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M. E. Guido, P. S. Nieto, D. J. Valdez; Circadian Clock Components and Outputs in a Mammalian Retinal Ganglion Cell Line. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5953.
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To investigate the potential capacity of mammalian retinal ganglion cells (RGCs) as autonomous circadian oscillators, we utilized a transformed mammalian retinal cell line (RGC-5) which has certain characteristics of RGCs based on expression of specific markers (thy-1, brn-3c, neuritin and NMDA receptor) and glutamate sensitivity (Krishnamoorthy et al., 2001). To do this, we assessed in these cultured cells, expression of clock proteins (Per, Cry and Clock), photopigments (rhodopsin and melanopsin) and a clock output, the key melatonin synthesizing enzyme, serotonin N-acetyl transferase (NAT).
RGC-5 were grown in basal culture medium DMEM and 10% BFS to about 80% of confluence, arrested during 24 h and synchronized by a 2 h-serum shock with 50% BFS at time 0 (zeitgeber time, ZT 0) according to Balsalobre's protocol (1998). Then, the medium was replaced by 10% BSF and cells were collected at different ZTs during the following 24 h. Cells were resuspended in 1% PBS buffer for protein immunocharacterization with specific clock protein antibodies by Western blot, or homogenized in TRIZol (Gibco) for RNA extraction and RT-PCR analysis.
We found that cells of the RGC-5 line present detectable levels of expression for Per1, Per2 and Cry proteins with a temporal variation (p<0.039 by ANOVA) exhibiting higher levels at ZTs 8-12 after synchronization by serum shock and lower values at ZTs 16-20. These cells did not show detectable levels of rhodopsin or melanopsin mRNAs but did express the Clock and AA-NAT transcripts.
The results show that cultures of RGC-5 cells contain the oscillatory circadian machinery expressing different clock proteins in a circadian manner, together with a clock output, the melatonin synthesizing enzyme NAT. Based on these observations, we may infer that the physiology of these mammalian retinal cells is also controlled by an endogenous circadian clock.
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