Effect of cell surface trypsinization on ethanolamine base exchange enzymatic activity of astrocyte primary cultures and derived spontaneously transformed cell lines.

Trypsinization of neonatal rat astrocyte primary cultures (normal cells) inhibited the activity of ethanolamine base exchange enzyme (EBEE) by 80%, whereas ethanolamine phosphotransferase (EPT) and choline base exchange (CBEE) enzymatic activities were not affected; subcellular fractionation demonstrated that trypsin treatment affected the intracellular EBEE activity. During trypsinization the enzyme was not taken up by cultured astrocytes but the cell surface was affected. In contrast, the same treatment did not alter EPT, CBEE and EBEE activities of spontaneously transformed cell lines derived from the primary cultures. However, treatment of the transformed cells with db-cAMP prior to trypsin, restored the pattern found in the primary culture, i.e. only EBEE activity was affected. These data suggest that a relationship exists between cell surface organization and intracellular EBEE activity in a culture system which possesses the property to control its own cell division or/and differentiation.

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines. Effect of dibutyryl cyclic AMP treatment.

Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.

Ethanolamine base exchange in astrocyte primary cultures: localization and developmental studies.

The enzymatic activities of ethanolamine base exchange (EBEE) and CDP-ethanolamine: 1,2-diacylglycerol ethanolamine phosphotransferase (EPT) were investigated during the growth of rat astrocyte primary cultures. From the 16th day, cells ceased to divide (2.0 X 10(6) cells per culture dish); the total phospholipid (PL) content increased 1.5 fold between the 16th and 24th day (0.20 to 0.30 mumol per mg protein) but the amount of ethanolamine phospholipid (28% of PL content) remained constant. Whereas the specific activity (pmol/min X mg protein) of EPT reached a plateau at 16 days in culture and remained constant (400) thereafter, that of EBEE increased up to the 19th day (190) and decreased gradually to a basal level (75) at the 24th day. EBEE activity was not detected in plasma membranes isolated from 16, 19 and 24 days astrocyte cultures. Sub-cellular fractionation and determination of EBEE specific activities showed that the 104 X 10(3) g fraction (P4) was 4.8 and 8.8 fold enriched at the 16th day and 24th day respectively as compared to the whole cell homogenate (50 and 75). The 7 X 10(3) g (P2) and 17 X 10(3) g (P3) fractions were 8.4 and 7.0 fold enriched respectively at the 19 day in culture. The percentages of the enzymatic activity in the different subcellular fractions were 30, 57.2 and 25.7 for P2 and 39.2, 2.6 and 39.8 for P4 at 16, 19 and 24 days in culture respectively. The activity remained constant in P3 (23%) and was negligible in P1 (6%). Ultrastructural studies revealed that P2 and P3 were enriched in mitochondria while P4 contained essentially microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid fatty acyl group composition in mitochondria, microsomes and plasma membranes isolated from rat astrocyte primary cultures: developmental studies.

The fatty acid composition of choline, ethanolamine and serine phospholipids (PC, PE and PS) in heavy mitochondria, light mitochondria, microsomes and plasma membranes isolated from 16-, 19- and 24-day-old cultured rat astrocytes was investigated; within this period cultures reached confluency and ceased to divide. Plasma membranes revealed a stable distribution of the major fatty acids (FA) present in PC, PE and PS. The FA pattern of PC was similar to that of PE and PS. Analysis of the FA composition of PC, PE and PS found in intracellular membranes indicates that each major FA displayed a typical variation depending on the organelle and the phospholipid studied. Schematically for both phospholipids, PC and PE, the palmitic acid content increased in most subcellular fractions between 16 and 24 days; this variation was compensated by the decrease of stearic acid. Arachidonic acid level was very low at 16 days and increased markedly up to 24 days; thus a decrease of the saturated to unsaturated FA ratio (delta:0/delta:1, 2, 4) occurred. In contrast PS, which was poor in arachidonic acid between 16 and 24 days, gave rise to delta:0/delta:1, 2, 4. These results suggest (1) a reorganisation of the phospholipid hydrophobic core in mitochondria and endoplasmic reticulum when density-dependent inhibition phenomena occurs, and (2) a 'buffering' mechanism which protects the plasma membrane from marked changes of its phospholipid FA composition.