Regulation of [³H]phorbol-12,13-dibutyrate binding sites in mouse neuroblastoma cells: Simultaneous down-regulation by phorbol esters and desensitization of their inhibition of muscarinic receptor function

The binding characteristics of [³H]phorbol-12,13-dibutyrate ([³H]PDBu) in mouse neuroblastoma N1E-115 cells were studied. The specific binding of [³H]PDBu to intact cells was saturable and to a homogeneous class of binding sites, with a K(d) of 21 nM. Phorbol 12-myristate-13-acetate and PDBu competed for [³H]PDBu binding whereas 4α-phorbol did not. The binding of [³H]PDBu to the cells was selective, as it was not affected by several agents that interact with various neurotransmitter receptors in N1E-115 cells. The density of the phorbol ester binding site decreased as the cell passage increased, although the K(d) of [³H]PDBu binding remained relatively constant. Upon exposure of the cells to 100 nM PDBu for 1 hr at 37°C, a translocation of the binding sites from the cytosol to the particulate fraction was observed. A similar pretreatment of the cells with 1 mM carbamylcholine, however, was ineffective. The specific binding of [³H]PDBu was down-regulated in both a time- and a concentration-dependent fashion by exposure of the cells to PDBu. When the cells were treated with 100 nM PDBu for 24 hr, the maximum binding site density of [³H]PDBu was decreased to 47% of control, with no change in the K(d). Recovery of [³H]PDBu binding after exposure to the phorbol ester for 24 hr was slow and incomplete, and was dependent on protein synthesis. The down-regulation of [³H]PDBu binding after pretreatment of the cells with PDBu for 24 hr was accompanied by an attenuation of the ability of phorbol 12-myristate-13-acetate to inhibit carbamylcholine-induced cyclic GMP formation as well as inositol phosphates accumulation in these cells, indicating desensitization of protein kinase C function.