Involvement of nuclear binding sites for melatonin in the regulation of IL-2 and IL-6 production by human blood mononuclear cells

Many functional studies show that melatonin plays a fundamental role in neuroimmunomodulation. In this paper, we have extended our studies on the influence of melatonin on IL-2 and IL-6 production by human peripheral blood mononuclear cells (PBMCs) by comparing the effects of the specific membrane receptor agonist S 20098, the RZR/ROR(alpha) receptor agonist CGP 52608, and structurally related thiazolidinediones. Melatonin bound to membranes as well as to nuclei of human PBMCs with about the same affinity (IC50 values around 5 nM). S 20098 bound to PBMC membranes but not to PBMC nuclei, although the affinity was at least 100 times lower than that of melatonin; this compound did not stimulate cytokine production. In contrast, all four CGP compounds did not bind to PBMC membranes, while binding to nuclei exhibited IC50 values comparable to those of melatonin. The thiazolidinediones activating the RZR/ROR(alpha) receptor (CGP 52608, CGP 53079) also increased IL-2 and IL-6 production. CGP 55644 had no effect on cytokine production and antagonized the effects of CGP 52608 on IL-2 and IL-6 production; moreover, CGP 55644 decreased the enhanced IL-2 production caused by melatonin. Results obtained in monocyte cultures resembled closely those shown in PBMCs. The results reported in this paper confirm the involvement of a nuclear mechanism in the melatonin effects on cytokine production in human PBMCs. We have also shown a synergistic effect of S 20098 and CGP 52608, suggesting a possible link between nuclear and membrane melatonin receptors in PBMCs.     

Distribution and characterization of the melatonin receptors in the hypothalamus and pituitary gland of three domestic ungulates

With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic suprachiasmatic nuclei are reportedly the main targets for the pineal hormone melatonin. However, it is not known if the conspicuous diversity in the distribution pattern of melatonin binding sites in these areas depicts differences in reproductive behavior observed in the seasonally breeding species in the temperate zones. We explored the distribution and the characteristics of melatonin binding sites in the hypothalamus and pituitary of three species (bovine, horse, and donkey) different in terms of seasonal reproductive competence. The topographical localization, investigated by in vitro autoradiography, revealed 2-[125I]iodomelatonin binding sites only in the pituitary gland in all three species, primarily in the pars tuberalis (PT), but also in the pars distalis (PD) and pars intermedia (PI). Kinetic, inhibition, and saturation studies, performed by means of in vitro binding, revealed presence of a single class high affinity binding sites. The Kd values, melatonin, and 2-iodomelatonin Ki values were in the low picomolar range. Coincubation with GTP gamma S inhibited 2-[125I]iodomelatonin binding, demonstrating that these putative receptors are linked to a G protein in their signal-transduction pathway. The hypothalamus was devoid of specific binding. In conclusion, the results suggest that in these species, the hypophysis may be a principal target for the melatonin action on the reproductive system.     

Melatonin binding in the house sparrow song control system: sexual dimorphism and the effect of photoperiod

Avian song is a sexually dimorphic behavior which is regulated seasonally. This regulation involves the construction and growth of song control structures: the high vocal center (HVC), nucleus robustus archistrialis (RA), nucleus magnocellularis anterior (MAN), and Area X. Song behavior and its neural correlates are controlled by steroid-dependent and independent processes. The avian circadian system is known to be involved in both daily processes and seasonal reproduction. A major part of this system is the circadian secretion of melatonin by the pineal gland. To determine possible interactions of the circadian and song control systems, the distribution and density of 2-[125I]iodomelatonin (IMEL) binding, an indicator of melatonin sensitivity, were determined in male and female house sparrow brains. Specific binding was found in visual system centers of both genders, but binding in HVC, RA, and Area X was present only in males. Binding in MAN was present in both sexes. Although the effects of short and long photoperiods on male house sparrow IMEL binding in song structures revealed no systematic changes, there were significant differences in binding under different photoperiods in HVC and RA. IMEL binding in the tectofugal nucleus rotundus, however, was consistently highest under short day conditions. IMEL binding in song control nuclei was independent of testicular influence, since castration did not affect it significantly. The data point to a role for the circadian system of house sparrows in song control, but a specific role for melatonin in the daily or seasonal regulation of the song control system in birds, could not be determined.     

Ovine arylalkylamine N-acetyltransferase in the pineal and pituitary glands: differences in function and regulation

The enzyme arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) has been conventionally linked with the biosynthesis of melatonin within the pineal gland and retina. This study establishes that AANAT messenger RNA (mRNA) and functional enzyme occurs within the pars tuberalis (PT) and to a lesser degree within the pars distalis (PD) of the sheep pituitary gland; expression in these tissues is approximately 1/15th (PT) and 1/300th (PD) of that in the ovine pineal gland. AANAT mRNA in the PT appears to be expressed in the same cells as the Mel1a receptor. No evidence was obtained to indicate that either PT or PD cells have the ability to synthesize melatonin, suggesting that this enzyme plays a different functional role in the pituitary. We also found that cAMP regulation of the abundance of AANAT mRNA differs between the PT and pineal gland. Forskolin (10 microM) has no effect on pineal AANAT mRNA levels, yet represses expression in the PT. This suppressive influence could be mediated by ICER (inducible cAMP response early repressor), which is induced by forskolin in both tissues. Although it appears that the specific function and regulation of AANAT in the pituitary gland differ from that in the pineal gland, it seems likely that AANAT may play a role in the broader area of signal transduction through the biotransformation of amines.     

The ovine pars tuberalis secretes a factor(s) that regulates gene expression in both lactotropic and nonlactotropic pituitary cells

The purpose of this study was to determine whether the cells of the ovine pars tuberalis (PT) secrete a factor(s) that can influence the activity of cells in the pars distalis (PD). By Northern blotting of total RNA isolated from PD cells that had been stimulated in the presence of cycloheximide (10 micrograms/ml), PT cell-conditioned medium was shown to induce a significant increase in the expression of the early response gene, c-fos, above both PD cell-conditioned and nonconditioned medium control levels (P < 0.05). Although forskolin (5 microM) induced a weak increase in c-fos expression in PD cells, the effect of PT medium conditioned in the presence of forskolin enhanced this expression more than additively (P < 0.05); furthermore, this effect was reversed by melatonin. These results are consistent with the release of a factor(s) from the PT, which for simplicity we have called tuberalin. This factor was released from PT cells in a time-dependent and cycloheximide-sensitive manner and was resistant to heating at 100 C for 10 min. Tuberalin activity could be size-fractionated using molecular size cut-off filters to produce activity in both the 1- to 10-kDa and more than 10-kDa size ranges. The activities in both of these fractions were sensitive to trypsin degradation and, therefore, appeared to be peptidergic. However, it was not clarified whether the biological activities were due to one or two components. Tuberalin also induced c-fos expression in other cell types, including GH3 and NIH3T3 cells. Dual labelling of PD cells by in situ hybridization using riboprobes for c-fos and PRL demonstrated that both the less than and more than 10-kDa fractions of tuberalin activated c-fos expression in some, but not all, lactotrophs in PD cell cultures, suggesting that a primary function of the PT is to regulate the activity of lactotrophs. This was supported further by enhanced secretion of PRL from PD cells in the presence of either PT-conditioned medium or PT cells in coculture. In addition, PT-conditioned medium was found to increase c-fos in a second cell type, which did not hybridize positively for PRL, indicating the existence of other endocrine interactions between the PT and PD.     

TOR: a new orphan receptor expressed in the thymus that can modulate retinoid and thyroid hormone signals

Vitamin A and other fat-soluble hormones and vitamins have important roles as modulators of essential biological processes such as homeostasis, development, differentiation, and oncogenesis and also as regulators of the immune system. The active form of vitamin A, retinoic acid, as well as vitamin D3 and thyroid hormones exert their actions by binding to specific nuclear receptors that represent one subfamily of the steroid/thyroid hormone receptor superfamily. To identify new members of the retinoid/thyroid hormone receptor subfamily that could play a role in the immune system, a screening of a T cell cDNA library was performed using a retinoid X receptor probe. A clone was isolated encoding a novel nuclear receptor expressed mainly in the thymus and T cell lines. This new receptor, TOR (thymus orphan receptor), is most closely related in both its DNA-binding domain and ligand-binding domain, 90% and 53%, respectively, to ROR alpha/RZR alpha and clusters with these two receptors and RZR beta in a phylogenetic tree, when both the DNA-binding domain and the ligand-binding domain sequences of nuclear receptors are compared. Thus, TOR is part of a subgroup of receptors, one of which has recently been reported to be activated by melatonin. TOR binds specifically to a direct repeat of the half-site sequence 5'-AGGTCA-3' with a four- or five-nucleotide spacer, DNA sequences that also serve as binding sites for thyroid hormone (TR), and retinoic acid receptors (RAR). In transient transfection experiments TOR does not activate a reporter gene carrying these sequences in the absence or the presence of any known nuclear receptor ligands. TOR, however, is able to repress TR and RAR activity on DR-4-TREs or DR-5-RAREs, respectively. Therefore, our data suggest that TOR, similar to COUP-TF, can negatively regulate retinoic acid and thyroid hormone signals. However, the response elements recognized by TOR and COUP-TF differ as do the expression patterns of these receptors. Thus, one important role of TOR could be to modulate retinoid and thyroid hormone signals in the thymus.     

Lesions of the melatonin- and androgen-responsive tissue of the dorsomedial nucleus of the hypothalamus block the gonadal response of male Syrian hamsters to programmed infusions of melatonin

The objective of this study was to characterize a site at which it is likely that melatonin mediates photoperiodic control of reproduction in the male Syrian hamster. The first experiment was a comparison of the distributions of iodomelatonin (IMEL)-binding sites and cells immunoreactive to androgen receptors (AR-ir) in the medio-basal hypothalamus (MBH). AR-ir cells extended throughout the MBH, whereas IMEL binding was restricted to the dorsomedial nucleus (DMN). Comparisons between IMEL binding and AR-ir on adjacent cryostat sections revealed a clear overlap between the IMEL-binding sites and a distinct subpopulation of AR-ir cells within the DMN. The second experiment examined whether lesions of these IMEL- and androgen-responsive cells affected the response of the hamsters to short-day (SD)-like infusions of melatonin. Animals received sham or bilateral electrolytic lesions of the IMEL-binding sites within the DMN of the hypothalamus (MBH-X). Animals were pinealectomized and 4 wk later fitted with an s.c. cannula for the daily infusion of either melatonin (50 ng/h) or saline (500 microliters/10 h). After 6 wk the animals with sham lesions showed gonadal atrophy and lower serum concentrations of LH and prolactin (PRL) after infusions with melatonin. In contrast, MBH-X animals given melatonin had large testes and long-day (LD)-like serum LH concentrations. Infusions of melatonin did, however, cause a significant decline in serum PRL level. This study shows that an intact MBH is essential for the expression of gonadotrophic but not lactotrophic responses to melatonin and/or photoperiod. It also suggests that cells responsive to both gondal steroids and melatonin may be involved in the seasonal variation in GnRH release, and indicates a site at which melatonin might influence sensitivity to steroid feedback, a hypothalamic function known to be regulated by photoperiod.     

Function of galanin in the anterior pituitary of estrogen-treated Fischer 344 rats: autocrine and paracrine regulation of prolactin secretion

Estrogen is a robust stimulator of galanin synthesis and secretion in the anterior pituitary. Galanin is colocalized in lactotrophs in the estrogen-treated anterior pituitary, and its roles in lactotroph function are still being elucidated. In the present studies, we quantified the phenotypes of estrogen-treated Fischer 344 rat anterior pituitary cells expressing the galanin gene by dual in situ hybridization. The total population of galanin-positive pituitary cells increased from undetectable levels to 16% of all cells after 2 weeks of estrogen treatment. More than 90% of the galanin-positive cells coexpressed PRL messenger RNA, and one-third of the lactotrophs expressed galanin messenger RNA. We hypothesized that galanin in the anterior pituitary may contribute to the heterogeneous secretion of PRL, and that one of the functions of galanin is to regulate PRL secretion in an autocrine/paracrine manner. To test this hypothesis, we performed the reverse hemolytic plaque assay combined with in situ hybridization to measure PRL secretion and galanin gene expression within the same individual cells. PRL secretion from galanin-positive lactotrophs was significantly greater than that from galanin-negative lactotrophs. Moreover, treatment with galanin antiserum significantly attenuated PRL secretion from galanin-positive cells, and treatment with galanin significantly enhanced PRL secretion from galanin-negative lactotrophs. In conclusion, these data provide direct evidence that galanin derived from the estrogen-treated anterior pituitary stimulates PRL secretion in both autocrine and paracrine manners.     

In vitro effects of CINC/gro, a member of the interleukin-8 family, on hormone secretion by rat anterior pituitary cells

We investigated the effects of CINC/gro on hormone secretion using normal rat anterior pituitary cells. In normal anterior pituitary cells, 10-100 ng/ml of CINC/gro significantly increased the secretion of PRL within 3 h of incubation, and two-fold enhancement of PRL secretion was induced by 100 ng/ml of CINC/gro within 24-h incubation, while the response of GH and ACTH secretions to CINC/gro was weak. On the other hand, CINC/gro suppressed basal LH and FSH secretions in a concentration-dependent manner. The percent inhibition of basal secretion by CINC/gro (50 ng/ml) within 24-h incubation was 70% for LH and 43% for FSH. Twenty-four-hour incubation with 100 ng/ml of IAP completely blocked the CINC/gro-stimulated PRL and GH secretions and CINC/gro's suppression of both basal LH and FSH secretions. These data demonstrate a new biological activity for CINC/gro and provide evidence for immune system regulation of anterior pituitary hormone secretion.     

The effects of GnRH and adrenergic agents on PRL and beta-endorphin secretion by porcine pituitary cells in vitro

The direct effects of alpha- and beta-adrenergic agents on PRL and beta-endorphin (beta-END) secretion in vitro by porcine pituitary cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomised (OVX) one month before slaughter. Ovariectomised gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2) and (3) oestradiol benzoate (EB; 2.5 mg/100 kg b.w.) at 30-36 h (OVX+EB I) and 60-66 h (OVX+EB II) before slaughter, respectively; and (4) progesterone (P4; 120 mg/100 kg b.w.) for 5 consecutive days before slaughter (OVX+P4). Isolated anterior pituitary cells were submitted to 3.5 h incubation in the presence of GnRH, alpha- and beta-adrenergic agonists [phenylephrine (PHEN) and isoproterenol (ISOP), respectively], or alpha- and beta-adrenergic blockers [phentolamine (PHENT) and propranolol (PROP), respectively]. The culture media were assayed for PRL (exp. I) and beta-endorphin-like immunoreactivity (beta-END-LI) (experiment II). In experiment I, GnRH did not influence PRL release by pituitary cells in all experimental groups. Some of tested doses of adrenergic agonists, PHEN and ISOP, increased PRL release from pituitary cells of OVX gilts, but not from those of OVX+EB I animals. In the OVX+EB II group, PHEN alone, but ISOP with PROP, potentiated PRL secretion by the cells. In OVX+P4 animals, PHEN alone or in combination with PHENT and also ISOP alone or with PROP enhanced PRL output from the cells. In experiment II, addition of GnRH increased beta-END-LI release from pituitary cells only in the OVX+EB II group. PHEN and PHENT potentiated beta-END-LI secretion by pituitary cells in OVX+EB II and OVX+P4 groups, while ISOP and PROP increased beta-END-LI secretion by the cells of OVX and OVX+EB II animals. In turn, in the OVX+EB I group, effect of PHENT and PROP on PRL secretion by pituitary cells was inhibitory. In conclusion, our results suggest that adrenergic agents can modulate PRL and beta-END secretion by porcine pituitary cells in a manner dependent on the hormonal status of gilts.     

Does alpha-melanocyte-stimulating hormone from the pars intermedia regulate suckling-induced prolactin release? Supportive evidence from morphological and functional studies

Recent evidence suggests that substances derived from the hypophyseal intermediate lobe (IL) play a crucial role in the regulation of suckling-induced PRL secretion. The purpose of the present study was to explore this possibility further by determining whether the suckling stimulus acutely increases the secretory activity of the IL and whether alpha MSH, a major secretory product of the IL, plays a specific role in suckling-induced PRL release. Light microscopic morphometric analysis of serial pituitary sections obtained from lactating rats revealed that as little as 1 min of suckling caused a significant increase in the proportion of the IL that was in secretory configuration (11.8 +/- 0.7% vs. 6.7 +/- 0.5%; 1-min suckled vs. nonsuckled control; mean +/- SE). Moreover, the fraction of the IL in secretory configuration continued to increase after 5 and 10 min of nursing (to 16.0 +/- 0.8% at 5 min and 18.2 +/- 0.7% at 10 min). In contrast, serum PRL was not significantly elevated above the control level after 1 min of suckling (18.1 +/- 13.5 vs. 9.9 +/- 6.5 ng/ml, 1-min suckled vs. control). In fact, a significant rise in PRL levels (to 314.4 +/- 19.4 ng/ml) could be detected only after 10 min of nursing. Thus, secretion by the IL in response to suckling preceded the release of adenohypophyseal PRL, suggesting that a secretory product(s) from the pars intermedia is involved in the modulation of nursing-induced PRL release. Having established a sequential temporal relationship between these two phenomena, we next investigated whether alpha MSH was the IL factor involved in the regulation of suckling-induced PRL secretion. To this end, lactating rats were injected either with antiserum to alpha MSH or preimmune serum and then allowed to nurse their pups. Serial blood samples were taken from the mothers 15, 30, 60, and 90 min after the litters were returned, and serum PRL was measured by RIA. We found that the suckling-induced rise in serum PRL was severely attenuated in animals that received anti-alpha MSH serum. This suppression was most evident at 15 min (70.1 +/- 13.4 vs. 323.5 +/- 127.0 ng/ml, antibody treated vs. preimmune serum control) and persisted throughout the entire 90-min test period. When taken together, our results suggest that suckling-induced PRL secretion is mediated at least in part by alpha MSH released from the hypophyseal IL.     

Effects of cannabinoids on prolactin and gonadotrophin secretion: involvement of changes in hypothalamic gamma-aminobutyric acid (GABA) inputs

CB1 cannabinoid receptors are located in hypothalamic nuclei and their activation alters several hypothalamic neurotransmitters resulting in, among other things, decreased prolactin (PRL) and luteinizing hormone (LH) secretion from the anterior pituitary gland. In the present study, we addressed two related objectives to further explore this complex regulation. First, we examined whether changes in gamma-aminobutyric acid (GABA) and/or dopamine (DA) inputs in the medial basal hypothalamus might occur in parallel to the effects resulting from the activation of CB1 receptors on PRL and gonadotrophin secretion in male rats. Thus, the acute administration of (-)-delta9-tetrahydrocannnabinol (delta9-THC) produced, as expected, a marked decrease in plasma PRL and LH levels, with no changes in follicle-stimulating hormone (FSH) levels. This was paralleled by an increase in the contents of GABA, but not of DA, in the medial basal hypothalamus and, to a lesser extent, in the anterior pituitary gland. The co-administration of delta9-THC and SR141716, a specific antagonist for CB1 receptors, attenuated both PRL and LH decrease and GABA increase, thus asserting the involvement of the activation of CB1 receptors in these effects. As a second objective, we tested whether the prolonged activation of these receptors might induce tolerance with regard to the decrease in PRL and LH release, and whether this potential tolerance might be related to changes in CB1-receptor binding and/or mRNA expression. The chronic administration of R-methanandamide (AM356), a more stable analog of anandamide, the putative endogenous cannabinoid ligand, produced a marked decrease in plasma PRL and LH levels, with no changes in FSH. The decreases were of similar magnitude to those caused by a single injection of this cannabimimetic ligand, thus suggesting the absence of tolerance. In parallel, the analysis of CB1-receptor binding and mRNA expression in several hypothalamic structures proved that the acute or chronic administration of AM356 did not affect either the binding or the synthesis of these receptors. In summary, the activation of CB1 receptors in hypothalamic nuclei produced the expected decrease in PRL and LH secretion, an effect which might be related to an increase in GABAergic activity in the hypothalamus-anterior pituitary axis. The prolonged activation of these receptors for five days did not elicit tolerance in terms of an attenuation in the magnitude of the decrease in PRL and LH, and, accordingly, did not alter CB1-receptor binding and mRNA levels in the hypothalamic nuclei examined.     

Endothelin is an autocrine regulator of prolactin secretion

The aim of this study was to establish the cellular source of ET-like peptides affecting PRL secretion. Fluorescence double label immunocytochemistry and confocal laser scanning microscopy were used to demonstrate cellular colocalization for PRL and endothelin-1 (ET1)-like immunoreactivities in the anterior lobe of the pituitary gland of rats. An ET-specific reverse hemolytic plaque assay was applied to demonstrate that lactotrophs are capable of releasing ET-like peptides. A PRL-specific reverse hemolytic plaque assay was used to assess the influence of the released endogenous ETs on PRL secretion. ET(A)-specific receptor antagonists BQ123 and BQ610, and endothelin convertase enzyme inhibitory peptide, [22Val]big ET1-(16-38), increased PRL secretion, whereas the ET(B) receptor-specific antagonist BQ788 was ineffective. The ET(A) antagonist BQ123-induced increase in PRL secretion followed a bell-shaped dose-response curve in cells obtained from female rats, whereas it followed a sigmoid curve in males. Frequency distribution of PRL plaque sizes using logarithmically binned data revealed two subpopulations of lactotrophs with differential responsiveness to endogenous ETs. These data demonstrate that a large proportion of lactotrophs is capable of expressing and secreting ET-like peptides in biologically significant quantities. As low pituitary cell density in reverse hemolytic plaque assay minimizes cell to cell communications, these findings constitute direct proof of autocrine regulation of PRL secretion by ET-like peptides.     

Modulation of prolactin receptors in the rat hypothalamus in response to changes in serum concentration of endogenous prolactin or to ovine prolactin administration

Specific binding of 125I-labeled rat prolactin (125I-rat PRL) to hypothalamic membranes was studied in Sprague-Dawley rats after ovine PRL administration and in relation to rat PRL serum variations induced by ectopic pituitary implants or by drugs which stimulate (domperidone) or inhibit (bromocriptine) PRL release. Repeated treatments with ovine PRL markedly increased specific binding values of 125I-rat PRL to hypothalamic membranes of female rats. Repeated treatments with domperidone also increased specific PRL binding in the hypothalamus. This effect was associated with an increase in PRL serum levels. Similar results were obtained in male rats after renal pituitary implants which resulted in a state of chronic hyperprolactinaemia. In contrast, a subchronic treatment with bromocriptine decreased specific PRL binding in the hypothalamus and concomitantly caused a sharp reduction in PRL serum levels. Scatchard analysis of data obtained from competition curves showed that the variations in the level of PRL binding to hypothalamic membranes were related to the number of PRL binding sites but not to the dissociation constant (Kd), which was unaffected by different treatments or by pituitary implantation. These results demonstrate a correlation between circulating concentrations of PRL and number of its receptors in the rat hypothalamus and give further support to the hypothesis that these binding sites may have a specific functional role in regulating the homeostasis of pituitary PRL secretion.     

Photoperiod-melatonin relay in deer

Long-lived mammals from cold and temperate climates, including many species of deer, express overt cycles in reproduction, moulting, fattening and other characteristics. These cycles persist under constant conditions, but are normally induced and entrained by the annual cycle in daylength. The photoperiod-relay involves the eyes, the suprachiasmatic nuclei (SCN) of the hypothalamus and the pineal gland which secretes melatonin only at night. The duration of daily melatonin secretion varies with daylength and provides an internal endocrine signal for the time-of-year. In deer, treatments with melatonin induce phase-shifts in all overt seasonal rhythms. Melatonin is thought to act on specific target cells in the brain and pituitary gland which express high affinity melatonin receptors. In sheep, micro-implants of melatonin placed in the mediobasal hypothalamus (MBH) induce a complete spectrum of short-day responses, while surgical disconnection of the pituitary gland blocks all photoperiodic responses except for the regulation of prolactin. These observations support the 'dual-site hypothesis' that melatonin acts primarily in the MBH to control gonadotrophin secretion and the reproductive axis, but acts primarily in the pituitary gland via the pars tuberalis, to control prolactin secretion and the pelage axis. This differential regulation helps explains how prolactin can be 'the hormone of summer' in all photoperiodic ungulates irrespective of their seasonal breeding characteristics.