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Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (Vte) and resistance (Rte) were measured and the equivalent short-circuit current (Isc = Vte/Rte) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 mumol/l) Isc was -71 +/- 11 (n = 118) microA/cm2. Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to +32 +/- 4 (n = 118) microA/cm2. In the presence of amiloride and indomethacin, prostaglandin E2 (1 mumol/l, s), known to induce Cl- secretion, generated an Isc of -143 +/- 8 (n = 92) microA/cm2. The arylaminobenzoate and Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) reduced Isc reversibly with a half-maximal inhibition (IC50) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC50 to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-L-cysteine (m) preincubation. Pretreatment with N-acetyl-L-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and L-homocysteine (m) were more effective and reduced IC50 to approximately 50 mumol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/-1-phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl- conductance in the thick ascending limb of the loop of Henle (TAL).(ABSTRACT TRUNCATED AT 250 WORDS)

Torasemide is a lipophilic loop diuretic which is largely metabolized in the liver and has an almost neutral pKa. Experiments were designed to address the questions of whether torasemide is secreted by the proximal tubule, and hence accumulates in tubular fluid, whether torasemide paralyses active transport in the cortical thick ascending limb of the nephron and hence reduces its ATP requirement, and finally whether torasemide is active in its protonated or unprotonated form. Intravenous torasemide, 10 mg/kg, induced a marked diuresis and natriure-sis and a moderate kaliuresis in antidiuretic rats. All effects were dose-dependently suppressed by intravenous probenecid, 20-80 mg/kg, indicating that torasemide is secreted by the anion secretory system of the proximal tubule. A time-dependent depolarization of the basolateral membrane of in vitro perfused rabbit cortical thick ascending limb segments was observed after removal of metabolic substrates and after addition of ouabain. This effect, caused by Na⁺ entry, K⁺ loss and cell swelling, was prevented when torasemide was added to the luminal perfusate before substrate removal or addition of ouabain, indicating that torasemide significantly reduced ATP consumption of the cortical thick ascending limb. To test whether protonated or unprotonated torasemide was the biologically active compound, cortical thick ascending limb segments were perfused over the pH range 6-8 and torasemide was added in the concentration range 0.01-10 µmol/l; active transport was measured as the equivalent short-circuit current. Half-maximal inhibition by torasemide was observed with 0.1 µmol/ 1 at pH 8.0 and with 3.7 µmol/l at pH 6.0, indicating that the unprotonated form of torasemide is biologically active and that changes in luminal pH have a profound effect on its concentration-response curve.

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