Autoradiography of high affinity uptake of catecholamines by primary astrocyte cultures

Uptake ofd,l-³H]norpinephrine (³H]NE and ³H]dopamine (³H]DA) by primary astrocyte cultures prepared from neonatal rat brains, which are 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 × 10⁻⁷M ³H]NE. Uptake of ³H]DA at 7.5 × 10⁻⁷M was 58% Na⁺ dependent, but total uptake of 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 °C, or 10⁻⁷ M desmethyllimipramine 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.