Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.