Synergism between phorbol ester and the Ca2+ ionophore A23187 on protein kinase C translocation, [3H]PDBu binding and adenosine A2-receptor activation in Jurkat cells.
Phorbol 12,13-dibutyrate (PDBu) enhances 5'-(N-ethylcarboxamido)-adenosine (NECA) stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in the human T-cell leukemia line, Jurkat. Addition of the Ca2+ ionophore A23187 lowered the EC50 value for PDBu from 49 to 7.1 nM. In binding experiments, where intact cells were incubated with [3H]PDBu at 37 degrees C, addition of A23187 increased the number of binding sites for the phorbol ester. Pretreatment of cells with A23187 was not sufficient to increase [3H]PDBu binding; A23187 had to be combined with phorbol ester in order to enhance [3H]PDBu binding. PDBu treatment translocated protein kinase from the cytosol to the membrane. This effect of the phorbol ester could be enhanced with A23187 whereas A23187 per se had no effect on protein kinase C distribution. From these data it is concluded that the synergism between A23187 and PDBu, monitored as enhancement of NECA-stimulated cAMP accumulation and increase in [3H]PDBu binding, is paralleled by translocation of the enzyme to the particulate fraction of the cells. The finding that cells where the cellular content of protein kinase C had been translocated to the membrane compartment bound more [3H]PDBu than control cells also suggests that [3H]PDBu binding to intact cells reflects the amount of membrane bound-, rather than the total cellular-enzyme content.
Nordstedt
C
C
Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden.
D000001:Calcimycin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis; D004357:Drug Synergism; D006801:Humans; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D011983:Receptors, Purinergic; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D000242:Cyclic AMP; D011493:Protein Kinase C