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The light-dark cycle is an environmental factor that influences immune physiology, and so, variations of the photoperiod length result in altered immune responsivity. Macrophage physiology comprises a spectrum of functions that goes from host defense to immune down-regulation, in addition to their homeostatic activities. Macrophages also play a key role in the transition from innate to adaptive immune responses. Met-enkephalin (MEnk) has been recognized as a modulator of macrophage physiology acting in an autocrine or paracrine fashion to influence macrophage activation, phenotype polarization and production of cytokines that would enhance lymphocyte activation at early stages of an immune response. Previously it was shown that splenic MEnk tissue content is reduced in rats exposed to constant light. In this work, we explored whether production of Met-enkephalin-containing peptides (MECPs) in cultured splenic macrophages is affected by exposure of rats to a constant light regime. In addition, we explored whether primary immune response was impaired under this condition. We found that in rats, 15 days in constant light was sufficient to disrupt their general activity rhythm. Splenic MEnk content oscillations and levels were also blunted throughout a 24-h period in animals subjected to constant light. In agreement, de novo synthesis of MECPs evaluated through incorporation of 35S-methionine was reduced in splenic macrophages from rats exposed to constant light. Moreover, MECPs immunocytochemistry showed a decrease in the intracellular content and lack of granule-like deposits in this condition. Furthermore, we found that primary T-dependent antibody response was compromised in rats exposed to constant light. In those animals, pharmacologic treatment with MEnk increased IFN-γ-secreting cells. Also, IL-2 secretion from antigen-stimulated splenocytes was reduced after incubation with naloxone, suggesting that immune-derived opioid peptides and stimulation of opioid receptors are involved in this process. Thus, the immune impairment observed from early stages of the response in constant light-subjected rats, could be associated with reduced production of macrophage-derived enkephalins, leading to a sub-optimal interaction between macrophages and lymphocytes in the spleen and the subsequent deficiency in antibody production. [ABSTRACT FROM AUTHOR]

Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII. [ABSTRACT FROM AUTHOR]

Since enkephalins discovery in 1975, several opioid peptides have been included in neuroscience research. Enkephalins have been involved in the homeostasis maintenance of the organism, mostly with cellular and molecular mechanisms implicated in antinociception and narcotic responses. Moreover, enkephalins have been shown to be involved in the control of stress, regulation of cardiovascular functions, modulating primary immune responses, in addition to cellular differentiation processes. As opioid peptides appear to modulate several bioactivities and physiological responses in organisms, this posits that several modifications should occur during their synthesis, cell release, and receptor binding in target cells. At present, it has been demonstrated that the endogenous opioid system (EOS), displays a circadian rhythm, in which its tissue content, presynaptic release, and receptor's number reaches its maximal concentration during the dark phase (24:00h) and the minimal during the early morning (05:00 h). Recently, our group reported that functional pinealectomy disrupts the enkephalin circadian rhythm and significantly reduces the tissue content of opioid peptides in the rat brain. However, the effect was shown to be specific to the hour along the 24h daytime. There were no significant changes during the light period, only during the dark period (01:00h), when the enkephalin tissue content decreased in the experimental group. The effect was reverted when pinealectomized rats were injected with single doses of melatonin (MEL) (150μg/kg i.p.). If the lack of melatonin in the rat brain significantly reduced the enkephalin tissue content, and its exogenous administration re-established the enkephalin tissue levels, it is possible that the hormone is involved in the enkephalin synthesis. In this paper we provide further evidence that supports the relation between melatonin and opioid peptides synthesis and release. In addition, we studied the effect of darkness and melatonin administration in enkephalin tissue levels. Finally, we analyzed the luzindole effect as a melatonin receptor antagonist in the pinealectomized rat brain. Subjects: Male Wistar rats were housed in a light and temperature controlled room. Water and pellet food were available ad libitum. This group was subdivided in: 1. Functional pinealectomy group (FP). Rats were housed individually during 15 days in a room with continuous light (<50lux). 2. FP rats were housed in a dark room during four or six hours. 3. FP rats were injected with melatonin (150, 300, 600μg/kg s.c.). 4. FP rats were injected with Luzindole (187.5, 375, 750μg/kg i.p.). After 30 min, the animals were injected with melatonin (150μg/kg). 5. FP rats were injected with melatonin (800μg/kg) and subjected to the in vitro release processes. The rats were sacrificed by decapitation and the blood collected for melatonin serum determination. The brains were removed and processed for an analytical preparative procedure for the enkephalin determination by radioimmunoassay technique. The in vitro release methodology was performed as follows: tissue samples were homogenized by applying 8 strokes with a Thomas grinder system. The homogenates were centrifuged at 4,000rpm, 4°C during 10 min. Supernatants were recovered and centrifuged at 12,000rpm at 4°C for 20 min. Supernatants were discarded and pellets were resuspended in the homogeneization buffer (1:9 w/v). Samples were placed on top of a Percoll gradient density (23%, 15%, and 10%) and centrifuged at 20,000 rpm at 4°C for 25min. The synaptosomal enriched fraction (15-23%) was obtained and diluted in 1mL of Krebs buffer (mM: NaCl 119, KCl 4.6, CaCl2·2H2O 1.25, KH2PO4 0.85 MgSO4 0.84, NaHCO3 24.8, sucrose 10). Buffer was gasified with a mixture of C02 95% and O2 5%, pH 7.4. 800μL aliquots were placed into plastic chambers. After 20 min of stabilization with Krebs buffer, three different superfusates were collected: 1. basal, 2. potassium [50mM], and 3. post-stimulus (Krebs buffer without potassium). Samples were collected into HCl 0.1N, boiled and subsequently loaded into Amberlite XAD-2 columns (8×0.7cm) for solid-phase peptide extraction. The flow rate was held constant at 0.5 mL min-1 and elution of the whole peptide fraction was carried out using a continuous gradient with absolute methanol. Eluted samples were lyophilized and resuspended in 2mL of distilled water and finally stored at -20°C for further quantification of IR-Enkephalin using standard radioimmunoassay procedures. The results showed that functional pinealectomy reduced the opioid tissue content in the different brain structures assayed. The lack of melatonin significantly decreased the enkephalin tissue content when compared to the control group. However, tissue levels of enkephalin material were completely restored after four and six hours of administration of different doses of exogenous melatonin administration to the rats. As continuous light decreases the melatonin content in the brain, darkness should be able to counteract the aforementioned effect. Our results showed that tissue levels of enkephalin material were increased over 200% and 300%, after exposing animals to a four or six-hour period of darkness, when compared to animals exposed to continuous light. Luzindole was used to abolish any melatonin activity via activation of its membrane brain receptors. Our experiments showed that different doses of the antagonist were not able to obliterate the increased content of opioid peptides induced with melatonin administration in the tested brain tissues. Finally, enkephalin release showed a significant decrease in pinealectomized rats, showing a complete restoration of released peptide levels as shown by control group, after hormone administration. Thus, melatonin appears to counteract the neuronal decrease of the releasable pool of opioid peptide within the nerve terminals, reaching the released rate values detected in the control group. Our results suggest four relevant features: 1. Functional pinealectomy induces a significant decrease in the tissue content of opioid peptide material that seems to correlate with a decrease in the presynaptic release of enkephalins. 2. Darkness and MEL administration counteracted the light induced effects, namely the decreased content of opioid peptide material in tissue and the reduced peptide release from nerve terminals. 3. Nonetheless, the presence of luzindole was not able to inhibit the synthesis of opioid peptides. Melatonin is a hormone that synchronizes cell activity with photoperiod. Beyond this physiological property, MEL has been related to neuropeptide synthesis. Previous reports have shown that surgical or functional pinealectomy reduces the Met-Enkephalin tissue content in the rat brain. Also, it has been demonstrated that MEL stimulates the Proopiomelanocortin (POMC) gene expression in the immune system. In this paper, we showed further evidence that melatonin participates in the biosynthetic regulation of opioid peptides. Dark stimulation after continuous light exposure on rats produced an increase of tissue content of enkephalin above 300%, when compared with tested animals subjected to functional pinealectomy. Similar results were obtained with the exogenous melatonin administration. These evidences support the hypothesis that hormone treatment may be able to interact with protein kinase C (in the active-state of the enzyme). In addition, the hormone could modulate the cellular responses of several transcription factors (c-Fos, c-Jun, AP-1) responsible for the enkephalin gene expression. On the other hand, rats exposed to MEL-receptor antagonist luzindole, injected before MEL administration, showed that the tissue content of opioid peptide was not totally abolished by the melatonin antagonist. It has been proposed that melatonin not only interacts with its membrane receptor, but it is able to cross the cytoplasmic membrane and interact directly with the intracellular signal transduction system via the biomolecules that operate within. In conclusion, the physiology of melatonin appears to control the synthesis and presynaptic release of the endogenous opioid system in the rat brain. [ABSTRACT FROM AUTHOR]