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Action potentials and membrane ion channels in clonal anterior pituitary cells. (Ca2" spike/Na4 spikee/Ca24-dependent K4 channel/corticotropin/,-endorphin).

Proc. Nati. Acad. Sci. USA Vol. 80, pp. 2086-2090, April 1983 Neurobiology

Action potentials and membrane ion channels in clonal anterior pituitary cells (Ca2" spike/Na4 spikee/Ca24-dependent K4 channel/corticotropin/,-endorphin) MICHAEL ADLER*§, BRENDAN S. WONGt, STEVEN L. SABOLt, NEIL BUSISt, MEYER B. JACKSONt, AND FORREST F. WEIGHT* *Laboratory of Preclinical Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852; tLaboratory of Biophysics, National Institute of Neurological and Communicative Disorders and Stroke; and *Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Bernhard Witkop, December 30, 1982 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 f8endorphin 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 AtT-20 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), and various endocrine cell lines derived from tumor sources (12, 13). More commonly, action potentials from endocrine cells were found to have both Na+ and Ca2" components (14-16). The presence of action potentials involving Ca2+ suggests that modulation of electrical activity by physiologically appropriate secretagogues and inhibitors may play a role in excitation-secretion coupling (11, 17). Since hormone release from AtT-20/D16-16 pituitary cells is stimulated by depolarization-dependent Ca2+ entry (4, 6), it is possible that these cells may also manifest regenerative ion conductance mechanisms. To examine this possibility, the elec-

trical properties of AtT-20/D16-16 cells were investigated by electrophysiological techniques using intracellular and patchclamp recordings. The results indicate that, under physiological conditions, AtT-20/D16-16 cells generate low-frequency spontaneous bursting action potentials. The action potentials have discrete Na+ and Ca2+ components and the bursts are separated by a Ca2+-sensitive afterhyperpolarization (AHP). Single channel recordings show that the AHP is mediated by Ca2+-dependent K+ channels that have a unitary conductance of 130 PS. MATERIALS AND METHODS AtT-20/D16-16 cells were subcloned from the parent line AtT20/D16v (4). The cells were grown at 37°C in humidified 10% C02/90% air in Dulbecco's modified Eagle's medium/10% fetal bovine serum. Electrophysiological Recordings. Intracellular recordings were made at 35-37°C by established procedures. The cells were impaled with 3 M KCl-filled microelectrodes (50-80 MW) and stimulated through the recording electrode via a bridge circuit. 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 (2224°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 were adjusted to pH 7.3-7.4 and an osmolality of 320-340 mOsm/kg. ,B-Endorphin Secretion. Cells were cultured to 80% confluence in 35-mm dishes. Dibutyryl cAMP was avoided in these experiments because chronic exposure has been found to reduce depolarization-dependent ,&endorphin secretion (8). Prior to the addition of catecholamines, the cultures were equilibrated for 30 min at 37°C in 2 ml of 25 mM Hepes-buffered medium (pH 7.4) containing crystalline bovine serum albumin at 1 mg/ml. At zero time, medium with catecholamines was added, and 0.2-ml aliquots were removed at 15, 30, 60, and 120 min for P-endorphin immunoreactivity assay (4). Protein was determined by the method of Lowry et al. (19). Abbreviations: AHP, afterhyperpolarization; Et4N+, tetraethylammonium; dV/dt, maximum rate of rise of the action potential. § Present address: Neurotoxicology Branch, USAMRICD, APG, MD

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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RESULTS Passive Electrical Properties. The resting potentials of the AtT-20/D16-16 cells used ranged from -30 to -74 mV (mean ± SEM = -52.9 ± 1.3 mV, n = 94). Cells with resting potentials positive to -30 mV were considered to be injured and therefore discarded. The input resistance and time constant, determined from 5- to 10-mV hyperpolarizing pulses, were 217.4 ± 22.8 Mfl and 14.1 ± 1.9 msec, respectively (n = 29). These values are similar to those reported for other pituitary cells (12, 15, 16). Hyperpolarizing responses in excess of 10 mV were accompanied by anomalous rectification that persisted in the absence of Na', Ca2+, or CF-. Delayed rectification was always observed on application of depolarizing current. Regenerative Activity. More than 95% of the cells displayed some form of spontaneous oscillatory activity in physiological solution. Under resting conditions, most cells generated lowfrequency bursting action potentials similar to those shown in Fig. 1. Each burst consisted of an initial depolarization, one to several brief spikes, a single slow spike, and an AHP. The burst frequency depended on applied current and thus on membrane potential, as shown in Fig. 1. In the absence of injected current, spontaneous bursting rates were 2.3 ± 0.1 Hz (n = 26). Burst frequencies were accelerated by depolarizing currents to a maximum of 8.2 ± 0.7 Hz and slowed by hyperpolarizing currents to a minimum of 1.4 ± 0.1 Hz. Frequencies of E

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FIG. 1. (a) Effect of membrane potential on burst frequency and configuration. The records are from the same cell under resting conditions (trace B) and after injection of a hyperpolarizing current (trace A) or depolarizing currents of increasing intensity (traces C-F). Membrane potentials are indicated by the scales to the left of the traces. (b) Plot of burst frequency as a function of applied current for this cell. Data represent means of three consecutive determinations.

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FIG. 2. Single burst cycles from a spontaneously active cell in physiological solution (trace A) and after superfusion with sucrose-substituted Na'-free solutions containing 1.8 mM Ca2+ (trace B), 0.9 mM Ca2+ (trace C), and 0.9 mM Ca2+/2 mM CoCl2 (trace D). Spontaneous firing ceased after removal of Na+ and records (traces B-D) were obtained after injection of a constant depolarizing current.

The effect of varying the Ca2+ concentration on Ca2+ spikes evoked from -55 mV by brief current pulses is shown in Fig. 3. Increasing the Ca2' concentration from 0.9 to 5.0 mM enhanced the overshoot and dV/dt of the Ca2+ spike and reduced its duration. The dependence of overshoot on external Ca2+ concentration was 19 mV per decade in the concentration range studied. This is considerably less than the Nernst value of 30.7 mV per decade. The deviation from the theoretical relationship may indicate low Ca2+ selectivity but more likely represents competition betweenthe inward Ca2+ current and outward currents. This is suggested by the finding that the Ca2+ spike overshoot can increase by >20 mV after addition of 5 mM tetraethylammonium ion (Et4N+) (see Fig. 6). Production of the AHP. The current underlying the AHP is carried by K+, as shown by its reversal near the expected K+ equilibrium potential (Fig. 4) and by the finding that the reversal potential shifted in the expected direction for different concentrations of external KV. The AHP is modulated, however, by Ca2+. This is shown by the dependence of the AHP on Ca2+ concentration (Fig. 3) and by its near total suppression after Ca2+ removal (see Fig. 7, trace C). A

The involvement of both Ca2" and K+ in the generation of the AHP suggests that it is mediated by a Ca24-dependent KV conductance (21). Direct evidence for the presence of Ca24-dependent KV channels in AtT-20/D16-16 cells comes from patchclamp recordings of single channel fluctuations (22). Trace A in Fig. 5 was obtained from an excised inside-out patch at a membrane potential of +25 mV. The patch electrode was filled with Ca2+-free saline and the internal membrane surface was bathed in a high KV solution containing 0.5 ,uM free Ca2'. In the presence of internal Ca2+ concentrations .10 nM, large outward currents were observed with single channel conductances of 120-130 pS. These channels were identified as Ca2'.dependent KV channels by their large unitary conductance, high KV selectivity, and sensitivity to Et4N+ (23, 24). Single channel currents from an intact cell bathed in control solution containing 1 AuM tetrodotoxin are shown in Fig. 5, trace B. In this record, Ca24-dependent KV channels are superimposed on spontaneous rhythmic Ca24 spikes generated presumably in regions outside the isolated patch. The channel openings occurred with high probability near the peak of the Ca2` spike but were relatively infrequent during the interspike interval. For 50 consecutive Ca2' spikes, >80% of the channel openings were observed within the spike duration, although the spikes comprised

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