17beta-Estradiol decreases R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS 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 17beta-estradiol in HEPES buffer (37 degrees 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 [(35)S]GTPgammaS binding. 17beta-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 17beta-estradiol to increase PKA and PKC was blocked by the estrogen receptor antagonist ICI 182,780 (1 microM). A selective PKA inhibitor (KT 5720, 60 nM) blocked 17beta-estradiol-stimulated PKA but NOT PKC activity. Conversely, the PKC inhibitor calphostin C (100 nM) blocked the increase in PKC activity produced by 17beta-estradiol but NOT the PKA response. The protein kinase inhibitors individually blocked the effects of 17beta-estradiol on R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding. By contrast, preincubation with the protein synthesis inhibitor cycloheximide (200 microM) or the mitogen activated protein (MAP) kinase kinase inhibitor PD 98059 (50 microM) was without effect. Incubation of hippocampus with 17beta-estradiol (50 nM, 60 min) caused the phosphorylation of a protein consistent with the 5-HT(1A) receptor. These studies demonstrate that 17beta-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.
Copyright 2002 S. Karger AG, Basel