Differentiation between amine transport and beta-adrenergic receptor-mediated binding in cultured mammalian cells.

We have found that several types of cultured mammalian cells, including both normal and transformed human, rat, and mouse cell lines, have an active transport system for a diverse group of structurally related compounds possessing an amine group and various types of aromatic ring structures. Ligands such as the beta-adrenergic antagonists (-)-[3H] dihydroalprenolol (DHA), (-)-[3H]propranolol, and (-)-[125I] iodocyanopindolol, and the tricyclic antidepressant [3H]imipramine, which are used to assess cell surface receptors for catecholamines and serotonin, appear to be actively transported into cells rather than simply bound to cell surface sites or accumulated by passive diffusion. DHA transport was competed by many structurally related amines including imipramine and certain alpha-and beta-adrenergic ligands, but not by catecholamines or serotonin. Ligand transport in HeLa cells was saturable at micromolar levels, selective, nonstereospecific, temperature- and pH-dependent, and sensitive to the ionophore monensin and the amine transport inhibitor reserpine, thus indicating dependence on a carrier system driven by a transmembrane proton gradient. In C6 glioma cells, amine transport was clearly distinguishable from beta-adrenergic receptor binding which could be measured with the recently developed hydrophilic beta-blocker (+/-)-[3H] 4-(3-tertiarybutylamino-2-hydroxy-propoxy)-benzimidazole-2-on hydrochloride (CGP-12177); binding of this ligand met rigorous pharmacological criteria, was not influenced by monensin or reserpine, and, therefore, did not appear to be transported. Membrane vesicles from HeLa and C6 cells transported DHA but not CGP-12177 via a MgATP-dependent mechanism which was inhibited by N,N'-dicyclohexylcarbodiimide, monensin, and reserpine, indicating a carrier system driven by a proton gradient maintained by a proton-pumping ATPase.