GLUCOCORTICOIDS DO NOT AFFECT INTRACELLULAR CALCIUM TRANSIENTS IN CORTICOTROPHS - EVIDENCE SUPPORTING AN EFFECT DISTAL TO CALCIUM INFLUX

The influx of extracellular calcium is a critical step involved in the stimulated release of adrenocorticotropin (ACTH) from pituitary corticotrophs. It has been proposed that the mechanism of early glucocorticoid feedback is mediated through inhibition of stimulus-evoked calcium transients. We tested this hypothesis using corticotrophic mouse pituitary AtT-20 cells by coevaluating secretory dynamics and cytosolic calcium transients. In static monolayer culture and in a dynamic microperifusion system, dexamethasone (DEX, 100 nM, 2 h) significantly inhibited ACTH secretion stimulated by corticotropin-releasing hormone (CRH, 100 nM). When ACTH was stimulated by KCl (56 mM) depolarization or the voltage-dependent calcium channel agonist, maitotoxin (MTX, 1 ng/ml), DEX did not inhibit secretion. In contrast, CRH-, KCl-, and MTX-stimulated ACTH secretion were significantly inhibited in static monolayer culture when cells were pretreated with the voltage-dependent calcium channel blocker, nifedipine (NIF, 1 mu M, 15 min), confirming the requirement for the influx of extracellular calcium. Intracellular calcium was measured under similar culture conditions in populations of cells grown on coverslips, utilizing the fluorescent calcium indicator, fura-2. DEX had no effect on basal, spike, or plateau calcium levels in response to CRH, KCl or MTX stimulation. For example, CRH stimulation resulted in an increase in intracellular calcium from a basal concentration of 90 +/- 3.1 nM (mean +/- SE) to a plateau of 222 +/- 8.7 nM, whereas the plateau after DEX was 225 +/- 4.1 nM. In contrast, NIF significantly lowered the stimulated calcium response to each secretagogue (spike and plateau). These results do not support the hypothesis that glucocorticoids suppress stimulated secretion of ACTH from corticotrophs through an effect on intracellular calcium transients. Instead, the data suggest that early glucocorticoid negative feedback occurs at a step(s) distal to the influx of extracellular calcium.