HIV-1 tat protein-induced rapid and reversible decrease in [ 3H]dopamine uptake: Dissociation of [3H]dopamine uptake and [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) binding in rat striatal synaptosomes

Jun Zhu, Charles F. Mactutus, David R. Wallace, Rosemarie M. Booze

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Abstract

Human immunodeficiency virus (HIV)-1 Tat protein plays a key role in the pathogenesis of both HIV-1-associated cognitivemotor disorder and drug abuse. Dopamine (DA) transporter (DAT) function is strikingly altered in patients with HIV-1-associated dementia and a history of chronic drug abuse. This study is the first in vitro evaluation of potential mechanisms underlying the effects of Tat protein on DAT function. Rat striatal synaptosomes were incubated with recombinant Tat1-86 protein, and [3H]DA uptake and the binding of [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) and [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)- piperazine (GBR 12935) were determined. Tat decreased [3H]DA uptake, [3H]WIN 35,428 binding, and [3H]GBR 12935 binding in a time-dependent manner. The potency of Tat for inhibiting [3H]DA uptake (Ki = 1.2 μM) was the same as that for inhibiting [ 3H]GBR 12935 binding but 3-fold less than that for inhibiting [ 3H]WIN 35,428 binding. Mutant Tat proteins did not alter [ 3H]DA uptake. Kinetic analysis of [3H]DA uptake revealed that Tat (1 or 10 μM) decreased the Vmax value and increased the Km value in a dose-dependent manner. The Vmax value, decreased by Tat (1 μM), returned to the control level after washout of Tat, indicating that the inhibitory effect of Tat on DA uptake was reversible. Saturation studies revealed that Tat decreased the Bmax value and increased the Kd value of [3H]WIN 35,428 binding, whereas Tat decreased the Bmax value of [3H]GBR 12935 binding, without a change in the Kd value. These findings provide new insight into understanding the pharmacological mechanisms of Tat-induced dysfunction of the DAT in the dopaminergic system in HIV-infected patients.

Original languageEnglish
Pages (from-to)1071-1083
Number of pages13
JournalJournal of Pharmacology and Experimental Therapeutics
Volume329
Issue number3
DOIs
StatePublished - 1 Jun 2009

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tat Gene Products
Corpus Striatum
Synaptosomes
HIV-1
Dopamine
Substance-Related Disorders
Dopamine Plasma Membrane Transport Proteins
Mutant Proteins
Dementia
methyl ester (1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid
GBR 12935
HIV
Pharmacology

Cite this

@article{6456c2d29ea047789f98b439f5a53146,
title = "HIV-1 tat protein-induced rapid and reversible decrease in [ 3H]dopamine uptake: Dissociation of [3H]dopamine uptake and [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) binding in rat striatal synaptosomes",
abstract = "Human immunodeficiency virus (HIV)-1 Tat protein plays a key role in the pathogenesis of both HIV-1-associated cognitivemotor disorder and drug abuse. Dopamine (DA) transporter (DAT) function is strikingly altered in patients with HIV-1-associated dementia and a history of chronic drug abuse. This study is the first in vitro evaluation of potential mechanisms underlying the effects of Tat protein on DAT function. Rat striatal synaptosomes were incubated with recombinant Tat1-86 protein, and [3H]DA uptake and the binding of [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) and [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)- piperazine (GBR 12935) were determined. Tat decreased [3H]DA uptake, [3H]WIN 35,428 binding, and [3H]GBR 12935 binding in a time-dependent manner. The potency of Tat for inhibiting [3H]DA uptake (Ki = 1.2 μM) was the same as that for inhibiting [ 3H]GBR 12935 binding but 3-fold less than that for inhibiting [ 3H]WIN 35,428 binding. Mutant Tat proteins did not alter [ 3H]DA uptake. Kinetic analysis of [3H]DA uptake revealed that Tat (1 or 10 μM) decreased the Vmax value and increased the Km value in a dose-dependent manner. The Vmax value, decreased by Tat (1 μM), returned to the control level after washout of Tat, indicating that the inhibitory effect of Tat on DA uptake was reversible. Saturation studies revealed that Tat decreased the Bmax value and increased the Kd value of [3H]WIN 35,428 binding, whereas Tat decreased the Bmax value of [3H]GBR 12935 binding, without a change in the Kd value. These findings provide new insight into understanding the pharmacological mechanisms of Tat-induced dysfunction of the DAT in the dopaminergic system in HIV-infected patients.",
author = "Jun Zhu and Mactutus, {Charles F.} and Wallace, {David R.} and Booze, {Rosemarie M.}",
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T1 - HIV-1 tat protein-induced rapid and reversible decrease in [ 3H]dopamine uptake

T2 - Dissociation of [3H]dopamine uptake and [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) binding in rat striatal synaptosomes

AU - Zhu, Jun

AU - Mactutus, Charles F.

AU - Wallace, David R.

AU - Booze, Rosemarie M.

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N2 - Human immunodeficiency virus (HIV)-1 Tat protein plays a key role in the pathogenesis of both HIV-1-associated cognitivemotor disorder and drug abuse. Dopamine (DA) transporter (DAT) function is strikingly altered in patients with HIV-1-associated dementia and a history of chronic drug abuse. This study is the first in vitro evaluation of potential mechanisms underlying the effects of Tat protein on DAT function. Rat striatal synaptosomes were incubated with recombinant Tat1-86 protein, and [3H]DA uptake and the binding of [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) and [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)- piperazine (GBR 12935) were determined. Tat decreased [3H]DA uptake, [3H]WIN 35,428 binding, and [3H]GBR 12935 binding in a time-dependent manner. The potency of Tat for inhibiting [3H]DA uptake (Ki = 1.2 μM) was the same as that for inhibiting [ 3H]GBR 12935 binding but 3-fold less than that for inhibiting [ 3H]WIN 35,428 binding. Mutant Tat proteins did not alter [ 3H]DA uptake. Kinetic analysis of [3H]DA uptake revealed that Tat (1 or 10 μM) decreased the Vmax value and increased the Km value in a dose-dependent manner. The Vmax value, decreased by Tat (1 μM), returned to the control level after washout of Tat, indicating that the inhibitory effect of Tat on DA uptake was reversible. Saturation studies revealed that Tat decreased the Bmax value and increased the Kd value of [3H]WIN 35,428 binding, whereas Tat decreased the Bmax value of [3H]GBR 12935 binding, without a change in the Kd value. These findings provide new insight into understanding the pharmacological mechanisms of Tat-induced dysfunction of the DAT in the dopaminergic system in HIV-infected patients.

AB - Human immunodeficiency virus (HIV)-1 Tat protein plays a key role in the pathogenesis of both HIV-1-associated cognitivemotor disorder and drug abuse. Dopamine (DA) transporter (DAT) function is strikingly altered in patients with HIV-1-associated dementia and a history of chronic drug abuse. This study is the first in vitro evaluation of potential mechanisms underlying the effects of Tat protein on DAT function. Rat striatal synaptosomes were incubated with recombinant Tat1-86 protein, and [3H]DA uptake and the binding of [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane (WIN 35,428) and [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)- piperazine (GBR 12935) were determined. Tat decreased [3H]DA uptake, [3H]WIN 35,428 binding, and [3H]GBR 12935 binding in a time-dependent manner. The potency of Tat for inhibiting [3H]DA uptake (Ki = 1.2 μM) was the same as that for inhibiting [ 3H]GBR 12935 binding but 3-fold less than that for inhibiting [ 3H]WIN 35,428 binding. Mutant Tat proteins did not alter [ 3H]DA uptake. Kinetic analysis of [3H]DA uptake revealed that Tat (1 or 10 μM) decreased the Vmax value and increased the Km value in a dose-dependent manner. The Vmax value, decreased by Tat (1 μM), returned to the control level after washout of Tat, indicating that the inhibitory effect of Tat on DA uptake was reversible. Saturation studies revealed that Tat decreased the Bmax value and increased the Kd value of [3H]WIN 35,428 binding, whereas Tat decreased the Bmax value of [3H]GBR 12935 binding, without a change in the Kd value. These findings provide new insight into understanding the pharmacological mechanisms of Tat-induced dysfunction of the DAT in the dopaminergic system in HIV-infected patients.

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