A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.
The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and of glial cells. The biosynthesis of lipids that occurs in the retina may be distinctly regulated in one neuronal type of cells with respect to another. To study the cell-type-specific aspects of lipid metabolism, a method for the separation of different retinal cell populations is needed. Herein, we describe a very simple procedure to isolate preparations highly enriched in specific retinal cell types that are suitable for in vitro biochemical assays. The method consists of selectively obtaining photoreceptors (PRC) and retina ganglion cells (RGC) from lyophilized chicken retinas using Scotch tape to assess, then, the in vitro incorporation of labeled precursors into phospholipid moieties. When their metabolic capability was assayed, it was found that these cell preparations maintain their enzyme activities intact to incorporate (32)P-phosphate into phospholipids in vitro at a similar rate as observed in fresh tissue after 1 h incubation. The highest proportion of labeling was observed in phosphatidylethanolamine (PE), followed by phosphoinositides (PIPs), phosphatidylcholine (PC) and phosphatidic acid (PA). Phosphatidate-phosphohydrolase (PAPase), a key enzyme of glycerolipid metabolism, exhibits similar levels of activity when assessed in fresh or frozen cell preparations, indicating that the lyophilization procedure does not significantly affect this activity. It is concluded that different cell populations obtained by the experimental procedure described herein, are useful to study the cellular metabolism and its regulation.
Guido
M E
ME
CIQUIBIC (CONICET)-Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000, Cordoba, Argentina. mguido@dqbfcq.uncor.edu
Bussolino
D F
DF
de Arriba Z
G A
GA
Deza
S
S
Pasquaré
S J
SJ
Giusto
N M
NM
Caputto
B L
BL
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002645:Chickens; D003902:Detergents / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism; D005612:Freeze Drying; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D050356:Lipid Metabolism; D008563:Membrane Lipids / Q000378:metabolism; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013048:Specimen Handling
D003902:Detergents; D005136:Eye Proteins; D008563:Membrane Lipids; D010743:Phospholipids; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D010711:Phosphatidate Phosphatase