Two phorbol ester receptor affinities in partially transformed human urothelial cells and decrease of receptor binding in desensitized cells

The presence of specific binding sites for phorbol esters was studied in a transformed but non-tumorigenic human urothelial cell line HCV-29 by assay of specific binding of 3H-phorbol-12,13-dibutyrate (3H-PDBu) to intact living cells. 3H-PDBu bound specifically to HCV-29 cells in a saturable and competitive manner. Scatchard plot analysis of specific binding yielded a curved plot consistent with two binding sites with Kd of 11 nM and 102 nM, respectively. At saturation the corresponding PDBu binding capacities (Bmax) were 8.8 pmol/10(6) cells (5.2 x 10(6) molecules bound per cell) and 2.8 pmol/10(6) cells (1.7 x 10(6) molecules bound per cell). 3H-PDBu binding was displaced by biologically active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein, but not by tumor promoters such as L-tryptophan, anthranilic acid and sodium saccharin. In cells desensitized by pretreatment with 1 microgram/ml (2 microM) TPA or PDBu for 24 h the level of binding was reduced to 28% of the level in non-exposed cells. The ability of desensitized cells to bind 3H-PDBu was gradually restored within 5-6 days. At the same time the cells became sensitive to the morphological alteration induced by PDBu. This suggests that desensitization of HCV-29 cells is due to a decreased receptor-ligand binding capacity probably associated with down regulation of the phorbol ester receptors.