RESUMEN

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.

Asunto(s)

Adaptación Fisiológica/fisiología , Química Encefálica/efectos de los fármacos , Agonistas de Dopamina/farmacología , Glicoproteínas de Membrana/agonistas , Proteínas de Transporte de Membrana/agonistas , Proteínas del Tejido Nervioso/agonistas , Proteínas Proto-Oncogénicas c-fos/genética , Simportadores/agonistas , Adaptación Fisiológica/efectos de los fármacos , Química Encefálica/fisiología , Línea Celular , Dopamina/metabolismo , Dopamina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Indoles/farmacología , Maleimidas/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Moduladores del Transporte de Membrana , Proteínas de Transporte de Membrana/antagonistas & inhibidores , Proteínas de Transporte de Membrana/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Norepinefrina/metabolismo , Norepinefrina/farmacología , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática , Fosforilación/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Simportadores/antagonistas & inhibidores , Simportadores/genética , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

The psychostimulants cocaine and amphetamine increase expression of the immediate early gene (IEG) c-fos indirectly, via D1 dopamine receptor activation. To determine whether dopamine transporter substrates and inhibitors can affect c-Fos expression directly, we investigated their effects on c-Fos protein and c-fos mRNA in HEK-293 (HEK) cells transfected with the human dopamine transporter (hDAT). In untransfected HEK cells, methylphenidate and cocaine produced a small but statistically significant increase in c-Fos, whereas dopamine and amphetamine did not. In hDAT cells, DAT substrates (dopamine, amphetamine) increased c-Fos immunoreactivity 6- and 3-fold (respectively). The DAT inhibitors cocaine, methylphenidate, and bupropion also increased c-Fos approximately 3-fold in hDAT cells. If coincubated with dopamine, the inhibitors attenuated dopamine-induced c-Fos in hDAT cells. The magnitude of c-fos mRNA induction by substrates and inhibitors paralleled induction of c-Fos protein immunoreactivity. The results indicate that substrates or inhibitors of the DAT can trigger induction of IEG expression in the absence of D1 dopamine receptor. For substrates, IEG induction is DAT-dependent, but for certain DAT inhibitors the cellular response can be elicited in the absence of the DAT in HEK cells. Oxidative stress may partly, but not fully, account for the DA-induced c-Fos induction as an inhibitor of oxidative stress Trolox C, attenuated DA-induced c-Fos induction. Protein kinase C (PKC) may also partially account for c-Fos induction as a specific inhibitor of PKC Bisindolylmaleimide I (BIS) attenuated DA-induced c-Fos by 50%. DAT substrate and inhibitor effects on IEGs, other fos-related antigens, and possible mechanisms that contribute to c-Fos induction warrant investigation in presynaptic neurons as a potential contribution to the long-term effects of psychostimulants.

Asunto(s)

Glicoproteínas de Membrana , Proteínas de Transporte de Membrana/fisiología , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas c-fos/metabolismo , Anfetamina/farmacología , Línea Celular , Cocaína/farmacología , Dopamina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Inhibidores de Captación de Dopamina/farmacología , Sinergismo Farmacológico , Humanos , Moduladores del Transporte de Membrana , Proteínas de Transporte de Membrana/antagonistas & inhibidores , Metilfenidato/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero

RESUMEN

Amyloid beta-peptide (Abeta) is a 42-43 amino acid peptide known to accumulate in Alzheimer's disease (AD) brain. We previously reported that the neurotoxicity caused by Abeta is a result of its associated free radicals, which can play an important role in generating oxidative stress. Abeta(25-35)-associated oxidative stress-induced neuronal death in vitro is well established by many laboratories, including ours. However, the oxidative stress-induced by the full-length [Abeta(1-42)] peptide is not well investigated. The protective effect of antioxidant vitamin E in full-length peptide-induced oxidative stress also has not been reported. Here, we report that the increased protein oxidation, reactive oxygen species (ROS) formation, and neurotoxicity induced by Abeta(1-42) in primary rat embryonic hippocampal neuronal culture are prevented by the free radical scavenger and antioxidant vitamin E. To test the hypothesis that vitamin E's protective effect may be due to inhibition of fibril formation, electron microscopy studies were undertaken. Vitamin E does not inhibit Abeta(1-42) fibril formation, suggesting that the neuroprotection afforded by this molecule stems from other processes, most probably through the scavenging of Ab-associated free radicals. These results may have implications on the treatment of Alzheimer's disease.