and enhanced IκBα/p65 signaling pathway. Inhibiting TAK1 activity by specific inhibitor completely prevented cardiac remodeling induced by RIP2 overexpression. This study further confirmed that RIP2 overexpression in NRCM could exacerbate PE-induced NRCM hypertrophy and TAK1 silence by specific siRNA could completely rescue RIP2 overexpression-mediated cardiomyocyte hypertrophy. Moreover, this study showed that RIP2 could bind to TAK1 in HEK293 cells, and PE could promote their interaction in NRCM. Surprisingly, we found that RIP2 overexpression caused spontaneous cardiac remodeling at the age of 12 and 18 months, which confirmed the powerful deterioration of RIP2 overexpression. Finally, we indicated that RIP2 global knockout attenuated pressure overload-induced cardiac remodeling via reducing TAK1/JNK1/2/P38 and IκBα/p65 signaling pathways. Taken together, RIP2-mediated activation of TAK1/P38/JNK1/2 and IκBα/p65 signaling pathways played a pivotal role in pressure overload-induced cardiac remodeling and spontaneous cardiac remodeling induced by RIP2 overexpression, and RIP2 inhibition might be a potential strategy for preventing cardiac remodeling.

scholarly journals GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis

2017 ◽  
Vol 474 (6) ◽  
pp. 983-1001 ◽  
Author(s):  
Darya Zibrova ◽  
Franck Vandermoere ◽  
Olga Göransson ◽  
Mark Peggie ◽  
Karina V. Mariño ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Energy Homeostasis ◽  
Vascular Dysfunction ◽  
Negative Regulator ◽  
Label Free ◽  
Hexosamine Biosynthesis ◽  
Underlying Mechanisms ◽  
And Function ◽  
In Cells

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

scholarly journals Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family

2005 ◽  
Vol 34 (2) ◽  
pp. 317-329 ◽  
Author(s):  
A Blondet ◽  
J Gout ◽  
P Durand ◽  
M Bégeot ◽  
D Naville
Keyword(s):  
Receptor Gene ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Hek293 Cells ◽  
Melanocortin 4 Receptor ◽  
Melanocortin 4 Receptor Gene ◽  
Sp Transcription Factor ◽  
First Time

The melanocortin-4 receptor (MC4-R) plays a key role in the hypothalamic control of food intake, lending importance to the understanding of the mechanisms that regulate its expression. To identify factors controlling the expression of the human (h) MC4-R gene, a fragment containing 1253 bp of the 5′-flanking region of the hMC4-R gene was isolated. A series of hMC4-R luciferase constructs were developed and used to transiently transfect HEK293 and GT1–7 cell lines, both expressing endogenous MC4-R mRNA. Deletion analysis of the 1253 bp fragment showed that the basal promoter activity is mainly restricted to the 179 bp upstream of the transcription start site in both cell types. Mutation of a putative Sp1-binding site located at position −76 bp resulted in a dramatic reduction of the luciferase activity in HEK293 and GT1–7 cells by 87 and 80% respectively. Both in vitro and in vivo studies (gel shift and chromatin immunoprecipitation analyses) revealed binding of both Sp1 and Sp3 to this site in HEK293 cells. Cotransfection with an Sp1 expression vector in Drosophila cells that do not express Sp1, in conjunction with treatment of HEK293 cells with mithramycin A, a specific inhibitor of Sp1, confirmed the role of Sp1. For the first time, we have demonstrated that the constitutive activity of the hMC4-R promoter is dependent upon Sp transcription factors.

Sign in / Sign up

Export Citation Format

Share Document