Resumen de Efecto de la melatonina sobre la concentración de péptidos oploides en el sistema inmune de la rata

Gilberto Matamoros Trejo, Miguel Asal, Marcela Valdés Tovar

• español

  La fisiología de la melatonina (MEL) se asocia con la regulación del sistema inmune. La inhibición farmacológica de la síntesis de MEL disminuye la respuesta humoral primaria desencadenada por un estímulo antigénico. Asimismo, la administración exógena de MEL antagoniza los efectos inmunosupresores de la corticosterona y del estrés agudo. La MEL induce la liberación de interleucina-12 a partir de los monocitos, lo que a su vez promueve la diferenciación de los linfocitos TH1. En estos linfocitos, la MEL estimula la liberación de interleucina-2 e interferón-g. Los receptores de la MEL presentes en la membrana plasmática y en el núcleo de los monocitos y los linfocitos son los responsables de producir la acción de la hormona. Se ha sugerido que el efecto inmunoestimulante de la MEL se produce por un mecanismo mediado por los péptidos opioides. Los linfocitos, los monolitos y las células polimorfonucleares producen y liberan péptidos opioides. En el SNC de la rata, la ausencia de MEL endógena rompe el ritmo circádico de las encefalinas y disminuye su concentración. Si la inhibición fisiológica de la síntesis endógena de MEL disminuye la concentración y liberación de encefalinas en el SNC de la rata, es factible suponer que el contenido de estos péptidos disminuye también en las células del sistema inmune. Por lo tanto, el objetivo de este trabajo fue estudiar la concentración de encefalinas en el bazo y el timo de la rata, en ausencia y presencia de melatonina.

  Los resultados obtenidos en el presente trabajo demuestran que en ausencia de MEL endógena se produjo una disminución significativa en la concentración de Met-encefalina, Leu-encefalina, Octapéptido y Heptapéptido en los órganos del sistema inmune. Por el contrario, en animales sometidos a luz continua a los cuales se administró MEL en forma exógena, el contenido de los cuatro péptidos aumentó significativamente tanto en el bazo como en el timo. Estos datos sugieren que la MEL estimula la síntesis de novo del precursor de las encefalinas y su procesamiento postraduccional hasta los péptidos biológicamente activos. En los animales control a los que se administró un exceso de MEL, no se produjo la sobre-expresión de las encefalinas. Estos datos nos indican que la relación fisiológica melatonina-opioides se encuentra sujeta a la homeostasis del organismo. Hasta la fecha, no se ha dilucidado el mecanismo molecular a través del cual la MEL promueve la síntesis de los péptidos opioides.

• English

  Melatonin (MEL) physiology has been related to the immune system regulation. The pharmacological inhibition of MEL synthesis decreased the primary antibody response produced by an antigenic stimulus. Moreover, the exogenous MEL administration antagonized the immunosupressive effects of corticosterone and acute stress in mice. Also, MEL induces the interleukin-12 release from monocytes and promotes the lymphocytes TH1 differentiation. In these lymphocytes, MEL stimulates the interleukin-2 and interferon-γ release. The MEL receptors localized in the plasma membrane and in the nucleus, either from monocytes and lymphocytes, are the MEL effector signals in the cell. It has been suggested that the immunoenhancing effect produced by the hormone could be mediated by an opioid mechanism. Several lines of evidence have shown that lymphocytes, monocytes, and polymorphonuclear cells have the biochemical machinery to produce and release opioid peptides. In the central nervous system (CNS), the endogenous melatonin absence disrupted the enkephalin circadian rhythm and its tissue content was decreased as well. If the MEL absence in the CNS decreased the enkephalin tissue content, it is possible to consider that the opioid peptides content also decreased in the immune system. The present study was performed to evaluate the effect of endogenous MEL absence over opioid peptides concentration in the thymus and spleen of the rat.

  Materials and methods Sixty male Wistar rats, weighing each 220-240 g, were housed under a 12 h. light: 12 h. dark cycle in a temperature controlled room (21 + 1°C); the illumination period started at 06:00. Water and food pellets were available ad libitum. This group was divided in six subgroups: a)Naïve control group: Ten animals were housed under a 12 h. light: 12 h. dark cycle. The darkness period started at 18:00. b)Control group + MEL: 10 animals kept with a 12 x 12 h. lightdarkness cycle were subcutaneously (s.c.) injected with melatonin (800 µg/kg) at 9:00. These rats were maintained four hours under light conditions before being sacrificed. c) Control group + vehicle: Ten control animals were s.c. injected (at 9:00) with the same volume used to dissolve the hormone (ethanol: isotonic saline solution). These rats were maintained four hours under light conditions before being sacrificed.

  d)Continuous light (CL): In order to reduce the melatonin plasma concentration, ten rats were kept in a room with continuous light during 15 days. Light intensity was ≤50 lux to avoid stress. e) Continuous light + MEL: Ten CL animals were injected with melatonin (800 µg/kg s.c.) at 9:00. These rats were maintained four hours under light conditions before being sacrificed. f) Continuous light + darkness: In order to enhance the melatonin plasma concentration, ten CL rats were kept in a dark room during four hours. The darkness period started at 9:00 and rats were sacrificed four hours later. Animals were sacrificed by decapitation and thymuses and spleens were dissected and subjected to preparative processes before enkephalin determination. Opioid peptides IR-ME, IR-LE, IRHE, IR-OC content was measured using a radioimmunoassay technique. Statistical differences between groups were established by one-way ANOVA test (α=0.05), and then calculated using Tukey HSD and Tamhane as post hoc tests. A p<0.05 level was accepted as significant. The concentration values were expressed as IR-peptide (pmol/mg protein).

  Results In this work, three main features were found: 1. The endogenous melatonin absence produced by a chronic lighting exposure significatively reduced (50%) the opioid peptides content in both thymus and spleen. 2. Melatonin administration to CL rats produced an enkephalin tissue content increase (>100%), in both lymphoid organs. 3. No changes were found in control groups after melatonin or vehicle administration.

  Discussion The immune system is susceptible to stress. Recent evidences in neuroimmunology have begun to define how mood alterations, stress, seasons, depression, and daily rhythms have profound effects on immune response through hormonal modulation. Several lines of evidence have suggested that immune system functions could be regulated by the melatonin-opioid peptides relation. In the present work, we found that endogenous melatonin absence significatively reduced the enkephalin content in both thymus and spleen of the rat. This reduction could be directly related to the cytokine and antibody production, since the primary response to an antigen could be mediated by opioids. Our results are related with those obtained for the CNS, where the melatonin absence significatively decreased the opioid peptide tissue content and disrupted the enkephalins circadian rhythm. It has been reported that MEL was able to induce the Proopiomelanocortin (POMC) synthesis in the immune system. These data suggest that MEL is related to the neuropeptides synthesis. However, the basic mechanisms underlying the melatonin effect over the opioid peptides synthesis remain unknown at present. The signaling pathway may include the union to MT1 membrane receptors that had been located in the thymus and spleen, and the activation of phospholipase C. These events may cause an increase of intracellular calcium and the activation of the protein kinase C (PKC). PKC can phosphorylate and activate other kinases as the mitogen-activated protein kinases (MAPK), which include the ERK and JNK families. The ERK1/2 and the JNK activate the expression of transcription factors such as c-Fos and c-Jun, which form a heterodimer called AP-1. This protein can modulate the expression of the Proenkephalin A gene as described for the CNS. There is experimental evidence about the involvement of the MT1 receptor with the activation of EKR and JNK enzymes. Moreover, MEL had been described to stimulate the DNA binding activity of AP-1. Furthermore, the JNK phosphorylation had been associated to the regulation of Proenkephalin A gene expression, mediated by c-Fos and c-Jun. The induction of Proenkephalin A gene expression would produce an increase of the tissue content of the enkephalins.