CONTROL OF ACTION-POTENTIALS AND CA2+ INFLUX BY THE CA2+-DEPENDENT CHLORIDE CURRENT IN MOUSE PITUITARY-CELLS

1. Perforated patch recording was used to examine the influence of the calcium-dependent chloride current (i(Cl)(Ca)) on Ca2+ action potentials in AtT-20 pituitary cells. The calculated chloride equilibrium potential (E(Cl)) was adjusted by changing either intracellular or extracellular [Cl-]. Action potential duration varied as a function of E(Cl). When E(Cl) was set at -21 mV, both spontaneous and evoked action potentials displayed a long plateau phase between -20 and -25 mV, which typically lasted for several seconds. Setting E(Cl) to more negative potentials resulted in briefer action potentials; at an E(Cl) of -52 mV, no plateau phase was evident. Spontaneous depolarization and action potential firing still occurred when E(Cl) was negative to firing threshold, which indicates that the slow depolarizing wave that precedes the firing of spontaneous action potentials does not require activation of I(Cl)(Ca). 2. In voltage clamp experiments the magnitude of I(Cl)(Ca) diminished slowly during a prolonged depolarization, over a time course that coincided with action potential termination. 3. Niflumic acid (100-mu-M) blocked I(Cl)(Ca) by 90% but had no effect on either K+ or Ca2+ currents. This concentration of niflumic acid eliminated the plateau phase, but did not prevent the firing, of Ca2+ action potentials. 4. Internal [Ca2+] was measured photometrically after loading cells with the Ca2+ indicator dye, Fura-2. Under voltage clamp conditions, concentrations of niflumic acid (30-100-mu-M) that blocked depolarization-evoked I(Cl)(Ca) had little or no effect on simultaneously recorded Ca2+ transients. Perforated patch recording from Fura-loaded cells showed that action potentials were temporally associated with transient increases in intracellular [Ca2+]. Niflumic acid (30-100-mu-M) disrupted the rhythmic firing of spontaneous action potentials and associated intracellular Ca2+ transients. 5. Fluorescent measurements of Ca2+ transients were also made in cells unperturbed by patch recording, and were used as a measure of action potential duration in the absence of experimental alteration of internal [Cl-]. Spontaneous Ca2+ transients were of long duration (approximately 2 s), which suggests that intracellular [Cl-] is relatively high (40-50 mM) in these cells. The spontaneous Ca2+ transients were inhibited by niflumic acid. 6. Niflumic acid up to 100-mu-M, had neglible effects on either basal or stimulated (by 2-mu-M-(+/-)-isoprenaline) hormone secretion, as shown by radioimmunoassay of adrenocortotrophic hormone release. 7. The results suggest that activation of I(Cl)(Ca) by Ca2+ influx during action potentials tend to maintain the membrane potential at a depolarized level, which enhances the Ca2+ influx through voltage-activated Ca2+ channels. Action potentials are terminated as the magnitude of I(Cl)(Ca) decreases. I(Cl)(Ca) thus acts to control the biphasic behaviour of membrane potential and Ca2+ influx, and appears to provide rhythmic control over the rise and fall of intracellular [Ca2+]. Block of this rhythmic behaviour by niflumic acid had little effect on hormone secretion, which suggests that secretion is not tightly coupled to the transient nature of either action potentials or Ca2+ influx.