IMPORTANCE OF RIBONUCLEOTIDE AVAILABILITY TO PROLIFERATING T-LYMPHOCYTES FROM HEALTHY HUMANS - DISPROPORTIONATE EXPANSION OF PYRIMIDINE POOLS AND CONTRASTING EFFECTS OF DE-NOVO SYNTHESIS INHIBITORS

Sensitive high performance liquid chromatography techniques, which dif ferentiate between purine and pyrimidine ribonucleoside and deoxyribon ucleoside triphosphates, were used to quantify pools in photohemagglut inin-stimulated T-lymphocytes (98% CD4(+) and CD8(+)) from healthy vol unteers. The importance of de novo synthesis and salvage was evaluated by incubating the cells with C-14-radiolabeled precursors (40 mu M), azaserine (20 mu M; a glutamine antagonist), and ribavirin (50 mu M; a n IMP dehydrogenase inhibitor). We confirmed that resting T-lymphocyte s meet their metabolic requirements by salvage. Noteworthy observation s were as follows. First, nucleotide pool expansion over 72 h is dispr oportionate, with that for purines (ATP and GTP) being a-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools, This supports an additional role for the latter in membra ne Lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion, Aza serine not only inhibited purine synthesis (confirmed by N-formylglyci namide polyphosphate accumulation), but also reduced expansion of pyri midine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and r educed pyrimidine pool expansion by 40% at 72 h. These findings underl ine the importance of pyrimidine ribonucleotide availability as well a s GTP synthesis de novo to proliferating T-lymphocytes. They also demo nstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis,