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

Abstract

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

Figure 1. Photomicrographs of phalloidin stained rat melanotrophs

Rat melanotrophs observed under phase contrast (A, × 40) and under fluorescence microscopy (B, × 40; C, × 100). Top panels show cells in control conditions, whereas lower panels show cells treated with CST (15 nm Sa and 15 nm Sb) for 1 h. Cell diameter is around 15 μm. Note that panels B and C display different cells.

Figure 2. Time-dependent changes in membrane capacitance with high [Ca²⁺]_i

A, changes in membrane capacitance in rat melanotrophs elicited with cytosol dialysis with 1 μm $C_{\text{i}}^{2+}$. The top trace is the control and the trace below is from a CST-treated cell (15 nm Sa and 15 nm Sb, treated for 1 h). Values adjacent to traces indicate resting capacitance. B, mean changes in membrane capacitance (%C_m) relative to resting C_m with high [Ca²⁺]_i. Changes in membrane capacitance are determined every 100 s after the start of cytosol dialysis with 1 μm $C_{\text{i}}^{2+}$. *Significant differences between the control and CST-toxin treated cells (Student's t test, P < 0.01). ○, CST toxin treated cells (+Toxin CST); ▾, cells treated with the temperature-denatured CST toxin (15 nm Sa and 15 nm Sb, treated for 1 h (+Denat. CST); •, control cells. Numbers adjacent to points indicate numbers of cells tested, bars indicate s.e.m.

Figure 3. Time-dependent changes in the rate of the secretory response (dC_m/dt) with high [Ca²⁺]_i

A, representative time-dependent changes in the rate of the secretory response (dC_m/dt) in control and in a CST-treated cell after cytosol dialysis with 1 μm $C_{\text{i}}^{2+}$. B, mean time-dependent changes in the rate of C_m change (dC_m/dt) determined at various time points after the start of cytosol dialysis with 1 μm ${\text{Ca}}_{\text{i}}^{2+}$ in control (•) and CST-treated cells (○). *Significant differences between the control and CST- treated cells (Student's t test, P < 0.01). Numbers adjacent to points indicate numbers of cells tested.

Figure 4. Time-dependent changes in membrane capacitance with low [Ca²⁺]_i

A, representative time-dependent changes in C_m in control (bottom trace) and in a CST-treated cell. Both cells were dialysed with a pipette-filling solution containing 5 mm EGTA with no added Ca²⁺. Values adjacent to traces indicate resting capacitance. B, mean changes in membrane capacitance (%C_m) relative to resting C_m with low [Ca²⁺]_i determined every 100 s after the start of cytosol dialysis. *Significant differences between the control (•) and CST-treated cells (○, Student's t test, P < 0.01). Differences between control cells and those treated with denatured CST (▾) were found not to be significantly different. Numbers adjacent to points indicate numbers of cells tested.