Using rapid kinetic techniques, we have determined the kinetics of zero-trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25OC in human erythrocytes which were pretreated with 2'-deoxycoformycin to inhibit deamination of adenosine. Both the K , and V, , , for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (K, about 0.2 pM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 pM adenosine, assure that, at extracellular concentrations of physiological relevance ( < I pM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 pM or higher, practically all adenosine transported into t h e cells is deaminated. When the concentration of adenosine was 0.1 pM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 rnin of incubation at 25OC. Adenosine (Ado) has many physiological functions among which are its participation in the metabolic regulation of coronary blood flow and lipolysis and the modulation of neuronal activity (Schrader, 1983;Berne et al., 1983;Wu and Phillis, 1984). Even though the effects of Ado involve a variety of different cells, its action is primarily inhibitory in nature and involves the interaction of Ado with several Ado-specific receptors on the cell surface which are coupled to membrane-associated adenylate cyclase (Schwabe, 1983;Newman and Levitzki, 1982;Yeung and Green, 1983). The source of Ado interacting with the receptors has not been identified unequivocally, neither is anythmg known about the mechanism and regulation of its release from cells, although hypoxia seems in some instances a stimulatory factor (Schrader, 1983;Berne et al., 1983). It is possible
MATERIALS AND METHODS
Human erythrocytes
Erythrocytes from freshly drawn blood were kindly supplied by Dr. J. Kersey (University of Minnesota) as a biproduct of lymphocyte isolation. The cells were thrice washed in cold saline containing 5 mM Tris-HC1, pH 7.4 (Tris-saline), and thereby further freed of residual lymphocytes and other cells by removal of the whitish interface layer. The washed cells were suspended in Trissaline to (4-7) x lo8 celldm1 as enumerated with a Coulter electronic counter.