A new class of inhibitors of cAMP-mediated Cl- secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance.

Previously we have shown that arylaminobenzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (Isc) induced by prostaglandin E2 (n = 7), vasoactive intestinal polypeptide (VIP, n = 5), adenosine (n = 3), cholera toxin (n = 4) and cAMP (n = 6), but not by ionomycin (n = 5) in distal rabbit colon half maximally (IC50) at 2 mumol/l from the mucosal and at 0.7 mumol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116 +/- 16 omega.cm2 to 136 +/- 21 omega.cm2 (n = 5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound within this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mumol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mumol/l 293 B had no effect on the membrane voltage across the basolateral membrane (Vbl) in non-stimulated crypt cells: -69 +/- 3 mV versus -67 +/- 3 mV (n = 10), whilst in the same cells 1 mmol/l Ba2+ depolarised Vbl significantly. However, 293 B depolarised Vbl significantly in the presence of 1 mumol/l forskolin: -45 +/- 4 mV versus -39 +/- 5 mV (n = 7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised Vbl from -40 +/- 5 mV to -30 +/- 4 mV (n = 19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mumol/l 293 B: -75 +/- 6 mV versus -75 +/- 6 mV (n = 6). Also 293 B had no effect on basal K+ conductance (n = 4). Hence, we conclude that 293 B inhibits the K+ conductance induced by cAMP. This conductance is apparently relevant for Cl- secretion and the basal K+ conductance is insufficient to support secretion.

Tubular effects of the diuretic torasemide.

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 natriuresis 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 mumol/l; active transport was measured as the equivalent short-circuit current.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon.

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)