Differential responses of the mammalian retinal ganglion cell line RGC-5 to physiological stimuli and trophic factors.
The rat retinal ganglion cell (RGC) line RGC-5 constitutes a widely used model for studying physiological processes in retinal cells. In this paper we investigated the expression of clock and immediately early genes, and calcium mediated responses to physiological stimuli in differentiated and mitotically active RGC-5 cells. To this end, we attempted to differentiate the RGC-5 cells with a variety of effectors classically used to induce morphological differentiation. No sign of morphological differentiation was observed after 24 h of treatment with BDNF (80 ng/mL), NGF (100 ng/mL) and retinoic acid (20 ng/mL), among others. Only staurosporine (SSP) was able to promote neurite outgrowth at concentrations ranging from 53.5 to 214 nM. However, apoptotic nuclei were seen at 24 h of treatment using DNA staining, and a few cells remained at 72 h post-treatment. Concentrations of SSP lower than 214 nM were partially effective in inducing cell differentiation. Dividing RGC-5 cells express the RGC marker Thy-1 and different clock genes such as Per1, Clock and Bmal1. When characterizing the responsiveness of proliferative RGC-5 cells we found that in most of them, brief pulses of 50% FBS induced c-Fos and PER1 expression. Subsets of RGC-5 cells displayed significant changes in intracellular Ca2+ levels by ATP (100 microM) but not by glutamate (100-200 microM) stimulation. On the basis of cell morphology, size and complexity and effector responsiveness it was possible to distinguish different subpopulations within the cell line. The results demonstrate that only SSP is effective in promoting RGC-5 morphological differentiation, though the treatment provoked cell death. Proliferative cells expressing the RGC marker Thy-1 and a number of clock genes, responded differentially to diverse physiological stimuli showing a rapid c-Fos and PER1 induction by FBS stimulation, and an increase in intracellular Ca2+ by ATP.
Nieto
Paula S
PS
CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.
Acosta-Rodríguez
Victoria A
VA
Valdez
Diego J
DJ
Guido
Mario E
ME
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D056926:CLOCK Proteins / Q000096:biosynthesis / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation; D005434:Flow Cytometry; D018698:Glutamic Acid / Q000494:pharmacology; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects; D051381:Rats; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044967:Serum
D009414:Nerve Growth Factors; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D056926:CLOCK Proteins; D002118:Calcium