Actin cytoskeleton depolymerization with Clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs

1. We measured membrane capacitance (C-m) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerises cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 mu M Ca-i(2+) increased C-m by 23 +/- 4% (n = 11) relative to the resting C-m 400 s after the start of patch rupture. In CST-treated cells the increase in C-m was 32 +/- 5% (n = 15), not significantly different from controls. The rate of C-m increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of C-m increase was 4.27 +/- 0.85 fF s(-1) (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s(-1) (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3. In control cells cytosol dialysis with 0 mu M Ca-i(2+) decreased C-m by 9 +/- 3% (n = 7), in CST-treated cells C-m increased by 11 +/- 3% (n = 7) relative to resting C-m 400 s after the start of cytosol dialysis. The rate of change in C-m remained constant (controls: -1 to -2 fF s(-1); CST treatment: 1-2 fF s(-1)). 4. Transient and sustained effects of CST treatment on changes in C-m at high or low [Ca2+](i), respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in C-m. Transient enhancement of the rate of C-m by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in C-m suggests cytoskeletal involvement, in endocytosis.