Characterization of Na+,K+-ATPase in cultured and separated neuronal and glial cells from rat cerebellum.

The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na+,K+-ATPase; EC 3.6.1.3) were examined in cultures and perikarya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synaptosomal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na+,K+-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3-15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the astrocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na+,K+-ATPase (apparent Ki values of about 10(-7) M and 10(-4)-10(-5) M, respectively), the high-affinity form accounting for 60-75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na+,K+-ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na+,K+-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na+,K+-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into account the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na+,K+-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of 86Rb into "live cells" was estimated as a measure of in situ ion pump activity.(ABSTRACT TRUNCATED AT 400 WORDS)