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-ribosylatjon factor (ARF)-activated and const itutive phospholipase D (PLD) activity in regulated exocytosis of pref ormed secretory granules in adrenal chromaffin and PC12 cells was exam ined. With use of digitonin-permeabilised cells, the effect of GTP ana logues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types, Exocytos is from cytosol-depleted permeabilised chromaffin cells was not increa sed by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Ad dition of bacterial PLD with demonstrably high activity in permeabilis ed chromaffin cells did not increase exocytosis in cytosol-depleted ch romaffin cells. Diversion of PLD activity from production of phosphati dic acid (PA) due to the presence of 4% ethanol did not inhibit exocyt osis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeab ilised chromaffin or PC12 cells. These results indicate that exocytosi s in these cell types does not appear to require a BFA-sensitive ARF a nd the triggering of exocytosis does not require PLD activity and form ation of PA. These findings rule out a general requirement for PLD act ivity during regulated exocytosis.