Rapid Uncoupling of Serotonin-1A Receptors in Rat Hippocampus by 17β-Estradiol in vitro Requires Protein Kinases A and C

Abstract

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.