Appreciating the physiology of astrocytes and their role in brain functions requires an understanding of molecules that activate these cells. Estradiol may influence astrocyte functions. We now report that estrogen altered intracellular calcium concentration ([Ca(2+)](i)) in neonatal astrocytes that expressed estrogen receptor (ER) mRNA in vitro. Western blotting revealed both ERalpha and ERbeta proteins in both the nuclear fractions and plasma-membrane fractions. Application of 17beta-estradiol (20 nm) to fura 2-loaded astrocytes in vitro stimulated [Ca(2+)](i) in 75% of astrocytes with an EC(50) of 12.7 +/- 3.1 nm. This rapid action of estradiol was blocked by the ER antagonist, ICI 182,780. The membrane-impermeable estradiol-BSA induced a [Ca(2+)](i) flux that was statistically similar to estradiol. Removal of extracellular Ca(2+) did not alter the effect of estradiol, but phospholipase C inhibitor U73122 (10 microm) and 2-aminoethoxydiphenyl borate (5 microm), an inhibitor of the inositol-1,4,5,-trisphosphate-gated intracellular Ca(2+) channel, significantly decreased the estradiol-induced [Ca(2+)](i) flux. Estradiol was unable to induce [Ca(2+)](i) flux in thapsigargin-depleted cells. These results indicate that estradiol mediates [Ca(2+)](i) flux in astrocytes through a membrane-associated ER that activates the phospholipase C pathway.

The brain synthesizes steroids de novo, especially progesterone. Recently estradiol has been shown to stimulate progesterone synthesis in the hypothalamus and enriched astrocyte cultures derived from neonatal cortex. Estradiol-induced hypothalamic progesterone has been implicated in the control of the LH surge. The present studies were undertaken to determine whether hypothalamic astrocytes derived from female neonatal or female postpubertal rats increased production of progesterone in response to an estradiol challenge. Estradiol induced progesterone synthesis in postpubertal astrocytes but not neonatal astrocytes. This estradiol action was blocked by the estrogen receptor antagonist ICI 182,780. Previously we had demonstrated that estradiol stimulates a rapid increase in free cytosolic Ca(2+) ([Ca(2+)](i)) spikes in neonatal cortical astrocytes acting through a membrane estrogen receptor. We now report that estradiol also rapidly increased [Ca(2+)](i) spikes in hypothalamic astrocytes. The membrane-impermeable estradiol-BSA construct also induced [Ca(2+)](i) spikes. Both estradiol-BSA and estradiol were blocked by ICI 182,780. Depleting intracellular Ca(2+) stores prevented the estradiol-induced increased [Ca(2+)](i) spikes, whereas removing extracellular Ca(2+) did not prevent estradiol-induced [Ca(2+)](i) spikes. Together these results indicate that estradiol acts through a membrane-associated receptor to release intracellular stores of Ca(2+). Thapsigargin, used to mimicked the intracellular release of Ca(2+) by estradiol, increased progesterone synthesis, suggesting that estradiol-induced progesterone synthesis involves increases in [Ca(2+)](i). Estradiol treatment did not change levels of steroid acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase, and sterol carrier protein-2 mRNAs as measured by quantitative RT-PCR, suggesting that in vitro, estradiol regulation of progesterone synthesis in astrocytes does not depend on transcription of new steroidogenic proteins. The present results are consistent with our hypothesis that estrogen-positive feedback regulating the LH surge involves stimulating local progesterone synthesis by hypothalamic astrocytes.

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A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.

Estradiol attenuates the ATP-induced increase of intracellular calcium concentration ([Ca2+]i) in rat dorsal root ganglion (DRG) neurons by blocking the L-type voltage gated calcium channel (VGCC). Since ATP is a putative nociceptive signal, this action may indicate a site of estradiol regulation of pain. In other neurons, 17β-estradiol (E2) has been shown to modulate L-type VGCC through a membrane estrogen receptor-group II metabotropic glutamate receptor (mGluR2/3). The present study investigated whether the rapid estradiol attenuation of ATP-induced increase [Ca2+]i requires the mGluR2/3. Previously we showed that DRG (L1-S3) express ERα, P2X3 and mGluR2/3 receptors. DRG were acutely dissociated by enzyme digestion and grown in short-term culture for imaging analysis. DRG neurons were stimulated twice, once with ATP (50 μM) for 5 seconds, and then again in the presence of E2 (100 nM) or E2 (100 nM) + LY341495 (100nM), an mGluR2/3 inhibitor. ATP induced transient increase in [Ca2+]i (216.3 ± 41.2 nM). This transient could be evoked several times in the same DRG neurons if separated by a 5 min washout. Treatment with estradiol significantly attenuated the ATP-induced [Ca2+]i in 60% of the DRG neurons, to 163.3±20.9 nM (p<0.001). Co-application of E2 and the mGluR2/3 inhibitor LY341495 blocked the 17β-estradiol-attenuation of the ATP-induced [Ca2+]i transient (209.1±32.2 nM, p>0.05). These data indicate that the rapid action of E2 in DRG neurons is dependent on the mGluR2/3, and demonstrate that membrane estrogen receptor-α initiated signaling involves tan interaction with mGluRs.

In women, pain symptoms and nociceptive thresholds vary with reproductive cycle suggesting the role of estrogen receptors (ERs) in modulating nociception. Our previous data strongly suggest interaction between ERs and ATP-induced purinergic (P2X3) as well as ERs and capsaicin-induced vanilloid (TRPV1) receptors at the level of dorsal root ganglion (DRG) neurons. In this study we investigated the expression of P2X3 and TRPV1 receptors by western blotting and immunohistochemistry in lumbosacral DRGs from wild type, estrogen receptor-α and estrogen receptor-β knockout mice. We found a significant decrease for both P2X3 and TRPV1 in ERαKO and ERβKO. This phenomenon was visualized in L1, L2, L4, and L6 levels for P2X3 receptors and in L1, L2, and S2 levels for TRPV1 receptors. This tan interaction between P2X3/TRPV1 and ERs expression in sensory neurons may represent a novel mechanism that can explain sex differences in nociception observed in clinical practice. The DRG is an important site of visceral afferent convergence and cross-sensitization and potential target for designing new anti-nociceptive therapies.

Emerging evidence support a role of purinergic P2X3 receptors in modulating nociceptive signaling in sensory neurons. Previously we showed that DRG neurons (L1-S1) express both ERα and ERβ receptors. In this study we investigated the expression of P2X3 receptors and the effect of 17β-estradiol (E2) on ATP-induced [Ca2+]i increase in DRG neurons collected from C57Bl/6J, ERαKO and ERβKO mice. Our data showed a significant decrease for P2X3 in ERαKO (all levels) and ERβKO (mostly observed in L1, L2, L4, and L6). Furthermore, 17β-estradiol (100 nM) significantly attenuated the ATP (10 μM)-induced [Ca2+]i in C57Bl/6J mice. ERs antagonist ICI 182,780 (1μM) blocked this attenuation. Homomeric P2X3 receptors are plentifully expressed in DRG neurons and contribute to nociceptive signals. α,β-me ATP which is a specific agonist of P2X2/3 receptors showed similar responses to the ATP-induced calcium increase in knock-out mice. A membrane-impermeable E-6-BSA (1μM) had the same effect as E2 suggesting action on the membrane. In DRG neurons from ERβKO and WT miceE2 attenuated the ATP/α,β-me ATP-induced [Ca2+]i fluxes but in DRG neurons from ERαKO mice, this hormone had no effect suggesting that this attenuation depends on membrane-associated ERα receptors. Together our data indicate an interaction between P2X3 and membrane-associated ERα in primary sensory neurons that may represent a novel mechanism to explain sex differences observed in clinical presentation of visceral nociceptive syndromes.