Transport of asymmetric dimethylarginine (ADMA) by cationic amino acid transporter 2 (CAT2), organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1).

Asymmetric dimethylarginine (ADMA), inhibiting the nitric oxide (NO) synthesis from L-arginine, is a known cardiovascular risk factor. Our aim was to investigate if ADMA and/or L-arginine are substrates of the human cationic amino acid transporters 2A (CAT2A, SLC7A2A) and 2B (CAT2B, SLC7A2B), the organic cation transporter 2 (OCT2, SLC22A2), and the multidrug and toxin extrusion protein 1 (MATE1, SLC47A1). We systematically investigated the kinetics of ADMA and L-arginine transport in human embryonic kidney (HEK293) cells stably overexpressing CAT2A, CAT2B, OCT2, or MATE1. Vector-only transfected HEK293 cells served as controls. Compared to vector control cells, uptake of ADMA and L-arginine was significantly higher (p < 0.05) in cells expressing CAT2B and OCT2 at almost all investigated concentrations, while cells expressing CAT2A only showed a significant uptake at concentrations above 300 µM. Uptake of MATE1 overexpressing cells was significantly (p < 0.05) higher at pH 7.8 and 8.2 than controls. Apparent V max values (nmol mg protein(-1) min(-1)) for cellular uptake of ADMA and L-arginine were ≈11.8 ± 1.2 and 19.5 ± 0.7 for CAT2A, ≈14.3 ± 1.0 and 15.3 ± 0.4 for CAT2B, and 6.3 ± 0.3 and >50 for OCT2, respectively. Apparent K m values (µmol/l) for cellular uptake of ADMA and L-arginine were ≈3,033 ± 675 and 3,510 ± 419 for CAT2A, ≈4,021 ± 532 and 952 ± 92 for CAT2B, and 967 ± 143 and >10,000 for OCT2, respectively. ADMA and L-arginine are substrates of human CAT2A, CAT2B, OCT2 and MATE1. Transport kinetics of CAT2A, CAT2B, and OCT2 indicate a low affinity, high capacity transport, which may be relevant for renal and hepatic elimination of ADMA or L-arginine.

Interaction of the cardiovascular risk marker asymmetric dimethylarginine (ADMA) with the human cationic amino acid transporter 1 (CAT1).

Elevated plasma concentrations of endogenously formed asymmetric (ADMA) and symmetric dimethyl-l-arginine (SDMA) are associated with adverse clinical outcomes. Our aim was to investigate the cellular uptake properties of ADMA by the human cationic amino acid transporter 1 (CAT1; SLC7A1). Human embryonic kidney cells (HEK293) stably overexpressing CAT1 (HEK-CAT1) and vector-transfected control cells (HEK-VC) were established to determine cellular uptake of labeled [(3)H]ADMA and [(3)H]l-arginine. Uptake of ADMA and l-arginine were significantly (p<0.001) higher in HEK-CAT1 than in HEK-VC at all investigated concentrations. Apparent V(max) values of cellular ADMA and l-arginine uptake by CAT1 were 26.9 ± 0.8 and 11.0 ± 0.2 nmol mg protein(-1) min(-1), respectively. K(m) values were 183 ± 21 µmoll(-1) (ADMA) and 519 ± 36 µmoll(-1) (l-arginine). Uptake of ADMA was inhibited by l-arginine and SDMA with IC(50) values (95% CI) of 227 (69-742) µmoll(-1) and 273 (191-390) µmoll(-1), respectively. ADMA and SDMA inhibited CAT1-mediated uptake of l-arginine with IC(50) values of 758 (460-1251) µmoll(-1) and 789 (481-1295) µmoll(-1), respectively. Efflux of ADMA was significantly increased in HEK-CAT1 cells as compared to HEK-VC (p<0.05). CAT1 mediates the cellular uptake of ADMA. In its physiological concentration range ADMA is unlikely to impair CAT1-mediated transport of l-arginine. Conversely, high (but still physiological) concentrations of l-arginine can inhibit CAT1-mediated cellular uptake of ADMA.

Characterization of beta-adrenoceptor antagonists as substrates and inhibitors of the drug transporter P-glycoprotein.

Transporter proteins such as P-glycoprotein are major determinants of intracellular drug concentrations. Moreover, inhibition or induction of transporters is an important mechanism underlying drug interactions in humans. However, very little is known whether beta-adrenoceptor antagonists are substrates and/or inhibitors of P-glycoprotein. Therefore, we investigated the P-glycoprotein-mediated transport of propranolol, metoprolol, bisoprolol, carvedilol and sotalol in P-glycoprotein-expressing Caco-2 monolayers and inhibition of P-glycoprotein-mediated digoxin transport by the beta-adrenoceptor antagonists. A significant inhibition of polarized, basal to apical drug transport by the P-glycoprotein inhibitor PSC-833 was observed for bisoprolol (0.5 and 5 microm) and carvedilol (0.5 microm). Moreover, propranolol and carvedilol inhibited P-glycoprotein-mediated digoxin transport with IC(50) values of 24.8 and 0.16 microm, respectively, whereas metoprolol and sotalol had no effect. Bisoprolol significantly inhibited directional digoxin transport at 50 and 250 microm by 31% and 44%, respectively. Taken together, P-glycoprotein is likely to be one determinant of bisoprolol and carvedilol disposition in humans. In addition, the beta-adrenoceptor antagonists propranolol and carvedilol significantly inhibit P-glycoprotein function thereby possibly contributing to drug interactions in humans (e.g. digoxin-carvedilol and cyclosporine-carvedilol).