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The electrophysiological properties of the mouse anterior pituitary cell line AtT-20/D16-16 were investigated with intracellular and patch-clamp techniques. Clonal AtT-20/D16-16 cells were found to be electrically excitable, with most cells exhibiting spontaneous bursting action potentials. The mean burst rates varied from 1.4 Hz at -55 mV to 8.2 Hz at -25 mV, showing an approximately linear frequency-current relationship in the low current range. The bursts consisted of one to several fast Na4 spikes superimposed on a slow pacemaker potential, followed by a Ca2+ spike and a Ca2+-sensitive afterhyperpolarization. Removal of either Na+ or Ca2+ from the bathing medium led to cessation of spontaneous activity and the appearance of arrhythmic firing patterns. Single channel recordings revealed the presence of Ca2+-dependent K+ channels with unitary conductances of =130 pS in physiological medium. These channels were activated by both intracellular Ca2+ and membrane depolarization. Addition of norepinephrine (10 ,LM) led to increases in burst frequency and f8-endorphin secretion mediated by activation of f3-adrenergic receptors. Our results, in conjunction with previous work, suggest that the Ca2+ that enters the cell during the burst may be involved in hormone secretion.Clonal AtT-20/D16v mouse anterior pituitary tumor cells synthesize, store, and secrete corticotropin (ACTH), f3-lipotropin, ,3-endorphin, and other peptides derived from the corticotropin/p3-endorphin common precursor protein (1-5). The coordinate secretion of corticotropin and P3-endorphin by AtT-20/ D16v cells, or by cells of other clones, is stimulated up to 30-fold by depolarization with 50-80 mM KCI (4, 6-8) and by 10 ,uM norepinephrine (5). Secretion stimulated by these compounds is dependent on extracellular Ca2" (5-8), indicating a requirement for Ca2" influx in the release process.Secretions from a number of endocrine systems have been reported to involve Ca2" entry through voltage-sensitive channels. Thus, action potentials in which Ca2" was identified as the principal charge carrier have been recorded from cells of the ,B-pancreatic islets (9, 10), the anterior pituitary gland (11) Unless stated otherwise, the cells were treated with 1 mM dibutyryl cAMP (Boehringer Mannheim) for 1-3 wk prior to recording. This procedure doubled the average cell diameter, facilitating microelectrode impalement, but did not appreciably alter the excitability of the cells (8).Patch-clamp recordings were made at room temperature (22-24°C) by the method of Hamill et al. (18). The patch electrode was fabricated on a microforge and had a resistance of 3-5 MfQ when filled with physiological saline.The control solution had the following composition (mM):NaCI, 145; KCI, 5.4; MgCl2, 0.8; CaCl2, 1.8; glucose,25; Hepes/ NaOH, 10. Na+-free solutions were prepared by isosmotic substitution of sucrose for NaCl. Ca2+-free solutions were prepared by omitting CaCl2 from the control solution and adding EGTA to 0.5 mM and additional MgCl2 to 2.8 mM. All solutions we...

The effects of extracellular and intracellular tetraethylammonium (TEA) ions on single Ca2+-activated K+ channels were studied in excised membrane patches from the anterior pituitary clone AtT-20/D16-16 with the patch-clamp technique. The presence of TEA on either surface of the membrane resulted in a decrease in the single-channel current. Dissociation constants at zero voltage for the TEA-receptor complex were calculated to be 52.2 mM and 0.08 mM for external and internal TEA, respectively. The high sensitivity of AtT-20/D16-16 cells to internal TEA is of considerable interest, since in other preparations, the greater TEA sensitivity for Ca2+-activated K+ channels has thus far been found to occur on the external membrane surface. Hill plot analysis of the dose-response data yielded a slope of 0.92, indicating a one-to-one stoichiometry for TEA-receptor binding. The blockade by TEA showed little voltage or current sensitivity over the membrane potential range studied, and could be fully reversed by washout in drug-free solution. The results suggest the presence of TEA receptors on both the external and internal membrane surfaces but with different binding affinities. Occupancy of either site by TEA leads to an apparent decrease in the single-channel conductance of Ca2+-activated K+ channels.