ACTH-secreting pituitary adenoma cells were cultured from specimens obtained by transphenoidal
hypophysectomy in five patients with
Cushing's disease. The majority of
adenoma cells (90%) stained specifically with antiserum against human
ACTH. The electrophysiological properties and response to
hormones of these cells were studied with intracellular recording techniques under current clamp and voltage clamp conditions. Most (80%) of the cells fired action potentials that were Ca2+-dependent inasmuch as they were blocked by Co2+ (5 mM) and by removal of Ca2+ from the medium, but were unaffected by
tetrodotoxin (0.3 mM) and by Na+ removal. The cells responded to factors known to stimulate
ACTH release, including high K+, CRF, and
angiotensin II (AII). High K+ (50 mM) induced a membrane depolarization in association with an increase in conductance. CRF (100 nM) produced a depolarization, a decrease in conductance, an increase in spike firing, and an increase in spike duration. Although AII was inactive in ordinary recordings, in cells loaded with
lithium (Li+) to promote the
phospholipid-dependent second messenger system, the
peptide produced an increase in spike firing and spike duration with no change in membrane potential. The combination of CRF and AII (CRF + AII; 100 nM each) in Li+-loaded cells caused a greater excitatory effect than either
peptide alone. Under voltage clamp, the response either to CRF or to CRF + AII could be attributed, at least in part, to the inhibition of a slow, voltage-dependent K+ current that is persistently active at resting potential. These results indicate that modulation of action potential firing may be an early step in the regulation of
ACTH release from pituitary cells by known
secretagogues. Since action potentials in these cells are associated with Ca2+ entry, the resulting changes in intracellular Ca2+ levels could mediate the effects of the
hormones on secretion.