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. 2006 Jun 14;71(12):1720-6.
doi: 10.1016/j.bcp.2006.03.011. Epub 2006 Mar 18.

The Cdc42 Inhibitor Secramine B Prevents cAMP-induced K+ Conductance in Intestinal Epithelial Cells

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The Cdc42 Inhibitor Secramine B Prevents cAMP-induced K+ Conductance in Intestinal Epithelial Cells

Henry E Pelish et al. Biochem Pharmacol. .
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Abstract

Cyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl- transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba2+-sensitive K+ channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca2+-mediated chloride secretion pathway, which requires a separate K+ channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K+ conductance in intestinal epithelial cells.

Figures

Fig. 1
Fig. 1
Secramine B inhibits cholera toxin-induced Cl secretion in T84 cell monolayers. (A) Structure of secramine B. (B) Time course for Cl secretion (Isc) in T84 monolayers induced by the addition of cholera toxin to the apical media at the indicated time (dotted line) in the presence of 25 μM secramine B (filled squares) or vehicle (0.2% DMSO, 0.25% BSA, open squares) added at 0 min (mean ± S.D., n = 2).
Fig. 2
Fig. 2
Secramine B inhibits cAMP-dependent Cl secretion and acts downstream of cholera toxin transport in T84 cell monolayers. T84 monolayers were incubated with vehicle (open squares) or 20 μM secramine B (filled squares) for 45 min followed by the addition of 10 μM forskolin (A) or 3 mM 8Br-cAMP (B) to the basolateral media at 0 min and the Isc was measured over time (mean ± S.D., n = 2). (C) Dose dependency of secramine B action on Cl secretory response elicited by 10 μM forskolin (mean ± S.D., n = 2). Secramine B has an IC50 of ~3.4 μM. (D) T84 cells were incubated with 10 μM forskolin (0 min) followed by 20 μM secramine B or vehicle at 15 min (dotted line) (mean ± S.D., n = 2).
Fig. 3
Fig. 3
Secramine B does not inhibit Cl secretion induced by carbachol. T84 cell monolayers were treated with vehicle (open squares) or 20 μM secramine B (filled squares) for 45 min followed by the addition of 100 μM carbachol to the basolateral media at 0 min (mean ± S.D., n = 2).
Fig. 4
Fig. 4
Secramine B selectively inhibits basolateral Ba2+-sensitive K+ conductance in T84 cell monolayers. (A) T84 monolayers were exposed to a symmetrical high Cl solution and basolaterally permeabilized with 20 μM amphotericin B. At 0 min, 20 μM secramine B or vehicle was added followed by 10 μM forskolin to the basolateral media (arrow). All measurements for the secramine B-treated monolayer were shifted by 8 μA/cm2. Data is representative of two independent experiments. (B) A basolaterally directed K+ gradient was established across T84 monolayers and the apical membrane was permeabilized with 20 μM amphotericin B. At 0 min, 20 μM secramine B or vehicle was added followed by 10 μM forskolin, 3 mM BaCl2, and 100 μM carbachol to the basolateral media (arrows, as indicated). Data is representative of four independent experiments. (C) A basolaterally directed K+ gradient was established across T84 monolayers and the apical membrane was permeabilized with 20 μM amphotericin B. Short circuit currents were recorded at the indicated voltage-clamped test potentials. The ordinate indicates the current difference measured on the same monolayer before and 10 min after 10 μM forskolin stimulation in the presence of 20 μM secramine B or vehicle. Data is representative of two independent experiments.
Fig. 5
Fig. 5
Pirl1 selectively inhibits cAMP-dependent Cl secretion in T84 cell monolayers. (A) Structures of pirl1 and pirl1.6. (B) T84 monolayers were incubated with vehicle (open squares), 20 μM pirl1 (filled squares), or pirl1.6 (filled circles) for 45 min followed by the addition of 10 μM forskolin to the basolateral media at 0 min and the Isc was measured over time (mean ± S.D., n = 2). (C) Dose dependency of pirl1 action on Cl secretory response elicited by 10 μM forskolin (mean ± S.D., n = 2). Pirl1 has an IC50 of ~28 μM. (D) Like (B), with 50 μM pirl1 and the addition of 100 μM carbachol to the basolateral media at 20 min (mean ± S.D., n = 2).

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