Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphorylation mechanisms. The aim of this study was to investigate the possible regulation of the human extraneuronal monoamine transporter, hEMT, by these mechanisms. The experiments were performed using HEK293 cells stably transfected with pcDNA3hEMT (293hEMT). The characteristics of hEMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) were studied by incubating the cells at 37 degrees C for 1 min with 200 nM [3H]MPP+. Uptake of [3H]MPP+ by 293hEMT cells was not affected or only slightly reduced by modulators of protein kinase A, protein kinase C, or protein kinase G. It was not affected by an inhibitor of protein tyrosine kinase and was reduced by mitogen-activated protein kinase inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was independent of extracellular Ca2+ and strongly reduced by Ca2+/calmodulin pathway inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced in the presence of non-selective phosphodiesterase inhibitors (IBMX, caffeine, theophylline). The effect of IBMX was independent of extracellular Ca2+ its IC50 was found to be 82.0 microM (66.2-101.6 microM; n=4), and its inhibitory effect resulted from a significant decrease in the maximal velocity of [3H]MPP+ uptake, with no change in the Michaelis-Menten constant. [3H]MPP+ uptake was reduced by 8-methoxy-methyl-IBMX, a selective inhibitor of the Ca2+/calmodulin-dependent phosphodiesterase (PDE1), but not by zaprinast, a selective inhibitor of PDE5. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced by protein tyrosine phosphatase inhibitors, by an alkaline phosphatase inhibitor and, by contrast. showed an increase in the presence of exogenous alkaline phosphatase. In conclusion, these results suggest that hEMT is regulated by phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state.