Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca(2+)-dependent K+ channel.
We evaluated the effects of the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), on Cl- secretion across T84 monolayers. 1-EBIO stimulated a sustained Cl- secretory response at a half-maximal effective concentration of 490 microM. Charybdotoxin (CTX) inhibited the 1-EBIO-induced short-circuit current (Isc) with an inhibitory constant (Ki) of 3.6 nM, whereas 293B, an inhibitor of adenosine 3',5'-cyclic monophosphate-activated K+ channels, had no effect on the current induced by 1-EBIO. In contrast, CTX failed to inhibit the 293B-sensitive forskolin-induced Isc. The above results suggested that 1-EBIO may be activating the basolateral membrane Ca(2+)-dependent K+ channel (KCa) in these cells. This was further confirmed using nystatin to permeabilize the apical membrane in the presence of a mucosa-to-serosa K+ gradient and determining the effects of 1-EBIO on the basolateral K+ current (IK). Under these conditions, 1-EBIO induced a large increase in IK that was blocked by CTX. In membrane vesicles prepared from T84 cells, 1-EBIO stimulated 86Rb+ uptake in a CTX-sensitive manner; the Ki for inhibition by CTX was 3.5 nM. Similar to our intact monolayer studies, this 86Rb+ uptake was not blocked by 293B. The effects of 1-EBIO on the KCa in T84 cells was determined in excised inside-out patches. 1-EBIO (100 microM) increased the product of the number of channels and the open channel probability from 0.09 +/- 0.03 to 1.17 +/- 0.27 (n = 8); this effect on KCa activity required a minimal level of free Ca2+. Similar to its effect on T84 cells, 1-EBIO stimulated a sustained Cl- secretory current in rat colonic epithelium, which was partially blocked by CTX. Finally, 1-EBIO stimulated a sustained Cl- secretory response in primary cultures of murine tracheal epithelium. We conclude that the benzimidazolone, 1-EBIO, stimulates Cl- secretion in secretory epithelia via the direct activation of a Kca. 1-EBIO is the first pharmacological opener of this important class of epithelial K+ channels to be identified.
Devor
D C
DC
Department of Cell Biology and Physiology, University of Pittsburgh, Pennsylvania 15261, USA. dd2+@pitt.edu
Singh
A K
AK
Frizzell
R A
RA
Bridges
R J
RJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D018999:Charybdotoxin / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D007413:Intestinal Mucosa / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D051379:Mice; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D014132:Trachea / Q000502:physiology*
D001562:Benzimidazoles; D002712:Chlorides; D015221:Potassium Channels; D002034:Bumetanide; D018999:Charybdotoxin; D005576:Colforsin; D000584:Amiloride; C102635:1-ethyl-2-benzimidazolinone; D012413:Rubidium; D002118:Calcium