Autoradiography of high affinity uptake of catecholamines by primary astrocyte cultures

Uptake of D.L-[3H]norepinephrine ([3H]NE) and [3H]dopamine ([3H]DA) by primary astrocyte cultures prepared from neonatal rat brains, which are greater than or equal to 95% glial fibrillary acidic protein (GFAP(+)), was studied by measuring accumulation of tritium label, and localizing such uptake at the cellular level by autoradiography. Uptake of [3H]NE was 95% Na+ dependent at 10(-7) M and 80% Na+ dependent at 7.5 X 10(-7) M [3H]NE. Uptake of [3H]DA at 7.5 X 10(-7) M was 58% Na+ dependent, but total uptake of [3H]DA was greater than uptake of [3H]NE. Autoradiography of cells incubated with 7.5 X 10(-7) M [3H]NE or [3H]DA showed that a high proportion of all the cells in these cultures had a grain density which was clearly above background. When Na+ was omitted from the medium, the temperature was lowered to 4 degrees C, or 10(-7) M desmethylimipramine or 10(-7) M amitryptyline were present, cellular grain density after exposure to both [3H]NE and [3H]DA was greatly reduced, to close to background levels. It also appeared necessary to have inhibitors of both monoamine oxidase (pargyline) and catecholamine-O-methyltransferase (tropolone) present to see clear cellular localization for [3H]DA. In the case of [3H]NE the presence of tropolone alone was adequate to observe cellular localization. These results confirm our previous findings of the existence of a high affinity uptake process for catecholamines in primary astrocyte cultures based on uptake properties, and in the present study also localizes such uptake to the major, astrocytic cell type.