Elba N. Pereyra

Neuroendocrine integrative mechanisms in mammalian pineal gland: Effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland.

Summary1.To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10–1000 nM NPY in the presence or absence of 10µM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA).2.NPY (10–1000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1000 nM), enhanced NE stimulation of melatonin production.3.NPY (1000 nM) impaired3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with3H-NE.4.These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.

Effect of naloxone on the nocturnal rise of rat pineal melatonin content

Rats exposed to 14 light: 10 h dark photoperiods were given naloxone hydrochloride (10 mg/kg) s.c. 2 h before and 5 h after the lights were turned off. A smaller nocturnal pineal melatonin peak, as well as a slower decrease from peak values were observed in naloxone-treated rats. Naloxone (up to 10(-4) M) did not affect either in vitro rat pineal melatonin content or 3H-labeled transmitter release in glands previously incubated with [3H]norepinephrine. Hence a central, rather than a peripheral opioid synapse appears to be involved in naloxone effect on nocturnal melatonin increase.

Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines.

Release and effect ofγ-Aminobutyric acid (GABA) on rat pineal melatonin productionin vitro

Summary1.3H-γ-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pinealsin vitro.2.The release of3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+.3.In a Ca2+-free medium3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted.4.Norepinephrine (NE; 10−6–10−4M) stimulated3H-GABA release from rat pineal explants in a dose-dependent manner.5.The activity of 10−5M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (α1- andα1/α2-adrenoceptor blockers, respectively) and was unaffected by propranolol (β-adrenoceptor blocker).6.Theα1-adrenoceptor agonist phenylephrine (10−7-10−5M) and theβ-adrenoceptor agonist isoproterenol (10−5M) mimicked the GABA releasing activity of NE, while 10−7M isoproterenol failed to affect it; theα2-adrenoceptor agonist clonidine (10−7–10−5M) did not modify3H-GABA release.7.The addition of 10−4M GABA or of the GABA transminase inhibitorγ-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures.8.GABA at concentrations of 10−5M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants.9.The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release.10.The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels.11.These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction withα1-adrenoceptors; (c) GABA inhibits melatonin productionin vitro via interaction with GABA type A receptor sites; and (d) GABAs effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.

Involvement of 5-lipoxygenase pathway in norepinephrine stimulation of rat pineal melatonin synthesis

The effect of lipoxygenase inhibition, leukotriene agonists and antagonists, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was examined in the rat pineal gland in organ culture. To study melatonin secretion pineal explants were incubated for 6 h in tissue culture medium 199 with the different drugs. Melatonin concentration in the pineal gland and the medium was measured by RIA. Exposure of explants to norepinephrine (NE) brought about a 2- to 5-fold increase in both parameters, an effect that was reduced but not abolished, by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 10(-5) M). Lilly 171883 (10(-5) M) or FPL 55712 (10(-5) M; both antagonists of leukotrienes) reduced NE-induced melatonin production. Neither NDGA nor Lilly 171883 affected melatonin production in the absence of NE. Leukotrienes C4 and D4 increased melatonin release to the media at all concentrations tested (1-1,000 nM) with a maximum effect at 1 nM (leukotriene C4) and 10 nM (leukotriene D4). Significantly higher tissue melatonin concentrations as compared to controls were observed after exposure of pineal explants to 1 and 100 nM of leukotriene C4, or 100 nM of leukotriene D4. Another 5-lipoxygenase metabolite, 5-HETE, increased pineal melatonin content at concentrations of 1, 10 and 100 nM whereas only 1,000 nM stimulated melatonin release. These results suggest that the 5-lipoxygenase pathway plays a significant role in NE-stimulated melatonin production by the rat pineal gland.

In vitro effects of adenohypophysial hormones on rat pineal melatonin content and release

The effect of adenohypophysial hormones on rat pineal melatonin content and release was examined in vitro. Medium concentration of radioimmunoassayable melatonin decreased after a 6 h exposure to 1-100 ng/ml FSH; pineal levels of melatonin were only decreased by 100 ng/ml FSH. LH (1-100 ng/ml) augmented significantly medium melatonin concentration, tissue levels being increased at 10 ng/ml LH. Parallel increases of explant and medium melatonin content were found after exposure to 1-100 ng/ml TSH. At the smallest concentration employed (1 ng/ml) prolactin increased melatonin content and release while at 100 ng/ml a significant depression of both parameters was found. Growth hormone (1-10 ng/ml) augmented melatonin levels in medium but failed to modify them at 100 ng/ml, although at this concentration tissue melatonin levels increased. ACTH did not modify pineal melatonin synthesis in vitro.

Molecular Aspects of Neuroendocrine Integrative Processes in the Pineal Gland

The mammalian pineal gland fulfills the criteria of a “neuroendocrine” transducer.1 It translates a neural language provided by norepinephrine (NE) released at the synaptic biophase to a hormone language,melatonin and perhaps endocrine active peptides. The pinealocytes are also “endocrineendocrine” transducers inasmuch as they convert an endocrine language, e.g. estradiol attaining the gland via the general circulation, to a different endocrine signal like melatonin. Additionally “endocrine-neural” transducer events occur in the pineal gland, as revealed by the significant modifications of the activity of the innery ting sympathetic pathway after several hormone treatments.1,2

Histamine inhibits adrenocortical cell proliferation but does not affect steroidogenesis.

Histamine (HA) is a neurotransmitter synthesized in most mammalian tissues exclusively by histidine decarboxylase enzyme. Among the plethora of actions mediated by HA, the modulatory effects on steroidogenesis and proliferation in Leydig cells (LCs) have been described recently. To determine whether the effects on LCs reported could be extrapolated to all steroidogenic systems, in this study, we assessed the effect of this amine on adrenal proliferation and steroidogenesis, using two adrenocortical cell lines as experimental models, murine Y1 cells and human NCI-H295R cells. Even when steroidogenesis was not modified by HA in adrenocortical cells, the biogenic amine inhibited the proliferation of H295R cells. This action was mediated by the activation of HRH1 subtype and an increase in the production of inositol phosphates as second messengers, causing cell-cycle arrest in the G2/M phase. These results indicate a new role for HA in the proliferation of human adrenocortical cells that could contribute to a better understanding of tumor pathology as well as to the development of new therapeutic agents.