M. Tena-Sempere

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.

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.

Interactions between serotoninergic and aminoacidergic pathways in the control of PRL secretion in prepubertal male rats

Prolactin secretion is controlled by the hypothalamus through different neurotransmitters which interact with multiple receptor subtypes. The discovery of different families of receptors for serotonin (5-HT1-5-HT7) and excitatory aminoacids (NMDA,KA,AMPA and metabotropic receptors) ilustrates the complexity of this regulation. Moreover, in the rat the role of different neurotransmitters changes during pubertal development. Present experiments were carried out to analyse the interactions between AMPA and serotoninergic receptors in the control of prolactin secretion in prepubertal male rats. For this purpose, 16 and 23-day old male rats were treated with 5-hydroxytryptophan (5-HTP, precursor of serotonin synthesis) plus fluoxetine (blocker of serotonin reuptake), 8-OH-DPAT (agonist of 5-HT1A receptors), DOI and α-Me-5-HT (agonists of 5-HT2 receptors), 1-phenylbiguanide (agonist of 5-HT3 receptors) alone or in combination with AMPA (agonist of AMPA receptors). The results obtained indicate that: (a) activation of 5-HT1A receptors stimulated PRL secretion on day 16 and inhibited it on day 23; activation of 5-HT2 receptors stimulated PRL secretion on days 16 and 23, whereas activation of 5-HT3 receptors inhibited PRL release only on day 23; (b) activation of AMPA receptors inhibited PRL secretion on day 23, but not on day 16 and (c) a cross-talk is apparent between 5-HT2 and AMPA receptors in the regulation of PRL secretion, the stimulatory effect of DOI being blocked by AMPA.ResumenLa secreción de prolactina se controla por el hipotálamo a través de diferentes neurotransmisores que interactúan con múltiples subtipos de receptores. El descubrimiento de diferentes receptores para serotonina agrupados en siete familias y aminoácidos excitatorios (NMDA, KA, AMPA y receptores metabotrópicos) ilustran la complejidad de esta regulación. Además, el papel de los diferentes neurotransmisores cambia en la rata durante el desarrollo puberal. Los experimentos se llevan a cabo para analizar las interacciones entre los receptores AMPA y los receptores serotoninérgicos en el control de la secreción de prolactina en ratas macho prepúberes. Para ello, machos de 16 y de 23 días de edad se tratan con 5-hidroxitriptófano (5-HTP, precursor de la síntesis de serotonina) más fluoxetina (bloqueante de la recaptación de serotonina), 8-OH-DPAT (agonista de los receptores 5-HT1A), DOI y α-Me-5-HT (agonistas de los receptores 5-HT2) 1-fenilbiguanida (agonista de los receptores 5-HT3) solos o en combinación con AMPA (agonista de los receptores AMPA). Los resultados obtenidos indican que: (a) la activación de los receptores 5-HT1A estimula la secreción de PRL el día 16 y la inhibe el día 23; la activación de los receptores 5-HT2 estimula la secreción de PRL el día 16 y 23, mientras que la activación de los receptores 5-HT3 inhibe la secreción de PRL sólo el día 23; (b) la activación de los receptores AMPA inhibe la secreción de PRL el día 23, pero no el día 16; y (c) hay una interrelación aparente entre los receptores 5-HT2 y los receptores AMPA en el control de la regulación de la secreción de PRL, como lo demuestra el hecho de que el efecto estimulador del DOI es bloqueado por AMPA.

Raloxifene effects upon the neuronal system controlling sexual receptivity in female rats

Selective estrogen receptor modulators (SERMs) constitute a new family of drugs with growing interest in the management of estrogen-associated pathology. Raloxifene is a SERM that mimics estrogen action on bone and blood lipid concentration but whether it acts as estrogen in the central nervous system remains to be fully established. In the present communication, we aimed at evaluating the estrogenic/antiestrogenic effects of raloxifene upon organization and activation of sexual receptivity, an estrogen-dependent event, in female rats. To this end, the effects of raloxifene, administered during the neonatal period, were compared with those of estrogen in terms of disruption of sexual receptivity in estrogen-progesterone-primed ovariectomized (OVX) female rats. In addition, the ability of raloxifene to induce sexual receptivity in progesterone-primed OVX females was analyzed. Similarly, the effects of the combined administration of estrogen and raloxifene were studied. Our results suggest that raloxifene does not act as estrogen upon the organization of the neuronal system involved in the control of sexual receptivity in female rats and exerted an antiestrogenic action in adult OVX estrogen-primed female rats.

Interactions between GABAergic and aminoacidergic pathways in the control of gonadotropin and GH secretion in pre-pubertal female rats

Present experiments were carried out in 23-day-old female rats to analyze the interaction between excitatory amino acids (EAAs) and gamma-aminobutyric acid (GABA) in the control of gonadotropin and GH secretion. For this purpose, serum concentrations of LH, FSH and GH were measured after injection of different agonists of EAA receptor subtypes [N-methyl-D-aspartate (NMDA); kainic acid (KA), ±α-amino-3-hydroxy- 5-methylisoxazole-4-propionic acid (AMPA)], antagonists of GABA receptors (bicuculline, phaclofen) or the combined administration of both types of drugs. The results obtained indicated that: 1) GABA has a minor physiological role in the control of LH and GH secretion, since neither LH nor GH serum concentrations changed after administration of bicuculline (antagonist of GABAA receptors) or phaclofen (antagonist of GABAB receptors); 2) GABA has a sex-specific physiological role in the control of FSH secretion in female rats, in which FSH secretion increases after phaclofen administration; 3) GH secretion was enhanced after administration of NMDA, KA and AMPA, while LH increased only after activation of NMDA receptors; 4) the stimulatory effect of NMDA on LH secretion was counteracted by administration of phaclofen; and 5) bicuculline and phaclofen reduced the ability of NMDA and AMPA to stimulate GH secretion. In conclusion, present experiments evidenced a physiological role of GABA, mediated by GABAB receptors, in the control of FSH secretion and a cross-talk between excitatory and inhibitory amino acids in the control of anterior pituitary secretion.

The pattern of testosterone replacement influences the recovery of the stimulatory effect of clonidine on growth hormone (GH) secretion in orchidectomized rats

It has previously been described that the growth hormone (GH) releasing effect of clonidine (CLO), an agonist of alpha2-adrenoreceptors, disappears after orchidectomy and is restored by testosterone replacement when started immediately after orchidectomy. In the present experiments, the effects of CLO on GH release was analysed in long-term (LTO; 12 weeks) and short-term (STO; 2 weeks) orchidectomized rats. In the first experiment, LTO males were implanted with silastic capsules containing testosterone 10 weeks after orchidectomy and killed 2 weeks later, 15 min after injection of CLO (150 microg/kg) or vehicle. In the second experiment, adult males were implanted with testosterone at the moment of orchidectomy and decapitated 2 or 12 weeks later, 15 min after vehicle or CLO administration. In addition, in order to evaluate the effects of orchidectomy and androgen replacement on alpha2 agonists GH release further, prepubertal males (21-days-old) implanted with testosterone or 5-alpha-androstane-3-alpha,17beta diol (alpha-diol) at the moment of orchidectomy were killed 2 weeks later, 15 min after ketamine-xylazine (an alpha2 agonist) administration. Finally, 10-day-old males (orchidectomized 72 h before) were decapitated 15 min after CLO or vehicle administration. Our results show that: (a) LTO and STO abolished the stimulatory effect of clonidine on GH secretion; (b) orchidectomy also abolished the stimulatory effect of clonidine in neonatal rats and that of xylazine in prepubertal males; (c) testosterone implanted at the moment of orchidectomy prevented the loss of the CLO effect in LTO and STO, but testosterone-delayed administration in LTO was unable to restore the effectiveness of CLO inducing GH release. We conclude that orchidectomy at all ages tested abolishes GH secretion induced by alpha2 agonists, which suggests that the functionality of alpha-adrenergic receptors involved in the control of GH secretion is critically dependent on a permanent exposure to testosterone in males.