Erythrocyte ghosts were loaded with pancreatic DNase I and fused with Y-1 adrenal tumor cells to test the possibility that this enzyme might inhibit the steroidogenic responses of the cells to ACTH and cyclic AMP. Fusion of erythrocyte ghosts loaded with DNase I, but not those containing albumin, ovalbumin, boiled DNase I, or DNase I with excess G-actin, inhibited the increase in production of 20 alpha-dihydroprogesterone produced by ACTH and dibutyryl cyclic AMP; inhibition was concentration-dependent with 50% inhibition by 3 X 10(7) molecules of DNase I per cell. It was found that inhibition by DNase I was exerted at the step in the steroidogenic pathway at which cholesterol is transported to mitochondria where steroidogenesis begins. This was shown by measuring transport of cholesterol into the inner mitochondrial membrane, by measuring the production of pregnenolone by isolated mitochondria and by demonstrating that DNase I was without effect on the conversion of pregnenolone to 20 alpha-dihydroprogesterone (an end-product of steroid synthesis). The actin content of Y-1 cells was measured by two methods based upon inhibition of DNase I and by SDS gels following centrifugation. The cells were found to contain 2-3 X 10(7) molecules of actin per cell of which two-thirds is present as G-actin. Since DNase I is known to bind to G-actin to give a one to one complex, these and other findings suggest that at least some of the G-actin in the cells may be necessary for the steroidogenic responses to ACTH and cyclic AMP.
Two approaches were used to study the possible role of calmodulin in the regulation of steroid synthesis by mouse adrenal tumor cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.