A counter-streaming centrifugation technique for the superfusion of adrenocortical cell suspensions stimulated by ACTH

A system is described for the superfusion of isolated adrenocortical cells. Cells are retained in suspension in a small conical tube by balancing the viscous force of flowing medium against an opposing centrifugal force. Fractions of the effluent medium have been collected over short time periods, for examination of the dynamic responses of the isolated cells to single injections and to continuous infusions of ACTH. The effect of cycloheximide has been studied and an overshoot phenomenon observed following its withdrawal, similar to that found when ACTH alone was continuously infused. The characteristics of the output rates of cyclic AMP and of corticosterone were very much alike in response to different amounts of injected ACTH, and showed that the cells remained responsive in this system for several hours. Large increases in the rate of steroidogenesis were apparent in this sytem within 24 seconds of ACTH administration. The rapidity of this effect makes unlikely the direct induction by ACTH of the synthesis of new protein for regulation of steroidogenesis, unless this protein can be assembled in an exceptionally short time. The data are however consistent with a model proposing the post-translational activation by ACTH of a pre-formed, but labile, protein. The half-life of this regulatory protein was estimated at 3 min in this system.