Extranuclear signaling by estrogen: role in breast cancer progression and metastasis.

The estrogen receptor (ERa) is implicated in the progression of breast cancer. Hormonal therapies which block ER functions or local and systemic estrogen production are currently used to treat ERa positive breast cancer. Hormonal therapy shows beneficial effects, however, initial or acquired resistance to endocrine therapies frequently occurs, and tumors recur as metastasis. Emerging evidence suggests in addition to exerting its well-studied nuclear functions, ERa also participates in extranuclear signaling that involve growth factor signaling components, adaptor molecules and the stimulation of cytosolic kinases. ERa extranuclear pathways have the potential to activate gene transcription, modulate cytoskeleton, and promote tumor cell proliferation, survival, and metastasis. Cytoplasmic/membrane ERa is detected in a subset of breast tumors and expression of extranuclear components ERa is deregulated in tumors. The extranuclear actions of ER are emerging as important targets for tumorigenic and metastatic control. Inhibition of ERa extranuclear actions has the potential to prevent breast tumor progression and may be useful in preventing ERa positive metastasis. In this review, we summarize the results of recent research into the role of ERa mediated extranuclear actions in breast tumorigenesis and metastasis.

A conditional tetracycline-regulated increase in Gamma amino butyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat.

Gamma amino butyric acid (GABA) is the main inhibitory neurotransmitter controlling LH-releasing hormone (LHRH) secretion in the mammalian hypothalamus. Whether alterations in GABA homeostasis within discrete regions of the neuroendocrine brain known to be targets of GABA action, such as the median eminence, can disrupt the ability of the LHRH releasing system to maintain reproductive cyclicity is not known but amenable to experimental scrutiny. The present experiments were undertaken to examine this issue. Immortalized BAS-8.1 astroglial cells were genetically modified by infection with a regulatable retroviral vector to express the gene encoding the GABA synthesizing enzyme glutamic acid decarboxylase-67 (GAD-67) under the control of a tetracycline (tet) controlled gene expression system. In this system, expression of the gene of interest is repressed by tet and activated in the absence of the antibiotic. BAS-8.1 cells carrying this regulatory cassette, and cultured in the absence of tet ("GAD on"), expressed abundant levels of GAD-67 messenger RNA and GAD enzymatic activity, and released GABA when challenged with glutamate. All of these responses were inhibited within 24 h of exposure to tet ("GAD off"). Grafting "GAD on" cells into the median eminence of late juvenile female rats, near LHRH nerve terminals, did not affect the age at vaginal opening, but greatly disrupted subsequent estrous cyclicity. These animals exhibiting long periods of persistent estrus, interrupted by occasional days in proestrus and diestrus, suggesting the occurrence of irregular ovulatory episodes. Administration of the tetracycline analog doxycycline (DOXY) in the drinking water inhibited GAD-67synthesis and restored estrous cyclicity to a pattern indistinguishable from that of control rats grafted with native BAS-8.1 cells. Animals carrying "GAD on" cells showed a small increase in serum LH and estradiol levels, and a marked elevation in serum androstenedione, all of which were obliterated by turning GAD-67 synthesis off in the grafted cells. Morphometric analysis of the ovaries revealed that both groups grafted with GABA-producing cells had an increased incidence of large antral follicles (>500 micrometer) compared with animals grafted with native BAS-8.1 cells, but that within this category the incidence of steroidogenically more active follicles (i.e. larger than 600 micrometer) was greater in "GAD on" than in "GAD off" rats. These results indicate that a regionally discrete, temporally controlled increase in GABA availability to LHRH nerve terminals in the median eminence of the hypothalamus suffices to disrupt estrous cyclicity in the rat, and raise the possibility that similar local alterations in GABA homeostasis may contribute to the pathology of hypothalamic amenorrhea/oligomenorrhea in humans.

The potential role of periovarian fat and leptin in initiation of puberty in the immature rat.

Twenty-four-day old female rats were injected with 25 microg recombinant leptin twice daily for 24, 48, 72, or 93 h. Periovarian fat contained significantly greater amounts of aromatase mRNA at 72 and 93 h and estradiol at 72 h after leptin treatment as compared to the ovary. On the other hand, there was an increase in testosterone at 72 h and progesterone at 72 and 93 h after leptin in the ovary as compared to periovarian fat. Cholesterol side chain cleavage and 17alpha-hydroxylase mRNA were found only in the ovary and not periovarian fat. Thus, periovarian fat has a greater capacity to synthesize estrogen than the ovary in the immature rat and adipose tissue may serve as the primary source of estrogen in the prepubertal period which results in the onset of puberty.

The role of glutamate and nitric oxide in the reproductive neuroendocrine system.

The preovulatory surge of gonadotropin releasing hormone (GnRH) is essential for mammalian reproduction. Recent work has implicated the neurotransmitters glutamate and nitric oxide as having a key role in this process. Large concentrations of glutamate are found in several hypothalamic nuclei known to be important for GnRH release and glutamate receptors are also located in these key hypothalamic nuclei. Administration of glutamate agonists stimulate GnRH and LH release, while glutamate receptor antagonists attenuate the steroid-induced and preovulatory LH surge. Glutamate has also been implicated in the critical processes of puberty, hormone pulsatility, and sexual behavior. Glutamate is believed to elicit many of these effects by activating the release of the gaseous neurotransmitter, nitric oxide (NO). NO potently stimulates GnRH by activating a heme containing enzyme, guanylate cyclase, which in turn leads to increased production of cGMP and GnRH release. Recent work has focused on identifying anchoring and (or) clustering proteins that target glutamate receptors to the synapse and couple the glutamate-NO neurotransmission system. The present review will discuss these new findings, as well as the role of glutamate and nitric oxide in important mammalian reproductive events, with a focus on the hypothalamic control of preovulatory GnRH release.

Estrogen-astrocyte-luteinizing hormone-releasing hormone signaling: a role for transforming growth factor-beta(1).

The purpose of this study was to identify factors from astrocytes that can regulate LHRH neurosecretion. Exposure of LHRH-secreting (GT1-7) cells to conditioned media (CM) from C6 glial cells and hypothalamic astrocytes (HA) stimulated LHRH release. Assays of C6 and HA CM revealed that transforming growth factor-beta(1) (TGF-beta(1)) and 3alpha-hydroxy-5alpha-pregnane-20-one (3alpha, 5alpha-THP), both known LHRH secretagogues, were present in CM and their levels increased in parallel to the LHRH-releasing activity of CM. In contrast, TGF-alpha was undetectable in C6 or HA CM. Ultrafiltration to remove peptides with molecular weights >10 kDa virtually abolished the LHRH-releasing ability of the HA CM. Furthermore, immunoneutralization with a panspecific THF-beta antibody dose-dependently attenuated the LHRH-releasing activity of the CM. Rat hypothalamus and GT1-7 cells were demonstrated to express TGF-beta receptors as well as furin, an enzyme that converts latent TGF-beta(1) to active TGF-beta(1). Estrogen receptor-alpha and ER-beta mRNA and protein were also demonstrated in HAs by reverse transcription-polymerase chain reaction and double immunofluorescence, and treatment with 17beta-estradiol (17beta-E(2)) increased both active and latent TGF-beta(1) levels in HA CM. The effect of 17beta-E(2) was completely blocked by the ER antagonist ICI8280. As a whole, these studies provide evidence of a previously undescribed 17beta-E(2)-TGF-beta(1)-LHRH signaling pathway.

Evidence that neuronal nitric oxide synthase but not heme oxygenase increases in the hypothalamus on proestrus afternoon.

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.

Bradykinin receptor localization and cell signaling pathways used by bradykinin in the regulation of gonadotropin-releasing hormone secretion.

In a previous publication we provided evidence of a novel neuronal pathway for the control of GnRH secretion by bradykinin. The action of bradykinin appeared to be exerted through the bradykinin B2 receptor. In this study we demonstrated that the bradykinin B2 receptor is densely localized in the arcuate nucleus, median eminence, organum vasculosum of the lamina terminalis, and preoptic area, regions known to be critical for the control of GnRH secretion. To determine the mechanism of action of bradykinin in stimulating GnRH release, we used immortalized GnRH (GT1-7) cells in vitro. Bradykinin stimulation of GnRH secretion from GT1-7 cells appears to involve activation of the phospholipase C signaling pathway and mobilization of extracellular and intracellular calcium stores. Evidence to support this contention was derived from the observations that incubation of the phospholipase C inhibitor, U-73122 with bradykinin, blocked the ability of bradykinin to stimulate release from GT1-7 cells. This effect was specific, as a nitric oxide synthase inhibitor and a cyclooxygenase inhibitor were found to have no effect on bradykinin-induced GnRH secretion, suggesting that nitric oxide and PGs do not mediate bradykinin effects. Pertussis toxin also had no effect on bradykinin action. This suggests that the bradykinin B2 receptor may be coupled to a pertussis toxin-insensitive G protein in GT1-7 cells. With respect to calcium involvement in bradykinin action, fura-2 calcium indicator studies revealed that bradykinin can rapidly increase intracellular Ca2+ levels in GT1-7 cells. A role for intracellular Ca2+ in bradykinin action was further suggested by the finding that an intracellular calcium chelator, 1,2-bis(O-aminophenoxy)]ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, significantly attenuated the effects of bradykinin on GnRH release. The elevation of intracellular calcium by bradykinin appears to be due to mobilization of calcium from the endoplasmic reticulum, as incubation of the Ca2+-adenosine triphosphatase inhibitor thapsigarin, which depletes endoplasmic reticulum Ca2+ stores, significantly attenuated bradykinin action on GnRH release. Extracellular calcium may also be involved in bradykinin action, as the L-type Ca2+ channel blockers verapamil and nifedipine had no effect on bradykinin-induced GnRH release, whereas the nonselective Ca2+ channel blocker, nickel chloride, attenuated bradykinin-induced GnRH release. Taken as a whole, these studies demonstrate that the bradykinin B2 receptor is densely localized in key hypothalamic nuclei responsible for regulation of GnRH release, and that the mechanism of bradykinin stimulation of GnRH secretion involves activation of the phospholipase C signaling pathway, with a critical role implicated for calcium in bradykinin action in GT1-7 cells.

Characterization of ionotropic glutamate receptors in rat hypothalamus, pituitary and immortalized gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells).

Evidence from various sources suggested that the Gonadotropin-Releasing Hormone (GnRH) neuron does not contain glutamate receptors. Northern analysis of the hypothalamus showed the presence of NMDAR1, GluR1, GluR4 and GluR6 mRNA, while the pituitary showed the presence of NMDAR1, GluR1 and GluR6 mRNA. Western blot analysis also showed the presence of NMDAR1 and GluR1 protein. Since there are relatively few GnRH neurons in the hypothalamus, and GT1-7 cells have been considered to be a GnRH neuronal cell line, GT1-7 cells were studied in detail. GT1-7 cells contained NMDAR1 mRNA levels as shown by Northern analysis but did not contain GluR1, GluR4, or GluR6 mRNA. They did not show the presence of NMDAR1 and GluR1 protein by Western analysis. In addition, GT1-7 cells showed no NMDA receptor binding using the competitive inhibitor CGP-39563 and the noncompetitive inhibitor MK-801. Likewise, no binding was detected for kainate receptors. However, a small amount of binding for AMPA receptors was found in GT1-7 cells. GT1-7 cells did not exhibit glutamate toxicity and NMDA failed to elicit inward currents using patch-clamp techniques, although GABA did induce currents in the cells. As a whole, these studies suggest that GT1-7 cells lack or possess only low levels of ionotropic glutamate receptors.

Regulation of the preovulatory gonadotropin surge by endogenous steroids.

Estradiol secreted by growing ovarian follicle(s) has been considered classically to be the neural trigger for the preovulatory surge of gonadotropins. The observation that the estradiol-induced gonadotropin surge in ovariectomized rats is of lesser magnitude and duration than that found in the cycling rat at proestrus has resulted in a search for other steroid regulators. Progesterone is a major regulator of the preovulatory gonadotropin surge. It can only act in the presence of an estrogen background, which is necessary for the synthesis of progesterone receptors. In the estrogen-primed ovariectomized rat, progesterone is able to initiate and enhance the gonadotropin surge to the magnitude observed on the day of proestrus and limit it to 1 day. The physiological role of progresterone in the induction of the preovulatory gonadotropin surge has been demonstrated by the attenuation of the progesterone-induced surge and the endogenous proestrus surge by progesterone receptor antagonist RU486 and the progesterone synthesis inhibitor trilostane. The promoter region of the follicle-stimulating hormone (FHS)-beta gene contains multiple progesterone response elements and progesterone brings about FSH release as well. The reduction of progesterone in the 5 alpha-position appears to be important for the regulation of progesterone secretion. Corticosteroids appear to play a significant role in the secondary FSH surge on late proestrus and early estrus.

Neuroendocrine mechanisms underlying the control of gonadotropin secretion by steroids.

There is considerable evidence that although estradiol may trigger the preovulatory surge of gonadotropins, progesterone is required for its full magnitude and duration and that glucocorticoids bring about selective follicle-stimulating hormone release. The luteinizing hormone-releasing hormone (LHRH) neuron does not have steroid receptors and is regulated by excitatory amino acid neurotransmission. Steroids do not appear to modulate excitatory amino acid receptors directly but increase release of glutamate in the preoptic area. This may be due to the suppression by steroids of the enzyme glutamatic acid decarboxylase67 that converts glutamate into GABA. NMDA receptors colocalize with nitric oxide synthase-containing neurons that surround the LHRH neurons in the preoptic area and intersect the LHRH fibers in the median eminence. Other potential novel pathways of LHRH release that are currently being explored include carbon monoxide generated by the action of heme oxygenase-2 on heme molecules and bradykinin acting via bradykinin B2 receptors.

Excitatory amino acid receptors and puberty.

Glutamate is an important excitatory signal in the hypothalamus for the steroid-mediated preovulatory gonadotropin surge. Steroids may exert this action by regulating glutamate receptor levels or glutamate release, or both. Work in our laboratory found no changes in NMDA and kainate receptor binding in the hypothalamus of castrated or castrated plus steroid-replaced male and female rats. Likewise, we found that NMDA and kainate binding did not change over the onset of puberty in the female rat. A competitive quantitative RT-PCR assay using exogenous internal standards was used to measure NMDAR1, GluR1, and beta-actin mRNAs levels. NMDAR1 and GluR1 expression was examined in the preoptic hypothalamic area and in the medial basal hypothalamus at Postnatal Days 10, 15, 20, 25, 30, 32, 34, 36, 40, and 63. A transient increase in GluR1 mRNA levels in the preoptic hypothalamic area was observed on Day 20, with all other time points showing comparable levels. NMDAR1 levels in the POA and medial basal hypothalamus did not change significantly at any of the time points; in contrast, however, AMPA receptor binding levels were increased in the hypothalamus at the time of puberty in the female rat. Thus, in addition to the previously reported elevation of glutamate release rates in the hypothalamus at the time of puberty, AMPA receptors may also be elevated and play a role in mediating glutamate regulatory effects on the timing of puberty in the female rat.

Evidence for a role of bradykinin neurons in the control of gonadotropin-releasing hormone secretion.

The present study provides evidence of a novel neuronal pathway for the control of GnRH secretion involving bradykinin neurons. Bradykinin neurons were shown by immunohistochemistry to be densely localized in several regions of the brain including the cortex, hippocampus and supraoptic nucleus, as well as two regions critical in the control of GnRH secretion, the organum vasculosum of the lamina terminalis and arcuate nucleus. Bradykinin dose-dependently stimulated GnRH release from male and proestrous female rat hypothalami in vitro. Antagonist studies revealed that bradykinin effects are mediated by the bradykinin B2 receptor. The effect of bradykinin on GnRH release is not mediated by the classical major transmitter, glutamate, as glutamate antagonists had no effect on bradykinin stimulation of GnRH release. Rather, bradykinin appears to act directly on the GnRH neuron as bradykinin stimulated GnRH release directly from immortalized GnRH (GT1-7) neurons in vitro, and immunoblot studies revealed that the bradykinin B2 receptor is present in GT1-7 neurons. The bradykinin B2 receptor was also demonstrated in the rat hypothalamus and pituitary by immunoblotting. Bradykinin-induced exocytosis of GnRH appears to involve activation of the PKC signaling pathway, as a PKC inhibitor blocked bradykinin-induced GnRH release. Finally, bradykinin neurons appear to be important mediators of steroid signals in the hypothalamus to produce the LH surge, as central administration of a B2 antagonist, but not a B antagonist, significantly attenuated the steroid-induced LH surge in the ovariectomized female rat.

Opioid-glutamate-nitric oxide connection in the regulation of luteinizing hormone secretion in the rat.

Opioid neurons are recognized to be an important component of the inhibitory "brake" in the CNS that restrains LHRH secretion. Opioid inhibition could be exerted directly on LHRH neurons, or it could be achieved via indirect mechanisms involving restrainment of excitatory "accelerator" neurons that facilitate LHRH release. The purpose of the present study was to explore the second hypothesis by investigating whether removal of opioid inhibition by administering the opioid antagonist, naloxone leads to enhanced activation of glutamate and nitric oxide (NO) neurons, which are known to be important excitatory "accelerator" components for the control of LHRH secretion. Naloxone administration (2.5 mg/kg) to adult male rats induced a significant elevation of serum LH levels at 20 min post injection. NOS activity in preoptic area (POA) and medial basal hypothalamic (MBH) fragments was demonstrated to be significantly elevated 20 min post naloxone injection. Administration of a glutamate (NMDA) receptor antagonist (MK-801, 0.2 mg/kg) abolished the naloxone-induced increase in NOS activity in the POA and MBH, with a corresponding block of the naloxone-induced LH release. Glutamate appears to only be involved in LH surge generation and not to regulate basal LH levels, as MK-801 had no effect on basal LH release. Because previous work by our laboratory and others have provided evidence that NO is a mediator of glutamate effects in the hypothalamus, these findings are interpreted to mean that opioid inhibition is mediated on glutamate neurons that are upstream of NO neurons. In support of this contention, we found that NMDA treatment enhanced NOS activity in the male rat POA and MBH fragments in vitro, an effect that was specific as it was completely blocked by the NMDA receptor antagonist, MK-801. Additionally, in vivo microdialysis studies revealed that naloxone treatment significantly enhances glutamate release in the preoptic area (POA) at 15 min post injection in conscious, unanesthetized, freely moving male rats. Release rates of the control amino acid, serine did not change significantly following naloxone injection. Taken as a whole, these findings provide evidence for an opioid-glutamate-NO pathway in the control of LHRH secretion, and they demonstrate the importance of "brake-accelerator" interactions in the control of LHRH and LH secretion.

Progesterone metabolite allopregnanolone in women with premenstrual syndrome.

OBJECTIVE: To evaluate the anxiolytic 3alpha-5alpha-reduced progesterone metabolite allopregnanolone in the luteal phase of the menstrual cycle in women with premenstrual syndrome (PMS) and controls. METHODS: Thirty-five women with prospectively documented PMS and 36 controls were evaluated. Serum progesterone and allopregnanolone levels were measured on days 19 and 26 of the cycle as determined by urinary LH detection kits. Analysis of variance and Student t tests were used to analyze the data. RESULTS: Allopregnanolone levels were significantly lower on day 26 in the PMS group than in controls (3.6 +/- 0.8 versus 7.5 +/- 1.3 ng/mL; P < .04). Significant differences in the ratio of the metabolite to progesterone also were noted, with a smaller ratio in the PMS subjects (0.9 +/- 0.3 versus 3.2 +/- 1.3 ng/mL; P < .05). There were no significant differences between the PMS and control groups with respect to serum progesterone levels. CONCLUSION: Subjects with PMS manifested lower levels of the anxiolytic metabolite allopregnanolone in the luteal phase when compared with controls. Diminished concentrations of allopregnanolone in women with PMS may lead to an inability to enhance gamma aminobutyric acid-mediated inhibition during states of altered central nervous system excitability, such as ovulation or physiologic or psychological stress. The lowered metabolite levels could contribute to the genesis of various mood symptoms of the disorder, such as anxiety, tension, and depression.

Gaseous transmitters and neuroendocrine regulation.

Recent work has demonstrated that the brain has the capacity to synthesize impressive amounts of the gases nitric oxide (NO) and carbon monoxide (CO). There is growing evidence that these gaseous molecules function as novel neural messengers in the brain. This article reviews the pertinent literature concerning the putative role of NO and CO as critical neurotransmitters and biological mediators of the neuroendocrine axis. Abundant evidence is presented which suggests that NO has an important role in the control of reproduction due to its ability to control GnRH secretion from the hypothalamus. NO potently stimulates GnRH secretion and also appears to mediate the action of one of the major transmitters controlling GnRH secretion, glutamate. Evidence is presented which suggests that NO stimulates GnRH release due to its ability to modulate the heme-containing enzyme, guanylate cyclase, which leads to enhanced production of the second messenger molecule, cGMP. A physiological role for NO in the preovulatory LH surge was also evidenced by findings that inhibitors and antisense oligonucleotides to nitric oxide synthase (NOS) attenuate the steroid-induced and preovulatory LH surge. CO may also play a role in stimulating GnRH secretion as heme molecules stimulate GnRH release in vitro, an effect which requires heme oxygenase activity and is blocked by the gaseous scavenger molecule, hemoglobin. Evidence is also reviewed which suggests that NO acts to restrain the hypothalamic-pituitary-adrenal (HPA) axis, as it inhibits HPA stimulation by various stimulants such as interleukin-1 beta, vasopressin, and inflammation. This effect fits a proinflammatory role of NO as it leads to suppression of the release of the anti-inflammatory corticosteroids from the adrenal. Although not as intensely studied as NO, CO has been shown to suppress stimulated CRH release and may also function to restrain the HPA axis. Evidence implicating NO in the control of prolactin and growth hormone secretion is also reviewed and discussed, as is the possible role of NO acting directly at the anterior pituitary. Taken as a whole, the current data suggest that the diffusible gases, NO and CO, act as novel transmitters in the neuroendocrine axis and mediate a variety of important neuroendocrine functions.

Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat.

The obese gene (ob) product, leptin, has recently been shown to be produced by adipocytes and to circulate in the plasma acting as a hormone to modulate appetite and metabolism. Intriguingly, the ob/ob mutant female mouse, which does not produce an active form of leptin due to a mutation of the ob gene, has been shown to be acyclic and sterile. This sterility can be reversed by treatment with recombinant leptin, but not by diet restriction--suggesting that leptin is required for normal reproductive function. The mechanism(s) whereby leptin modulates reproductive function are unknown; however, it is possible that leptin could directly regulate reproductive tissues. To determine whether endocrine and neuroendocrine tissues could be targets for leptin action, we examined whether these tissues express the leptin receptor mRNA by utilizing reverse-transcription polymerase chain reaction (RT-PCR) analysis in selected tissues from the male and female rat. The results revealed that the leptin receptor mRNA transcript is highly expressed in the ovary, uterus and testis, moderately expressed in the hypothalamus and anterior pituitary, with low to no expression in the adrenal. The RT-PCR results were confirmed by Northern analysis. Furthermore, immortalized GnRH (GT1-7 and NLT) neurons and ovarian granulosa cells were also demonstrated by RT-PCR analysis to express the leptin receptor, suggesting that GnRH neurons and steroid-producing cells of the ovary could be targets for leptin action. Immunohistochemical studies revealed dense immunolocalization of the leptin receptor in the choroid plexus, and interestingly, in the arcuate nucleus/median eminence of the female rat--a key sit in the control of feeding and reproduction. Finally, treatment of the ob/ob mouse with recombinant leptin (0.15 mg/kg/day x 2 weeks) was found to markedly upregulate side chain cleavage and 17 alpha-hydroxylase mRNA levels in the ovary, demonstrating that leptin, acting either through a direct or indirect mechanism, can regulate gene expression in reproductive tissues.

Regulation of gonadotropin-releasing hormone and luteinizing hormone secretion by AMPA receptors. Evidence for a physiological role of AMPA receptors in the steroid-induced luteinizing hormone surge.

Recent work has demonstrated that glutamate functions as a major transmitter involved in the regulation of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in female animals, although the specific receptors and mechanisms mediating its effects have not been completely worked out. The purpose of the present study, therefore, was to examine the role of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptor in the control of GnRH and LH secretion in female animals. Toward this end, the dose- and steroid-dependent effects of AMPA on GnRH and LH secretion in female rats were investigated using both in vitro and in vivo approaches, and the role of AMPA receptors in the production of the steroid-induced LH surge was also assessed. The results of the study revealed that central administration of AMPA resulted in a stimulation of LH release in the estrogen-primed ovariectomized adult rat. AMPA was also found to potently stimulate GnRH release in vitro from mediobasal hypothalamic (MBH) fragments obtained from estrogen-primed ovariectomized adult rats, and this effect was blocked by the selective AMPA receptor antagonist, NBQX. The mechanism of action of AMPA appeared to differ from that of N-methyl-D-aspartate (NMDA) as AMPA, in contrast to NMDA, failed to elevate nitric oxide synthase activity in the hypothalamus. The effect of AMPA on LH secretion was demonstrated to be steroid dependent, as central administration of AMPA stimulated LH release in estrogen-primed ovariectomized rats, but was inhibitory to LH release in non-estrogen-primed ovariectomized rats. In contrast, AMPA stimulated GnRH release equally well from MBH fragments obtained from estrogen-primed or non-estrogen-primed ovariectomized rats. The different effects of AMPA on LH release may be due to different pituitary sensitivities between the two models, or alternatively, AMPA may stimulate the release of LH inhibitory factors in the ovariectomized rat in the absence of estrogen. Finally, a physiological role for AMPA receptors in the production of the steroid-induced LH surge was suggested, based on the finding that central administration of the selective AMPA receptor antagonist, NBQX, into the third cerebroventricle significantly attenuated the steroid-induced LH surge in the ovariectomized adult female rat.