Inhibiting PI3K or PKA did not affect E2 mediated dopamine efflux. The presence of intracellular kinase inhibitor Belinostat Ca2 is required for E2 mediated dopamine efflux Although we have controlled for dopamine flux specifi cally through the DAT through the use of DAT and nore pinephrine selective transporter inhibitors, the addition of these inhibitors does not account for the possibility of exocytotic release of dopamine which is dependent on extracellular Inhibitors,Modulators,Libraries Ca2. Intracellular Ca2 is also an important second messenger signal that is required to activate Ca2 dependent PKC isoforms. Compared to 9 min 10 9 M E2 treatment, preincubating the cells for 10 min in 0 Ca2 medium containing 5 mM EGTA did not inhibit E2 induced dopamine efflux, but instead actually increased dopamine efflux.

However, the prior emptying Inhibitors,Modulators,Libraries of intracel lular stores of Ca2 with thapsigargin did reverse E2 medi ated dopamine efflux. Vesicular Inhibitors,Modulators,Libraries release of dopamine is not involved in E2 mediated dopamine efflux We then further examined the mechanisms involved in the E2 induced movement of dopamine to the outside of PC12 cells. To confirm that vesicular release of dopamine is not involved in E2 mediated dopamine efflux mecha nism, we preincubated our cells with reserpine, a vesicular prese Effects of three physiological estrogens on dopamine efflux and trafficking of the DAT and ERs Changes in DAT membrane presence and functioning could be an important mechanism for alterations in neu rochemical signaling by several physiological Inhibitors,Modulators,Libraries estrogens monoamine transporter inhibitor which causes emptying of dopamine from VMATs.

Figure 3 shows that the inhibition of vesicular release does not inhibit subse quent E2 induced dopamine efflux, further confirm ing that the E2 mediated dopamine efflux that we have observed is specifically via the DAT. We found that the dopamine efflux resulting from treatment with reserpine alone compared to the control Inhibitors,Modulators,Libraries are similar indicating that basal and reserpine control are not different from one another. We also noted that inhibiting VMATs signifi cantly increased E2 mediated dopamine efflux. p. Therefore, we first monitored the concentra tion dependent effects of a 9 min physiological estrogen treatment on dopamine efflux. E2, caused dopamine efflux at 10 14 M followed by a return to baseline, and then another peak of dopamine efflux at the higher concentrations.

E1 and E3, did not cause dopamine efflux at the tested concentrations at 9 min but at 10 13 and 10 10 M E1 significantly inhibited dopamine citation efflux. E3 also did not cause dopamine efflux, but did cause inhibition at 10 15, and 10 9 M concentra tions with no effect at other concentrations. These bimo dal concentration effects of estrogens on dopamine efflux are typical of nongenomic actions that we have described before on these and other cell types.