PROTEIN-PHOSPHORYLATION IN THE REGULATION OF INSULIN-SECRETION - THE USE OF SITE-DIRECTED INHIBITORY PEPTIDES IN ELECTRICALLY PERMEABILIZEDISLETS OF LANGERHANS

We have used electrically permeabilised rat islets of Langerhans to in vestigate the role of protein phosphorylation in the regulation of ins ulin secretion using pseudosubstrate inhibitory peptides for cyclic AM P-dependent protein kinase (PKA) and for protein kinase C (PKC). The p rotein kinase inhibitor (PKI) peptide, PKI(6-22), completely inhibited the effects of cyclic AMP on islet PKA activity in vitro, on endogeno us protein phosphorylation and on insulin secretion. This peptide had no significant effect on islet PKC activity in vitro, on Ca2+-induced protein phosphorylation and on secretory responses to Ca2+ or to the P KC activator, 4 beta-phorbol myristate acetate (PMA). The PKC pseudo-s ubstrate inhibitory peptide, PKC(19-36), caused a marked inhibition of islet PKC activity in vitro and inhibite PMA-induced insulin secretio n without affecting secretory responses to cyclic AMP and Ca2+. These results demonstrate that PKA- and PKC-induced protein phosphorylation is obligatory for cyclic AMP- and PMA-stimulated insulin secretion, re spectively, and suggest that there is little ''crosstalk'' between the response elements of the secretory pathways to the different second m essengers, at least after the generation of the messengers within the beta-cells.