Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells.
The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.
Glenn
D E
DE
Physiological Laboratory, University of Liverpool, England, UK.
Thomas
G M
GM
O'Sullivan
A J
AJ
Burgoyne
R D
RD
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002417:Cattle; D019439:Chromaffin Cells / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D016716:PC12 Cells / Q000502:physiology*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats
D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors