Long-lived cytoplasmic factors that suppress adrenal steroidogenesis

Adrenal cells secrete steroids after stimulation with corticotropin (ACTH), whereas cells reconstructed by fusing adrenal cell nuclei to fibroblast cytoplasms are temporarily (2-3 wk) unresponsive to ACTH. In this report, we characterize this inhibition by using 'cybrids' (cytoplasmic hybrids) isolated by either genetic selection or a new procedure that utilizes the fluorescence-activated cell sorter and the vital mitochondrial dye rhodamine 123. Such cybrids, which contain both adrenal and fibroblast cytoplasmic components, are unable to produce steroids, suggesting the existence of cytoplasmic inhibitory factors. In order to elucidate this cytoplasmic inhibition of steroidogenesis, techniques are described that test the contribution of fibroblast mitochondria to this phenomenon. The first technique utilizes purified mitochondria, isolated from chloramphenicol (CAP)-resistant fibroblasts, to confer CAP resistance on an otherwise sensitive adrenal cell. The resulting CAP(r) cells, termed mitochondrial transformants, are responsive to ACTH. The second technique utilizes a procedure for isolating small fragments of cytoplasm (microcytospheres) from fibroblasts. Microcytospheres, which do not contain mitochondria, are stained with rhodamine 18, a vital membrane dye, and then fused to unstained adrenal cells. The fusion products are then isolated with the fluorescence-activated cell sorter. Approximately 30% of the fusion products are inhibited in their ability to respond to ACTH. These results suggest that the fibroblast cytoplasm contains nonmitochondrial long-lived inhibitory factors that temporarily suppress steroidogenic function in adrenal cells.