Cellular localization and functional expression of P‐glycoprotein in rat astrocyte cultures

Ronaldson, Patrick T.; Bendayan, Moı̈se; Gingras, Diane; Piquette-Miller, Micheline; Bendayan, Reina

doi:10.1111/j.1471-4159.2004.02417.x

Cited by 86 publications

(71 citation statements)

References 71 publications

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“…However, different cell types used for these experiments may account for the different results. Although the majority of data in literature indicates the localization of P-gp in caveolae (Rolfe et al, 2005;Ronaldson et al, 2004), other reports suggest lipid rafts but non-caveolar localization of this efflux transporter (Radeva et al, 2005) or localization both in caveolae and non-caveolae membrane domains (Jodoin et al, 2003). Lipid rafts are membrane microdomains that are enriched with cholesterol; thus, cholesterol depletion by MbCD is an effective strategy to disrupt lipid rafts.…”

Section: Discussionmentioning

confidence: 99%

Intact Lipid Rafts Regulate HIV-1 Tat Protein-Induced Activation of the Rho Signaling and Upregulation of P-Glycoprotein in Brain Endothelial Cells

Zhong

Hennig

Toborek

2009

J Cereb Blood Flow Metab

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The Rho signaling has an essential function in human immunodeficiency virus (HIV)-1-mediated disruption of the integrity of the blood-brain barrier (BBB). However, it is unknown how membrane domains, such as lipid rafts, can influence HIV-1-mediated activation of the Rho pathway and how these processes can affect the expression of the efflux transporters at the BBB level. This study is focused on the function of HIV-1 protein Tat in activation of the Rho signaling and upregulation of P-glycoprotein (P-gp) in human brain endothelial cells. Treatment with Tat markedly elevated GTPRhoA levels and the potential downstream effectors, such as myosin phosphatase target subunit 1 and myosin light chain. In addition, Tat upregulated expression and promoter activity of P-gp as well as its efflux function. Inhibition of the Rho signaling cascade effectively blocked P-gp overexpression at the level of promoter activity. Disruption of lipid rafts by depletion of membrane cholesterol by methyl-beta-cyclodextrin, but not caveolin-1 silencing, also abolished Tat-mediated RhoA activation and P-gp upregulation. The present data indicate the critical function of intact lipid rafts and the Rho signaling in HIV-1-mediated upregulation of P-gp and potential development of drug resistance in brain endothelial cells.

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Section: Discussionmentioning

confidence: 99%

Intact Lipid Rafts Regulate HIV-1 Tat Protein-Induced Activation of the Rho Signaling and Upregulation of P-Glycoprotein in Brain Endothelial Cells

Zhong

Hennig

Toborek

2009

J Cereb Blood Flow Metab

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“…Primary cultures of rat astrocytes were prepared as described previously (Ronaldson et al, 2004a). All procedures were carried out in accordance with the University of Toronto Animal Care Committee and the Province of Ontario Animals for Research Act.…”

Section: Methodsmentioning

confidence: 99%

“…Our laboratory has also characterized P-gp functional expression in a rat microglia cell line (MLS-9) (Lee et al, 2001), cultured rat astrocytes (Ronaldson et al, 2004a), and in an immortalized rat brain microvessel endothelial cell line (RBE4) (Bendayan et al, 2002).…”

mentioning

confidence: 99%

HIV-1 Viral Envelope Glycoprotein gp120 Triggers an Inflammatory Response in Cultured Rat Astrocytes and Regulates the Functional Expression of P-Glycoprotein

2006

Self Cite

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In this work, we examined the ability of gp120, a human immunodeficiency virus-1 (HIV-1) viral envelope glycoprotein, to trigger the innate immune response in astrocytes, an HIV-1 brain cellular target, and we investigated the functional expression of the ATP-binding cassette membrane transporter P-glycoprotein (P-gp) in primary cultures of rat astrocytes treated with gp120 or cytokines [tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), and IL-6]. Standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium and D-mannitol uptake assays confirmed that HIV-1 96ZM651 gp120 treatment did not alter cell viability or membrane permeability. Semiquantitative reversetranscriptase polymerase chain reaction analysis and enzymelinked immunosorbent assay demonstrated increased TNF-␣, IL-1␤, and IL-6 mRNA and protein expression in cultures treated with HIV-1 96ZM651 gp120, suggesting in vitro activation of immune responses. Cytokine secretion was detected when CXCR4 but not CCR5 was inhibited with a specific antibody, implying that cytokine secretion is primarily mediated via CCR5 in astrocytes triggered with HIV-1 96ZM651 gp120. P-gp protein expression was increased in astrocyte cultures exposed to TNF-␣ (2.9-fold) or IL-1␤ (1.6-fold) but was decreased profoundly in the presence of IL-6 (8.9-fold), suggesting that IL-6 is primarily involved in modulating P-gp expression. In parallel, after HIV-1 96ZM651 gp120 treatment, immunoblotting analysis showed a significant decrease in P-gp expression (4.7-fold). Furthermore, the accumulation of two P-gp substrates, digoxin and saquinavir (an HIV-1 protease inhibitor), was enhanced (1.5-to 1.8-fold) in HIV-1 96ZM651 gp120-treated astrocyte monolayers but was not altered by P-gp inhibitors [e.g., valspodar (PSC833) and elacridar (GF120918)], suggesting a loss of transport activity. Taken together, these data imply that HIV-1 96ZM651 gp120 or cytokine treatment modulate P-gp functional expression in astrocytes, which may lead to complex drug-transporter interactions during HIV-1 encephalitis-associated immune responses.

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“…Primary cultures of rat astrocytes were prepared as described previously (Ronaldson et al, 2004). All procedures were performed in accordance with the University of Toronto Animal Care Committee and the Province of Ontario Animals for Research Act.…”

Section: Immunocytochemistry For Immunocytochemistry In Adult Spraguementioning

confidence: 99%

Glutamate Transporter Coupling to Na,K-ATPase

Rose

Koo²,

Antflick³

et al. 2009

Journal of Neuroscience

279

232

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Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The ␣1-␣3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using

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Cellular localization and functional expression of P‐glycoprotein in rat astrocyte cultures

Cited by 86 publications

References 71 publications

Intact Lipid Rafts Regulate HIV-1 Tat Protein-Induced Activation of the Rho Signaling and Upregulation of P-Glycoprotein in Brain Endothelial Cells

Intact Lipid Rafts Regulate HIV-1 Tat Protein-Induced Activation of the Rho Signaling and Upregulation of P-Glycoprotein in Brain Endothelial Cells

HIV-1 Viral Envelope Glycoprotein gp120 Triggers an Inflammatory Response in Cultured Rat Astrocytes and Regulates the Functional Expression of P-Glycoprotein

Glutamate Transporter Coupling to Na,K-ATPase

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