No abstract

Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in "pure" oligodendrocyte cultures. "Pure" oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside-positive oligodendrocytes and produce "myelin" membranes (Fressinaud et al., 1990) after 6-10 days in subcultures (30-35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and "pure" oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase. In "pure" oligodendrocyte cultures, about half of cells were labeled with anti-GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in "pure" oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 kDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, "pure" oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl-adenosine 3':5'-cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.

Energy dispersive x-ray fluorescence and atomic absorption spectroscopy were used to determine the concentrations of Mg, Ca, Mn, Fe, Zn, and Cu in primary cultures of astroglial cells from chick embryo cortex in chemically defined serum-free growth medium. The intracellular volume of cultured glia was determined to be 8.34 microliter/mg protein. Intracellular Mn, Fe, Zn, and Cu in these cells were ca. 10-200 microM, or 20-200 times the concentrations in the growth medium. Mg2+ was 7 mM in glial cells, only four-fold higher than in growth medium. Glutamine synthetase (GS), compartmentalized in glia, catalyzes a key step in the metabolism of neurotransmitter L-glutamate as part of the glutamate/glutamine cycle between neurons and glia. Hormones (insulin, hydrocortisone, and cAMP) added to growth medium differentially altered the activity of GS and the intracellular level of Mn(II), but not Mg(II). These findings suggest the possibility that glutamine synthetase activity could be regulated in brain by the intracellular levels of Mn(II) or the ratio of Mn(II)/Mg(II), which may in turn be controlled indirectly by means of transport processes that respond to hormones or secondary metabolic signals.

Primary cultures of neurons and glial cells (astroblasts) prepared from brains of 8-day-old and 15-day-old chick embryos, respectively, were grown for periods between 3 and 19 days. Specific activity of lactate dehydrogenase (LDH) increased in both types of cultures as a function of time and was always significantly higher in glial cells than in neurons. Glial cell extracts were found to contain predominantly the anaerobic isoenzymatic form of LDH (LDH-M4), and this pattern did not change over a period of 19 days. Cultured neurons contained predominantly the aerobic isoenzymatic form LDH-H4, and there was a progressive appearance of all other isoenzymes over an 8-day period. These results support the hypothesis of a different energy metabolism in neurons and glia.