Estrogens Suppress Spinal Endomorphin 2 Release in Female Rats in Phase with the Estrous Cycle

Abstract

Background/aims: Male and female rats differ in their ability to utilize spinal endomorphin 2 (EM2; the predominant mu-opioid receptor ligand in spinal cord) and in the mechanisms that underlie spinal EM2 analgesic responsiveness. We investigated the relevance of spinal estrogen receptors (ERs) to the in vivo regulation of spinal EM2 release.

Methods: ER antagonists were administered directly to the lumbosacral spinal cord of male and female rats, intrathecal perfusate was collected, and resulting changes in EM2 release were quantified using a plate-based radioimmunoassay.

Results: Intrathecal application of an antagonist of either estrogen receptor-α (ERα) or the ER GPR30 failed to alter spinal EM2 release. Strikingly, however, the concomitant blockade of ERα and GPR30 enhanced spinal EM2 release. This effect was sexually dimorphic, being absent in males. Furthermore, the magnitude of the enhancement of spinal EM2 release in females was dependent upon estrous cycle stage, suggesting a relationship with circulating levels of 17β-estradiol. The rapid onset of enhanced EM2 release following intrathecal application of ERα/GPR30 antagonists (within 30-40 min) suggests mediation via ERs in the plasma membrane, not the nucleus. Notably, both ovarian and spinally synthesized estrogens are essential for membrane ER regulation of spinal EM2 release.

Conclusion: These findings underscore the importance of estrogens for the regulation of spinal EM2 activity and, by extension, endogenous spinal EM2 antinociception in females. Components of the spinal estrogenic mechanism(s) that suppress EM2 release could represent novel drug targets for improving utilization of endogenous spinal EM2, and thereby pain management in women.

Fig. 1

Blockade of ERα/GPR30 disinhibits spinal EM2 release in proestrous females but not males. The spinal intrathecal space was perfused at 5 µl/min using the push-pull method as described in Materials and Methods. MPP (ERα antagonist) and G15 (GPR30 antagonist) were administered via the inflow spinal cannula. The EM2 content of perfusate was quantified using a competitive radioimmunoassay. Vertical bars represent spinal EM2 release at three time points: baseline, 10–20 min after drug treatment, and 30–40 min after drug treatment. Data are expressed as fmol EM2 per 10-min period. * p < 0.01 for EM2 release in proestrous females before vs. 30–40 min after intrathecal administration of MPP and G15 (n = 4–5).

Fig. 2

The magnitude of change in EM2 release following spinal ERα/GPR30 blockade is stage of estrous cycle dependent. One-way ANOVA showed a main effect of estrous stage on change in spinal EM2 release (F_{2, 10} = 14.73; p = 0.001). Post hoc tests revealed that the magnitude of enhanced EM2 release was significantly greater in proestrus vs. diestrus or estrus (* p < 0.05 for both comparisons). Data are expressed as fmol EM2 above basal release 30–40 min after intrathecal application of ERα/GPR30 antagonists (n = 4–5).

Fig. 3

ERα and GPR30 co-immunoprecipitate. Lane A: ERα immunoprecipitate contains GPR30 immunoreactivity. The specificity of the ≈50-kDa GPR30 Western signal is shown in lane B by the >80% reduction in signal intensity when the same immunoprecipitated sample is Western blotted using preadsorbed anti-GPR30 antibody. Data shown for one of three replications.

Fig. 4

Disinhibition of EM2 release following intrathecal MPP (ERα antagonist) and G15 (GPR30 antagonist) is abolished by intrathecal pretreatment (1 h) with the aromatase inhibitor fadrozole (FAD) in proestrous females, or by bilateral ovariectomy (OVX). Vertical bars represent spinal EM2 release (fmol per 10 min) at three time points: baseline, 10–20 min after drug treatment (ERα/GPR30 blockade), and 30–40 min after drug treatment. * p < 0.01 for EM2 release in proestrous females before vs. 30–40 min after intrathecal administration of MPP and G15 (n = 4–6).