Wang Ying-Jie, Gregory Roland B, Barritt Greg J
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia.
Biochem J. 2002 Apr 1;363(Pt 1):117-26. doi: 10.1042/0264-6021:3630117.
The roles of the filamentous actin (F-actin) cytoskeleton and the endoplasmic reticulum (ER) in the mechanism by which store-operated Ca(2+) channels (SOCs) and other plasma-membrane Ca(2+) channels are activated in rat hepatocytes in primary culture were investigated using cytochalasin D as a probe. Inhibition of thapsigargin-induced Ca(2+) inflow by cytochalasin D depended on the concentration and time of treatment, with maximum inhibition observed with 0.1 microM cytochalasin D for 3 h. Cytochalasin D (0.1 microM for 3 h) did not inhibit the total amount of Ca(2+) released from the ER in response to thapsigargin but did alter the kinetics of Ca(2+) release. The effects of cytochalasin D (0.1 microM) on vasopressin-induced Ca(2+) inflow were similar to those on thapsigargin-induced Ca(2+) inflow, except that cytochalasin D did inhibit vasopressin-induced release of Ca(2+) from the ER. Cytochalasin D (0.1 microM) inhibited vasopressin-induced Mn(2+) inflow (predominantly through intracellular messenger-activated non-selective cation channels), but the degree of inhibition was less than that of vasopressin-induced Ca(2+) inflow (predominantly through Ca(2+)-selective SOCs). Maitotoxin- and hypotonic shock-induced Ca(2+) inflow were enhanced rather than inhibited by 0.1 microM cytochalasin D. Treatment with 0.1 microM cytochalasin D substantially reduced the amount of F-actin at the cell cortex, whereas 5 microM cytochalasin D increased the total amount of F-actin and caused an irregular distribution of F-actin at the cell cortex. Cytochalasin D (0.1 microM) caused no significant change in the overall arrangement of the ER [monitored using 3',3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] in fixed cells] but disrupted the fine structure of the smooth ER and reduced the diffusion of DiOC(6)(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.
利用细胞松弛素D作为探针，研究了丝状肌动蛋白（F-肌动蛋白）细胞骨架和内质网（ER）在原代培养的大鼠肝细胞中储存操纵性Ca(2+)通道（SOCs）和其他质膜Ca(2+)通道激活机制中的作用。细胞松弛素D对毒胡萝卜素诱导的Ca(2+)内流的抑制作用取决于处理浓度和时间，0.1μM细胞松弛素D处理3小时时抑制作用最大。细胞松弛素D（0.1μM，处理3小时）不抑制毒胡萝卜素刺激内质网释放的Ca(2+)总量，但改变了Ca(2+)释放的动力学。细胞松弛素D（0.1μM）对血管加压素诱导的Ca(2+)内流的影响与对毒胡萝卜素诱导的Ca(2+)内流的影响相似，只是细胞松弛素D确实抑制了血管加压素诱导的内质网Ca(2+)释放。细胞松弛素D（
