Action potentials and membrane ion channels in clonal anterior pituitary cells.

Abstract

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...

Control of action potentials and Ca2+ influx by the Ca(2+)‐dependent chloride current in mouse pituitary cells.

“…IA c). As previously reported (Adler et al 1983 (Fig. 1 C, open symbols) Step potential (mV) simultaneously with perforated patch recording and fluorescence photometry.…”

“…Both acutely dissociated and clonal anterior pituitary cells spontaneously fire Ca2+ action potentials (Kidokoro, 1975;Taraskevich & Douglas, 1987; Suprenant, 1982;Adler, Wong, Sabol, Busis, Jackson & Weight, 1983;Mollard, Vacher, Guerin, Rogowski & Dufy, 1987;Schlegel, Winiger, Mollard, Vacher, Wuarin, Zahnd, Wollheim & Dufy, 1987). One mechanism by which secretion is thought to be controlled is by modulation of Ca2+ influx though voltagegated Ca2+ channels.…”

“…For example, somatostatin, which inhibits secretion (Patel & Srikant, 1986), depresses intracellular Ca2+ transients that are associated with spontaneous action potentials (Schlegel et al 1987). Conversely, fi-adrenergic agonists, which stimulate secretion and cause a rise in intracellular Ca21 (Luini et al 1985), have been reported to increase the frequency of action potential firing (Suprenant, 1982;Adler et al 1983). …”

Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes.

“…In addition, 3-adrenergic agonists enhance the frequency of action potential generation in AtT-20 cells, which has been associated with an increase in transient calcium currents across the cell membrane (9,10). This increase in calcium currents has been correlated with the ability of 3-adrenergic agonists to release ACTH from these tumor cells (9,10).…”

“…8-Adrenergic agonists, which stimulate cAMP-dependent protein kinase activity and the secretion of ACTH from AtT-20 cells (7), also depolarize AtT-20 cells and increase the frequency of action potentials in these corticotrophs (9,10). This electrical activity has been associated with transient calcium currents (9,10), and (3-adrenergic agonists and forskolin, a direct activator at adenylate cyclase, increase cytosolic calcium levels in AtT-20 cells (11). In addition to cAMP and calcium, activators of protein kinase C evoke ACTH release (12), and recent studies (13) have suggested a role for phosphatidylinositol turnover in hormone secretion from corticotrophs.…”