PubMed1973
1. The effects of changes of ionic environment upon corticosteroid production by rabbit adrenal glands have been investigated in vitro using a superfusion technique and on-line steroid analysis by an automated fluorescence method. In some experiments micro-electrode recordings of adrenocortical transmembrane potentials were made concomitantly with measurement of steroid output.2. Adrenocorticotrophic hormone (ACTH), 10 m-u./ml., induced a sevenfold increase in corticosteroid production rate in normal Krebs solution.3. The steroidogenic response to ACTH was not impaired after omission of [K](o) for 1 hr but was inhibited following exposure to K(+)-free medium for 3 hr. Increase of [K](o) tenfold to 47 mM increased the basal but not the ACTH-stimulated output of corticosteroid whereas raising [K](o) twentyfold to 94 mM enhanced both the basal and ACTH-stimulated steroid production rate. In K(+)-free solution the adrenocortical cells hyperpolarized from - 67 to - 86 mV; subsequently on addition of ACTH they depolarized. Reintroduction of K(+) restored the membrane potential.4. Omission of Ca(2+) partially depolarized the cells but only affected the steroidogenic response to ACTH in the presence of EDTA. A threefold increase of [Ca](o), to 7.68 mM, had no effect on either membrane potentials or steroid formation, but increasing [Ca](o) tenfold to 25.6 mM partially blocked ACTH action. Increasing [Mg](o) twentyfold to 22.6 mM had little effect on ACTH-stimulated corticosteroid output and Sr 2.56 mM, in substitution for Ca(2+), supported ACTH action, but La, 0.25 mM, completely blocked the steroidogenic effect of ACTH.5. Replacement of NaCl, 118 mM by choline chloride, 118 mM, was without effect on ACTH-induced steroidogenesis, whereas LiCl, 118 mM, reduced it by 50%. NaF, 1 and 10 mM, inhibited ACTH-induced steroidogenesis by approximately 60%.6. Nupercaine, 10(-4)M, inhibited the steroid response to ACTH with no effect upon membrane potentials: increasing the nupercaine concentration to 10(-3)M inhibited the steroid response and depolarized the cells. Ouabain, 10(-5)M, induced complete depolarization and suppression of the steroidogenic response to ACTH.7. Action-potential-like changes in membrane potential appeared in cells exposed to ACTH in a K(+)-free medium. The amplitude of the action potentials ranged from 10 to 60 mV according to cell, with a frequency up to 36/min; the frequency tended to increase with time. Tetrodotoxin, 10(-6) g/ml., did not inhibit ACTH-induced action potentials in K(+)-free medium.8. These observations are discussed in relation to the ionic requirements for the steroidogenic action of ACTH. The results further emphasize the dissociation of membrane polarization and the secretion of steroid. The mechanism of output of steroid hormone from the adrenocortical cell may thus differ fundamentally from the secretory mechanisms in other, particle-storing cells.