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 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.
Turner
N A
NA
Institute for Cardiovascular Research, University of Leeds, England.
Walker
J H
JH
Ball
S G
SG
Vaughan
P F
PF
D054713:Bryostatins; D002227:Carbazoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008301:Maleimides / Q000494:pharmacology; D008934:Mitogens / Q000494:pharmacology; D009447:Neuroblastoma / Q000473:pathology; D009638:Norepinephrine / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D054713:Bryostatins; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D008301:Maleimides; D008934:Mitogens; D010703:Phorbol Esters; C081021:Go 6976; C046785:bryostatin 1; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D009638:Norepinephrine; C064755:Ro 31-7549