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Gonadal steroids. Serotonin Serotonin receptors Serotonin agonists Protein kinases Hippocampus
Abstract
17 beta-Estradiol decreases R(+)8-OH-DPAT-stimulated [ssS]GTP(gamma)S binding [an index of serotonin-lA (5-HT^sub 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^sub 1A^ receptors. Hippocampus from ovariectomized rats was incubated with 170-estradiol in HEPES buffer (37deg 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]GTP(gamma)S binding. 17 beta-Estradiol (50 nM) increased PKA and PKC activities approximately 2- to 3fold. PKC activity was elevated at 10, 30 and 60 min, whereas PKA activity was increased at 10 and 30 min. The ability of 170 beta-estradiol to increase PKA and PKC was blocked by the estrogen receptor antagonist ICI 182,780 (1 (mu)M). A selective PKA inhibitor (KT 5720, 60 nM) blocked 17 beta-estradiol-stimulated PKA but not PKC activity. Conversely, the PKC inhibitor calphostin C (100 nM
blocked the increase in PKC activity produced by 17 betaestradiol but not the PKA response. The protein kinase inhibitors individually blocked the effects of 170 beta-estradiol on R(+)8-OH-DPAT-stimulated [35S]GTP(gamma)S binding. By contrast, preincubation with the protein synthesis inhibitor cycloheximide (200 (mu)M) or the mitogen activated protein (MAP) kinase kinase inhibitor PD 98059 (50 (mu)M) was without effect. Incubation of hippocampus with 17 beta-estradiol (50 nM, 60 min) caused the phosphorylation of a protein consistent with the 5-HT ^sub 1A receptor. These studies demonstrate that 17 beta-estradiol acts on estrogen receptors locally within the hippocampus through nongenomic mechanisms to activate PKA and PKC, phosphorylate 5-HT^sub 1A^ receptors and uncouple them from their G proteins.
Introduction
A large body of evidence exists documenting the rapid effects of estrogen on neuronal functions [ 1-3]. These responses occur at rates which are not in accordance with classical genomic mechanisms of hormone action and/or are not dependent on protein synthesis. As examples, 17 betaestradiol rapidly (1) increases extracellular signal-regulated kinase phosphorylation in cortical explants [4] and (2) desensitizes t-opioid and GABA^sub Beta^ receptors in the guinea pig hypothalamus [5-7]. Of particular interest to our laboratory...