CYTOCHALASIN D

Guard cells rapidly adjust their plasma membrane surface area while responding to osmotically induced volume changes. Previous studies have shown that this process is associated with membrane internalization and remobilization. To investigate how guard cells maintain membrane integrity during rapid volume changes, the effects of two membrane trafficking inhibitors on the response of intact guard cells of Vicia faba to osmotic treatments were studied. Using confocal microscopy and epidermal peels, the relationship between the area of a medial paradermal guard-cell section and guard-cell volume was determined. This allowed estimates of guard-cell volume to be made from single paradermal confocal images, and therefore allowed rapid determination of volume as cells responded to osmotic treatments. Volume changes in control cells showed exponential kinetics, and it was possible to calculate an apparent value for guard-cell hydraulic conductivity from these kinetics. Wortmannin and cytochalasin D inhibited the rate of volume loss following a 0-1.5 MPa osmotic treatment. Cytochalasin D also inhibited volume increases following a change from 1.5 MPa to 0 MPa, but wortmannin had no effect. Previous studies showing that treatment with arabinanase inhibits changes in guard-cell volume in response to osmotic treatments were confirmed.

We investigated the mechanosensitivity of voltage-gated K+ channel (VGPC) currents by using whole-cell patch clamp recording in rat trigeminal ganglion (TG) neurons. On the basis of biophysical and pharmacological properties, two types of VGPC currents were isolated. One was transient (I(K,A)), the other sustained (I(K,V)). Hypotonic stimulation (200 mOsm) markedly increased both I(K,A) and I(K,V) without affecting their activation and inactivation kinetics. Gadolinium, a well-known blocker of mechanosensitive channels, failed to block the enhancement of I(K,A) and I(K,V) induced by hypotonic stimulation. During hypotonic stimulation, cytochalasin D, an actin-based cytoskeletal disruptor, further increased I(K,A) and I(K,V), whereas phalloidin, an actin-based cytoskeletal stabilizer, reduced I(K,A) and I(K,V). Confocal imaging with Texas red-phalloidin showed that actin-based cytoskeleton was disrupted by hypotonic stimulation, which was similar to the effect of cytochalasin D. Our results suggest that both I(K,A) and I(K,V) are mechanosensitive and that actin-based cytoskeleton is likely to regulate the mechanosensitivity of VGPC currents in TG neurons.

Our previous report has revealed that PKC activation by 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake activity of serotonin transporter (SET), via an indirect mechanism unknown, but not likely via direct phosphorylation of SET by PKC (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). To elucidate whether PKC can directly phosphorylate SET in vivo, FLAG-tagged SET (FLAG-SET) was expressed in COS-7 cells and the TPA-induced incorporation of (32)P into immunoprecipitated FLAG-SET was examined. PKC activation with TPA caused no phosphorylation of FLAG-SET expressed in COS-7 cells. On the other hand, morphological change associated with the disruption of filamentous actin (F-actin) was seen in TPA-treated COS-7 cells. Therefore, we studied the effects of cytochalasin D, an inhibitor of actin polymerization, on the uptake activity of the serotonin transporter (SET) to elucidate whether the actin cytoskeleton modulates the SET uptake activity. The treatment with cytochalasin D inhibited the uptake activity of both native and recombinant SET in a concentration-dependent manner. Eadie-Hofstee analysis revealed that cytochalasin D down-regulated the recombinant SET uptake activity by reducing the V(max), but not the K(m), mimicking the result observed in TPA-induced inhibition of SET activity (Sakai et al.