It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non-classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) a and/or b to mediate such responses is lacking, the hippocampal-derived cell line HT22 was stably transfected with either ERa (HTERa) or ERb (HTERb). In HTERa and HTERb cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17b-estradiol treatment. The ER antagonist ICI 182, 780 (1 lM) and the MEK inhibitor, PD98059 (50 lM) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERa and HTERb cells with the b-amyloid peptide (25-35) (10 lM) resulted in a significant decrease in cell viability. Pre-treatment for 15 min with 10 nM 17b-estradiol resulted in a 50% increase in the number of living cells in HTERa and HTERb cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17b-estradiol-mediated protection. This study demonstrates that both ERa and ERb can couple to rapid signaling events that mediate estrogen-elicited neuroprotection.

It is well documented that estrogen mediates responses by both genomic and nongenomic mechanisms, both of which are important for cell survival. Because direct evidence showing that the estrogen receptors (ERs) alpha and/or beta can activate rapid signaling that may mediate neuroprotection is lacking, the hippocampal-derived cell line, HT22, was stably transfected with ERalpha (HTERalpha), ERbeta (HTERbeta), or a mutated form of ERalpha (HTERalphaHE27), which lacks the ability to mediate ER element-mediated transcription. Treatment of HT22, HTERalpha, HTERbeta, and HTERalphaHE27 cells with glutamate (5 mM) resulted in a significant decrease in cell viability. Pretreatment for 15 min with 10 nM 17beta-estradiol resulted in a 50% increase in the number of living cells in HTERalpha and HTERbeta cells but not in HT22 cells. The ER antagonist ICI 182,780 and the MEK inhibitor PD98059 prevented 17beta-estradiol-mediated protection. In HTERalphaHE27 cells, 17beta-estradiol rapidly phosphorylated ERK2 (within 15 min), in the absence of estrogen response element-mediated transcription. Treatment of HTERalphaHE27 cells with 10 nM 17beta-estradiol partially reversed the cell death produced by glutamate treatment. This study demonstrates that activation of either ERalpha or ERbeta can result in neuroprotection and that activation of the MAPK pathway is an important part of the neuroprotective mechanism.

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Previously our laboratory has shown that 17β-estradiol in vivo rapidly decreases R(+)-8-OH-DPAT-stimulated [³⁵S]GTPγS binding (a measure of the initial biochemical event in the intracellular signaling pathway associated with 5-HT_1A receptors) in the hippocampus, frontal cortex and amygdala. Studies were designed to determine if 17β-estradiol also acts in vitro on estrogen receptors in the hippocampus and frontal cortex to decrease 5-HT_1A receptor function. Hippocampus and frontal cortex were dissected from ovariectomized rats and incubated for up to 3 h with various estrogens and antiestrogens; membrane homogenates were prepared for R(+)-8-OH-DPAT-stimulated [³⁵S]GTPγS binding assays. 17β-Estradiol (10^–6 M) decreased the maximal response in the R(+)-8-OH-DPAT-stimulated [³⁵S]GTPγS binding assay in a time-dependent manner (observed at 30, 60 and 120 min) in both hippocampus and frontal cortex. The hormone, however, did not alter the EC₅₀ of R(+)-8-OH-DPAT. When hippocampus and frontal cortex were incubated in graded concentrations of 17β-estradiol for 1 h, the calculated EC₅₀ was approximately 2.5 × 10^–8 M in both brain regions. The nonestradiol estrogen diethylstilbestrol also decreased 5-HT_1A receptor function while the less potent estrogens 17α-estradiol and estriol were inactive at 5 × 10^–8 M. The estrogen receptor antagonist ICI 182,780 potently and completely blocked the effects of 17β-estradiol on 5-HT_1A receptor function with an apparent K_B of approximately 10^–9 M. These data demonstrate clearly that estrogens can act on estrogen receptors located in hippocampus and frontal cortex of ovariectomized rats to produce rapid heterologous decreases in 5-HT_1A receptor function.

17β-Estradiol decreases R(+)8-OH-DPAT-stimulated [³⁵S]GTPγS binding [an index of serotonin-1A (5-HT_1A) receptor coupling] through the activation of estrogen receptors. We hypothesize that this occurs as a result of activation of protein kinase A (PKA) and/or protein kinase C (PKC) and phosphorylation of 5-HT_1A receptors. Hippocampus from ovariectomized rats was incubated with 17β-estradiol in HEPES buffer (37°C). Cytosolic and membrane fractions were prepared to assess PKA and PKC activities, respectively. In separate experiments, membranes were prepared to measure R(+)8-OH-DPAT-stimulated [³⁵S]GTPγS binding. 17β-Estradiol (50 nM) increased PKA and PKC activities approximately 2- to 3-fold. PKC activity was elevated at 10, 30 and 60 min, whereas PKA activity was increased at 10 and 30 min. The ability of 17β-estradiol to increase PKA and PKC was blocked by the estrogen receptor antagonist ICI 182,780 (1 µM). A selective PKA inhibitor (KT 5720, 60 nM) blocked 17β-estradiol-stimulated PKA but not PKC activity. Conversely, the PKC inhibitor calphostin C (100 nM) blocked the increase in PKC activity produced by 17β-estradiol but not the PKA response. The protein kinase inhibitors individually blocked the effects of 17β-estradiol on R(+)8-OH-DPAT-stimulated [³⁵S]GTPγS binding. By contrast, preincubation with the protein synthesis inhibitor cycloheximide (200 µM) or the mitogen activated protein (MAP) kinase kinase inhibitor PD 98059 (50 µM) was without effect. Incubation of hippocampus with 17β-estradiol (50 nM, 60 min) caused the phosphorylation of a protein consistent with the 5-HT_1A receptor. These studies demonstrate that 17β-estradiol acts on estrogen receptors locally within the hippocampus through nongenomic mechanisms to activate PKA and PKC, phosphorylate 5-HT_1A receptors and uncouple them from their G proteins.