2010, Number 2
La melatonina induce la síntesis y liberación de las encefalinas en el cerebro de la rata
Asai M, Valdés-Tovar M, Matamoros-Trejo G, García D
Full text
Language: Spanish
References: 47
Page: 123-131
PDF size: 148.54 Kb.
ABSTRACT
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.
Material and methods: 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.µ 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.
REFERENCES