Effects of latrunculin reveal requirements for the actin cytoskeleton during secretion from mast cells

To investigate the role of the actin cytoskeleton in exocytosis, we have te sted the effects of latrunculin B, a microfilament-disrupting drug, on secr etion from intact and permeabilised rat peritoneal mast cells. The toxin st rongly inhibited secretion from intact cells (attached or in suspension) re sponding to a polybasic agonist, compound 48/80. However, this effect was r evealed only after a profound depletion of actin filaments. This was achiev ed by a long (1 h) exposure of cells to the drug before activation, togethe r with its presence during activation. Maximal inhibition of secretion by s uch treatment was 85% at 40 mug/ml latrunculin B. These results indicate th at minimal actin structures are essential for the exocytotic response. In c ontrast, stimulus-induced cell spreading was prevented by latrunculin (5 mu g/ml) applied either before or after activation. The effects of the toxin o n intact cells were fully reversible. The responses of permeabilised cells were affected differentially: secretion induced by calcium was more sensiti ve to latrunculin than that induced by GTP-gamma -S. The calcium response, therefore, is more dependent upon the integrity of the actin cytoskeleton t han the response induced by GTP-gamma -S. Again, maximal inhibitory effects (similar to 65 and 25% at 40 mug/ml) were observed only when cells were ex posed to the toxin both before and after permeabilisation. Since the permea bilised cells system focuses on the final steps of exocytosis, the incomple te inhibition suggests that actin plays a modulatory rather than a central role at this stage. Cell Motil. Cytoskeleton 48:37-51, 2001. (C) 2001 Wiley -Liss, Inc.