Dihydrocytochalasin B

The differentiated phenotype of rabbit articular chondrocytes was modulated in primary culture by treatment with 1 microgram/ml retinoic acid (RA) and reexpressed in secondary culture by treatment with the microfilament-disruptive drug dihydrocytochalasin B (DHCB) in the absence of RA. Because the effective dose of DHCB (3 microM) did not elicit detectable cell rounding or retraction, the nature and extent of microfilament modification responsible for induction of reexpression was evaluated. The network of microfilament stress fibers detected with rhodamine-labeled phalloidin in primary control chondrocytes was altered by RA to a "cobblestone" pattern of circularly oriented fibers at the cell periphery. Subsequent treatment with DHCB resulted in rapid changes in this pattern before overt reexpression. Stress fibers decreased in number and were reoriented. Parallel arrays of long fibers that traversed the cell were evident, in addition to fiber fragments and focal condensations of staining. Immunofluorescent staining of intermediate filaments revealed a marked decrease in complexity and intensity during RA treatment but no change during reexpression. An extended microtubular architecture was present throughout the study. These results clearly identify microfilaments as the principal affected cytoskeletal element and demonstrate that their modification, rather than complete disruption, is sufficient for reexpression. The specificity of DHCB and the reorientation of these filaments before reexpression of the differentiated phenotype suggests a causative role in the mechanism of reexpression.

Differentiation of preadipose 3T3-F442A cells into adipose cells is accelerated by the addition of dihydrocytochalasin B. The effect of the drug on 3T3-C2 cells is more marked: these cells are practically unable to differentiate in the absence of H2CB but a long-term exposure to the drug enables the cells to accumulate lipid droplets in medium supplemented with fetal calf serum and insulin. During their differentiation under these conditions the 3T3-C2 cells develop markers typical of adipose cells: glycerophosphate dehydrogenase, ATP-citrate lyase, fatty acid synthetase and glycerophosphate acyltransferase.

In vivo calcium absorption was studied in normal and rachitic chicks. Cytochalasin B (CB) at a concentration of 25 microgram/ml added to the medium inside the duodenal lumen inhibited calcium absorption (20 min) from 82.5 +/- 1.9% of calcium absorbed in the controls to 59.2 +/- 3% in normal and from 70.0 +/- 2.3% to 47.0 +/- 2.1% in rachitic chicks. In vitro studies by everted ileal sacs of young rabbits also showed an inhibition of active transport of calcium due to CB. Whereas in the controls the ratio of 45Ca concentrations in serosal and mucosal media (60 min) was 7.2 +/- 0.32, the ratios were 5.24 +/- 0.52; 4.40 +/- 0.36; 3.40 +/- 0.42; 5.77 +/- 0.52; 1.38 +/- 0.08; and 1.06 +/- 0.02 in the presence of CB at concentrations of 5, 10 and 25 microgram/ml; colchicine 10(-4)M, Na citrate 0.02M, and heat-devitalized conditions, respectively. 45Ca concentration in the mucosal scrapings was also affected. It showed an increase from controls (15,101 +/- 404 cpm/mg) and correlated with CB concentration: 17,378 +/- 489, 19,015 +/- 1000, and 20,201 +/- 362 at 5, 10, and 25 microgram/ml, respectively. Dihydrocytochalasin B also inhibited active calcium transport and caused an increase in 45Ca concentration in the mucosal scrapings. Correlated electron microscopic studies showed certain changes in the brush border, especially in some actin microfilaments in the terminal web region. It seems that these morphological alterations may be related to transcytoplasmic movement of calcium.