Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.
We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.
W R
WR
Koopmann
R C
RC
Jackson
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006636:Histamine Release; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology*
D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D007770:L-Lactate Dehydrogenase; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium