Temporal gene expression of melanopsin (Opn4), Clock, Cry and Per and regulation by melatonin in Danio rerio cells / Expressão gênica temporal de Melanopsina (Opn4), Clock, Cry e Per e sua regulação por melatonina em células de Danio rerio

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

The presence of melanopsin mRNA in ZEM-2S embryonic cells was determined through PCR, followed by sequencing. PCR experiments for melatonin receptors with ZEM-2S cell cDNA suggested the presence of the MT2 subtype. Bands corresponding to the expected weight for MT1 or Mel 1C were not identified. The identity of the MT2 receptor in ZEM-2S was confirmed through sequencing. We have also determined that the six Cry genes known in Danio rerio are expressed in ZEM-2S embryonic cells. When ZEM-2S cells were submitted to a light:dark (12L:12D) cycle, melanopsin expression presented two peaks, one at the beginning of the light phase (ZT3), the other at the beginning of the dark phase (ZT12). These peaks of expression remained when the cells were kept under constant darkness, and interestingly, a significant rise in expression was found in all ZTs when compared with the corresponding ZTs of cells kept under the light:dark cycle. Melanopsin did not exhibit a rhythmic expression in ZEM-2S cells in none of the conditions. However, there is a tendency of a rhythm in cells kept under 12L:12D, which disappears under constant darkness. Melatonin pulse seems to stimulate the expression during the subjective dark phase, but without any statistical significance. Clock mRNA did not present a rhythm in ZEM-2S cells kept either under 12L:12D, constant darkness or constant darkness with a melatonin pulse. However, there is a tendency of a rise in expression during the dark phase and during the subjective darkness, which is abolished by the melatonin pulse. Per 1 and Cry 1b mRNAs presented a robust rhythmicity in cells kept under 12L:12D. There is a significant rise three hours before the beginning of the light phase (ZT21), and sharp fall during the dark phase. Under constant darkness, Per1 and Cry1b rhythmicity, although present, was greatly attenuated. Melatonin pulse was not able to recover the amplitude observed under 12L:12D, moreover, it abolished rhythmicity of both genes. After melatonin pulse, Clock, Per1 and Cry1b genes in ZEM-2S cells lost the rhythmic expression, which still persisted under constant darkness. It is possible that 48 melatonin, as observed in other preparations, inhibits the phosphorylation of CREB in ZEM-2S cells, reducing the activation of the Clock genes promoters. Anyway, one could interpret that melatonin brings the Clock genes to the same level, therefore resetting the rhythm, independently of the phase. This study brings important contributions to the understanding of peripheral Clock physiology and opens new perspectives for future investigations of the underlying mechanisms of rhythms in isolated cells and their regulation by hormones and light.

ASSUNTO(S)

embryonic cells melatonin células embrionárias melatonina biological rhythms ritmos biológicos

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