Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters.
1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction.
Lynge
J
J
Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark.
Juel
C
C
Hellsten
Y
Y
D000241:Adenosine / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D005110:Extracellular Space / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D033703:Nucleoside Transport Proteins; D051381:Rats; D017208:Rats, Wistar; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000494:pharmacology
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; C444719:adenosine transporter; D000249:Adenosine Monophosphate; D013868:Thioinosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C001789:4-nitrobenzylthioinosine; D000241:Adenosine