Dopamine and norepinephrine transporter-dependent c-Fos production in vitro: relevance to neuroadaptation.
Cocaine, methylphenidate and other drugs that block dopamine transport indirectly promote immediate early gene expression, via dopamine-mediated activation of D1 dopamine receptors. Increased expression of the immediate early gene (IEG) c-fos, initiates a cascade of intracellular events that may underlie neuroadaptive changes following repeated exposure to the drugs. We investigated whether substrates (dopamine, norepinephrine) of the human dopamine (hDAT) and norepinephrine (hNET) transporters can directly induce c-Fos protein in HEK-293 (HEK) cells transfected with the hDAT and hNET and whether PKC modulators affect this process. Dopamine and norepinephrine robustly induced c-Fos immunofluorescence in both hDAT and hNET cells, but not in untransfected HEK-293 cells, demonstrating that catecholamine-induced c-Fos induction was DAT- and NET-dependent. The PKC activator PMA induced c-Fos in hDAT, hNET and HEK cell lines indicating that PKC stimulated c-Fos independent of transporters. The PKC inhibitor bisindolylmaleimide I (BIS) significantly increased c-Fos expression in hDAT cells, but not in hNET or HEK-293 cells, suggesting that inhibition of DAT-mediated phosphorylation results in c-Fos induction. BIS pretreatment abolished norepinephrine-induced c-Fos expression hNET but not dopamine-induced c-Fos induction in hDAT cells. In conclusion, induction of c-Fos by dopamine and norepinephrine requires the presence of hDAT and hNET but the contributions of hDAT and hNET to c-Fos induction is distinguishable on the basis of differing responses to a PKC inhibitor. These findings present a cell system and methodology for investigating the potential contribution of monoamine transporters to pre-synaptic neuroadaptation.
Yatin
Servet M
SM
Harvard Medical School, New England Primate Research Center, Division of Neurochemistry, One Pine Hill Drive, Southborough, MA 01772-9102, USA.
Miller
Gregory M
GM
Madras
Bertha K
BK
D000222:Adaptation, Physiological / Q000187:drug effects / Q000502:physiology*; D001923:Brain Chemistry / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D018491:Dopamine Agonists / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics*; D027981:Symporters / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018491:Dopamine Agonists; D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D016760:Proto-Oncogene Proteins c-fos; C493227:SLC6A2 protein, human; C493214:SLC6A3 protein, human; D027981:Symporters; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine; D009638:Norepinephrine