1-EBIO stimulates Cl - secretion by activating a basolateral K + channel in the mouse jejunum

Abstract

We investigated the effects of 1-ethyl-2-benzimidazolinone (1-EBIO) on ion transport in the mouse jejunum through the use of the short-circuit (Isc) current technique and the application of the patch-clamp technique to isolated jejunal crypts. In HCO3- Ringer's, 1-EBIO stimulated a dose-dependent (EC50 964 micromol/l), bumetanide-sensitive increase in Isc consistent with stimulation of Cl- secretion. In contrast, in Cl(-)-free HCO3-Ringer's containing glucose, 1-EBIO (500 micromol/l) did not increase the phloridzin (100 micromol/l) sensitive Isc, suggesting that electrogenic Na+ absorption was unaltered. Measurement of the membrane potential (Vm) with the perforated-patch technique indicated that in isolated crypts, 1-EBIO caused a reversible hyperpolarization of Vm and an increase in the change in Vm associated with step changes in bath K+, consistent with an increase in K+ conductance. In on-cell patch experiments with KCI Ringer's in the patch pipette and crypts bathed with NaCl Ringer's, 1-EBIO (500 micromol/l) increased the open probability (NPo; 0.01+/-0.01 to 0.45+/-0.11, n=7) of an inwardly rectified intermediate conductance (g) channel. In inside-out patches with KCl Ringer's in the patch pipette and KCI Ringer's containing 100 nmol/l Ca2+ in the bath, the current-voltage relationship of the channel was inwardly rectified (g of 10 and 52 pS at -Vp of 100 and -100 mV, respectively) and reversed at 0 mV (n=5). Replacement of bath K+ with Na+ shifted the reversal potential toward the equilibrium potential for K+. In the presence of 1-EBIO, reducing the bath Ca2+ from 200 nmol/l to nominally Ca(2+)-free conditions decreased NPo from 0.90+/-0.27 to 0.07+/-0.03 (n=3). We conclude that in the mouse jejunum, I-EBIO does not stimulate electrogenic Na+ absorption. It does, however, stimulate secretion primarily through the activation of a basolateral, intermediate conductance Ca(2+)-sensitive K+ channel.

DCEBIO stimulates Cl− secretion in the mouse jejunum

“…However, 1-EBIO, the parent molecule of DCEBIO (61), was less potent than DCEBIO. 1-EBIO, too, increased I sc in a concentration-dependent manner with a V max of 86 Ϯ 8 A/cm 2 , an EC 50 of 862 Ϯ 150 M and Hill coefficient of 1.6 Ϯ 0.3 (n ϭ 8 -11 per data point, N ϭ 7, data not shown) as has been demonstrated by us and others (14,28).…”

“…Tissues were voltage clamped to 0 mV (Biodesign, South Campus Electronics, University of Otago), and I sc was continuously recorded with a MacLab data-acquisition system (ADInstruments, Castle Hill, Australia) (3,5,28). Initially, tissues were rinsed three times with NaCl Ringer solution.…”

“…Electrophysiological measurements-Ussing chamber experiments. The Ussing chamber short-circuit current experiments were performed as previously described (3,5,28). Once the tissues were isolated and pinned, the tissues were glued (Loctite 454; Henkel Loctite Adhesives, Welwyn Garden City, UK) to plastic annuli (0.7 cm 2 ) and mounted in modified Ussing chambers.…”

“…1-EBIO is a known activator of K ϩ channels (34), in particular the intermediate-conductance Ca 2ϩ -dependent K ϩ channel (IK Ca , KCNN4) (14,28,33,69). 1-EBIO has been reported to stimulate intracellular levels of cAMP in native tissue (11,39); however, others do not concur (67).…”

“…1-Ethyl-2-benzimidazolinone (1-EBIO), another benzimidazolone, has been demonstrated to stimulate Cl Ϫ secretion in several epithelia, including the T84 colonic cell line (14), mouse jejunum (28) and colon (11,38), rat colon (14,69), and Calu-3 human airway cells (16), for example. 1-EBIO is a known activator of K ϩ channels (34), in particular the intermediate-conductance Ca 2ϩ -dependent K ϩ channel (IK Ca , KCNN4) (14,28,33,69).…”