The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

Abstract

The effects of 17beta-estradiol (E(2)) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E(2) at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) alpha, ERbeta, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of (3)H-DA from pre-loaded cells; a 9-15 min 10(-9 )M E(2) treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E(2)-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose-responses for E(2) were non-monotonic, also characteristic of non-genomic estrogenic actions. ERalpha siRNA knockdown abolished E(2)-mediated DA efflux, while ERbeta knockdown did not, and GPR30 knockdown increased E(2)-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERalpha is the predominant mediator of E(2)-mediated DA efflux, with inhibitory contributions from GPR30 and ERbeta. E(2) also caused trafficking of ERalpha to the plasma membrane, trafficking of ERbeta away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERalpha is largely responsible for non-genomic estrogenic effects on DAT activity.

Figure 1

E₂ dopamine efflux time course and dose-response curve. (A) 10⁻⁹M E₂ time course (B) physiologically relevant E₂ dose response curve at 9 minutes; *=significant compared to 0.005% ethanol control (p<0.05); n=24 in four experiments.

Figure 2

E₂-dendrimer-elicited dopamine efflux time course. (●) 10⁻⁹ M E₂ dendrimer equivalents in mins compared to (○) 10⁻⁹ M E₂ dopamine efflux time course. *=p<0.05 significance of control (0.005% ethanol) compared to non-conjugated dendrimer (the E₂ dendrimer control), #=p<0.05 from E₂ treatment. n=18 in three experiments

Figure 3

Effects of siRNA on protein expression and DA efflux. (A) Immunoblot analysis of ERα, ERβ, and GPR30 proteins due to siRNA knockdown. PC12 cells were electroporated without (−) and with (+) Dharmacon SMART pool rat siRNA transcripts for ERα, ERβ, and GPR30. Protein expression was examined in whole cell lysates after 72 hrs. (B) Effect of 10⁻⁹ M E₂ treatment for 9 mins on DA efflux from siRNA-transfected cells compared to ethanol control, non-transfected, and random sequence-transfected cells. *=p<0.05 significance compared to 0.005% ethanol control, #=p<0.05 from non-transfected, ^=p<0.05 from random sequence pool. n=18 in three experiments

Figure 3

Effects of siRNA on protein expression and DA efflux. (A) Immunoblot analysis of ERα, ERβ, and GPR30 proteins due to siRNA knockdown. PC12 cells were electroporated without (−) and with (+) Dharmacon SMART pool rat siRNA transcripts for ERα, ERβ, and GPR30. Protein expression was examined in whole cell lysates after 72 hrs. (B) Effect of 10⁻⁹ M E₂ treatment for 9 mins on DA efflux from siRNA-transfected cells compared to ethanol control, non-transfected, and random sequence-transfected cells. *=p<0.05 significance compared to 0.005% ethanol control, #=p<0.05 from non-transfected, ^=p<0.05 from random sequence pool. n=18 in three experiments

Figure 4

Effects of selective agonists and antagonists for ERα, ERβ, and GPR30 on 9 min dopamine efflux compared with % of 10⁻⁹ M E₂ treatment. Horizontal dashed lines represent standard error around 100%; solid line represents the mean of 10⁻⁹ M E₂ treatment set at 100%. (A) PPT is an ERα-, DPN an ERβ-, and G-1 a GPR30-selective ligand. (B) Effect of selective ligands in combination with E₂ compared to E₂ alone. MPP is an ERα- and ICI 182 780 an ERα- and ERβ-antagonist. The G-1 vehicle control is DMSO+EtOH, 0.005% ethanol is the control for PPT, and 0.005% ethanol is the control for both E₂ and DPN. *=p<0.05 significance compared to control, #=p<0.05 from E₂ treatment. n=18 in three experiments

Figure 5

Quantitative plate assay measuring membrane and total ERα, ERβ, and GPR30 immunoreactive protein levels after a 9 minute 10⁻⁹ M E₂ treatment. *=p<0.05 significance compared to 0.005% ethanol control, #=p<0.05 from membrane levels. n=50 in 3 experiments