The use of isolated fish opercular epithelium as a model tissue for studying intrinsic activities of loop diuretics

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Abstract
Isolated opercular epithelia of killifish (Fundulus heteroclitus). were mounted in an Ussing chamber. The epithelia displayed a transepithelial electrical potential difference (PD) of 10.6 ± 0.3 mV (sea‐water side negative) and a short‐circuit current (SCC) of 72.1 ±2.1 μA cm‐2. The electrical resistance was 160 ± 3 Ω cm2(mean ± SE, n 269). The unidirectional flux of36C1 from blood side to sea‐water side compared well with the SCC. No net flux of22Na or24Na across the epithelium was observed. Raising of cyclic AMP levels by theophylline, 3‐isobutyl‐1‐methyl‐xanthine, isoprenaline and forskolin, increased SCC and PD. Prostaglandins PGE2and to some extent PGF2αinhibited SCC and PD. Inhibition of Na+‐K+‐ATPase by ouabain and orthovanadate reduced SCC and PD. Pretreatment of the epithelium with the stilbene disulphonic acid (DIDS) did not prevent the action of orthovanadate. Different types of diuretics were tested, but only the loop diuretics bumetanide, piretanide, and furosemide, rapidly and strongly inhibited PD and SCC and unidirectional36C1 serosal to mucosal flux. Dose‐response curves for these agents were parallel and EC50values for effects on SCC were 40, 52, and 295 μM, respectively. The same relative activities of these diuretics have been seen in the renal thick ascending limb of Henle's loop (TALH). It is concluded that the killifish opercular skin responds to hormonal stimuli and various pharmacological agents in a manner similar to that of mammalian renal TALH. It should therefore be a useful model for studies of the modes of action and the structure‐activity relationships of diuretics which act by inhibition of chloride transport or Na+‐K+‐ATPase activity.