E. Aguilar

LEPTIN116-130 STIMULATES PROLACTIN AND LUTEINIZING HORMONE SECRETION IN FASTED ADULT MALE RATS

Leptin is a hormone secreted by adipocytes with an important role in the control of feeding behavior and neuroendocrine function. Leptin stimulates in vivo LH secretion in fasted female rats and in vitro PRL secretion. Recent data indicate that leptin116–130, an active fragment of the native molecule, exerts effects similar to those of the native peptide on body weight and food intake. The present study was carried out to determine whether this fragment is also able to stimulate LH and PRL secretion. Adult male rats fasted for 5 days were injected with saline or leptin116–130 (15 µg i.c.v.) and LH and PRL concentrations were measured thereafter at 15-min intervals during a 150-min period. Administration of leptin116–130 increased the frequency of LH pulses (2.0 ± 0.26 vs. 1.20 ± 0.37/150 min; p ≤ 0.05), mean LH levels (0.24 ± 0.06 vs. 0.07 ± 0.03 ng/ml; p ≤ 0.05), LH pulse amplitude (0.33 ± 0.10 vs. 0.10 ± 0.05 ng/ml; p ≤ 0.05) and the net LH secretion estimated by area under curve (AUC: 36 ± 8.5 vs. 9 ± 3.9 ng/ml/150 min; p ≤ 0.01). In addition leptin116–130 increased the frequency of PRL pulses (2.83 ± 0.48 vs. 1.33 ± 0.21/150 min; p ≤ 0.05), trough PRL levels (5.71 ± 0.99 vs. 2.72 ± 0.32 ng/ml; p ≤ 0.01), mean PRL levels (13.02 ± 0.92 vs. 5.89 ± 0.51 ng/ml; p ≤ 0.01) and net PRL secretion (AUC: 1,625 ± 171 vs. 658 ± 46 ng/ml/150 min; p ≤ 0.05). In conclusion, these data show that leptin116–130 stimulates LH and PRL secretion in fasted adult male rats.

Nitric Oxide Stimulates Growth Hormone Secretion in vitro through a Calcium- and Cyclic Guanosine Monophosphate-Independent Mechanism

In the last years, nitric oxide (NO) has emerged as an important intra- and intercellular transmitter involved in the control of the hypothalamic-pituitary axis, and NO synthase (NOS) has been identified in pituitary cells. To determine the role of NO in the control of GH secretion acting directly at the pituitary level, we have studied GH release by hemipituitaries incubated in the presence of different concentrations (10–7–10–3 M) of sodium nitroprusside (SNP), a potent NO donor. We found that SNP (10–4–10–3 M) stimulated GH release. This effect was mediated by the release of NO since it was abolished in the presence of hemoglobin, a scavenger of NO, but preserved in the presence of rhodanese + sodium thiosulfate (inactivators of cyanides generated from SNP). To analyze the participation of cyclic guanosine monophosphate (cGMP), the second messenger for a wide range of NO actions, in SNP-stimulated GH secretion, hemipituitaries were incubated in the presence of 8-bromo-cGMP (8-Br-cGMP; 10–7–10–3 M). In addition, hemipituitaries were stimulated with SNP plus oxadiazoloquinoxaline (OQD) or LY 83,583 (inhibitors of guanylyl cyclases). We found that 8-Br-cGMP was ineffective in eliciting GH release, and that the stimulatory effect of SNP was maintained in presence of OQD and LY 83,583. Finally, to analyze calcium dependence, the SNP effect was studied in hemipituitaries incubated in free medium calcium, in the presence of nifedipine and verapamil (blockers of calcium channels) and after depletion of intracellular Ca2+ stores with caffeine. We found that the SNP-induced GH secretion is also detected after incubation of hemipituitaries in free calcium medium, in the presence of nifedipine and verapamil, and after caffeine preincubation. We conclude that NO stimulates GH secretion in vitro through a specific calcium-cGMP-independent mechanism.

Regulation of Growth Hormone Secretion byα -Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Receptors in Infantile, Prepubertal, and Adult Male Rats1

Excitatory amino acids, such as glutamate, constitute a major transmitter system in the control of hypothalamic-pituitary function. Different subtypes of glutamate receptors, such as N-methyl-D-aspartic acid and kainate receptors, have been involved in the control of GH secretion. Other excitatory amino acid receptor subtypes, as alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), amino-4-phosphobutyric acid, and metabotropic receptors, have been identified, yet their role in the control of neuroendocrine function remains to be completely characterized. The purpose of this study was to assess the potential involvement of AMPA receptors in the control of GH secretion. In a first set of experiments, neonatal (5 and 10 days) and prepubertal (23 days) male rats were injected with AMPA (1, 2.5, or 5 mg/kg) or the antagonist of AMPA receptors, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg). Serum GH concentrations significantly increased 15 min after i.p. administration of AMPA in both neonatal and prepubertal male rats. In addition, serum GH concentrations decreased after NBQX treatment. The stimulatory effect of AMPA was abolished by pretreatment with the blocker of nitric oxide synthase, nitro(w)-arginine-methyl ester (40 mg/kg), and was partially counteracted by the simultaneous administration of GH-releasing hormone (500 microg/kg). Moreover, AMPA was unable to elicit in vitro GH secretion by hemipituitaries from prepubertal males, pointing out that the hypothalamus is probably the site of action for the reported stimulatory action of AMPA on GH release. In a second set of experiments, the effects of AMPA and NBQX were tested in adult male rats. As in prepubertal animals, AMPA significantly increased GH secretion in adult males, whereas NBQX (20 or 40 nmol), administered through intracerebroventricular injection, induced a significant decrease in the amplitude of GH pulses. In conclusion, our data indicate that AMPA receptors have a physiological stimulatory role in the control of GH secretion in male rats throughout the life span. This effect depends on appropriate nitric oxide synthesis during the prepubertal age. In addition, AMPA receptors appear to modulate pulsatile GH secretion in adulthood.

Possible Direct Effect of Serotonin on Pituitary Prolactin Secretion: in vivo and in vitro Studies

In this work we analyze the possibility of serotonin (5-HT)-releasing prolactin (PRL) through a direct action at the pituitary level. 5-HT (2 mg/kg i.v.) stimulates PRL secretion in hypophysectomized autotransplanted animals (HAG) significantly and this effect was not influenced by pretreatment with the dopaminergic antagonist domperidone. In perifused pituitaries, 5-HT administration (0.01, 0.1 and 1 microM for 90 min, or 1, 10, 100 microM for 15 min) was ineffective in stimulating PRL release. In pituitaries obtained from animals previously treated with the neurotoxic 5,7-dihydroxytryptamine (5,7-DHT) or vehicle and incubated in the presence of 5-HT (2.5, 5 and 10 microM), no response in PRL secretion was observed. These results suggested that 5-HT does not release PRL through a direct pituitary action, and that the effect observed in HAG animals could be mediated through the release of a PRL-releasing factor after 5-HT administration.

In vitro, nitric oxide (NO) stimulates LH secretion and partially prevents the inhibitory effect of dopamine on PRL release

In recent years nitric oxide (NO) has emerged as an important intra- and intercellular transmitter involved in the control of hypothalamic-pituitary axis. In order to discriminate the potential actions of NO at hypothalamic or pituitary level in the control of PRL and LH release, we have studied PRL and LH secretion by dispersed pituitary cells obtained from males, cycling and lactating females in the presence of 1) sodium nitroprusside (SNP), a NO donor; 2) cyclic guanosine monophosphate (cGMP), the second messenger for a wide range of NO actions; 3) Nw-nitro-L-arginine methyl ester (NAME), a competitive inhibitor of NO synthase (NOS) and 4) oxadialoquinoxalione (OQD) and LY 83,583, antagonists of guanylyl cyclases. We found that SNP (at doses of 100 and 500 μmol) stimulated LH and FSH release and partially blocked the inhibitory action of dopamine (50 and 100 nmol) on prolactin secretion. These effects were not mimicked by cGMP and remained in the presence of OQD and LY 83,583. NAME alone had no significant effect on hormone secretion. These results suggest that NO plays a role in the control of gonadotropins and prolactin secretion acting directly at the pituitary level and that these effects are mediated by mechanisms other than changes in cGMP levels.

Activation of AMPA receptors inhibits prolactin and estradiol secretion and delays the onset of puberty in female rats.

Previous experiments have evidenced the neuroendocrine role of AMPA receptors. Present studies were carried out to obtain information on the role of these receptors in the control of the onset of puberty. To this end, female rats were i.c.v. injected with vehicle or AMPA (agonist of AMPA receptors: 0.1 or 0.5 nmol/day) between 26 and 30 days (Experiment 1), or 30 and 34 days (Experiment 2) of age. Serum concentrations of PRL, LH and estradiol were measured before drug administration, 10 min after the last injection, at vaginal opening (VO) and at first estrus (FE) presentation. In both experiments, AMPA administration inhibited PRL and estradiol secretion without affecting LH release. When AMPA was administered between 26 and 30 days a significant delay in the day of vaginal opening was observed. These results confirmed the inhibitory effect of AMPA on PRL secretion and suggests a role of AMPA receptors in the control of puberty onset.

The role of nitric oxide in the control of basal and LHRH-stimulated LH secretion

The gaseous transmitter nitric oxide (NO) appears to be involved in the control of LH secretion and in the modulation of LH responses after stimulation with luteinizing hormone releasing hormone (LHRH), excitatory amino acids (EAAs) and leptin. The regulatory action of NO in the control of LH secretion includes modulation of LHRH release, changes in hypothalamic-pituitary blood flow and direct effects at pituitary level. To determine the net balance of these actions we evaluated (1) the effects of systemic administration of sodium nitroprusside (SNP, a NO donor) and Nw-nitro-L-arginine methyl ester (NAME, a blocker of NO synthase) on basal and LHRH-stimulated LH secretion in intact and ovariectomized females; and (2) the effects of SNP and NAME on LH secreted by dispersed pituitary cells. Finally, since NO is involved in the stimulatory effect of excitatory amino acids (EAAs) on LH secretion, we analyzed the effects of different inhibitors of NO synthase (NOS) in the LH response to kainic acid (KA), an agonist of kainate receptors, in male and female rats, neonatally injected with estradiol that show an increased sensitivity to EAAs. We found that NAME (40 and 60 mg/kg) increases LH secretion in intact and ovariectomized females, while SNP had no effect. The effect of NAME was not mediated through a direct action at pituitary level, since the basal and LHRH-stimulated LH release remained unchanged in presence of NAME. Similarly, basal and LHRH-stimulated LH secretion from dispersed pituitary cells were unaffected by NAME. Finally, the stimulatory effects of KA on LH release were not abolished by NOS inhibitors. In conclusion, our results provide evidence that the global action of NOS inhibitors is an increase in basal LH secretion, through a mechanism that remains to be fully characterized. In addition, our data demonstrate that the KA-stimulated LH secretion is not mediated by an increase in NO generation.

Role of serotoninergic receptors in gonadotropin secretion in male rats

Experimental data concerning the role of serotonin (5-HT) in the control of gonadotropins secretion remain controversial: different receptors subtypes should mediate the serotoninergic action. In the present work we analyzed in prepubertal and adult male rats the FSH and LH responses after administration of the following drugs: 5-hydroxy-L-tryptophan (5-HTP), precursor of 5-HT synthesis, the 5-HT1 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), the 5-HT2 receptor agonists α-methylserotonin maleate (α-CH3 -HT) and (±)-DOI hydrochloride (DOI), the 5-HT3 agonists, quipazine-N-methyldimaleate (QUIP), 2 methyl-serotonin maleate (2-CH3-HT), trimethylserotonin iodide (5-HTQ) and 1-phenylbiguanide (PHE). We found that: a) FSH secretion in adult males was stimulated by 5-HTP, inhibited by 5-HT2 agonists and unaffected by 5-HT3 agonists; b) LH secretion in adult males was stimulated by 5-HTP and α-CH3-HT, while DOI and 5-HT3 agonists were ineffective; c) in prepubertal males, 5-HT2 and 5-HT3 agonists inhibited LH release. All these results taken together suggest that: (1) 5-HT2 and 5-HT3 receptors are involved in the control of gonadotropin secretion; (2) FSH and LH secretion are differently modulated by agonists of 5-HT2 and 5-HT3 receptors; (3) the 5-HT2 agonists α-CH3-HT and DOI elicit different responses; and (4) the responses obtained vary with animal age, since the inhibitory effect of 5-HT2 and 5-HT3 agonists on LH secretion was observed only in prepubertal males.

Mechanism of reproductive deficiency in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) show multiple endocrine disorders. In the present work, specific reproductive modifications were analysed using normotensive Wistar-Kyoto rats (WKY) as controls. SHR showed delayed vaginal opening and first estrus presentation, regular vaginal cycles and released a normal number of ova each cycle. When compared with controls, SHR showed a decrease in the percentage of successful pregnancies (69% vs. 86% in WKY) and in the litter size (7.83 +/- 0.5 vs. 10.41 +/- 0.5). In SHR, progesterone plasma levels were significantly increased during the days 1-14 of pregnancy, and on the 5th day of pregnancy the plasma concentrations of LH but not of FSH were enhanced. Mortality during the first month of life was higher in SH (50%) than in control (24%) strain. When the SH females were mated with Wistar or WKY males, the percentage of pregnancies rose up to 95%. On the contrary, Wistar or WKY females mated with SH males showed a decrease in the percentage of pregnancies (62.5% and 50%, respectively). Besides, the litter sizes were significantly reduced in Wistar females mated with SH males. Newborn SH suckled immediately after birth from a WKY mother showed a significant reduction in the mortality during the first month of life (8% vs. 50%). In conclusion, our results suggest that changes in fertilization and/or implantation processes of SH rats were responsible for the reduced pregnancy rate, whereas the increased neonatal mortality could be due to lactation activity of SH mothers.

Ovarian Role in the Modulation of Pituitary Responsiveness to Growth Hormone-Releasing Hormone in Rats

The influence of ovarian function on the pituitary responsiveness to growth hormone-releasing hormone (GHRH) was studied by measuring GH levels in serum before and after the injection of GHRH 1-29 NH2 (5 micrograms/kg i.v.) in female Wistar rats under sodium pentobarbital anesthesia (50 mg/kg i.p.). In the first experiment, GHRH was administered to 90-day-old females in different phases of the estrous cycle (in the morning and afternoon of proestrus and in the morning of estrus, diestrus 1 and diestrus 2). In the second experiment, females were ovariectomized or sham-operated on day 23 and analyzed on days 30, 45, 60 and 90. Pituitary growth hormone (GH) content in females ovariectomized or sham-operated on day 23 was also analyzed at different ages. In the third experiment, females ovariectomized or sham-operated on day 83 were tested on day 90. Results showed that (a) pituitary responsiveness to GHRH changes throughout the estrous cycle is maximal in diestrus and minimal in proestrus; (b) GHRH stimulated GH secretion in intact and ovariectomized females on days 45, 60 and 90, but not on day 30; (c) the age increase of GH responsiveness to GHRH administration was slightly reduced in animals ovariectomized on day 23; (e) the age increase of pituitary GH content was similar in control and ovariectomized females; and (d) ovariectomy on day 83 reduced pituitary responsiveness to GHRH more effectively than ovariectomy on day 23.(ABSTRACT TRUNCATED AT 250 WORDS)