ENHANCEMENT OF CA2-SY5Y) CELLS BY PHORBOL ESTERS WITH AND WITHOUT ACTIVATION OF PROTEIN-KINASE-C( CHANNEL CURRENTS IN HUMAN NEUROBLASTOMA (SH)

The effects of phorbol esters on Ca2+ channel currents in human neurob lastoma SH-SY5Y cells were studied using whole-cell patch-clamp record ings. Bath application of 12-O-tetradecanoylphorbol-13-acetate (TPA) o r phorbol 12, 13-dibutyrate (PDBu; 100 nM to 1 mu M), known activators of protein kinase C (PKC), enhanced Ca2+ channel currents in a voltag e-dependent manner similar to that of Bay K 8644. TPA also enhanced Ca 2+ channel currents during cell dialysis with the PKC pseudosubstrate, PKC(19-36), and in cells which had been pre-incubated with 500 nM sta urosporine, and which were exposed to staurosporine during recordings. Application of 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD; 100 nM ), which does not activate PKC, caused current enhancement similar to the effects of TPA. However, intracellular dialysis of TPA was without effect on Ca2+ channel currents. Residual Ca2+ channel currents recor ded after exposure to 1 mu M omega-conotoxin GVIA were still enhanced by TPA, but in the presence of either Bay K 8644 (5 mu M) or nifedipin e (5 mu M), TPA was without effect. When cells were pre-incubated for 10 min at 37 degrees C with 100 nM TPA, currents subsequently recorded in its absence were enhanced as compared to untreated cells; 5 mu M n ifedipine still inhibited currents to the same degree. This enhancemen t was not mimicked by 4 alpha-PDD, and was inhibited by staurosporine. Our results indicate that acute applications of phorbol esters (at co ncentrations commonly used to activate PKC) enhance L-type Ca2+ channe l currents in SH-SY5Y cells via a PKC-independent mechanism which appe ars similar to that induced by Bay K 8644. By contrast, pre-incubation with TPA enhances both L- and N-type currents via activation of PKC.