Evidence That A mEr Mediates Estradiolevoked Dopamine Release From Female Rat Striatal Tissue

Rapid effects of estradiol on all three major dopamine (DA) systems—nigrostriatal, mesolimbic, and tuberoinfundibular—have been reported. For example, an early study (14) showed that relatively high concentrations of E and a synthetic estrogen, diethylstil-bestrol (DES) produced a concentration-dependent (0.1-20 mM) release of DA and nore-pinephrine (NE) from in vitro hypothalamic preparations, whereas its biologically inactive enantiomer, 17a-estradiol, was ineffective. The effect was observed following a delay of approximately 20-40 min for DA, suggesting mediation of a possible membrane mechanism. In the nigrostriatal DA system, in which the corpus striatum (CS) is the site of major dopaminergic innervation, a variety of behavioral and functional indices are dependent on the gonadal status of the animal, particularly upon E levels. For instance, E has been reported to influence rapidly striatal DA release, DA receptor concentration, and behavior mediated by the striatum (15-17). Becker (18,19) showed that physiological concentrations (0.22-3.7 nM) of E or DES rapidly (within 20 min) potentiated, the amphetamine-stimulated striatal DA release from striatal tissue of ovariectomized (ovx) rats as measured by microdialysis. This rapid E effect was correlated to rapid effects of E on rat rotational behavior. 17a-estradiol (17a-E) had much less effect, indicating a stereospecific effect of E. This rapid effect of E is sexually dimorphic because it was absent in the striatal tissue of intact male rats (20). A subsequent study also revealed that

Fig. 1. Structure of novel ligands to study steroid membrane actions. Crystallographically observed structure of the estradiol molecule in a perpendicular view to the phenolic ring; modified from Keasling and Schneler (33). For details see text.

E acutely inhibits (within 30 min) the striatal D2-DA receptor binding (21), which might be owing to a rapid conversion of high to low affinity D2-DA receptors, as shown by Levesque and DiPaolo (22). Recently, a rapid stimulation of striatal dopamine synthesis by E but not by 17a-E, was reported in ovx rats in vivo within 15 min of physiological doses of the steroid injected subcutaneously (23,24). In addition, incubation of striatal slices in vitro with 1 nM E (but not 17a-E) evoked a twofold increase in the Ki of one form of the tyrosine hydroxylase (TH) enzyme for DA, suggesting a decrease in TH susceptibility to end-product inhibition, presumably owing to phosphorylation of the enzyme (23,24). However, all these findings were obtained using free E, which can diffuse inside the cells; therefore, the results may not be owing to a membrane-mediated event. In the mesolimbic system, a nongenomic effect of E on DA release from the nucleus accumbens studied by in vivo microdialysis was also observed (25). E hemisuccinate infusion resulted in an initial increase in K+-simulated DA release within 2 min, followed by a later increase at about 1 h after the infusion of the steroid, which, according to the authors, was probably secondary to a genomic stimulation.

Thus, to differentiate between a certain outer-membrane effect of E and one in which the diffusion of this liposoluble steroid can confound those initial membrane events, we decided to study the acute release of DA by estradiol, using the impermeable E-6-BSA conjugate (Fig. 1). To address this issue we utilized an in vitro superfusion system to perifuse CS fragments derived from female rats in specific phases of the estrous cycle, as determined by daily vaginal smears. For technical details, see our previous publications (26,27). Figure 2 shows that E-6-BSA rapidly stimulates (within the first 10 min of infusion at interval #4) basal DA release from CS fragments derived from proestrous rats killed in the morning (9 am). Noteworthy, the isomer of E-6-BSA, the 17a-E-6-BSA conjugate (Fig. 1), did not stimulate the basal DA release using an identical concentration of 10 nM in parallel experiments. The fact that BSA does not stimulate the release of DA

Fig. 2. E-6-BSA stimulates dopamine release from CS fragments from Proestrous rats. Proestrous rats were sacrificed at 900 h and the CS fragments were distributed in four chambers. Two chambers were stimulated with E-6-BSA and two chambers with 17a-E-6-BSA. The experiment was repeated four times; however, in the fourth repetition, two chambers received BSA instead of 17a-E-6-BSA. The data indicate no effect of BSA (31-66; 42-53; 50-18; 25-20; 10-22; 6-24; 14-24, 10-15 pg/mg/min for intervals 1-8 in the two chambers, respectively) and a robust response to E-6-BSA during proestrus. In all figures, values are X ± SE. The stars indicate significant differences between values at intervals 3 and 5. The arrow indicates the time when the infusion of the hormones occurred (starting after interval 3 and ending at interval 4). The same applies to Figs. 3 and 4.

Fig. 2. E-6-BSA stimulates dopamine release from CS fragments from Proestrous rats. Proestrous rats were sacrificed at 900 h and the CS fragments were distributed in four chambers. Two chambers were stimulated with E-6-BSA and two chambers with 17a-E-6-BSA. The experiment was repeated four times; however, in the fourth repetition, two chambers received BSA instead of 17a-E-6-BSA. The data indicate no effect of BSA (31-66; 42-53; 50-18; 25-20; 10-22; 6-24; 14-24, 10-15 pg/mg/min for intervals 1-8 in the two chambers, respectively) and a robust response to E-6-BSA during proestrus. In all figures, values are X ± SE. The stars indicate significant differences between values at intervals 3 and 5. The arrow indicates the time when the infusion of the hormones occurred (starting after interval 3 and ending at interval 4). The same applies to Figs. 3 and 4.

(see Fig. 2 legend), together with the lack of effect of 17a-E-6-BSA, indicates that the stimulatory action of the complex is owing to estradiol and not BSA. Interestingly, CS fragments from diestrous and estrous rats are not or less responsive to E-6-BSA, respectively, as shown in Table 1.

In ovx rats (more than 14 d), an optimal effective concentration of the complex (10 nM) evoked a small but not significant increase in DA release (Fig. 3) in spite of a robust response of the preparation to a depolarizing dose of K+ (30 mM). To test if the reduction of E levels causes the decrease in responsiveness of the CS fragments to the complex, ovx rats were treated with estradiol benzoate for 2 d (0.5 ^g on d 1, increased to 1 ^g on d 2) and sacrificed on d 3 (at 9 am). This treatment partially recovers the response of the CS to E-6-BSA (Fig. 4), suggesting a crucial role for E in the function of its own membrane receptor in CS neural membranes; this was probably owing to a genomic activation of the neurons leading to the production of specific transcripts involved in the synthesis of mERs.

The rapid increase in basal DA release by E-6-BSA from striatal fragments derived from proestrous rats is dose-dependent, with a maximal effective concentration of10 nM, which induces close to a sixfold increase in DA release over control pretreatment values (Fig. 5). The ED50 is about 5 nM and a minimal but significant response was observed with

Table 1

Differential Response to E-6-BSA-Evoked DA release from CS Fragments Superfused In Vitro

Table 1

Differential Response to E-6-BSA-Evoked DA release from CS Fragments Superfused In Vitro

Condition

Dose (nM)

Number of Chambersa

Percent Response

1. Diestrus

Was this article helpful?

0 0

Post a comment