Biomedical subjects
Publications and source records attributed to A M Etgen.
Treatment of ovariectomized female rats with estrogen for 2 days reduces alpha2-adrenoceptor binding density by 25%, increases G protein coupled receptor kinase (GRK) activity by 50% and elevates GRK 2 protein levels by 50% in the frontal cortex. These results suggest that estrogen may decrease alpha2-adrenoceptor expression in the frontal cortex of female rats by regulating GRK 2.
In many mammalian species, the ovarian steroid hormones estradiol (E(2)) and progesterone (P) act in the hypothalamus and preoptic area to coordinate the timing of female sexual receptivity with ovulation. We study lordosis behavior, an important component of sexual receptivity in rats, and its regulation by E(2) and P as a model system for understanding how hormonal modulation of synaptic neurotransmission influences reproductive physiology and behavior. Our findings suggest that E(2) and P extensively regulate synaptic communication involving the catecholamine norepinephrine (NE) in the hypothalamus. Estrogen priming shifts the balance of postsynaptic NE receptor signaling in the hypothalamus and preoptic area away from beta-adrenergic activation of cAMP synthesis toward alpha(1)-adrenergic signaling pathways. Attenuation of beta-adrenergic signal transduction is achieved by receptor-G-protein uncoupling, apparently due to stable receptor phosphorylation. E(2) modification of alpha(1)-adrenergic signaling includes both increased expression of the alpha(1B)-adrenoceptor subtype and a dramatic, P-induced reconfiguration of the biochemical responses initiated by agonist activation of alpha(1)-adrenoceptors. Among these is the emergence of alpha(1)-adrenergic receptor coupling to cGMP synthesis. We also present evidence that estrogen promotes novel, functional interactions between insulin-like growth factor-1 (IGF-1) and alpha(1)-adrenergic receptor signaling in the hypothalamus and preoptic area. Thus, estrogen amplification of signaling mediated by alpha(1)-adrenoceptors is multifaceted, involving changes in gene expression (of the alpha(1B)-adrenoceptor), switching of receptor linkage to previously inactive intracellular pathways, and the promotion of cross talk between IGF-1 and NE receptors. We propose that this hormone-dependent remodeling of hypothalamic responses to NE maximizes reproductive success by coordinating the timing of the preovulatory release of gonadotropins with the period of behavioral receptivity in female rodents.
Previous studies showed that repeated estrogen treatment reduces the ability of the 5-HT(1A) receptor agonist, 8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT), to inhibit lordosis behavior of female rats. The present study evaluated the effects of repeated estrogen treatment on lordosis behavior and 5-HT(1A) receptor binding and coupling to G protein in the hypothalamus-preoptic area using the agonist ligand [3H]-8-OH-DPAT, which binds selectively to G-protein-coupled 5-HT(1A) receptors. Rats were injected twice with 25 or 50 microg of estradiol benzoate (EB) 7 days apart followed by 500 microg of progesterone (P) 48 h after the second EB injection. Controls received a single injection of 25 or 50 microg EB followed 48 h later by 500 microg of P. Four hours after P, 0.15 mg/kg 8-OH-DPAT was injected, and lordosis behavior examined for 30 min. Rats treated twice with EB showed significantly less 8-OH-DPAT inhibition of lordosis behavior than rats receiving a single EB injection. For receptor binding, rats received EB without P treatment. None of the estrogen treatments reduced [3H]-8-OH-DPAT binding density or affinity in the hypothalamus-preoptic area or hippocampus. These studies suggest that estrogen modulates 5-HT(1A) agonist potency without a measurable change in 5-HT(1A) receptor density or coupling to G protein.
GABAA receptor activation augments electrically-stimulated release of norepinephrine (NE) from rat brain slices. Because this effect is not observed in synaptoneurosomes, GABA probably acts on inhibitory interneurons to disinhibit NE release. To determine whether opioids or adenosine influence GABA-augmented NE release, hypothalamic and cortical slices from female rats were superfused with GABA or vehicle in the presence and absence of 10 microM morphine or 100 microM adenosine. GABA augments [3H]NE release in the cortex and hypothalamus. Morphine alone has no effect on [3H]NE release, but attenuates GABA augmentation of [3H]NE release in both brain regions. Adenosine alone modestly inhibits [3H]NE release in the cortex, but not in the hypothalamus. Adenosine inhibits GABA-augmented [3H]NE release in both brain regions. The general protein kinase inhibitor H-7, augments [3H]NE release in both brain regions and may have additive effects with GABA in cortical slices. These results implicate opioid and adenosine interneurons and possibly protein kinases in regulating GABAergic influences on NE transmission.
Recently, we demonstrated that estradiol (E(2)) modulates cross-talk between protein tyrosine kinases and norepinephrine (NE) receptor signaling in the hypothalamus (HYP) and preoptic area (POA), brain areas that govern female reproductive function. We are now investigating the identity of protein tyrosine kinase(s) that modify NE receptor signaling in the HYP and POA. Incubation of POA and HYP slices with insulin-like growth factor I (IGF-I), which signals via a receptor (IGF-IR) with endogenous tyrosine kinase activity, enhances NE-stimulated cAMP accumulation only in tissue derived from ovariectomized, E(2)-primed animals. JB-1, an antagonist for IGF-IR, prevents the IGF-I enhancement of NE-stimulated cAMP accumulation in both POA and HYP slices. IGF-I enhances NE-stimulated cAMP accumulation via modulation of alpha(1)-adrenoceptor potentiation of adenylyl cyclase. Binding studies in membranes demonstrate that ovariectomized, E(2)-primed animals show a significant increase in the density of [(125)I]IGF-I-binding sites in both POA and HYP compared with ovariectomized control animals. Neither the IC(50) for [(125)I]IGF-I displacement by IGF-I nor the levels of IGF-I binding proteins in serum or brain tissue are affected by E(2). RIA results showed that E(2) does not modify serum or brain IGF-I levels. These results indicate that E(2) regulation of NE receptor function in the POA and HYP involves increased expression of IGF-IR, and that after E(2) treatment, IGF-IR activation augments alpha(1)-adrenoceptor signaling.
Estrogen increases evoked norepinephrine release in the hypothalamus of female rodents, in part by reducing the ability of alpha2-adrenoceptors to act as negative feed-back inhibitors of norepinephrine release. Estrogen enhancement of norepinephrine release in the hypothalamus correlates with decreased coupling of the alpha2-adrenoceptor to G protein. To determine the mechanism by which estrogen uncouples alpha2-adrenoceptors from G protein, we tested the hypothesis that estrogen increases alpha2-adrenoceptor phosphorylation. Short-term activation of endogenous serine/threonine phosphatases with protamine or treatment with exogenous phosphatase restored alpha2-adrenoceptor coupling to G protein to control levels in hypothalami from estrogen-exposed female rats. Additional experiments examined whether estrogen alters G protein-coupled receptor kinase expression or activity or serine/threonine phosphatase activity. These proteins are involved in G protein-coupled receptor phosphorylation, internalization, and recycling. Estrogen exposure reduced G protein-coupled receptor kinase mRNA, protein, and activity in the hypothalamus. Furthermore, estrogen treatment reduced serine/threonine phosphatase activity in the hypothalamus. Analysis of ligand binding in subcellular fractions demonstrated that estrogen decreases the fraction of internalized alpha2-adrenoceptors in the hypothalamus.Therefore, estrogen promotes norepinephrine release in the hypothalamus by stabilizing alpha2-adrenoceptor phosphorylation, uncoupling the receptor from G protein. Estrogen may stabilize alpha2-adrenoceptor phosphorylation by inhibiting receptor internalization and dephosphorylation.
These studies examined the functional interactions between adrenergic G-protein coupled receptors and protein tyrosine kinases in the preoptic area and hypothalamus, brain regions that regulate reproductive function in female rats, and evaluated whether in vivo treatment with estradiol for 2 days modulates the cross-talk between these two signaling pathways. In hypothalamic slices genistein, a general tyrosine kinase inhibitor, enhances norepinephrine-stimulated cAMP synthesis independent of estradiol treatment. Genistein appears to act by increasing beta-adrenoceptor signaling. At high norepinephrine concentrations, estradiol potentiates genistein enhancement of the cAMP response in hypothalamic slices. This interaction between estradiol and genistein appears to involve modification of alpha(2)-adrenoceptor signaling mechanisms. In preoptic area slices, genistein enhancement of norepinephrine-stimulated cAMP synthesis is only observed in estradiol-treated rats. In this brain region, genistein enhances cAMP accumulation by modifying alpha(1)- and/or alpha(2)-adrenoceptor rather than beta-adrenoceptor signaling. Genistein amplification of norepinephrine-stimulated cAMP synthesis is not mediated by interactions with estrogen receptors, or by regulation of adenylyl cyclase or phosphodiesterase activities. At the concentration used, genistein inhibits tyrosine phosphorylation in slices from both brain regions. Daidzein, an inactive analogue of genistein, fails to enhance the norepinephrine-stimulated cAMP response in either brain region independent of hormone treatment. These results suggest that protein tyrosine kinases regulate adrenergic responses in the hypothalamus and preoptic area. Moreover, the functional interaction between adrenergic G-protein coupled receptor signaling and protein tyrosine kinases is modified in a brain region and receptor subtype specific manner by estradiol.
Activation of beta-adrenoceptors in the hypothalamus (HYP) and preoptic area (POA) inhibits both gonadotropin release and reproductive behaviour in female rats. Exposure of female rats for 48 h to physiologically relevant doses of oestrogen attenuates beta-adrenoceptor function in the HYP and POA as indicated by reduced isoproterenol (beta-adrenoceptor agonist) stimulation of adenylyl cyclase activity. Reduced beta-adrenoceptor coupling to G protein in the HYP-POA from oestrogen-exposed female rats correlates with attenuation of beta-adrenoceptor function. To examine potential mechanisms underlying receptor-G protein uncoupling, initial experiments tested the hypothesis that oestrogen attenuation of beta-adrenoceptor function in the HYP and POA involves receptor phosphorylation. Activation of endogenous serine/threonine phosphatases with protamine restores agonist-stimulated cAMP accumulation in HYP slices from oestrogen-exposed female rats to control levels. Additional experiments examined whether oestrogen-induced changes in beta-adrenoceptor binding density and/or subcellular localization correlate with the attenuation of beta-adrenoceptor function in the HYP and POA. Oestrogen treatment does not alter total beta-adrenoceptor binding density in the HYP or POA. However, oestrogen significantly reduces cell surface binding of the hydrophilic beta-adrenoceptor antagonist [3H] CGP 12177 to intact HYP and POA slices. At the same time, oestrogen decreases the fraction of beta-adrenoceptors localized in a light vesicle fraction following sucrose density gradient centrifugation. Therefore, oestrogen attenuates beta-adrenoceptor signalling in the HYP-POA by uncoupling the beta-adrenoceptor from G protein, perhaps by promoting receptor phosphorylation. Furthermore, a significant fraction of beta-adrenoceptors in the HYP and POA are no longer accessible to hydrophilic ligands, but are not internalized. Thus, physiological doses of oestrogen may facilitate reproductive behaviour and gonadotropin release, in part, by stabilizing beta-adrenoceptor phosphorylation in the HYP and POA, thereby uncoupling the receptors from G protein.
This review focuses on findings from our laboratory regarding mechanisms by which the ovarian steroid hormones, estradiol (E2) and progesterone (P), act in the hypothalamus (HYP) to regulate the expression of lordosis, an important component of female reproductive behavior in rats and many other species. The first section summarizes recent work suggesting that cGMP, perhaps via P-receptor activation, may be an intracellular mediator of the facilitatory actions of a variety of hormones and neurotransmitters on lordosis behavior in E2-primed rats. In the second section, we focus on E2 and P regulation of norepinephrine (NE) neurotransmission in the HYP. We review evidence that ovarian hormones act both peripherally and centrally to determine whether NE is released in the HYP in response to copulatory stimuli. We also suggest that the steroid milieu determines the cellular responses of hypothalamic neurons to released NE, favoring the activation of pathways implicated in the facilitation of both lordosis behavior and the preovulatory gonadotropin surge. It is likely that E2 and P have similar actions on other neurotransmitter and neuromodulator systems, thereby maximizing the probability that females are sexually receptive during the periovulatory period.
Gonadal steroid hormones can markedly affect seizure susceptibility. Ovariohysterectomized female rats given ovarian steroid hormone supplements were used to evaluate the effects of ovarian steroids on epileptiform activity in hippocampal slices in vitro and on flurothyl-induced seizures in vivo. Seizure susceptibility was compared in the entorhinal cortex (EC) and CA1 regions of the hippocampus perfused with Mg(2+)-free medium, which leads to epileptiform discharges caused by a relief of voltage-dependent NMDA receptor block. After in vivo treatment with 500 microg of progesterone for 2 h prior to slice preparation, the latency to onset of low Mg(2+)-induced epileptiform activity of slices was significantly prolonged compared to slices from controls. In contrast, progesterone replacement accelerated the development of epileptiform activity in the CA1 region. Neither estrogen alone (2 x 2 microg of estradiol benzoate, 48 and 24 h prior to the experiment), nor a combined treatment with estrogen plus progesterone, significantly affected seizure susceptibility in either CA1 or the EC. There were no consistent effects of estrogen or progesterone, alone or in combination, on flurothyl-induced seizures in vivo. The data suggest that in vitro, progesterone alters seizure susceptibility in a site- and seizure model-specific fashion. The differential effects of progesterone may be due to differential expression of progesterone receptor isoforms or metabolites in specific brain areas suggesting that selective modulation of NMDA receptor-dependent epileptiform activity may play a role in hormonal effects on epileptogenesis.
The ovarian hormones estradiol (E(2)) and progesterone (P) facilitate rat lordosis behavior in part by regulating the expression of and signal transduction by adrenoceptors in the hypothalamus (HYP) and preoptic area (POA). The major adrenoceptor subtype mediating E(2) and P facilitation of lordosis is the alpha(1)-adrenoceptor. In the present studies, we tested the hypotheses that (1) alpha(1)-adrenoceptors in the HYP enhance lordosis responses by activating the nitric oxide (NO)-cGMP signaling pathway, and (2) coupling of alpha(1)-adrenoceptors to this signal transduction pathway is hormone-dependent. Basal levels of cGMP were significantly higher in HYP and POA slices from animals treated with E(2) and P when compared with slices from ovariectomized controls or females treated with only E(2) or P. When slices of HYP and POA from ovariectomized female rats were incubated with norepinephrine or the selective alpha(1)-adrenoceptor agonist phenylephrine, cGMP accumulation was observed only if slices had been derived from females treated with both E(2) and P before experimentation. Moreover, alpha(1)-adrenoceptor stimulation of cGMP synthesis was blocked by an inhibitor of NO synthase, confirming that these receptors act by NO-mediated stimulation of soluble guanylyl cyclase. Behavioral studies demonstrated further that the cell-permeable cGMP analog 8-bromoadenosine-cGMP reverses the inhibitory effects of the alpha(1)-adrenoceptor antagonist prazosin on lordosis behavior in E(2)- and P-treated female rats. Thus, the NO-cGMP pathway mediates the facilitatory effects of alpha(1)-adrenoceptors on lordosis behavior in female rats, and previous exposure of the HYP and POA to both E(2) and P are required to link alpha(1)-adrenoceptors to this pathway.
Activation of alpha1-adrenergic receptors has been linked to the control of blood pressure, neuroendocrine secretion, reproductive behavior and mood. The present study describes the distribution of alpha1B-adrenergic receptor immunoreactivity in female rat brain regions involved in stress and neuroendocrine function. The pattern of immunolabeling seen resembles that obtained in previous in situ hybridization studies. Several hypothalamic areas that control pituitary function showed intense fiber and/or cell immunolabeling, including the paraventricular nucleus of the hypothalamus, the supraoptic nucleus, and the median eminence. Some regions such as the arcuate nucleus, the median eminence, and dorsal hypothalamus exhibit intense labeling of axonal varicosities, while other regions exhibit only perikarya immunolabeling. alpha1B-adrenergic receptor immunoreactivity was also observed in large pyramidal neurons of layer V of the cerebral cortex, the frontal cortex showing a particularly strong immunoreactivity. Virtually all thalamic regions were labeled, especially the lateral and ventral areas. In addition, labeled cells were present in hippocampus, the medial septum, the horizontal and vertical limbs of the diagonal band of Broca, and the caudate putamen. Finally, some midbrain and hindbrain regions important for motor function were immunoreactive. Because ligands specific for alpha1-adrenergic receptor subtypes are not available, the present immunocytochemical study not only addresses the subcellular and regional distribution of alpha1B-adrenergic receptors but may also provide clues about receptor subtype-specific function.
The purpose of this study was to test the hypothesis that cGMP acts as a progesterone substitute to facilitate lordosis in oestrogen-primed rats. Female Sprague-Dawley rats underwent stereotaxic surgery to place a 26-gauge guide cannula into the third ventricle. Bilateral ovariectomy was done at the same time as stereotaxic surgery. Five days later ovariectomized rats were primed with 2 microg estradiol benzoate 24 and 48 h prior to behaviour testing. Some animals were further injected with 200 microg progesterone 4 h before behaviour testing. A nitric oxide synthase inhibitor infused into the third ventricle before progesterone administration significantly reduced lordosis performance. 8-Bromo-cGMP, a cell permeable cGMP analogue, or saline vehicle was infused into the third ventricle of hormone-primed animals approximately 4 h prior to the first of 3-h behaviour tests. This cGMP analogue facilitated lordosis behaviour. We next used KT5823, a highly specific inhibitor of protein kinase G (PKG), to test the hypothesis that cGMP action is mediated by this kinase. In this experiment, KT5823 was infused 15 min before progesterone. KT5823 significantly decreased lordosis behaviour. RU486, a progesterone receptor antagonist, was used to assess whether the stimulatory effects of cGMP are mediated through the progesterone receptor. Oestrogen-primed animals were injected with 5 mg of RU486 or vehicle 60 min before infusion with 8-bromo-cGMP. RU486 significantly attenuated cGMP-facilitated lordosis behaviour. These data show that cGMP facilitates lordosis through activation of PKG and the progesterone receptor.
GABAA receptor activation augments stimulated release of 3H-norepinephrine (NE) in brain slices from female rats. This effect is not blocked by acetazolamide or MK-801, indicating that permeability of the GABAA chloride channel to bicarbonate ions and NMDA receptor activation do not mediate GABA-induced NE release. Furthermore, GABA augments 3H-NE release from slices, but not from isolated nerve terminals (synaptosomes), indicating that interneurons mediate GABA effects on 3H-NE release.
These studies determined whether diabetes and estradiol treatment altered norepinephrine (NE) release from hypothalamus, preoptic area (POA), and cortical slices from ovariectomized (OVX) female rats. Animals were sacrificed 12 days after the onset of streptozotocin-induced diabetes and 48 h following vehicle or estradiol injection. Brain slices were preloaded with 3H-NE, and release was evoked twice (S and S2) by electrical stimulation. Diabetes increased hypothalamic NE release during S1 regardless of the administration of vehicle or estradiol. Neither estradiol treatment nor diabetes alone affected NE release during S2 in the hypothalamus or POA. Estradiol treatment elevated NE release in the POA during S2 but only in diabetic animals. Moreover, estradiol elevated cortical NE release during S2 regardless of the presence or absence of disease. We also examined whether alpha2-adrenoceptor regulation of NE release was influenced by diabetes or hormone treatment. Enhancement of NE release by alpha2-adrenoceptor antagonism was evident in all 3 brain regions. However, alpha2-adrenoceptor regulation of NE release was unaffected by diabetes and hormone treatment. These findings suggest that diabetes alters NE release in the hypothalamus/POA of female rats. Additionally, this work identifies a novel action of estradiol to enhance stimulated NE release in the cortex of female rats.
Estrogen acts in the brain to regulate female reproductive physiology and behavior, and protein kinase C (PKC) is estrogen-regulated in many estrogen-responsive tissues. We examined whether estrogen regulates PKC in the hypothalamus (HYP) and preoptic area (POA), brain regions which mediate estrogenic control of female reproductive function. PKC activity in tissue from hormone-treated and control female rats was measured, in the presence of phorbol ester and calcium, by quantifying 32p incorporation into a substrate peptide. PKC catalytic activity increased significantly in POA tissue extracts from estradiol-treated, ovariectomized (OVX) female rats but not in HYP or cortical extracts. Phorbol ester potentiation of cAMP accumulation also was examined to determine whether the ability of PKC to potentiate adenylyl cyclase activity was affected by estrogen. PKC stimulation potentiated forskolin-induced cAMP accumulation to a greater degree in POA, but not HYP, slices from estrogen-treated OVX female rats. PKC enzyme levels were examined using phorbol-12,13-dibutyrate binding assays and immunoblots. Estrogen treatment did not change phorbol ester binding affinity or the density of binding sites in the POA or HYP. Immunoblots for the alpha, beta, and gamma PKC isoforms combined, or the gamma PKC isoform alone, did not detect differences between hormone-treated and control OVX female rats. Therefore, estrogen treatment increased PKC catalytic activity in the POA of OVX female rats but not in the HYP. However, the increased PKC catalytic activity was not correlated with detectable changes in the level of the alpha, beta, or gamma PKC isoforms or in the density of phorbol ester binding sites.
Nitric oxide (NO) has been suggested to play a crucial role in the regulation of lordosis behavior via stimulation of guanylyl cyclase to synthesize cyclic GMP. Whalen and Lauber (1986, Neurosci. Biobehav. Rev. 10, 47-53) hypothesized that hormones and pharmacological agents known to facilitate lordosis in estrogen-primed rodents act through cyclic GMP. The compound 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) has been shown to selectively inhibit NO-stimulated cyclic GMP production. In the present study, we investigated the effects of ODQ on lordosis behavior. Female rats were implanted with a guide cannula aimed at the lateral or third ventricles by stereotaxic surgery, and their ovaries were bilaterally removed. Five days later, animals were injected subcutaneously with 2 microg estradiol benzoate at 48 and 24 hr, and 200 microg progesterone 4 hr before behavioral testing. ODQ or vehicle (1 microl) was administered at the time of progesterone treatment or 20 min before lordosis testing. ODQ significantly decreased lordosis quotients and the quality of lordosis at both intervals of drug infusion. Locomotor activities, measured by line crossing and rearing, were not affected by ODQ. ODQ also inhibited cyclic GMP accumulation in response to NMDA stimulation in hypothalamic and cerebellar slices in vitro. We conclude that cyclic GMP produced by NO generation is an important modulator of female rat sexual behavior.
These studies examined the regulation by GABA of norepinephrine release from hypothalamus, preoptic area and frontal cortex. Using superfused brain slices from female rats, we show that 100 microM GABA enhances both basal and electrically stimulated release of 3H-norepinephrine in all three brain regions. The GABAA agonist muscimol (100 microM) significantly augments 3H-norepinephrine release, but it is somewhat less effective than GABA. The GABAB agonist baclofen has little or no effect on basal 3H-norepinephrine efflux. GABA also augments both the magnitude and duration of electrically evoked 3H-norepinephrine release in slices from all three brain regions. GABA facilitation of electrically stimulated 3H-norepinephrine release is mediated through GABAA receptors as evidenced by its blockade by 10 microM bicuculline, a GABAA antagonist, but not by 200 microM 2-OH-saclofen, a GABAB antagonist. These data show that the inhibitory amino acid neurotransmitter GABA enhances both basal and evoked release of 3H-norepinephrine in brain slices from female rats. These effects are predominantly mediated by GABAA receptors. GABA modulation of hypothalamic norepinephrine release may play a role in the regulation of gonadotropin secretion and reproductive behaviors such as lordosis.