Chloride transport inhibition by various types of loop diuretics in fish opercular epithelium

Isolated opercular epithelia of killifish (Fundulus heteroclitus), mounted in an Ussing chamber, were used to study the effects of various diuretics on chloride transport, measured as short-circuit current (SCC). The acidic 'loop' diuretics, ethacrynic acid and azosemide, and the basic 'loop' diuretics, muzolimine and MK 447, reduced SCC and exhibited similar dose-effect curves, with EC50s for SCC of 64, 17, greater than 500 and 224 microM, respectively. The alkaline diuretic tizolemide (HOE 740) and the p-COOH-analogue of sulphanilamide were inactive, suggesting that the chloruretic effects of these agents are of a thiazide type. The method can thus discriminate between the effects of loop and thiazide types of diuretics, but not between those of structurally highly different 'loop' diuretics of an acidic and basic nature. Monomethylation of the SO2NH2 group of bumetanide had no effect on the activity of this agent whereas dimethylation reduced it fourfold. The (-)enantiomers of the 'loop' diuretics indacrinone and ozolinone were four and greater than 100 times more active, respectively, than the (+)forms. These results are in accordance with those obtained for the same drugs in the mammalian kidney, and point to the presence of a highly specific binding site for these diuretics. Attempts were also made to explore the prerequisites for binding of the loop diuretic to the active site. Pretreatment of the opercular epithelium with an alpha-L-fucose-binding lectin did not prevent the inhibitory actions of furosemide and indacrinone. Probenecid and (+)ozolinone, both of which block organic anion transport, did not prevent the effects of bumetanide and (-)ozolinone.