Cation requirements of basal and ATP-regulated dopamine transport in rat pheochromocytoma cells

The transport of dopamine into presynaptic nerve terminals is the primary mechanism for the termination of dopaminergic neurotransmission. This transport process has recently been found to be composed of two components, a basal dopamine transport pathway which exists in the absence of extracellular ATP and an ATP-regulated moiety which comprises approximately 66% of the total transport system [Cao C. J. et al. (1990) Biochem. Pharmac. 39, R9-R14; Cao C. J. et al. (1989) Biochemistry 8, 207-220; Dunigan C. D. and Shamoo A. E. (1995) Neuroscience 65, 1-4; Eshleman A. et al. (1995) Life Sci. 56, 1613-1621]. Using a rat pheochromocytoma cell line and a Krebs bicarbonate buffering system, the present study examined the effect of several cations on both basal and ATP-regulated dopamine transport. In the absence of extracellular ATP, dopamine transport had an absolute dependence on the presence of Na+, but exhibited no requirement for Mg2+. Kinetically, the addition of 120 mM NaCl increased the Vmax of basal dopamine transport by approximately 150%. In contrast, the ATP-regulated dopamine transport pathway displayed a different sensitivity to Na+ and was completely dependent upon the presence of Mg2+. The addition of 1.2 mM MgSO4 increased the Vmax of transport in the presence of 0.7 mM extracellular ATP by 222%. Both basal and ATP-regulated transport were unaffected by the removal of either Ca2+ or K+ from the assay buffer. When the effects of ouabain, a potent inhibitor of Na+, K(+)-ATPase, were tested in the rat pheochromocytoma cell model, it was found that concentrations of ouabain as high as 1 mM were ineffective at inhibiting either the basal or ATP-regulated dopamine transport components. These results imply that the Na+ gradient supplied by Na+, K(+)-ATPase is not the sole provider of energy needed to drive either transport process. The ionic requirements of the basal and ATP-regulated dopamine transport pathways demonstrate the distinction between the two transport processes. In addition, the ionic dependency profile of the ATP-regulated moiety has provided some mechanistic insights into ATP-regulated catecholamine uptake, as the absolute Mg2+ requirement and the ineffectiveness of Ca2+ argues against the involvement of either purinergic receptors or a Ca(2+)-dependent, Mg(2+)-independent ectokinase in the ATP-regulated transport system.