Abstract
Cyclin-dependent kinase (Cdk) 5 reduces the rewarding properties of psychostimulants by dampening postsynaptic dopamine (DA) receptor signaling. Cdk5 is also present in midbrain DA neurons, where the DA transporter (DAT) is localized and limits DA neurotransmission by removing extracellular DA. Here, we tested the hypothesis that Cdk5 could also affect the disposition of DA by regulating DAT activity. Incubation of rat dorsal striatal (dSTR) synaptosomes with the Cdk5 inhibitors roscovitine, olomoucine, and 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(2-pyridyl)benzenesulfonamide (GW8510) or the inactive congener iso-olomoucine resulted in a rapid, concentration-dependent inhibition of specific [3H]DA uptake. However, roscovitine was the only inhibitor that did not also decrease [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN35,428) binding to dSTR DATs. Roscovitine-induced inhibition of dSTR [3H]DA uptake was explained by decreased maximal uptake velocity, without a change in cell-surface DAT levels. Roscovitine did not enhance [3H]DA release mediated by either DAT reverse-transport or Ca2+ channels in dSTR slices. Instead, roscovitine enhanced spontaneous [3H]DA outflow and inhibited DAT-mediated [3H]DA reaccumulation into dSTR slices. To explore the involvement of Cdk5 in roscovitine-induced down-regulation of DAT activity, Cdk5 protein was knocked down via Cdk5-small interfering RNA by as much as 86% in porcine aortic endothelial cells stably expressing human (h)DATs. However, Cdk5 depletion did not alter hDAT activity. Taken together, our results suggest that roscovitine inhibits DAT activity independently of Cdk5; therefore, results obtained with such inhibitors should be interpreted with caution. Our study is the first to demonstrate that Cdk5 inhibitors reduce brain DAT activity via a mechanism that is independent of DAT trafficking and reverse-transport.
- DA, dopamine
- DAT, dopamine transporter
- AMPH, amphetamine
- MAPK, p44/42 mitogen-activated protein kinase
- Cdk, cyclin-dependent kinase
- DARPP-32, dopamine and cAMP-regulated phospho-protein of relative molecular mass 32,000
- dSTR, dorsal striatal/striatum
- PAE, porcine aortic endothelial
- YFP, yellow fluorescent protein
- HA, hemagglutinin epitope
- h, human
- siRNA, small interfering RNA
- PMA, 12-myristate 13-acetate
- DMSO, dimethyl sulfoxide
- WIN35,428, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane
- PP2A-A α/β, protein phosphatase 2A-A α/β
- Cy, cyanine
- NT-siRNA, Dharmacon siGENOME nontargeting 3 siRNA
- BSA, bovine serum albumin
- CMF-PBS, Ca2+/Mg2+-free phosphate-buffered saline
- Vmax, maximal transport velocity
- Km, substrate affinity
- ANOVA, analysis of variance
- S, stimulation
- GW8510, 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(2-pyridyl)benzenesulfonamide.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants F32 DA-025397, DA-004216, DA-015050, DA-014204].
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This work has been previously presented in poster form: Price DA and Zahniser NR (2008) Rapid regulation of rat brain dopamine transporter by cyclin-dependent kinase 5 inhibitors. Soc Neurosci Abstr 34:134.19.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- Received April 10, 2009.
- Accepted July 23, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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