Stilbene disulfonate and phenylanthranilic acid derivatives block Cl- transport and ACTH secretion from mouse AtT-20 clonal corticotrophs. This study was to determine whether antagonism of Na+/K+/Cl- co-transport by loop diuretics (furosemide or bumetanide) and thereby Cl- entry could block ACTH secretion. Activity of the Na+/K+/Cl- co-transporter (symport) was measured as ouabain-insensitive, loop diuretic-sensitive uptake of 86Rb. Ouabain-insensitive uptake (80% of total 86Rb uptake) was linear over 10 min and was markedly reduced by furosemide. Substitution of Na+ by choline or Cl- by gluconate resulted in an 82 and 55% decrease, respectively, in 86Rb uptake. Furosemide and bumetanide incompletely inhibited 86Rb uptake (maximal inhibition of 60% and 69%, respectively; IC50: 1.6 x 10(-5) and 3 x 10(-6) M, respectively). Forskolin did not affect the activity of the Na+/K+/Cl- co-transporter but both basal and forskolin-stimulated secretion of ACTH were inhibited by furosemide (IC50 of 5 x 10(-4) M; maximal inhibition: 50%). Bumetanide did not affect forskolin-induced cAMP synthesis. Use of cyclamate or gluconate in place of Cl- also resulted in the inhibition of basal and forskolin-stimulated ACTH secretion. The data support the belief that inhibition of Cl- entry into AtT-20 cells by way of an Na+/K+/Cl- co-transporter can result in the inhibition of ACTH secretion but that other anion entry mechanisms may also be coupled to the secretory response in these cells.