CIRCADIAN REGULATION OF PHOSPHOLIPID BIOSYNTHESIS IN CHICK RETINAL GANGLION CELLS
M. E. Guido, E. Garbarino-Pico, M. A. Contin and A. R. Carpentieri
CIQUIBIC-Dpto. de Química Biológica, Facultad de Ciencias Químicas, UNC, 5000 Córdoba, Argentina
mguido@dqb.fcq.unc.edu.ar
Purpose: We recently reported that chicken retinal ganglion cells (RGCs) display daily variations in the biosynthesis of glycerophospholipids in constant darkness. It was unclear if this rhythmicity was driven by this population itself or by other retinal cells. In this context, the aim of the present work was to further investigate the circadian regulation of the phospholipid metabolism under different illumination conditions as well as the autonomous capacity of these cells to synthesize phospholipids when they are maintained in culture.
Methods: Chicks entrained to a 12:12 h LD cycle were released to DD, constant light (LL) or maintained under the previous LD cycle for 48 h. Then, chicks were killed at different times, RGC preparations were obtained from lyophilized retinas (Guido et al., 1999) and the in vivo labeling of phospholipids was assessed after the intraocular injection of 32P-orthophosphoate or 3H-glycerol for 1 h (Guido et al., 2001). RGCs obtained from 8 day-old chicken embryos by Thy-1 antibody immunopurification (Brocco and Panzetta, 1997) were cultured in B27 supplemented DMEM (GIBCO) for several hours and synchronized by a medium exchange at time 0 and pulsed with 32P-phosphate at different phases for 30 min each.
Results: Here we show that ganglion cells present circadian oscillations in the 32P-phospholipid labeling both in vivo in constant light and in cultures of immunopurified embryonic cells. In vivo, there was greater 32P-orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol was the utmost 32P-labeled lipid at all times examined displaying maximum levels during the subjective day and dusk. Furthermore, cultures of retinal ganglion cells, free of other cellular types and synchronized by medium exchange, displayed a circadian fluctuation in the phospholipid labeling.
Conclusions: The results demonstrate that retinal ganglion cells contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.
Supported by: Fundación Antorchas, FONCyT, CONICET, SeCyT-UNC, CAEN-ISN and Agencia Córdoba Ciencia.
