2-Fluoroadenosine uptake by erythrocytes and endothelial cells studied by 19F-NMR.
Transport and phosphorylation of 2-fluoroadenosine (F-AR) were studied in human erythrocytes and porcine aortic endothelial cells by 19F-nuclear magnetic resonance (NMR) spectroscopy. F-AR (590 microM) added to a human erythrocyte suspension (15% hematocrit) was rapidly incorporated into adenine nucleotides at a rate of 38 nmol.min-1.ml red blood cells-1. Intracellular F-AR could be distinguished from extracellular F-AR due to a chemical shift difference of 0.43 +/- 0.03 ppm (n = 5 experiments). Compared with F-AR, fluoro-ATP purified by high-performance liquid chromatography (HPLC) exhibited a chemical shift of -0.052 ppm, which was too small to differentiate intracellular F-AR and fluoro-ATP in vivo. F-AR uptake was decreased by inhibition of membrane transport with dipyridamole (25 microM) or blockade of adenosine kinase by iodotubercidin (10 microM). The time course of F-AR uptake suggested that the rate-limiting step was not membrane transport but the intracellular phosphorylation by adenosine kinase. In porcine aortic endothelial cells grown on microcarrier beads and perfused within the magnet, there was a linear relation between the F-AR concentration applied (2, 4, 8, or 32 microM) and net uptake measured (27-827 pmol.min-1.mg-1). Intra- and extracellular fluoroadenine compounds were separated by 0.12 ppm, and HPLC analysis confirmed F-AR conversion to fluoroadenine nucleotides. Our findings demonstrate that cellular transport and metabolism of F-AR can be noninvasively studied and analyzed by 19F-NMR.
Decking
U K
UK
Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Alves
C
C
Spahr
R
R
Schrader
J
J
D000241:Adenosine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005461:Fluorine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013552:Swine
C034064:2-fluoroadenosine; C087189:2-fluoro-ADP; C038022:2-fluoro-ATP; D005461:Fluorine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000241:Adenosine