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

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Abstract
Previously we have shown that arylamino-benzoates 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 μmol/l from the mucosal and at 0.7 μmol/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 Ω·cm2 to 136±21 Ω·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 with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 μmol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 μmol/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 μmol/l forskolin: −45±4mV 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 μmol/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.

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