Adenosine metabolism in phytohemagglutinin stimulated human lymphocytes

The association of a human genetic deficiency of adenosine deaminase (AD) activity with combined immunodeficiency prompted a study of the effects of adenosine (A) and of inhibition of A.D. activity on human lymphocyte transformation and a detailed study of A. metabolism throughout phytohemagglutinin (PHA) induced blastogenesis. The A.D. inhibitor, coformycin, at a concentration that inhibited A.D. activity more than 95%, or 50 μM A., did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid precipitable material. The combination of coformycin and A., however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in PHA stimulated lymphocytes. Incubation of lymphocytes with PHA for 72 hr produced a 12 fold increase in the rate of deamination and a 6 fold increase in phosphorylation of A. by intact lymphocytes. There was no change in the apparent affinity for A. with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 hr after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased A. metabolism was not due to changes in total enzyme activity; after 72 hr in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for A. kinase, 0.92, and decreased for A.D., 0.44. As much as 38% of the initial lymphocyte A.D. activity accumulated extracellularly after a 72 hr culture with PHA. In PHA stimulated lymphocytes, the principal route of A. metabolism was phosphorylation at less than 5 μM A. and deamination at concentrations greater than 5 μM. In unstimulated lymphocytes, deamination was the principal route of A. metabolism over the range of A. concentrations studied (0.5-250 μM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on A.D. activity for the metabolism of A. and may account for the observed sensitivity of mitogen stimulated lymphocytes to the toxic effects of exogenously supplied A. in the presence of the A.D. inhibitor coformycin. A case of immunodeficiency disease is reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased 6 fold after 72 hr culture with PHA. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 hr of culture.