Maintenance of the filamentous actin cytoskeleton is necessary for the activation of store-operated Ca2+ channels, but not other types of plasma-membrane Ca2+ channels, in rat hepatocytes

Abstract

The roles of the filamentous actin (F-actin) cytoskeleton and the endoplasmic reticulum (ER) in the mechanism by which store-operated Ca2+ channels (SOCs) and other plasma-membrane Ca2+ channels are activated in rat hepatocytes in primary culture were investigated using cytochalasin D as a probe. Inhibition of thapsigargin-induced Ca2+ inflow by cytochalasin D depended on the concentration and time of treatment, with maximum inhibition observed with 0.1μM cytochalasin D for 3h. Cytochalasin D (0.1μM for 3h) did not inhibit the total amount of Ca2+ released from the ER in response to thapsigargin but did alter the kinetics of Ca2+ release. The effects of cytochalasin D (0.1μM) on vasopressin-induced Ca2+ inflow were similar to those on thapsigargin-induced Ca2+ inflow, except that cytochalasin D did inhibit vasopressin-induced release of Ca2+ from the ER. Cytochalasin D (0.1μM) inhibited vasopressin-induced Mn2+ inflow (predominantly through intracellular messenger-activated non-selective cation channels), but the degree of inhibition was less than that of vasopressin-induced Ca2+ inflow (predominantly through Ca2+-selective SOCs). Maitotoxin- and hypotonic shock-induced Ca2+ inflow were enhanced rather than inhibited by 0.1μM cytochalasin D. Treatment with 0.1μM cytochalasin D substantially reduced the amount of F-actin at the cell cortex, whereas 5μM cytochalasin D increased the total amount of F-actin and caused an irregular distribution of F-actin at the cell cortex. Cytochalasin D (0.1μM) caused no significant change in the overall arrangement of the ER {monitored using 3′,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] in fixed cells} but disrupted the fine structure of the smooth ER and reduced the diffusion of DiOC6(3) in the ER in live hepatocytes after photobleaching. It is concluded that (i) the concentration of cytochalasin D is a critical factor in the use of this agent as a probe to disrupt the cortical F-actin cytoskeleton in rat hepatocytes, (ii) a reduction in the amount of cortical F-actin inhibits SOCs but not intracellular messenger-activated non-selective cation channels, and (iii) inhibition of the activation of SOCs and reduction in the amount of cortical F-actin is associated with disruption of the organization of the ER.