Differential responses in RGC-5 cells to different physiological stimuli
Paula S. Nieto, Diego J. Valdez, Victoria A. Acosta and Mario E. Guido.
CIQUIBIC-Dpto. de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, Argentina
paula@mail.fcq.unc.edu.ar
Purpose: To characterize the responses of mammalian retinal ganglion cells to different physiological stimuli (ATP, glutamate and light) by calcium imaging microscopy and protein expression. 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). Besides, we measured the mRNA expression of the key melatonin synthesizing enzyme, serotonin N-acetyl transferase (AA-NAT) and the ability of RGC-5 cells to modulate the activity of such enzyme.
Methods: RGC-5 were grown in basal culture medium DMEM and 10% BFS to reach the proper confluence. For calcium imaging measurements, cells were grown until 40 % of confluence and incubated with fura-2 AM during 30 min at 25ºC in the dark. For western blot (WB), cells were grown to 90 % of confluence, arrested during 36 h and synchronized by a 2 h-serum shock (50% FBS at time 0). Then, the medium was replaced by 10% FBS. Cells were resuspended in 1% PBS buffer for protein immunocharacterization with specific antibodies or enzyme activity, and homogenized in TRIZol (Gibco) for RNA extraction and RT-PCR assay.
Results: We found that the RGC-5 cells express the clock proteins Per1, Per2 and Cry and show expression of AA-NAT mRNA, but we did not find detectable enzyme activity levels. Nevertheless, RGC-5 extracts can inhibit the intrinsic AA-NAT activity of retinal photoreceptor cells suggesting the existence of an endogenous enzyme inhibitor present in this cell line.
In addition, RGC-5 cells show differential calcium responses to ATP stimulation: some of them exhibit a high amplitude positive response; other cells display a low amplitude response, while a third population shows a null response. Surprisingly, we could not measure detectable responses to glutamate stimulation (100 and 200 uM); however, cells treated with a brief white light pulse (30 s) showed an increase in the intracellular levels of calcium whereas white cold light stimulation induces a change of c-Fos expression as compared with dark controls.
Conclusions: The current results show that RGC-5 cells express the AA-NAT mRNA but the enzyme seems to be affected by an intrinsic inhibitor present in the cells. RGC-5 cells displays heterogeneous calcium responses to ATP and null responses to glutamate stimulation. Preliminary results reveal calcium responses to a pulse of white light and changes in the expression of c-Fos by WB.
