Protein phosphorylation in permeabilized pancreatic islet cells.
A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D007328:Insulin; D011506:Proteins; D011494:Protein Kinases; D004072:Digitonin; D002118:Calcium