Autoradiography of high affinity uptake of catecholamines by primary astrocyte cultures

Abstract

Uptake of d,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.