PHORBOL ESTER-ENHANCED NORADRENALINE SECRETION CORRELATES WITH THE PRESENCE AND ACTIVITY OF PROTEIN-KINASE C-ALPHA IN HUMAN SH-SY5Y NEUROBLASTOMA-CELLS

The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes alpha, epsilon, and zeta on noradrenaline (NA) secretion fro m human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibito r Ro 31-7549 inhibited carbachol-evoked NA release (IC50 0.6 mu M) but not 100 mM K+-evoked release. In addition, Ro 31-7549 inhibited the e nhancement of carbachol- and K+-evoked release after pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) for 8 min, with IC 50 values of 0.7 and 2.4 mu M, respectively. Immunoblotting studies sh owed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13- dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-alpha and PKC-epsilon but not PKC-zeta. Under these conditions, the acute TP A enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to e ither PDBu or bryostatin-1, conditions that caused down-regulation of PKC-alpha, but not PKC-epsilon or zeta. The PKC inhibitor Go-69766 (2 mu M), which has been shown to inhibit selectively PKC-alpha and beta in vitro, also inhibited the TPA enhancement of carbachol- and K+-evok ed NA release by >50%. These data suggest that in SH-SY5Y cells, the a bility of TPA to enhance carbachol- and K+-evoked NA secretion is due to activation of PKC-alpha.