RESUMO
Our studies indicate that tritiated 12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) produced by the reduction of the C-20 aldehyde with sodium [3H]borohydride is recognized by the same cellular site as is unlabeled 12-O-tetradecanoylphorbol-13-acetate (TPA). None of the concentrations of TPA used in these studies had an effect on the cell number and viability of human peripheral blood lymphocytes (HPBL) when incubated up to 1 hr at temperatures of 37 and 4 degrees as compared to untreated controls. [3H]TPA was not significantly metabolized by these cells after 1 hr at 37 degrees. Examination of the binding of [3H]TPA with simultaneous examination of uptake of tritiated thymidine ([3H]dThd) in parallel cultures demonstrated a close correlation between the apparent binding constant (0.94 X 10(8) M-1) and the activation constant for TPA stimulation of [3H]-dThd incorporation (0.95 X 10(-8) M). Binding of [3H]TPA was examined in two experimental conditions in which TPA-induced mitogenesis was inhibited: (a) preincubation of HPBL at 37 degrees for 24 hr causes a decrease of [3H]dThd uptake of 50% and an apparent loss of binding sites for [3H]TPA; and (b) glucocorticoid inhibition of [3H]dThd uptake in HPBL by 50%, however, did not reduce [3H]TPA binding. Our data suggest that cellular receptors either at the membrane or in the cytoplasm exist for TPA in HPBL. Alterations in binding of TPA to these receptors may account for the decrease in mitogenic response in preincubation experiments.
