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Estrogen modulates the action of nitric oxide in the medial preoptic area on luteinizing hormone and prolactin secretion
Life Sciences 74 (2004) 2049 – 2059 www.elsevier.com/locate/lifescie
Estrogen modulates the action of nitric oxide in the medial preoptic area on luteinizing hormone and prolactin secretion A.S. Moreno, C.R. Franci * Departamento de Fisiologia, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeira˜o Preto - SP, Brazil Received 2 June 2003; accepted 22 September 2003
Abstract Several substances work as neuromediators of the estrogen direct and indirect (throught glial cells or interneurons) action on luteinizing hormone- releasing hormone (LH-RH) neurons in medial basal hypothalamus and medial preoptic area (MPOA).Angiotensin II (AII) in the MPOA stimulates the LH and it inhibits PRL secretion in some situations. On the other hand, the effect of excitatory aminoacids on LH and PRL surges during proestrus as well LH surge induced by steroids depend on nitric oxide (NO). In the present study we investigated the participation of MPOA endogenous NO on gonadotropin and PRL secretion mediated by estrogen and AII. Plasma LH, FSH and PRL was determinated in estrogen primed and unprimed ovariectomized Wistar rats that received microinjection of AII or saline into the MPOA, associated or not with a previous microinjection of an inhibitor for NOS. Our results show the following: 1- there was no change in plasma FSH in estrogen- primed or umprimed ovarictomized related with microinjections of AII or NO antagonist in the MPOA; 2- the increase in LH secretion after ovariectomy depends on, at least in part, NO activity in the MPOA; 3- estrogen may have an indirect negative feedback action on LH-RH neurons in the MPOA through NO; 4- the stimulatory action of AII in the MPOA on LH secretion in ovariectomized rats treated with estrogen depends on NO; 5- NO in the MPOA stimulates or inhibits PRL secretion depending on the absence or presence of estrogen, respectively; 6- the inhibitory action of AII into the MPOA on PRL secretion does not seem to depend on NO. D 2003 Elsevier Inc. All rights reserved. Keywords: Angiotensin II; Medial preoptic area; Gonadotropin; Prolactin; Nitric oxide
* Corresponding author. Tel.: +55-16-602-3022; fax: +55-16-633-0017. E-mail address: [email protected] (C.R. Franci). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.049
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Introduction The discovery of a renin-angiotensin system in the brain (Ganten and Speck, 1978), the high content of angiotensin II (AII) in the hypothalamus and median eminence (Brownfield et al., 1982) and large number of AII receptors in the central nervous system and pituitary (Hauger et al., 1982) indicate that AII has a neuroendocrine action in the control of pituitary function. The action of AII on the brain and pituitary has been shown to alter the secretion of corticotropin, growth hormone, thyrotropin, prolactin (PRL) and gonadotropins (Franci et al., 1990a,b, 1997; Gaillard et al., 1981; Ganong, 1993; McCann, 1991; Phillips, 1987; Saavedra, 1992; Steele, 1992; Steele et al., 1982). Evidence has been accumulated for a central stimulatory action of AII on the control of luteinizing hormone-releasing hormone (LH-RH) / luteinizing hormone (LH) in female rats (Franci et al., 1990a,b, 1997; Steele, 1992; Dornelles and Franci, 1998b; Steele et al., 1985). Also, AII has a dual action on the control of PRL secretion, i.e. a central inhibitory action and a stimulatory action in the pituitary (McCann, 1991; Saavedra, 1992; Steele, 1992). Estrogen facilitated and losartan, an antagonist of AT1 subtype AII receptors, blocked the AII stimulatory and inhibitory action in the medial preoptic area (MPOA) on LH and PRL secretion, respectively (Dornelles and Franci, 1998a). Nitric oxide (NO) acts as a neuromediator in the control of LH-RH secretion (Bonavera et al., 1993, 1994; Moretto et al., 1993; Rettori et al., 1993a,b). NO neurons are present in some hypothalamic nuclei, including important sites for LH-RH regulation such as the organum vasculosum laminae terminalis (OVLT), MPOA, median preoptic nucleus (MnPO), ventromedial hypothalamus (VMH), and arcuate nucleus / median eminence (ARC/ME). There was no double labeling for nitric oxide synthase (NOS) / LH-RH in the hypothalamic neurons. In the OVLT, MnPO and MPOA, LH-RH neurons have been frequently found surrounded by NOS neurons, indicating a possible contact between them (Aguan et al., 1996; Bhat et al., 1995; Grossman et al., 1994; Herbison et al., 1996). NO neurons stimulate the release of a large number of hypothalamic polypeptides, including LH-RH (Rettori et al., 1993a) and prolactin releasing factors (Rettori et al., 1994). NO participates in the control of the LH surge induced by steroids and mediates the stimulatory effect of excitatory amino acids on LH and PRL surges during estrous cycle (Bonavera et al., 1993, 1994). The aim of this work was to investigate the participation of MPOA endogenous NO on gonadotropin and PRL secretion mediated by estrogen and AII.
Material and methods Animals and treatments Female Wistar rats weighing 180–200 g were ovariectomized and kept under conditions of controlled temperature (22–24 jC) and luminosity (lights on from 7:00 to 19:00 h), with free access to water and food. After two weeks, a stainless steel guide cannula was implanted into the MPOA using a stereotaxic apparatus and the following coordinates: 0.8 mm lateral to the sagittal line; 2.2 mm anterior to the bregma; and 8.0 mm ventral to the skullcap. The cannula was fixed to the skull with two stainless steel screws and dental cement and fitted with a mandril to prevent obstruction. About one week after surgery and 24 h before the experiment, a silastic catheter was inserted into
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the jugular vein by a previously described technique (Harms and Ojeda, 1974) under anesthesia with 2.5% tribromoethanol (Aldrich-USA, 1 ml/100 g body weight, i.p.). The animals were injected subcutaneously with estradiol benzoate (Schering–Brazil, 25 Ag/day) or vehicle (corn oil) during three days before the experiment. The first two surgeries were performed under thiopental anesthesia (Abbott, 4.0 mg/100 g body weight, i.p.) and the animals received a prophylactic antibiotic dose (Veterinary pentabiotic, Fontoura-Whythe-Brazil). Experimental procedure The experiments were started between 8:00 and 9:00 h. Heparinized blood samples (0.6 ml) were collected through the jugular catheter 20 minutes before (basal), immediately before (time zero) and 15, 30 and 60 minutes after microinjection of AII (Sigma Chemical - USA, 100 pmoles) or saline microinjection (0.15 M NaCl) into the MPOA. After each blood collection, the removed volume was replaced with the same volume of isotonic saline. Plasma was obtained by refrigerated blood centrifugation and stored at 20 jC for later hormone determination by radioimmunoassay. Ten minutes before the second blood collection (time zero), Nw-nitro-L-arginine, (L-NNA, Sigma-USA, 10 ng/Al), an inhibitor of NOS, or saline (0.15 M NaCl) was microinjected into the MPOA. The brains were removed at the end of the experiment for confirmation of the cannula position in the MPOA. Only animals whose cannula was positioned in the MPOA were used for analysis of plasma hormones. Radioimmunoassay Plasma LH, FSH and PRL concentrations were determined by double antibody radioimmunoassay (RIA) using specific standards and antibodies of the National Institute of Diabetes and Digestive and Kidney Diseases (NIADDK, USA). The nonspecific antibody, anti-rabbit gammaglobulin, was produced in a goat in our laboratory. The smallest detectable doses were 0.08 ng/ml for the LH-RP3 standard, 0.2 ng/ml for the FSH-RP2 standard, and 0.2 ng/ml for the PRL-RP3 standard. The intra-assay coefficients of variations were 3.5%, 3% and 3% for LH, FSH and PRL, respectively. The interassay coefficients of variation were 15%, 10.7% and 11% for LH, FSH and PRL, respectively. Statistics The results were submitted to analysis of variance for repeated measures and to the Newman–Keuls multiple comparison test, with the level of significance set at p < 0.05.
Results Plasma LH was lower in ovariectomized animals treated with estradiol- OVX + E2 (Fig. 2) than in untreated animals - OVX (Fig. 1). This result shows the known negative feedback mechanism mediated by estradiol in gonadotropin release. Microinjection of L-NNA into the MPOA at time minus 10 minutes significantly reduced plasma LH at times 0 and 30 minutes in ovariectomized animals (Fig. 1) and this
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Fig. 1. Plasma luteinizing hormone (LH) before and after microinjection of saline (NaCl 0.15 M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15 M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in ovariectomized rats (OVX). Values are means F standard error. The number of animals in each group is in parenthesis (n = ). a p < 0.05 vs 20 minutes (group L-NNA), bp < 0.05 vs 20 minutes (group L-NNA /AII). Arrows: 1- first microinjection (at time 10 minutes) of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
Fig. 2. Plasma luteinizing hormone (LH) before and after microinjection of saline (NaCl 0.15 M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15 M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in estrogen - primed ovariectomized rats (OVX + E2). Values are means F standard error. The number of animals in each group is in parenthesis (n= ). ap < 0.05 vs 20 minutes (group L-NNA/AII), bp < 0.05 vs 20 minutes (group AII). Arrows: 1- first microinjection (at time 10 minutes) of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
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inhibitory action of L-NNA was not changed by AII microinjection. There was no difference in plasma LH between groups L-NNA and AII+ L-NNA. However, plasma LH was lower in these two groups than control group. Plasma LH was not changed by microinjection (at minus 10 minutes) of NaCl or L-NNA into the MPOA of ovariectomized animals treated with estradiol (Fig. 2). However, microinjection of AII at time zero caused a significant increase in plasma LH at 30 minutes compared to the minus 20 minutes time point or compared to the other groups. This LH secretion in response to AII microinjection into the MPOA of ovariectomized rats treated with estrogen was reduced by previous microinjection of LNNA. There was no significant difference in plasma LH between control, L-NNA and AII+ L-NNA groups. The microinjections of NaCl, L-NNA or AII, separately or in combination, did not change plasma FSH in ovariectomized animals treated or not with estrogen (data not shown). Fig. 4 shows that plasma PRL was higher in animals treated with estradiol than in untreated animals (Fig. 3), the known stimulatory action of estrogen on prolactin secretion. In ovariectomized animals, L-NNA microinjection into the MPOA at time minus 10 minutes reduced plasma PRL starting at time zero, while microinjection of AII at time zero significantly reduced plasma PRL at times 30 and 60 minutes (Fig. 3). The combination of L-NNA and AII also caused a reduction in plasma PRL similar to that caused by each of these substances separately. There was no significant
Fig. 3. Plasma prolactin (PRL) before and after microinjection of saline (NaCl 0.15 M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15 M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in ovariectomized rats (OVX). Values are means F standard error. The number of animals in each group is in parenthesis (n= ). ap < 0.05 vs 20 minutes (group AII), bp < 0.001 vs 20 e 0 minutes (group AII), cp < 0.05 vs 20 minutes (group L-NNA), dp < 0.05 vs 20 minutes (group L-NNA/ AII). Arrows: 1- first microinjection (at time 10 minutes) of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
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Fig. 4. Plasma prolactin (PRL) before and after microinjection of saline (NaCl 0.15M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in estrogen - primed ovariectomized rats (OVX + E2). Values are means F standard error. The number of animals in each group is in parenthesis (n= ). a p < 0.05 vs 20 minutes (group AII), bp < 0.05 vs 20 minutes (group L-NNA /AII), cp < 0.05 vs 60 minutes (group d 20, 0 and 15 minutes (group L-NNA). Arrows: 1- first microinjection 0(at time minus10 minutes) L-NNA), p < 0.001 vs of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
difference between the groups AII, L-NNA and L-NNA + AII that showed plasma PRL lower than control group. Microinjection of L-NNA into the MPOA at time minus 10 minutes significantly increased plasma PRL at times 30 and 60 minutes in ovariectomized animals treated with estrogen (Fig. 4). Thus, the results obtained suggest that the NO of the MPOA has an inhibitory action on PRL secretion at time 15 minutes compared to times minus 20 and 0 minutes in ovariectomized animals treated with estrogen. However, the stimulatory effect of L-NNA microinjection did not occur when AII was microinjected after it, causing a reduction in plasma PRL starting at time 15 minutes. The inhibitory effect of AII prevailed regardless of the blockade of NO.
Discussion Microinjection of L-NNA into the MPOA reduced plasma LH in ovariectomized rats but not in ovariectomized rats treated with estrogen. The increased LH secretion occurring in ovariectomized rats due to the suppression of the negative feedback action of estrogen involves the stimulatory action of NO on the MPOA. Treatment of ovariectomized rats with estrogen (replacement of the negative feedback action of estrogen) caused a reduction in plasma LH, which was not changed by NOS blockade. Thus, it appears that estrogen reduces LH secretion (by negative feedback) in part by inhibiting the action of NO. NOS inhibition had no effect on ovariectomized rats treated with estrogen because the action of NO had probably, been suppressed by the steroid itself.
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Our experiments were conducted in the morning, i.e., outside the time of peak LH secretion induced by estrogen in ovariectomized rats. Thus the possible neutralization of the NOergic pathway seems to be related to the negative feedback mechanism of action of estrogen on LH secretion. The action of NO on the positive feedback mechanism appears to be different. NO has been shown to have a stimulatory action on the peak of LH secretion induced by steroids in ovariectomized rats and to mediate the stimulatory effect of excitatory amino acids on phasic LH release by stimulating LH-RH release (Bonavera et al., 1993). According to a previously proposed model based on studies with medial basal hypothalamus (MBH), LH-RH release is under the control of NO, GABA and noradrenaline, among other neuromediators. NO may have a direct stimulatory action on LH-RH neurons and an indirect inhibitory action via gabaergic neurons (Seilicovich et al., 1995). A study of the excitatory amino acids action on the control of LH secretion did propose a model involving excitatory neurotransmitters with an accelerating function and inhibitory neurotransmitters with a braking function regulated by steroid hormones. The opioid neurons would be the major component of the inhibitory brake, which would also involve the participation of GABA and of neuropeptide K. The accelerating mechanism would consist of glutamate, nitric oxide, neuropeptide Y, catecholamines, and galanin. The preovulatory surge of LH would occur due to the action of steroid hormones inhibiting the components of the brake and permitting the activation of the accelerating components (Brann and Mahesh, 1997). In addition, leptin has stimulating action on LH release probably mediated by NO (Borowiec et al., 2002; McCann et al., 2003). The preoptic area has been classically identified as a site for the control of cyclic secretion and the MBH as a site for the control of tonic secretion of gonadotropin in female rats (Halasz and Gorski, 1967). Estrogen receptive interneurons were shown necessary to communicate estrogen signals to LHRH neurons. There was no detection of estrogen receptor-alpha expression in LHRH neurons in preoptic area, only in surrounding neurons (Herbison, 1998; Herbison and Theodosis, 1992). However, estrogen receptor-beta immunoreactivity was identified in LH-RH neurons in preoptic and the authors discuss the involvement of estrogen receptor-beta in negative feedback regulation of the expression of LH-RH gene (Hrabovszky et al., 2000a,b). Our results support the hypothesis of negative feedback action of estrogen on the preoptic area and this action could be mediated by NO. Then, in addition to a possible direct action of estrogen on LH-RH neurons through estrogen receptors-beta, as previously reported (Hrabovszky et al., 2000a,b), there may also be an indirect action through the inhibition of NO. The LH-RH neurons in the preoptic area do not have NOS but are surrounded by neurons that release NO (Bhat et al., 1995). These neurons free of the estrogen suppressor action could stimulate LH-RH neurons in ovarietomized rats. NOS inhibition would reduce LH secretion, as was the case in our experiments. The decrease of LH secretion provoked by L-NNA was not modified by AII in ovariectomized rats. However, the stimulatory action of AII in the MPOA of ovariectomized rats treated with estrogen on LH secretion was reduced by blockade of NO synthesis by L-NNA.It was verified that stimulatory or inhibitory effects of AII on LH secretion depend on gonadal steroids (Steele et al., 1985). The effect of AII is fleeting in absence of estrogen as refereed in earlier study (Dornelles and Franci, 1998a,b) and in present study. The stimulatory action of AII in the MPOA on LH secretion demonstrated previously (Dornelles and Franci, 1998a,b; Steele, 1987, 1992) occurs through AT1 receptors since losartan inhibits this response (Dornelles and Franci, 1998a). The decrease of this response by L-NNA indicates that, at least, part of the AII effect may be mediated by NO.
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We detected no changes in FSH secretion related to microinjection of AII or NO antagonist in the MPOA. Other studies also found no relationship between FSH secretion and the cerebral action of AII (Franci et al., 1990a,b; Dornelles and Franci, 1998a,b) or NO (Rettori et al., 1993a). However, a possible stimulatory effect of NO on the pituitary for FSH secretion did occur when dispersed cells of pituitaries from male rats, cycling female rats and lactating females were placed in contact with sodium nitroprusside (Gonzalez and Aguilar, 1999). Thus, it seems that NO has an action on the pituitary but not on the brain for FSH secretion. Furthermore, our results represent further evidence for a differentiated control of gonadotropin secretion as discussed by other authors (McCann et al., 1983; Yu et al., 1990, 1997). In the present study we observed that plasma PRL was reduced by microinjection of AII or of an inhibitor of NO synthesis (LNNA) into the MPOA of ovariectomized rats, indicating a stimulatory action of NO. The inhibitory action of AII seems to be independent of NO of the MPOA since it was not altered by LNNA. Other investigators have shown that intracerebroventricular injection of sodium nitroprusside (a NO generator) increases plasma PRL in a dose-dependent manner in male rats because it inhibits the tyrosine hydroxilase activity in the median eminence (Gonzalez et al., 1998). NO modulates dopaminergic tubero-infundibular neurons activity and contributes for its inhibition on proestrous afternoon (Yen and Pan, 1998). On the other hand, AII reduced while LNNA increased PRL secretion in ovariectomized rats treated with estrogen, indicating an inhibitory action of NO. The inhibitory action of AII was not altered by LNNA. The inhibitory action of AII in the MPOA, mediated by type AT1 receptors has been shown previously (Dornelles and Franci, 1998a,b) and our results show that it seems to be independent of NO in the MPOA. Dopaminergic neurons in arcuate nucleus present AT1 receptors whose mRNA expression is induced by estrogen and progesterone (Johren et al., 1997). AT1 receptors content is inversely related with PRL secretion (Seltzer et al., 1993). Therefore, AII and NO act on the MPOA through two distinct neural pathways for the control of PRL secretion under the conditions studied. There is no information in the literature about the action of NO in the MPOA for PRL secretion. Only some in vitro studies have shown that NO inhibits PRL release by the pituitary from male rats and by dispersed pituitary cells of male rats, cycling female rats and lactating female rats (Gonzalez and Aguilar, 1999; Duvilanski et al., 1995, 1996). Our results indicate that NO in the MPOA stimulates or inhibits PRL secretion in the absence or presence of estrogen, respectively. This dual action of NO permits a modulatory mechanism of PRL secretion that may function as a brake against a greater fall in PRL secretion due to the absence of estrogen or against a greater increase in the presence of estrogen. This does not rule out the estrogen actions on PRL secretion by other mechanisms that do not depend on NO. In conclusion: 1 - AII or NO antagonist in the MPOA does no change FSH secretion in estrogenprimed or umprimed ovarictomized; 2 - the increase in LH secretion after ovariectomy depends, at least in part, on the activity of NO in the MPOA; 3 - estrogen may have an indirect negative feedback action on LH-RH neurons in the MPOA through NO; 4 - the stimulatory action of AII in the MPOA on LH secretion in ovariectomized rats treated with estrogen depends on NO; 5 - the NO of the MPOA stimulates or inhibits PRL secretion depending on the absence or presence of estrogen, respectively; 6 - the inhibitory action of AII into the MPOA on PRL secretion does not seem to depend on NO.
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Acknowledgements We are grateful to Sonia A. Zanon for technical assistance and FAPESP and CNPq for financial support. References Aguan, K., Mahesh, V.B., Ping, L., Bhat, G., Brann, D.W., 1996. Evidence for a physiological role for nitric oxide in the regulation of the LH surge: Effect of central administration of antisense oligonucleotides to nitric oxide synthase. Neuroendocrinology 64, 449 – 455. Bhat, G.K., Mahesh, V.B., Lamar, C.A., Ping, L., Aguan, K., Brann, D.W., 1995. Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing hormone neurons and co-localization with N-methylD-aspartate receptors. Neuroendocrinology 62, 187 – 197. Bonavera, J.J., Sahu, A., Kalra, P.S., Kalra, S.P., 1994. Evidence in support of nitric oxide (NO) involvement in the cyclic release of prolactin and LH surges. Brain Research 660, 175 – 179. Bonavera, J., Sahu, A., Kalra, S.P., 1993. Evidence that nitric oxide may mediate the ovarian steroid induced luteinizing hormone surge involvement of excitatory amino acids. Endocrinology 133, 2481 – 2487. Borowiec, M., Wasilewska-Dziubinska, E., Chmielowska, M., Wolinska-Witort, E., Baranowska, B., 2002. Effects of leptin and neuropeptide Y (NPY) on hormones release in female rats. Neuroendocrinology Letters 23, 149 – 154. Brann, D.W., Mahesh, V.B., 1997. Excitatory aminoacid: Evidence for a role in the control of reproduction and anterior pituitary hormone secretion. Endocrine Reviews 18, 678 – 700. Brownfield, M.S., Reid, I.A., Ganten, D., Ganong, W.F., 1982. Differential distribution of immunoreactive angiotensin and angiotensin-converting enzyme in rat brain. Neuroscience 7, 1759 – 1769. Dornelles, R.C., Franci, C.R., 1998a. Action of AT1 subtype angiotensin II receptors of the medial preoptic area on gonadotropins and prolactin release. Neuropeptides 32, 51 – 55. Dornelles, R.C., Franci, C.R., 1998b. Alpha- but not beta-adrenergic receptors mediate the effect of angiotensin II in the medial preoptic area on gonadotropin and prolactin secretion. European Journal of Endocrinology 138, 583 – 586. Duvilanski, B.H., Zambruno, C., Lasaga, M., Pisera, D., Seilicovich, A., 1996. Role of nitric oxyde / cyclic GMP pathways in the inhibitory effect of GABA and dopamine on prolactin release. Journal of Neuroendocrinology 8, 909 – 913. Duvilanski, B.H., Zambruno, C., Seilicovich, A., Pisera, D., Lasaga, M., Dias, M.C., Belova, N., Rettori, V., McCann, S.M., 1995. Role of nitric oxide in control of prolactin release by the adenohypophysis. Proceedings of the National Academy of Sciences USA 92, 170 – 174. Franci, C.R., Anselmo-Franci, J.A., McCann, S.M., 1990a. Angiotensin II antiserum decreases luteinizing hormone-releasing hormone in the median eminence and preoptic area of the rat. Brazilian Journal of Medical and Biological Research 23, 899 – 901. Franci, C.R., Anselmo-Franci, J.A., McCann, S.M., 1990b. Opposite effects of central immunoneutralization of A II or atrial natriuretic peptide on luteinizing hormone release in ovariectomized rats. Neuroendocrinology 51, 683 – 687. Franci, C.R., Anselmo-Franci, J.A., McCann, S.M., 1997. Angiotensinergic neurons physiologically inhibit prolactin growth hormone and thyroid-stimulating hormone but not adrenocorticotropic hormone release in ovariectomized rats. Peptides 18, 971 – 976. Gaillard, R.C., Grossman, A., Gilles, G., Rees, L.H., Besser, G.M., 1981. Angiotensin II stimulates the release of ACTH from dispersed rat anterior pituitary cells. Clinical Endocrinology 15, 573 – 578. Ganong, W.F., 1993. Blood pituitary and brain rennin-angiotensin system and regulation of secretion of anterior pituitary. Frontiers in Neuroendocrinology 14, 233 – 249. Ganten, D., Speck, G., 1978. The brain renin-angiotensin system a model for the synthesis of peptides in the brain. Biochemistry Pharmacology 27, 2379 – 2389. Gonzalez, D., Aguilar, E., 1999. In vitro nitric oxide (NO) stimulates LH secretion and parcially prevents the inhibitory effect of dopamine on PRL release. Journal of Endocrinology Investigation 22, 772 – 780. Gonzalez, M.C., Llorente, E., Abreu, P., 1998. Sodium nitroprusside inhibits the tyrosine hydroxylase activity of the median eminence in the rat. Neuroscience Letters 204, 133 – 136. Grossman, A.B., Rosmanith, W., Kabegting, E., Cadd, G., Clifton, D., Steiner, R., 1994. The distribution of hypothalamic
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nitric oxide synthase mRNA in relation to gonadotropin-releasing-hormone hormone neurons. Journal of Endocrinology 140, R5 – R8. Halasz, B., Gorski, R., 1967. Gonadotropin hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus. Endocrinology 80, 608 – 622. Harms, P.G., Ojeda, S.R., 1974. A rapid and simple procedure for chronic cannulation of the rat jugular vein. Journal of Applied Physiology 36, 391 – 392. Hauger, R.L., Aquilera, G., Baukal, A., Catt, K.J., 1982. Characterization of angiotensin II receptors in the anterior pituitary gland. Mollecular Cell Endocrinology 25, 203 – 213. Herbison, A.E., Simonian, S., Norris, P., Emson, P., 1996. Relationship of neuronal nitrtic oxide synthase immunoreactivity to GnRH neurons in the ovariectomized and intact female rat. Journal of Neuroendocrinology 8, 73 – 82. Herbison, A.E., 1998. Multinodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocrine Reviews 19, 302 – 330. Herbison, A.E., Theodosis, D.T., 1992. Localization of estrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxilase, cholecystokinin or luteinizing hormone-releasing hormone in male and female rats. Neuroscience 50, 283 – 298. Hrabovszky, E., Shughrue, P.J., Merchenthaler, I., Hajszan, T., Carpenter, C.D., Liposits, Z., Petersen, S.L., 2000a. Detection of estrogen receptor- h messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of rat brain. Endocrinology 141, 3506 – 3509. Hrabovszky, E., Steinhauser, A., Barabas, K., Shughrue, P.J., Petersen, S.L., Merchenthaler, I., Liposits, Z., 2000b. Estrogen receptor- h immunoreactivity in luteinizing hormone-releasing hormone neurons of rat brain. Endocrinology 142, 3261 – 3264. Johren, O., Sanvitto, G.L., Egidy, G., Saavedra, J.M., 1997. Angiotensin II AT1 receptor mRNA expression is induced by estrogen and progesterone in dopaminergic neurons of the female rat arcuate nucleus. Journal of Neuroscience 17, 8283 – 8292. McCann, S.M., 1991. Neuroregulatory peptides. In: Motta, M. (Ed.), Brain Endocrinology, 1 – 29. McCann, S.M., Haens, G., Mastronardi, C., Walczewska, A., Karath, S., Rettori, V., Yu, W.H., 2003. The role of nitric oxide (NO) in control of LH-RH release that mediates gonadotropin release and sexual behaviour. Current Pharmaceutical Design 9, 381 – 390. McCann, S.M., Mizunuma, H., Samson, W.K., Lumpkin, M.D., 1983. Differential hypothalamic control of FSH secretion a review. Psychoneuroendocrinology 8, 299 – 308. Moretto, M., Lopez, E., Negro-Vilar, A., 1993. Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 133, 2399 – 2402. Phillips, M.I., 1987. Functions of angiotensin in the central nervous system. Annual reviews of Physiology 49, 413 – 445. Rettori, V., Belova, N., Dees, W.L., Nyberg, C., Gimeno, M., McCann, S.M., 1993a. Role of nitric oxide in control of luteinizing hormone-releazing hormone release in vivo and in vitro. Proceedings of the National Academy of Sciences USA 90, 10130 – 10134. Rettori, V., Belova, N., Gimeno, M., McCann, S.M., 1994. Inhibition of nitric oxide synthase in the hypothalamus blocks the increase in plasma prolactin induced by intraventricular injection of interleukin-1 alpha in the rat. Neuroimmunomodulation 1, 116 – 120. Rettori, V., Kamat, A., McCann, S.M., 1993b. Nitric oxide mediates the stimulation of luteinizing-hormone releasing hormone release induced by glutamic acid in vitro. Brain Research Bulletin 33, 501 – 503. Saavedra, J.M., 1992. Brain and pituitary angiotensin. Endocrine Reviews 13, 329 – 380. Seilicovich, A., Duvilanski, B.H., Pisera, D., Theas, S., Gimeno, M., Rettori, V., McCann, S.M., 1995. Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing g-aminobutyric acid. Proceedings of the National Academy of Sciences USA 92, 3421 – 3424. Seltzer, A., Tsutsumi, K., Shigematsu, K., Saavedra, J.M., 1993. Reproductive hormones modulate angiotensin II AT1 receptors in dorsomedial arcuate nucleus of the female rats. Endocrinology 133, 939 – 941. Steele, M.K., McCann, S.M., Negro-Vilar, A., 1982. Modulation by dopamine and estradiol of central effects of angiotensin II on pituitary hormone release. Endocrinology 111, 722 – 729. Steele, M.K., 1987. Effects of angiotensins injected into various brain areas on LH release in female rats. Neuroendocrinology 46, 401 – 405. Steele, M.K., 1992. The role of brain angiotensin II in the regulation of luteinizing hormone and prolactin secretion. Trends in Endocrinology and Metabolism 3, 295 – 301.
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Steele, M.K., Gallo, R.V., Ganong, W.F., 1985. Stimulatory or inhibitory effects of A II upon LH secretion in ovariectomized rats a function of gonadal steroids. Neuroendocrinology 40, 210 – 216. Yen, S.H., Pan, J.T., 1998. Progesterone advances the diurnal rhythm of tuberoinfundibular dopaminergic neuronal activity and the prolactin surge in ovariectomized estrogen-primed rats and in intact proestrus rats. Endocrinology 139, 1602 – 1609. Yu, W.H., Karath, S., Walczewska, A., Sower, S.A., McCann, S.M., 1997. A hypothalamic follicle stimulating hormonereleasing decapeptide in the rat. Proceedings of National Academy of Sciences USA 94, 9499 – 9503. Yu, W.H., Millar, R.P., Milton, S.C.F., Milton, R.C.L., McCann, S.M., 1990. Selective FSH-releasing activity of [D-Trp9] GAP 1 – 13 comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs. Brain Research Bulletin 25, 867 – 873.
Estrogen modulates the action of nitric oxide in the medial preoptic area on luteinizing hormone and prolactin secretion A.S. Moreno, C.R. Franci * Departamento de Fisiologia, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeira˜o Preto - SP, Brazil Received 2 June 2003; accepted 22 September 2003
Abstract Several substances work as neuromediators of the estrogen direct and indirect (throught glial cells or interneurons) action on luteinizing hormone- releasing hormone (LH-RH) neurons in medial basal hypothalamus and medial preoptic area (MPOA).Angiotensin II (AII) in the MPOA stimulates the LH and it inhibits PRL secretion in some situations. On the other hand, the effect of excitatory aminoacids on LH and PRL surges during proestrus as well LH surge induced by steroids depend on nitric oxide (NO). In the present study we investigated the participation of MPOA endogenous NO on gonadotropin and PRL secretion mediated by estrogen and AII. Plasma LH, FSH and PRL was determinated in estrogen primed and unprimed ovariectomized Wistar rats that received microinjection of AII or saline into the MPOA, associated or not with a previous microinjection of an inhibitor for NOS. Our results show the following: 1- there was no change in plasma FSH in estrogen- primed or umprimed ovarictomized related with microinjections of AII or NO antagonist in the MPOA; 2- the increase in LH secretion after ovariectomy depends on, at least in part, NO activity in the MPOA; 3- estrogen may have an indirect negative feedback action on LH-RH neurons in the MPOA through NO; 4- the stimulatory action of AII in the MPOA on LH secretion in ovariectomized rats treated with estrogen depends on NO; 5- NO in the MPOA stimulates or inhibits PRL secretion depending on the absence or presence of estrogen, respectively; 6- the inhibitory action of AII into the MPOA on PRL secretion does not seem to depend on NO. D 2003 Elsevier Inc. All rights reserved. Keywords: Angiotensin II; Medial preoptic area; Gonadotropin; Prolactin; Nitric oxide
* Corresponding author. Tel.: +55-16-602-3022; fax: +55-16-633-0017. E-mail address: [email protected] (C.R. Franci). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.049
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Introduction The discovery of a renin-angiotensin system in the brain (Ganten and Speck, 1978), the high content of angiotensin II (AII) in the hypothalamus and median eminence (Brownfield et al., 1982) and large number of AII receptors in the central nervous system and pituitary (Hauger et al., 1982) indicate that AII has a neuroendocrine action in the control of pituitary function. The action of AII on the brain and pituitary has been shown to alter the secretion of corticotropin, growth hormone, thyrotropin, prolactin (PRL) and gonadotropins (Franci et al., 1990a,b, 1997; Gaillard et al., 1981; Ganong, 1993; McCann, 1991; Phillips, 1987; Saavedra, 1992; Steele, 1992; Steele et al., 1982). Evidence has been accumulated for a central stimulatory action of AII on the control of luteinizing hormone-releasing hormone (LH-RH) / luteinizing hormone (LH) in female rats (Franci et al., 1990a,b, 1997; Steele, 1992; Dornelles and Franci, 1998b; Steele et al., 1985). Also, AII has a dual action on the control of PRL secretion, i.e. a central inhibitory action and a stimulatory action in the pituitary (McCann, 1991; Saavedra, 1992; Steele, 1992). Estrogen facilitated and losartan, an antagonist of AT1 subtype AII receptors, blocked the AII stimulatory and inhibitory action in the medial preoptic area (MPOA) on LH and PRL secretion, respectively (Dornelles and Franci, 1998a). Nitric oxide (NO) acts as a neuromediator in the control of LH-RH secretion (Bonavera et al., 1993, 1994; Moretto et al., 1993; Rettori et al., 1993a,b). NO neurons are present in some hypothalamic nuclei, including important sites for LH-RH regulation such as the organum vasculosum laminae terminalis (OVLT), MPOA, median preoptic nucleus (MnPO), ventromedial hypothalamus (VMH), and arcuate nucleus / median eminence (ARC/ME). There was no double labeling for nitric oxide synthase (NOS) / LH-RH in the hypothalamic neurons. In the OVLT, MnPO and MPOA, LH-RH neurons have been frequently found surrounded by NOS neurons, indicating a possible contact between them (Aguan et al., 1996; Bhat et al., 1995; Grossman et al., 1994; Herbison et al., 1996). NO neurons stimulate the release of a large number of hypothalamic polypeptides, including LH-RH (Rettori et al., 1993a) and prolactin releasing factors (Rettori et al., 1994). NO participates in the control of the LH surge induced by steroids and mediates the stimulatory effect of excitatory amino acids on LH and PRL surges during estrous cycle (Bonavera et al., 1993, 1994). The aim of this work was to investigate the participation of MPOA endogenous NO on gonadotropin and PRL secretion mediated by estrogen and AII.
Material and methods Animals and treatments Female Wistar rats weighing 180–200 g were ovariectomized and kept under conditions of controlled temperature (22–24 jC) and luminosity (lights on from 7:00 to 19:00 h), with free access to water and food. After two weeks, a stainless steel guide cannula was implanted into the MPOA using a stereotaxic apparatus and the following coordinates: 0.8 mm lateral to the sagittal line; 2.2 mm anterior to the bregma; and 8.0 mm ventral to the skullcap. The cannula was fixed to the skull with two stainless steel screws and dental cement and fitted with a mandril to prevent obstruction. About one week after surgery and 24 h before the experiment, a silastic catheter was inserted into
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the jugular vein by a previously described technique (Harms and Ojeda, 1974) under anesthesia with 2.5% tribromoethanol (Aldrich-USA, 1 ml/100 g body weight, i.p.). The animals were injected subcutaneously with estradiol benzoate (Schering–Brazil, 25 Ag/day) or vehicle (corn oil) during three days before the experiment. The first two surgeries were performed under thiopental anesthesia (Abbott, 4.0 mg/100 g body weight, i.p.) and the animals received a prophylactic antibiotic dose (Veterinary pentabiotic, Fontoura-Whythe-Brazil). Experimental procedure The experiments were started between 8:00 and 9:00 h. Heparinized blood samples (0.6 ml) were collected through the jugular catheter 20 minutes before (basal), immediately before (time zero) and 15, 30 and 60 minutes after microinjection of AII (Sigma Chemical - USA, 100 pmoles) or saline microinjection (0.15 M NaCl) into the MPOA. After each blood collection, the removed volume was replaced with the same volume of isotonic saline. Plasma was obtained by refrigerated blood centrifugation and stored at 20 jC for later hormone determination by radioimmunoassay. Ten minutes before the second blood collection (time zero), Nw-nitro-L-arginine, (L-NNA, Sigma-USA, 10 ng/Al), an inhibitor of NOS, or saline (0.15 M NaCl) was microinjected into the MPOA. The brains were removed at the end of the experiment for confirmation of the cannula position in the MPOA. Only animals whose cannula was positioned in the MPOA were used for analysis of plasma hormones. Radioimmunoassay Plasma LH, FSH and PRL concentrations were determined by double antibody radioimmunoassay (RIA) using specific standards and antibodies of the National Institute of Diabetes and Digestive and Kidney Diseases (NIADDK, USA). The nonspecific antibody, anti-rabbit gammaglobulin, was produced in a goat in our laboratory. The smallest detectable doses were 0.08 ng/ml for the LH-RP3 standard, 0.2 ng/ml for the FSH-RP2 standard, and 0.2 ng/ml for the PRL-RP3 standard. The intra-assay coefficients of variations were 3.5%, 3% and 3% for LH, FSH and PRL, respectively. The interassay coefficients of variation were 15%, 10.7% and 11% for LH, FSH and PRL, respectively. Statistics The results were submitted to analysis of variance for repeated measures and to the Newman–Keuls multiple comparison test, with the level of significance set at p < 0.05.
Results Plasma LH was lower in ovariectomized animals treated with estradiol- OVX + E2 (Fig. 2) than in untreated animals - OVX (Fig. 1). This result shows the known negative feedback mechanism mediated by estradiol in gonadotropin release. Microinjection of L-NNA into the MPOA at time minus 10 minutes significantly reduced plasma LH at times 0 and 30 minutes in ovariectomized animals (Fig. 1) and this
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Fig. 1. Plasma luteinizing hormone (LH) before and after microinjection of saline (NaCl 0.15 M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15 M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in ovariectomized rats (OVX). Values are means F standard error. The number of animals in each group is in parenthesis (n = ). a p < 0.05 vs 20 minutes (group L-NNA), bp < 0.05 vs 20 minutes (group L-NNA /AII). Arrows: 1- first microinjection (at time 10 minutes) of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
Fig. 2. Plasma luteinizing hormone (LH) before and after microinjection of saline (NaCl 0.15 M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15 M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in estrogen - primed ovariectomized rats (OVX + E2). Values are means F standard error. The number of animals in each group is in parenthesis (n= ). ap < 0.05 vs 20 minutes (group L-NNA/AII), bp < 0.05 vs 20 minutes (group AII). Arrows: 1- first microinjection (at time 10 minutes) of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
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inhibitory action of L-NNA was not changed by AII microinjection. There was no difference in plasma LH between groups L-NNA and AII+ L-NNA. However, plasma LH was lower in these two groups than control group. Plasma LH was not changed by microinjection (at minus 10 minutes) of NaCl or L-NNA into the MPOA of ovariectomized animals treated with estradiol (Fig. 2). However, microinjection of AII at time zero caused a significant increase in plasma LH at 30 minutes compared to the minus 20 minutes time point or compared to the other groups. This LH secretion in response to AII microinjection into the MPOA of ovariectomized rats treated with estrogen was reduced by previous microinjection of LNNA. There was no significant difference in plasma LH between control, L-NNA and AII+ L-NNA groups. The microinjections of NaCl, L-NNA or AII, separately or in combination, did not change plasma FSH in ovariectomized animals treated or not with estrogen (data not shown). Fig. 4 shows that plasma PRL was higher in animals treated with estradiol than in untreated animals (Fig. 3), the known stimulatory action of estrogen on prolactin secretion. In ovariectomized animals, L-NNA microinjection into the MPOA at time minus 10 minutes reduced plasma PRL starting at time zero, while microinjection of AII at time zero significantly reduced plasma PRL at times 30 and 60 minutes (Fig. 3). The combination of L-NNA and AII also caused a reduction in plasma PRL similar to that caused by each of these substances separately. There was no significant
Fig. 3. Plasma prolactin (PRL) before and after microinjection of saline (NaCl 0.15 M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15 M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in ovariectomized rats (OVX). Values are means F standard error. The number of animals in each group is in parenthesis (n= ). ap < 0.05 vs 20 minutes (group AII), bp < 0.001 vs 20 e 0 minutes (group AII), cp < 0.05 vs 20 minutes (group L-NNA), dp < 0.05 vs 20 minutes (group L-NNA/ AII). Arrows: 1- first microinjection (at time 10 minutes) of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
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Fig. 4. Plasma prolactin (PRL) before and after microinjection of saline (NaCl 0.15M) or Nw-nitro-L-arginine (L-NNA) at 10 minutes and saline (NaCl 0.15M) or angiotensin II (AII) at time zero into the medial preoptic area (MPOA) in estrogen - primed ovariectomized rats (OVX + E2). Values are means F standard error. The number of animals in each group is in parenthesis (n= ). a p < 0.05 vs 20 minutes (group AII), bp < 0.05 vs 20 minutes (group L-NNA /AII), cp < 0.05 vs 60 minutes (group d 20, 0 and 15 minutes (group L-NNA). Arrows: 1- first microinjection 0(at time minus10 minutes) L-NNA), p < 0.001 vs of NaCl or L-NNA; 2- second microinjection (at time 0 minutes) of NaCl or AII.
difference between the groups AII, L-NNA and L-NNA + AII that showed plasma PRL lower than control group. Microinjection of L-NNA into the MPOA at time minus 10 minutes significantly increased plasma PRL at times 30 and 60 minutes in ovariectomized animals treated with estrogen (Fig. 4). Thus, the results obtained suggest that the NO of the MPOA has an inhibitory action on PRL secretion at time 15 minutes compared to times minus 20 and 0 minutes in ovariectomized animals treated with estrogen. However, the stimulatory effect of L-NNA microinjection did not occur when AII was microinjected after it, causing a reduction in plasma PRL starting at time 15 minutes. The inhibitory effect of AII prevailed regardless of the blockade of NO.
Discussion Microinjection of L-NNA into the MPOA reduced plasma LH in ovariectomized rats but not in ovariectomized rats treated with estrogen. The increased LH secretion occurring in ovariectomized rats due to the suppression of the negative feedback action of estrogen involves the stimulatory action of NO on the MPOA. Treatment of ovariectomized rats with estrogen (replacement of the negative feedback action of estrogen) caused a reduction in plasma LH, which was not changed by NOS blockade. Thus, it appears that estrogen reduces LH secretion (by negative feedback) in part by inhibiting the action of NO. NOS inhibition had no effect on ovariectomized rats treated with estrogen because the action of NO had probably, been suppressed by the steroid itself.
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Our experiments were conducted in the morning, i.e., outside the time of peak LH secretion induced by estrogen in ovariectomized rats. Thus the possible neutralization of the NOergic pathway seems to be related to the negative feedback mechanism of action of estrogen on LH secretion. The action of NO on the positive feedback mechanism appears to be different. NO has been shown to have a stimulatory action on the peak of LH secretion induced by steroids in ovariectomized rats and to mediate the stimulatory effect of excitatory amino acids on phasic LH release by stimulating LH-RH release (Bonavera et al., 1993). According to a previously proposed model based on studies with medial basal hypothalamus (MBH), LH-RH release is under the control of NO, GABA and noradrenaline, among other neuromediators. NO may have a direct stimulatory action on LH-RH neurons and an indirect inhibitory action via gabaergic neurons (Seilicovich et al., 1995). A study of the excitatory amino acids action on the control of LH secretion did propose a model involving excitatory neurotransmitters with an accelerating function and inhibitory neurotransmitters with a braking function regulated by steroid hormones. The opioid neurons would be the major component of the inhibitory brake, which would also involve the participation of GABA and of neuropeptide K. The accelerating mechanism would consist of glutamate, nitric oxide, neuropeptide Y, catecholamines, and galanin. The preovulatory surge of LH would occur due to the action of steroid hormones inhibiting the components of the brake and permitting the activation of the accelerating components (Brann and Mahesh, 1997). In addition, leptin has stimulating action on LH release probably mediated by NO (Borowiec et al., 2002; McCann et al., 2003). The preoptic area has been classically identified as a site for the control of cyclic secretion and the MBH as a site for the control of tonic secretion of gonadotropin in female rats (Halasz and Gorski, 1967). Estrogen receptive interneurons were shown necessary to communicate estrogen signals to LHRH neurons. There was no detection of estrogen receptor-alpha expression in LHRH neurons in preoptic area, only in surrounding neurons (Herbison, 1998; Herbison and Theodosis, 1992). However, estrogen receptor-beta immunoreactivity was identified in LH-RH neurons in preoptic and the authors discuss the involvement of estrogen receptor-beta in negative feedback regulation of the expression of LH-RH gene (Hrabovszky et al., 2000a,b). Our results support the hypothesis of negative feedback action of estrogen on the preoptic area and this action could be mediated by NO. Then, in addition to a possible direct action of estrogen on LH-RH neurons through estrogen receptors-beta, as previously reported (Hrabovszky et al., 2000a,b), there may also be an indirect action through the inhibition of NO. The LH-RH neurons in the preoptic area do not have NOS but are surrounded by neurons that release NO (Bhat et al., 1995). These neurons free of the estrogen suppressor action could stimulate LH-RH neurons in ovarietomized rats. NOS inhibition would reduce LH secretion, as was the case in our experiments. The decrease of LH secretion provoked by L-NNA was not modified by AII in ovariectomized rats. However, the stimulatory action of AII in the MPOA of ovariectomized rats treated with estrogen on LH secretion was reduced by blockade of NO synthesis by L-NNA.It was verified that stimulatory or inhibitory effects of AII on LH secretion depend on gonadal steroids (Steele et al., 1985). The effect of AII is fleeting in absence of estrogen as refereed in earlier study (Dornelles and Franci, 1998a,b) and in present study. The stimulatory action of AII in the MPOA on LH secretion demonstrated previously (Dornelles and Franci, 1998a,b; Steele, 1987, 1992) occurs through AT1 receptors since losartan inhibits this response (Dornelles and Franci, 1998a). The decrease of this response by L-NNA indicates that, at least, part of the AII effect may be mediated by NO.
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We detected no changes in FSH secretion related to microinjection of AII or NO antagonist in the MPOA. Other studies also found no relationship between FSH secretion and the cerebral action of AII (Franci et al., 1990a,b; Dornelles and Franci, 1998a,b) or NO (Rettori et al., 1993a). However, a possible stimulatory effect of NO on the pituitary for FSH secretion did occur when dispersed cells of pituitaries from male rats, cycling female rats and lactating females were placed in contact with sodium nitroprusside (Gonzalez and Aguilar, 1999). Thus, it seems that NO has an action on the pituitary but not on the brain for FSH secretion. Furthermore, our results represent further evidence for a differentiated control of gonadotropin secretion as discussed by other authors (McCann et al., 1983; Yu et al., 1990, 1997). In the present study we observed that plasma PRL was reduced by microinjection of AII or of an inhibitor of NO synthesis (LNNA) into the MPOA of ovariectomized rats, indicating a stimulatory action of NO. The inhibitory action of AII seems to be independent of NO of the MPOA since it was not altered by LNNA. Other investigators have shown that intracerebroventricular injection of sodium nitroprusside (a NO generator) increases plasma PRL in a dose-dependent manner in male rats because it inhibits the tyrosine hydroxilase activity in the median eminence (Gonzalez et al., 1998). NO modulates dopaminergic tubero-infundibular neurons activity and contributes for its inhibition on proestrous afternoon (Yen and Pan, 1998). On the other hand, AII reduced while LNNA increased PRL secretion in ovariectomized rats treated with estrogen, indicating an inhibitory action of NO. The inhibitory action of AII was not altered by LNNA. The inhibitory action of AII in the MPOA, mediated by type AT1 receptors has been shown previously (Dornelles and Franci, 1998a,b) and our results show that it seems to be independent of NO in the MPOA. Dopaminergic neurons in arcuate nucleus present AT1 receptors whose mRNA expression is induced by estrogen and progesterone (Johren et al., 1997). AT1 receptors content is inversely related with PRL secretion (Seltzer et al., 1993). Therefore, AII and NO act on the MPOA through two distinct neural pathways for the control of PRL secretion under the conditions studied. There is no information in the literature about the action of NO in the MPOA for PRL secretion. Only some in vitro studies have shown that NO inhibits PRL release by the pituitary from male rats and by dispersed pituitary cells of male rats, cycling female rats and lactating female rats (Gonzalez and Aguilar, 1999; Duvilanski et al., 1995, 1996). Our results indicate that NO in the MPOA stimulates or inhibits PRL secretion in the absence or presence of estrogen, respectively. This dual action of NO permits a modulatory mechanism of PRL secretion that may function as a brake against a greater fall in PRL secretion due to the absence of estrogen or against a greater increase in the presence of estrogen. This does not rule out the estrogen actions on PRL secretion by other mechanisms that do not depend on NO. In conclusion: 1 - AII or NO antagonist in the MPOA does no change FSH secretion in estrogenprimed or umprimed ovarictomized; 2 - the increase in LH secretion after ovariectomy depends, at least in part, on the activity of NO in the MPOA; 3 - estrogen may have an indirect negative feedback action on LH-RH neurons in the MPOA through NO; 4 - the stimulatory action of AII in the MPOA on LH secretion in ovariectomized rats treated with estrogen depends on NO; 5 - the NO of the MPOA stimulates or inhibits PRL secretion depending on the absence or presence of estrogen, respectively; 6 - the inhibitory action of AII into the MPOA on PRL secretion does not seem to depend on NO.
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