Pharmacological characteristics of high-affinity serotonin uptake systems established through gene transfer.

Inactivation of a neurotransmitter, after its stimulated release, via high-affinity uptake mechanisms is an essential regulatory step of neurotransmission in both the central and peripheral nervous systems. To initiate explorations of the molecular mechanisms and the underlying biochemical architecture of high-affinity neurotransmitter uptake systems, we have used gene transfer technology to establish and identify novel cellular models that express these systems. Human genomic DNA was transfected into mouse L-M fibroblasts and two independently arising, clonal cell lines (L-S1 and L-S2) have been identified as expressing high-affinity serotonin (5-HT) uptake systems. The 5-HT uptake characteristics of L-S1 and L-S2 are essentially comparable (in terms of Na+ dependence, temperature sensitivity, imipramine antagonizability, kinetic saturability and high affinities) and those of L-S1 have been reported previously. Furthermore, competition studies utilizing catecholamine neurotransmitters and their amino acid precursors demonstrated that these systems are highly specific for 5-HT. Several known inhibitors of high-affinity 5-HT uptake systems (including amitriptyline, desipramine, fluoxetine, imipramine, nortriptyline, tryptamine, 5-methoxytryptamine and N-acetyl 5-methoxytryptamine) were assessed in terms of their respective potencies to inhibit 5-[3H]HT uptake by L-S1 and L-S2 cells. For L-S1 cells, the rank order of inhibitor potencies is imipramine greater than amitriptyline greater than fluoxetine greater than desipramine = nortriptyline greater than tryptamine greater than 5-methoxytryptamine greater than N-acetyl-5-methoxytryptamine. For L-S2, the rank order is similar to that of L-S1 except that fluoxetine is more potent than amitriptyline.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a genetically reconstituted high-affinity system for serotonin transport.

By transfecting mouse fibroblast L-M cells with human genomic DNA, we have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-[3H]HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (iii) both Na+ and temperature dependences; (iv) kinetic saturability; and (v) high affinity for 5-HT (Km = 0.39 +/- 0.10 microM; Vmax = 2.14 +/- 0.55 pmol/min per mg of protein). This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, we postulate that the observed 5-HT-uptake system resulted from the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neurotransmitter-uptake systems as well as substrates for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.