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

Helena H. Chowdhury, Michel R. Popoff * and Robert Zorec

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.

In control cells, cytosol dialysis with 1 µM Ca²⁺_i 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).

In control cells cytosol dialysis with 0 µM Ca²⁺_i 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).

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.

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