Abstract Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphor- ylation mechanisms. The aim of this study was to inves- tigate the possible regulation of the human extraneuronal monoamine transporter, hEMT, by these mechanisms. The experiments were performed using HEK293 cells stably transfected with pcDNA3hEMT (293 hEMT ). The character- istics of hEMT-mediated uptake of [ 3 H]1-methyl-4-phenyl- pyridinium ([ 3 H]MPP + ) were studied by incubating the cells at 37°C for 1 min with 200 nM [ 3 H]MPP + . Uptake of [ 3 H]MPP + by 293 hEMT 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 [ 3 H]MPP + by 293 hEMT cells was independent of extracel- lular Ca 2+ and strongly reduced by Ca 2+ /calmodulin path- way inhibitors. Uptake of [ 3 H]MPP + by 293 hEMT cells was strongly reduced in the presence of non-selective phos- phodiesterase inhibitors (IBMX, caffeine, theophylline). The effect of IBMX was independent of extracellular Ca 2+ , its IC 50 was found to be 82.0 μM (66.2–101.6 μM; n=4), and its inhibitory effect resulted from a significant de- crease in the maximal velocity of [ 3 H]MPP + uptake, with no change in the Michaelis-Menten constant. [ 3 H]MPP + uptake was reduced by 8-methoxy-methyl-IBMX, a selec- tive inhibitor of the Ca 2+ /calmodulin-dependent phospho- diesterase (PDE1), but not by zaprinast, a selective in- hibitor of PDE5. Uptake of [ 3 H]MPP + by 293 hEMT 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 regu- lated by phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state. Keywords Extraneuronal monoamine transporter · Protein kinases · Protein phosphatases · Phosphodiesterase · Ca 2+ /calmodulin pathways Introduction The inactivation of released neurotransmitters is essential for synaptic and junctional signal transmission. For cate- cholamines such as noradrenaline and adrenaline, their in- activation is accomplished by active and specialised trans- membrane transport systems. These transporters remove catecholamines from the synaptic cleft or blood stream, thus decreasing the concentration of these amines at the receptor level. Several transport systems for catecholamines have been described. The first to be recognised were up- take 1 , a sodium-driven, high-affinity, cocaine-sensitive neu- ronal transporter, and uptake 2 , a sodium-independent, low- affinity, high-capacity, corticosterone-sensitive extraneu- ronal transporter. More recently, other sodium-indepen- dent transmembrane transporters were found to accept cat- echolamines as substrates. These include the organic cation transporter type 1 (OCT1; Breidert et al. 1998) and the or- ganic cation transporter type 2 (OCT2; Gründemann et al. 1998a). Uptake 2 is found in sympathetically innervated tissues (e.g. myocardium, vascular smooth muscle and glandular cells; Trendelenburg 1988), in central nervous system glia (Staudt et al. 1993; Russ et al. 1996; Streich et al. 1996) and in the retina (Rajan et al. 2000). This transporter was first described more than 30 years ago (Iversen 1965), but its primary structure was only recently elucidated (Grün- demann et al. 1998b; Kekuda et al. 1998). The two inde- F. Martel · E. Keating · C. Calhau · D. Gründemann · E. Schömig · I. Azevedo Regulation of human extraneuronal monoamine transporter (hEMT) expressed in HEK293 cells by intracellular second messenger systems Naunyn-Schmiedeberg’s Arch Pharmacol (2001) 364 : 487–495 DOI 10.1007/s002100100476 Received: 25 April 2001 / Accepted: 19 July 2001 / Published online: 20 September 2001 ORIGINAL ARTICLE F. Martel (✉) · E. Keating Department of Biochemistry, Faculty of Medicine, 4200-319 Porto, Portugal e-mail: fmartel@med.up.pt, Tel.: +351-22-5095694, Fax: +351-22-5502402 F. Martel · C. Calhau · I. Azevedo Institute of Pharmacology and Therapeutics (U38-FCT), Faculty of Medicine, 4200-319 Porto, Portugal D. Gründemann · E. Schömig Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany © Springer-Verlag 2001