Increases in acetylcholine release produced by phorbol esters are not mediated by protein kinase C at motor nerve endings

Recent work from our laboratory has demonstrated that phorbol esters known to stimulate protein kinase C (PKC) also stimulate acetylcholine (ACh) secretion by an action at a strategic component of the secretory apparatus [Redman et al. (1997) J Physiol (Lond) 501:41-48]. In an attempt to determine whether the stimulatory effects of phorbols are mediated by PKC, we examined the effects of several PKC antagonists on ACh release promoted by phorbol 12,13-dibutyrate (PDBu) at the frog neuromuscular junction. PKC antagonists that act at the ATP binding site (C3 domain) were examined for their ability to antagonize the stimulatory action of PDBu. Neither the non selective PKC inhibitor, staurosporine (at concentrations as high as 1 μM), nor its more selective derivative, GF109203X (at concentrations as high as 10 μM), attenuated the stimulatory effects of PDBu. PKC antagonists that act at the phorbol ester binding site (C1 domain) were examined for their ability to antagonize the stimulatory action of PDBu. Neither sphingosine (500 μM) nor calphostin C (25 μM) reduced the stimulatory actions of PDBu on ACh release. These results suggest that a presynaptic protein possessing a phorbol ester receptor and not the enzyme PKC is the target site for the stimulatory effects of phorbol esters at motor nerve endings.