The human erythrocyte ghost: a new experimental model for studying adenosine transport

Previous work on adenosine transport has always had problems with the interference of adenosine metabolism, due to its high metabolic rate and because the enzymes involved are consistently present in most tissues. A new experimental model for studying adenosine transport in human erythrocyte ghosts is presented in this work: Human erythrocyte ghosts were sealed in the presence of erythro-3(2-hydroxynonyl)adenine and P1-P5-di(adenosine)5'-pentaphosphate, inhibitors of adenosine deaminase and adenosine kinase, respectively. These ghosts proved to lack adenosine metabolism when incubated in [U-14C]adenosine at 10 microM concentration at lack 37 degrees C for 60 min. Ghosts were 99.4% sealed in the correct orientation and had constant intracellular water volume. With these characteristics, the erythrocyte ghost preparation has many advantages for studying adenosine transport without adenosine metabolism interference. Adenosine transport was studied following the technique of W. R. Lieb and W. D. Stein [(1974) Biochim. Biophys. Acta 373, 165-177, 178-196.] Experiments to study Zero-trans influx and efflux, equilibrium exchange, and infinite-trans influx and efflux are presented. Adenosine transport did not behave linearly in any of these experimental procedures. Adenosine basic kinetic constants, calculated according to the procedure of Lieb and Stein, were R1----2 = 4.1 X 10(-4), R2----1 = 3.97 X 10(-4), Ree = 1.94 X 10(-4), Roo = 6.08 X 10(-4), K1----2 = 125.67 microM, and K2----1 = 84.36 microM. Lieb and Stein rejection criteria were used to distinguish a simple pore from a simple carrier. The data accumulated indicate that adenosine transport is carried out by a system that satisfies the criteria used for the simple carrier model. Asymmetric behavior was observed indicating lower affinity of the carrier for adenosine influx, although Vmax values for influx and efflux were similar.