Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats.

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17ß-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Estrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2.

The role of classical estrogen receptors (ERs) in priming female reproductive behavior has been studied previously; however, the participation of this receptor during activation of estrous behavior has not been extensively studied. The purpose of this work was to test the possibility that the facilitation of lordosis behavior in estrogen-primed rats by progesterone (P) and its 5α- and 5ß-reduced metabolites, gonadotropin-releasing hormone (GnRH), leptin, prostaglandin E2 (PGE2) and vagino-cervical stimulation (VCS) involves interactions with classical ERs by using the selective ER modulator, tamoxifen. To further assess the role of ERs, we also explored the effects of the pure ER antagonist, ICI182780 (ICI), on estrous behavior induced by P and GnRH. Ovariectomized, estrogen-primed rats (5µg estradiol benzoate 40h earlier) were injected intraventricularly with the above-mentioned compounds, or they received VCS. All compounds and VCS effectively facilitated estrous behavior when tested at 60, 120 or 240min after infusion or application of VCS. Intraventricular infusion of tamoxifen (5µg), 30min before, significantly attenuated estrous behaviors induced in estradiol-primed rats by P, most of its 5α- and 5ß-reduced metabolites, GnRH, and PGE2, but not by VCS. Although there was a trend for reduction, tamoxifen did not significantly decrease lordosis in females treated with 5ß-pregnan-3,20-dione. ICI also inhibited lordosis behavior induced by P and GnRH at some testing intervals. These results suggest that activation of classical ERs participates in the triggering effects on estrous behavior induced by agents with different chemical structures that do not bind directly to ERs.

Phasic synaptic incorporation of GluR2-lacking AMPA receptors at gonadotropin-releasing hormone neurons is involved in the generation of the luteinizing hormone surge in female rats.

Reproductive success depends on a robust and appropriately timed preovulatory luteinizing hormone (LH) surge, which is induced by the activation of gonadotropin-releasing hormone (GnRH) neurons in response to positive feedback from increasing estrogen levels. Here we document an increase in postsynaptic GluR2-lacking Ca2+ -permeable AMPA-type glutamate receptors (CP-AMPARs) at synapses on GnRH neurons on the day of proestrus in rats, coincident with the increase in estrogen levels. Functional blockade of CP-AMPARs depressed the synaptic responses only on the day of proestrus and concomitantly attenuated the LH surge. Thus, the phasic synaptic incorporation of postsynaptic CP-AMPARs on GnRH neurons is involved in the generation of the LH surge.

Effects of global ischemia and estradiol pretreatment on phosphorylation of Akt, CREB and STAT3 in hippocampal CA1 of young and middle-aged female rats.

Transient global ischemia induces selective, delayed neuronal death of pyramidal neurons in the hippocampal CA1. Whereas long term treatment of middle-aged female rats with estradiol at physiological doses ameliorates neuronal death, the signaling pathways that mediate the neuroprotection are, as yet, unknown. Protein kinase B (Akt) and downstream transcription factors, the cAMP response element binding protein (CREB) and signal transducer and activator of transcription (STAT3) are critical players in cellular survival following injury. The present study was undertaken to determine whether long term estradiol alters the phosphorylation status and activity of Akt, STAT3 and CREB in ovariohysterectomized, middle-aged and young female rats subjected to global ischemia. Irrespective of either hormone or ischemic condition, middle-aged females exhibited lower levels of p-CREB and higher levels of Akt and STAT3 in CA1 than young females, as assessed by Western blot. In middle-aged animals, ischemia increased the phosphorylation status/activity of Akt and STAT3, and decreased the phosphorylation status/activity of CREB in the hippocampal CA1. Whereas estradiol did not detectably alter the phosphorylation status/activity of Akt or STAT3, it prevented the ischemia-induced decrease in nuclear p-CREB. Similar results were observed for the young females. Collectively, these data demonstrate that CREB, STAT3, and Akt are involved in the molecular response to global ischemia and that age influences the status of CREB, STAT3 and Akt activity in CA1 under physiological as well as pathological conditions, further emphasizing the importance of including older rodents in neuroprotection studies.

Oestradiol and insulin-like growth factor-1 reduce cell loss after global ischaemia in middle-aged female rats.

Whereas the ability of oestradiol and insulin-like growth factor (IGF)-1 to afford neuroprotection against ischaemia-induced neuronal death in young female and male rodents is well established, the impact of IGF-1 in middle-aged animals is largely unknown. The present study assessed the efficacy of oestradiol and IGF-1 with respect to reducing neuronal death after transient global ischaemia in middle-aged female rats after 8 weeks of hormone withdrawal. Rats were ovariohysterectomised and implanted 8 weeks later with an osmotic mini-pump delivering IGF-1 or saline into the lateral ventricle. Some rats also received physiological levels of oestradiol by subcutaneous pellet. Two weeks later, rats were subjected to global ischaemia or sham operation. Surviving hippocampal CA1 neurones were quantified. Ischaemia produced massive CA1 cell death compared to sham-operated animals, which was evident at 14 days. Significantly more neurones survived in animals treated with either oestradiol or IGF-1, but simultaneous treatment produced no additive effect. IGF-1, an endogenous growth factor, may be a clinically useful therapy in preventing human brain injury, with neuroprotective equivalence to oestradiol but without the harmful side-effects.

GPR30 differentially regulates short latency responses of luteinising hormone and prolactin secretion to oestradiol.

Rapid, nongenomic actions of 17beta-oestradiol (E(2)) on hypothalamic neurones that may be relevant to reproductive function were described decades ago. The orphan G protein-coupled receptor, GPR30, was recently shown to bind oestrogens and to trigger rapid signalling in vitro, and is expressed in several rat and human brain regions, including the hypothalamus. We used two complementary approaches to investigate the role of GPR30 in hypothalamic responses to E(2) that are relevant to reproductive physiology. Serial blood sampling after the acute administration of the selective GPR30 agonist G1 was used to assess the role of GPR30 in short latency negative-feedback inhibition of luteinising hormone (LH) secretion and facilitation of prolactin secretion in ovariohysterectomised female rats. In vivo RNA interference (RNAi), mediated by adeno-associated virus-expressing small hairpin RNA (shRNA) infused into the mediobasal hypothalamus, was used to study the effects of GPR30 knockdown on these rapid responses to E(2). Longer-term actions of E(2) on female sexual behaviour (lordosis) were also examined in female rats subjected to in vivo RNAi. Administration of E(2) or G1 triggered a short latency surge of prolactin secretion, and animals subjected to GPR30 RNAi showed significantly less E(2)-dependent prolactin release than animals receiving control virus. G1 did not mimic E(2) negative-feedback inhibition of LH secretion, and GPR30 RNAi did not interfere with E(2) suppression of LH or facilitation of lordosis behaviour. These findings suggest that activation of GPR30 promotes short latency prolactin secretion but does not mediate E(2) negative-feedback inhibition of LH secretion or E(2) facilitation of female reproductive behaviour.

Chronic estradiol treatment increases CA1 cell survival but does not improve visual or spatial recognition memory after global ischemia in middle-aged female rats.

Transient global ischemia induces selective, delayed neuronal death in the hippocampal CA1 and cognitive deficits. Physiological levels of 17beta-estradiol ameliorate ischemia-induced neuronal death and cognitive impairments in young animals. In view of concerns regarding hormone therapy in postmenopausal women, we investigated whether chronic estradiol treatment initiated 14 days prior to ischemia attenuates ischemia-induced CA1 cell loss and impairments in visual and spatial memory, in ovariohysterectomized (OVX), middle-aged (9-11 months) female rats. To determine whether the duration of hormone withdrawal affects the efficacy of estradiol treatment, hormone treatment was initiated immediately (0 week), 1 week, or 8 weeks after OVX. Age-matched, OVX and gonadally intact females were studied at each OVX interval. Ischemia was induced 1 week after animals were pretested on a variety of behavioral tasks. Global ischemia produced significant neuronal loss in the CA1 and impaired performance on visual and spatial recognition. Chronic estradiol modestly but significantly increased the number of surviving CA1 neurons in animals at all OVX durations. However, in contrast with previous results in young females, estradiol did not preserve visual or spatial memory performance in middle-aged females. All animals displayed normal locomotion, spontaneous alternation and social preference, indicating the absence of global behavioral impairments. Therefore, the neuroprotective effects of estradiol are different in middle-aged than in young rats. These findings highlight the importance of using older animals in studies assessing potential treatments for focal and global ischemia.

Antagonists of the protein kinase A and mitogen-activated protein kinase systems and of the progestin receptor block the ability of vaginocervical/flank-perineal stimulation to induce female rat sexual behaviour.

Brief vaginocervical stimulation using a glass rod (VCS) combined with manual flank-perineal stimulation (FS) rapidly (within 5 min) induced both receptive and proceptive behavioural responses to males in ovariectomised, oestrogen-primed rats. This receptive-proceptive response to males, resulting from a single brief (5-s duration) instance of manual VCS + FS, declined markedly within 4 h. However, the decline was prevented if the females were mounted by males immediately after the manual VCS + FS and 2 h later. We tested the participation of the cAMP-dependent protein kinase A system and the mitogen-activated protein kinase (MAPK) system in the response to VCS + FS by infusing either 100 ng of Rp-adenosine 3',5'-cyclic monophosphorothiate triethylamonium salt (a protein kinase A blocker) or 3.3 microg of PD98059 (a MAPK blocker) i.c.v. 15 min prior to VCS + FS. Both inhibitors blocked the ability of VCS + FS to induce the proceptive-receptive responses to males at all testing intervals. In experiment 2, systemic administration of 5 mg of RU486 1 h before VCS + FS also blocked the ability of VCS + FS to induce the proceptive-receptive responses to males. The present findings suggest that both VCS + FS and mating stimuli provided by males release neurotransmitters and neuromodulators that trigger the protein kinase A and the MAPK signalling systems, which interact with the progestin receptor to rapidly (within 5 min) induce proceptive-receptive behaviour in females.

Voltage-dependent calcium channels in ventromedial hypothalamic neurones of postnatal rats: modulation by oestradiol and phenylephrine.

Oestradiol actions in the hypothalamus play an important role in reproductive behaviour. Oestradiol treatment in vivo induces alpha(1b)-adrenoceptor mRNA and increases the density of alpha(1B)-adrenoceptor binding in the hypothalamus. Oestradiol is also known to modulate neuronal excitability, in some cases by modulating calcium channels. We assessed the effects of phenylephrine, an alpha(1)-adrenergic agonist, on low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium channels in ventromedial hypothalamic (VMN) neurones from vehicle- and oestradiol-treated female rats. Whole-cell and gramicidin perforated-patch recordings were obtained, with barium as the charge carrier. In the absence of phenylephrine, oestradiol treatment increased the magnitude of LVA currents compared to controls, but had no effect on HVA currents. Phenylephrine enhanced HVA currents in a significantly greater proportion of neurones from oestradiol-treated rats (76%) than from vehicle-treated (41%) rats. The L-channel blocker nifedipine abolished this oestradiol effect on phenylephrine-enhanced HVA currents. Preincubating slices with the N-type channel blocker omega-conotoxin GVIA completely blocked the phenylephrine response, suggesting that the N-type channel is essential. Phenylephrine also stimulated LVA currents in approximately two-thirds of neurones in slices from both vehicle- and oestradiol-treated rats. Our data show that oestradiol increases LVA currents in the VMN. Oestradiol also amplifies alpha(1)-adrenergic signalling by increasing the proportion of neurones showing phenylephrine-stimulated HVA currents mediated by N- and L-type calcium channels. In this way, oestradiol may increase excitatory responses to arousing adrenergic inputs to VMN neurones governing oestradiol-dependent reproductive behaviour.

Regulation of soluble guanylyl cyclase activity by oestradiol and progesterone in the hypothalamus but not hippocampus of female rats.

Oestradiol and progesterone act in the hypothalamus to coordinate the timing of lordosis and ovulation in female rats in part through regulation of nitric oxide (NO) and cyclic guanosine monophosphate (cyclic GMP) signalling pathways. Soluble guanylyl cyclase is an enzyme that produces cyclic GMP when stimulated by NO and plays a crucial role in the display of lordosis behaviour. We examined the effects of oestradiol and progesterone on the stimulation of cyclic GMP synthesis by NO-dependent and independent activators of soluble guanylyl cyclase in preoptic-hypothalamic and hippocampal slices. Ovariectomised Sprague-Dawley rats were injected with oestradiol (2 microg oestradiol benzoate, s.c.) or vehicle for 2 days. Progesterone (500 microg, s.c.) or vehicle was injected 44 h after the first dose of oestradiol. Rats were killed 48 h after the first oestradiol or vehicle injection, and hypothalamus and hippocampus were obtained. NO-dependent activation of soluble guanylyl cyclase was induced by NO donors, sodium nitroprusside or diethylamine NONOate; NO-independent activation of soluble guanylyl cyclase was induced with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole and 5'-cyclopropyl-2-[1-2fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyridine-4-ylamine. The NO-dependent activators of soluble guanylyl cyclase produced a concentration-dependent increase in cyclic GMP accumulation and induced significantly greater cyclic GMP accumulation in preoptic-hypothalamic slices from animals treated with oestradiol and progesterone than in slices from rats injected with vehicle, oestradiol or progesterone alone. Hormones did not modify soluble guanylyl cyclase activation by NO-independent stimulators or influence NO content in preoptic-hypothalamic slices. Oestradiol and progesterone did not affect activation of soluble guanylyl cyclase in hippocampal slices by any pharmacological agent, indicating a strong regional selectivity for the hormone effect. Thus, oestradiol and progesterone, administered in vivo, enhance the ability of NO to activate soluble guanylyl cyclase in brain areas modulating female reproductive function without an effect on production of NO itself.

Somatosensory stimuli evoke norepinephrine release in the anterior ventromedial hypothalamus of sexually receptive female rats.

We used in vivo brain microdialysis to determine the role of specific copulatory stimuli in mating-induced release of norepinephrine in the lateral ventromedial hypothalamus (VMH) of hormone-treated, sexually receptive female rats. Ovariectomized rats implanted with a unilateral guide cannula aimed at the ventrolateral VMH received systemic injections of estradiol benzoate daily for 2 days before and progesterone 4 h before the initiation of a 1-h behavioural test. Dialysis probes were lowered immediately after progesterone administration, and 20-min dialysis samples were collected until 1 h after the termination of behavioural testing. Norepinephrine content of dialysates was quantified by high performance liquid chromatography with electrochemical detection. During mating tests with male rats, dialysate levels of norepinephrine increased significantly over baseline in sexually receptive females with probe placements in the anterior but not posterior VMH. Norepinephrine levels were unchanged if rats were nonreceptive, even if males mounted vigorously and probes were located in the anterior VMH. Hormone-treated females that were placed on male-soiled bedding for 1 h showed no changes in dialysate levels of norepinephrine. Similarly, females in which vaginocervical stimulation was prevented by a vaginal mask failed to show increased levels of norepinephrine in dialysates collected from the anterior VMH, even if they displayed high levels of lordosis behaviour. Thus, the release of norepinephrine is not a result of executing the lordosis posture. The findings suggest that mating-induced increases in norepinephrine release in hormone-treated, sexually receptive rats are confined to the anterior VMH and that somatosensory rather than chemosensory stimuli evoke norepinephrine release. Moreover, experiments with vaginal masks indicate that vaginocervical stimulation is necessary for mating-evoked norepinephrine release in the anterior VMH.

Inhibition of lordosis behavior by intrahypothalamic infusion of a protein kinase G antagonist.

Previous experiments demonstrated that intracerebroventricular infusion of the protein kinase G inhibitor KT5823 inhibits lordosis behavior in hormone-treated female rats. Present studies show that KT5823 attenuates lordosis in a dose-dependent manner when infused bilaterally into the ventromedial hypothalamus. Thus, activation of protein kinase G in the ventromedial hypothalamus is necessary for the expression of hormone-dependent lordosis behavior in female rats.

Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology.

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.

Modulation of GABA-augmented norepinephrine release in female rat brain slices by opioids and adenosine.

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.

Receptor phosphorylation mediates estradiol reduction of alpha2-adrenoceptor coupling to G protein in the hypothalamus of female rats.

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.

Estrogen increases G protein coupled receptor kinase 2 in the cortex of female rats.

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.

Estrogen modulates 5-HT(1A) agonist inhibition of lordosis behavior but not binding of [(3)H]-8-OH-DPAT.

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.

Insulin-like growth factor-1 regulation of alpha(1)-adrenergic receptor signaling is estradiol dependent in the preoptic area and hypothalamus of female rats.

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.

Evidence that oestradiol attenuates beta-adrenoceptor function in the hypothalamus of female rats by altering receptor phosphorylation and sequestration.

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.

Tyrosine kinase effects on adrenoceptor-stimulated cyclic AMP accumulation in preoptic area and hypothalamus of female rats: modulation by estradiol.

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.