A role for protein kinase C subtypes alpha and epsilon in phorbol-ester-enhanced K(+)- and carbachol-evoked noradrenaline release from the human neuroblastoma SH-SY5Y.
Protein kinase C (PKC) consists of a family of closely related subtypes which differ in their localization and activation properties. Our previous studies have suggested a role for PKC in the regulation of noradrenaline (NA) release from the human neuroblastoma SH-SY5Y. Here we have used two approaches to characterize the PKC subtypes present in SH-SY5Y cells. Firstly, the PCR was used to show that SH-SY5Y cells contain mRNA encoding PKC subtypes alpha, beta, gamma, delta, epsilon and zeta. Secondly, immunoblotting showed that SH-SY5Y cells express PKC subtypes alpha, epsilon and zeta at the protein level. Prolonged (48 h) exposure of cells to the phorbol ester phorbol 12-myristate 13-acetate (PMA; 100 nM) resulted in a marked decrease in the amounts of PKC-alpha and PKC-epsilon, with no change in levels of PKC-zeta. Prolonged PMA treatment had no significant effect on K(+)-evoked NA release from SH-SY5Y cells, whereas carbachol-evoked release was increased 2.2-fold. However, prolonged exposure to PMA completely inhibited the ability of acute (12 min) PMA treatment to enhance both K(+)- and carbachol-evoked NA release. The specific PKC inhibitor RO 31-7459 (10 microM) was found to inhibit K(+)- and carbachol-evoked release by 27% and 68% respectively. RO 31-7549 also completely inhibited the ability of acute PMA treatment to enhance release. These data suggest that PKC-alpha and/or PKC-epsilon play an essential role in the regulation of PMA-enhanced K(+)- and carbachol-evoked NA release in SH-SY5Y cells.
Turner
N A
NA
Unit of Cardiovascular Studies, University of Leeds, U.K.
Rumsby
M G
MG
Walker
J H
JH
McMorris
F A
FA
Ball
S G
SG
Vaughan
P F
PF
D001483:Base Sequence; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D005796:Genes; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000201:enzymology*; D009638:Norepinephrine / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000145:classification / Q000276:immunology / Q000502:physiology*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007211:Indoles; D008301:Maleimides; D010703:Phorbol Esters; D002217:Carbachol; D011493:Protein Kinase C; D011188:Potassium; D009638:Norepinephrine; C064755:Ro 31-7549