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L Pinilla, M Tena-Sempere, R Aguilar and E Aguilar

OBJECTIVE: The stimulatory and inhibitory effects of N-methyl-D-aspartic acid (NMDA) and kainic acid on prolactin (PRL) secretion have been correlated with the serum prolactin concentrations before drug administration. In the present experiments, we analysed the role of NMDA and kainic acid in PRL secretion in females with different serum concentrations of PRL. METHODS: Hypoprolactinaemic females were obtained by ovariectomy or after administration of diethyldithiocarbamate (an inhibitor of dopamine-beta-hydroxylase). Chronic hyperprolactinaemia was induced by neonatal administration of testosterone or oestradiol and acute hyperprolactinaemia was induced either by administration of alpha-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase) or by ether exposure. To analyse the role of dopamine in the effects of NMDA, we measured pituitary concentrations of dopamine after NMDA treatment and the effects of pretreatment with domperidone. RESULTS: (1) NMDA, but not kainic acid, stimulated PRL release in cyclic females. This effect was independent of serum PRL concentrations and was not accompanied by a decrease in pituitary concentrations of dopamine. (2) NMDA did not change PRL secretion in neonatally androgenized females, whereas NMDA and kainic acid inhibited PRL release in neonatally oestrogenized females. The inhibitory effects of NMDA and kainic acid were blocked by domperidone. (3) Kainic acid inhibited PRL secretion in prepubertal hyper- and hypoprolactinaemic rats. (4) Hyperprolactinaemia induced by ether stress was counteracted by administration of NMDA and kainic acid. CONCLUSIONS: (a) NMDA has a dual effect on prolactin secretion that is independent of prior prolactin concentrations and of dopamine activity, but kainic acid is only inhibitory. (b) The stimulatory or inhibitory effects of NMDA and kainic acid on PRL secretion were not strictly related to basal PRL concentrations and necessarily involved a change in the secretion of prolactin releasing factors, as no correlations were observed between changes in pituitary concentrations of dopamine and serum PRL concentrations. (c) Females rendered hyperprolactinaemic by neonatal administration of testosterone or oestradiol responded differently after NMDA administration. (d) NMDA and kainic acid blocked the mechanisms involved in stress-induced PRL secretion.

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E Trimiño, L Pinilla and E Aguilar

We have analyzed the mechanisms involved in ovarian failure after administration of gonadotropin hormone-releasing hormone agonists (GnRH-A) or antagonists (GnRH-ANT). Ovarian and uterine weights, serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol and pituitary FSH and LH contents were measured in Wistar female rats injected from 1–15 or 90–104 days of age with the agonist d-Ala6-d-Gly10-GnRH or the GnRH-ANT Org. 30276. Vaginal opening, first estrous presentation, vaginal smears and reproductive capacity were also analyzed. In both neonatal and adult females GnRH-A induced pituitary desensitization and reduced ovarian and uterine weights and estradiol serum concentrations. Therefore, serum gonadotropin concentrations were increased in adults and decreased in neonatal females. Puberty occurrence and reproductive function remain unaltered after neonatal treatment with GnRH-A. In neonatal females, FSH and LH pituitary content and FSH serum concentrations decreased at the end of treatment with GnRH-ANT. The effects on LH and estradiol secretion depended on the pattern of treatment. Interestingly enough, both vaginal opening and first estrous presentation were precipitated by GnRH-ANT administration. Normal reproductive function was observed in adults. We conclude that:

(i) pituitary desensitization of receptors occurred in both neonatal and adult females after chronic administration of GnRH-A;

(ii) the ovarian failure observed in adults that is accompanied by increased serum concentrations of gonadotropins was probably due to an inhibitory effect of GnRH-A directly on the ovaries;

(iii) the blockade of GnRH action shortly after birth with GnRH-ANT precipitated the onset of puberty; possibly the antagonist blocks some suppressive effects of endogenous LHRH;

(iv) the effects of neonatal administration of GnRH-A or GnRH-ANT were transitory.

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L. Pinilla, F. J. López and E. Aguilar


Pituitary responsiveness to GHRH (1-29) NH2 (GHRH, 5 μg/kg iv) was analysed under sodium pentobarbital anesthesia (50 mg/kg ip), on days 30 and 90 in male rats orchidectomized or sham-operated 7 days earlier. Other groups of rats were orchidectomized or shamoperated on day 23 and tested on days 30, 45, 60 and 90. In the sham-operated animals, GHRH stimulated GH secretion on day 90, but not on day 30. GHRH-induced secretion was similar on days 45, 60 and 90 in orchidectomized and sham-operated animals. Orchidectomy on day 83 reduced this GHRH-induced GH secretion on day 90. In contrast, orchidectomy on day 23 enhanced the pituitary responsiveness to GHRH a week later. These results suggest that the increase in pituitary responsiveness to GHRH with age is independent of the testicular function and that the effect of orchidectomy depends on both the age of the rats at orchidectomy and the time elapsed between the orchidectomy and the administration of GHRH.

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E Aguilar, M Tena-Sempere and L Pinilla

The effect of different androgens and estradiol on pituitary responsiveness to growth hormone releasing hormone was studied in intact and orchidectomized adult male Wistar rats, by injecting subcutaneously immediately after orchidectomy for two weeks with testosterone, dihydrotestosterone, 5-α androstane, 3-α, 17 β-diol or estradiol dissolved in olive oil (in doses of 0.2 or 2.0 mg·kg−1·day−1) or vehicle. Pituitary responsiveness was tested in pentobarbital anaesthetized rats by measuring growth hormone plasma levels at different times after administration of growth hormone releasing hormone (1-29) NH2. We found that: (a) High doses of testosterone, dihydrotestosterone and 5-α androstane, 3α, 1 7 β-diol restored gonadotropin plasma concentrations and organ weights altered by orchidectomy; (b) both pituitary growth hormone content and concentration remained unaffected after orchidectomy or androgen replacement and decreased significantly after estradiol injection; (c) orchidectomy significantly reduced growth hormone-stimulated growth hormone releasing hormone secretion; (d) treatment with 5-α androstane, 3-α, 1 7 β-diol increased more than testosterone or dihydrotestosterone both the peak concentration and the mean growth hormone secretion after growth hormone releasing hormone stimulation: (e) no differences were observed in the treatment with testosterone or dihydrotestosterone; (f) estradiol given at a dose of 0.2 mg·kg−1·day−1 increased pituitary responsiveness to growth hormone releasing hormone. These results demonstrated that testosterone and 5-α androstane, 3-α, 17 β-diol, which do not differ in their action on pituitary growth hormone content, increased the pituitary responsiveness to growth hormone releasing hormone differently and that the low pituitary responsiveness to growth hormone releasing hormone previously described in prepubertal animals was not due mainly to the secretion of 5-α androstane, 3-α, 1 7 β-diol.

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M Tena-Sempere, L Pinilla and E Aguilar

Tena-Sempere M, Pinilla L, Aguilar E. Orchidectomy selectively increases follicle-stimulating hormone secretion in gonadotropin-releasing hormone agonist-treated male rats. Eur J Endocrinol 1995;132: 357–62. ISSN 0804–4643

The pituitary component of the feedback mechanisms exerted by testicular factors on gonadotropin secretion was analyzed in adult male rats treated with a potent gonadotropin-releasing hormone (GnRH) antagonist. In order to discriminate between androgens and testicular peptides, groups of males were orchidectomized (to eliminate androgens and non-androgenic testicular factors) or injected with ethylene dimethane sulfonate (EDS), a selective toxin for Leydig cells (to eliminate selectively androgens) and treated for 15 days with vehicle or the GnRH antagonist Ac-d-pClPhe-d-pClPhe-d-TrpSer-Tyr-d-Arg-Leu-Arg-Pro-d-Ala-NH2CH3COOH (Org.30276, 5 mg/kg/72 hours). Serum concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured 7 and 14 days after the beginning of treatment. We found that: in males treated with GnRH antagonist, orchidectomy or EDS treatment did not induce any increase in LH secretion; and orchidectomy, but not EDS treatment, increased FSH secretion in GnRH-treated males. The present results show that negative feedback of testicular factors on LH secretion is mediated completely through changes in GnRH actions. In contrast, a part of the inhibitory action of the testis on FSH secretion is exerted directly at the pituitary level. It can be hypothesized that non-Leydig cell testicular factor(s) inputs at different levels of the hypothalamic–pituitary axis in controlling LH and FSH secretion.

Manuel Tena-Sempere, Department of Physiology, Faculty of Medicine, University of Córdoba, 14004 Córdoba, Spain

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F. López, D. Gónzalez and E. Aguilar

Abstract. To analyze a possible direct action of serotonin (5-hydroxytryptamine) at pituitary level in GH secretion, two experimental models were used: hypophysectomized autografted rats and perifused pituitaries. Adult male rats were hypophysectomized and their own pituitaries placed under the right kidney capsule. Ten days later an intra-atrial cannula was inserted. The next day, blood samples were obtained before and every 10 min during a 2 h period after the injection of saline or 5-hydroxytryptamin (1 or 2 mg/kg iv). Plasma volume was replaced with saline. Both doses of 5-hydroxytryptamine elicit a strong release of GH, the effect being dose-dependent. In pituitaries perifused with 5-hydroxytryptamine (100 μm during 115 min or 1, 10 and 100 μm during 15 min), a significant release of GH was also observed. These results suggested that 5-hydroxytryptamine may stimulate GH secretion through a direct pituitary action.

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F Gaytan, C Bellido, R Aguilar, C Morales, N van Rooijen and E Aguilar

Nitric oxide (NO) is generated from the guanidine group of L-arginine by NO synthases (NOS) in a wide variety of tissues, including endocrine organs. In order to discriminate between central and local effects of NO-related agents on the pituitary-testicular axis, adult rats were injected intraperitoneally with 1 g/kg body weight (bw) L-arginine methyl ester (L-AME, an exogenous substrate of NOS), 0.5 mg/kg bw sodium nitroprusside (SNP, an NO donor) or vehicle (0.9% NaCl) or intratesticularly with 2 mg/testis L-AME, 2 micrograms/testis SNP or 25 microliters vehicle, and killed at 60 or 120 min after treatment. Both intraperitoneal and intratesticular administration of L-AME had the same effects: a decrease in the serum concentrations of LH and testosterone and in those of testosterone in the testicular interstitial fluid. However, treatment with SNP was more effective when given intratesticularly, inducing a decrease in serum and interstitial fluid testosterone concentrations, without significant changes in LH concentrations. Furthermore, when rats were injected intraperitoneally with 4 mg L-AME (the same dose as that given intratesticularly), serum LH concentrations were not changed. In addition, L-AME administration was not effective in modifying serum LH concentrations in castrated rats. To test the possible role of Leydig cells, the effects of systemic administration of L-AME were studied in rats depleted of Leydig cells by treatment with ethylene dimethane sulphonate. In these animals L-AME significantly decreased serum LH concentrations. To study the role of macrophages in this system, rats depleted of testicular macrophages by the liposome-suicide approach were injected intraperitoneally (1 g/kg bw) or intratesticularly (2 mg/testis) with L-AME or vehicle, 10 days after macrophage depletion, and killed at 120 min after treatment. The effects of L-AME on serum LH concentrations were blocked when the drug was administered intratesticularly.

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E Aguilar, M Tena-Sempere, R Aguilar, D Gonzalez and L Pinilla

The role of N-methyl-D-aspartate (NMDA) in the control of prolactin (PRL) secretion was analysed in prepubertal male rats. In experiment 1, males of different ages were decapitated after administration of NMDA or vehicle. In experiment 2, 30-day-old males were killed at different times after administration of vehicle, NMDA, MK-801 (a non-competitive NMDA antagonist) or NMDA plus MK-801. In experiment 3, 23-day-old males were sham-orchidectomized or orchidectomized. Orchidectomized males were or were not implanted with Silastic capsules containing different amounts of testosterone. On day 30, the animals were decapitated after administration of vehicle, NMDA or MK-801. In experiment 4, 30-day-old male rats were decapitated after being injected with vehicle, NMDA, Nw-nitro-L-arginine methyl ester (NAME) (an inhibitor of nitric oxide (NO) synthase), or NMDA plus NAME. Serum PRL concentrations, and dopamine pituitary and hypothalamic content were measured. In experiment 5, males pretreated with vehicle or NAME were killed after administration of the precursor of serotonin synthesis 5-hydroxytryptophan (5-HTP), the 5-HT1 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) or the 5-HT2 agonist (+/-) 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). Finally, the effects of NMDA, NAME and sodium nitroprusside (SNP) were tested in dispersed adenohypophyseal cells. We found that: (1) antagonism of NMDA receptors with MK-801 decreased PRL secretion in intact, orchidectomized and orchidectomized-testosterone treated male rats; (ii) NMDA inhibited PRL release in vivo through an increase in dopamine release and this effect was potentiated by NAME and prevented by testosterone; (iii) NMDA inhibited PRL, secretion in vitro and this effect was observed in presence of both SNP and NAME; (iv) NAME blocked the stimulatory effects of 5-HTP and DOI on PRL secretion. We conclude that endogenous glutamate stimulates PRL release and that NO might have a pivotal role in the mechanisms involved in the control of PRL release, inhibiting the release of dopamine and modulating the effects of NMDA and 5-HT.

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L Pinilla, LC Gonzalez, M Tena-Sempere and E Aguilar

OBJECTIVE: Excitatory amino acids and serotonin are involved in the control of gonadotropin secretion. The actions of these neurotransmitters are interconnected and recently we have reported that 5-HT(1) and 5-HT(2) receptor agonists blunted (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal rats. The present experiments were carried out to analyze the effects of activation of different 5-hydroxytryptamine (5-HT) receptor subtypes on gonadotropin secretion and their role in the N-methyl-d-aspartate (NMDA)-stimulated LH release. DESIGN AND METHODS: We analyzed the gonadotropin secretion after manipulation of serotoninergic and aminoacidergic systems and their interactions in 5-, 16- and 23-day-old male and female rats. To this end, serum LH and FSH concentrations were measured in rats treated with 5-hydroxytryptophan methyl ester (5-HTP) (a precursor of 5-HT synthesis) plus Fluoxetine (Fx, a blocker of 5-HT reuptake), d,l-p-chlorophenyl-alanine methyl ester (PCPA, a blocker of 5-HT synthesis), R-(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of 5-HT(1A) receptors), (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT, agonists of 5-HT(2) receptors), and 1-Phenylbiguanide (1-PHE an agonist of 5-HT(3) receptors). In addition, the effects of 8-OH-DPAT and DOI on NMDA-stimulated LH secretion were analyzed. RESULTS: Neither the activation nor blockade of the serotoninergic system modified LH secretion. Basal gonadotropin secretion remained unchanged in 23-day-old male and female rats after activation of 5-HT(1A), 5-HT(2) and 5-HT(3) receptors. The stimulatory effect of NMDA on LH secretion was blocked in both sexes after activation of the serotoninergic system, through specific 5-HT(1) and 5-HT(2) receptor agonists. CONCLUSIONS: Activation of serotoninergic receptors decreased the stimulatory effect of NMDA on LH secretion in prepubertal male and female rats.

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M Tena-Sempere, ML Barreiro, E Aguilar and L Pinilla

OBJECTIVE: Raloxifene is a non-steroidal selective estrogen receptor modulator (SERM) that mimics estrogenic activity on bone density and blood lipid concentration without uterotropic actions. Previous data from our laboratory indicated that, as is the case for estrogen, neonatal administration of raloxifene disturbed normal differentiation of the hypothalamic circuitries governing the gonadotropic axis. In contrast, raloxifene did not act in the same way as estrogen does on the neuronal systems controlling sexual receptivity in the female rat. At present, however, the mechanisms for these organizing effects of raloxifene are not completely elucidated. DESIGN AND METHODS: To analyze this phenomenon, female rats were injected daily with raloxifene (50, 100, 250 or 500 microg/rat per day) between days 1 and 5 of age. On day 23, hypothalamic gonadotropin-releasing hormone (LHRH) mRNA expression was assessed, and pituitary and plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured in basal and LHRH-stimulated conditions. In addition, LH and FSH responses to ovariectomy were evaluated in raloxifene-treated females. Finally, we monitored the ability of neonatal administration of a potent LHRH agonist ([d-Ala(6),d-Gly(10)]-LHRH ethylamide; 0.01 microg/kg per 12 h on days 1-5) to counteract the effects of raloxifene. RESULTS: Our analyses demonstrated that prepubertal rats (23-day-old females) treated neonatally with raloxifene showed decreased hypothalamic LHRH mRNA expression levels, reduced pituitary content of LH and FSH, reduced basal and LHRH-stimulated LH secretion in vivo and in vitro, and decreased response to ovariectomy. In addition, adult females treated neonatally with raloxifene showed anovulation and reduced serum LH levels; these effects were not prevented by the simultaneous administration of a LHRH agonist. CONCLUSION: In conclusion, our data demonstrate that neonatal administration of raloxifene can disrupt the programming of hypothalamic-pituitary-ovarian axis function. Reduced LH secretion, under basal and LHRH-stimulated conditions and after ovariectomy, is probably related to decreased LHRH expression, reduced pituitary LH content and/or decreased pituitary responsiveness to hypothalamic LHRH.