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

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Abstract
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‐lsoquinolinesulphonyl)‐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.

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