Az óragének és a környezeti fényviszonyok szerepe az N-acetiltranszferáz génexpressziójának szabályozásában csirke retinában = The role of clock-genes and environmental lighting conditions in the regulation of N-acetyltransferase gene expression in the chicken retina

Csirke retina es tobozmirigy sejtekben a melatonin (MT) szintezis kulcsenzimenek, az arilalkilamin N-acetiltranszferaznak genje (cAanat) co-expresszalodik az oragenekkel. Emellett a dopamin (DA) a fő katekolamin a retinaban, mely neuroregulatorkent hat. Tanulmanyoztuk: 1. a cBmal1 es cAanat mRNS expressziok dinamikajat retinalis/pinealis sejtekben in vivo es in vitro RT-PCR-ral. A cBmal1 mRNS csucsa a fenyes fazisban volt Zeitgeber idő (ZT) 8 es 10 kozott, megelőzve az a cAanat expresszio ejszakai emelkedeset ZT 16-17-nal. A retinalis/pinealis cBmal1 es cAanat mRNS ritmusainak fazisai arra utalnak, hogy kapcsolat van az expressziok kozott; 2. locked nucleinsav tartalmu cBmal1 antisense oligonukleotidok (AON) hatasat a cAanat transzkriptumokra es az MT termelesre pinealocita kulturaban, melyet szuperfuzios rendszerben transzfektaltunk. Az AON, mely vagy az RNaz H-t aktivalta, vagy a transzlacios masineriahoz valo kotődest blokkolta szignifikansan csokkentette a cAanat transzkripciojat es az MT szekreciojat jelezven a cBmal1 kulcsszerepet az indol metabolizmus szabalyozasaban; 3. ket fő DA-receptor csalad (D1 es D2) szerepet a circadian retinalis MT ritmus szabalyozasaban eye-cup modellen. A D2 agonista Quinpirol szignifikansan csokkentette az MT szekreciojat sotet fazisban, mig a D1 agonista SKF hatastalan volt, amely arra utal, hogy a DA gatlo hatast fejt ki a retinalis MT szekreciora (reciprok negativ feed-back), melyet elsősorban a D2 receptorok medialnak. | In chicken retinal and pineal cells gene of arylalkylamine N-acetyltransferase (cAanat), the key enzyme of melatonin (MT) synthesis is co-expressed with clock genes. In addition, dopamine (DA) is the major catecholamine in the retina serving as a neuroregulator. We studied: 1. the temporal profile of cBmal1 and cAanat mRNA expressions in retinal/pineal cells in vivo and in vitro using RT-PCR. cBmal1 mRNA had a peak in the light phase between Zeitgeber time (ZT) 8 and 10, preceding the nocturnal increase in cAanat expression at ZT 16 to 17. The phase of the rhythms of retinal/pineal cBmal1 and cAanat mRNAs suggests a link between their expressions; 2. the effect of cBmal1 antisense oligonucleotides (AONs) containing locked nucleic acid on cAanat transcripts and MT production in cultured pinealocytes transfected in superfusion system. These AONs synthesized for activating RNase H or blocking the binding of the translation machinery were able to reduce significantly cAanat transcription and MT secretion indicating the key role of cBmal1 in the regulation of indole metabolism; 3. the role of the two major DA-receptor families (D1 and D2) in the regulation of circadian retinal MT rhythm on an eye-cup model. Quinpirole (D2 agonist) reduced significantly MT secretion in dark phase, whereas SKF (D1 agonist) was ineffective suggesting that DA exerts an inhibitory effect on retinal MT secretion (reciprocal negative feed-back), which is mediated primarily via D2 receptors.