Phorbol Ester‐Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C‐α in Human SH‐SY5Y Neuroblastoma Cells

Abstract

The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes alpha, epsilon, and zeta on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC(50) 0.6 microM) but not 100 mM (K+)-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and (K+)-evoked release after pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) for 8 min, with IC50 values of 0.7 and 2.4 microM, respectively. Immunoblotting studies showed 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 TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-alpha, but not PKC-epsilon or zeta. The PKC inhibitor Gö-6976 (2 microM), which has been shown to inhibit selectively PKC-alpha and beta in vitro, also inhibited the TPA enhancement of carbachol- and (K+)-evoked NA release by > 50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and (K+)-evoked NA secretion is due to activation of PKC-alpha.