Extracellular ATP stimulates norepinephrine uptake in PC12 cells.

This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with synaptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase. In the present study, we examined the high-affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 microM) increased uptake 1 by approximately 36%. This increase could be mimicked by adenosine-5'-O-(3-thiotriphosphate) tetralithium salt (ATP gamma S), an analogue of ATP which can be utilized by protein kinases, and not by 5'-adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP, GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATP gamma S, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATP gamma S blocked the activation produced by ATP gamma S alone. Kinetic analysis showed that ATP gamma S pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase.