Basolateral potassium (IKCa) channel inhibition prevents increased colonic permeability induced by chemical hypoxia

Abstract

Major liver resection is associated with impaired intestinal perfusion and intestinal ischemia, resulting in decreased mucosal integrity, increased bacterial translocation, and an increased risk of postoperative sepsis. However, the mechanism by which ischemia impairs intestinal mucosal integrity is unclear. We therefore evaluated the role of Ca(2+)-sensitive, intermediate-conductance (IK(Ca)) basolateral potassium channels in enhanced intestinal permeability secondary to chemical hypoxia. The effects of chemical hypoxia induced by 100 μM dinitrophenol (DNP) and 5 mM deoxyglucose (DG) on basolateral IK(Ca) channel activity and whole cell conductance in intact human colonic crypts, and paracellular permeability (G(S)) in isolated colonic sheets, were determined by patch-clamp recording and transepithelial electrical measurements, respectively. DNP and DG rapidly stimulated IK(Ca) channels in cell-attached basolateral membrane patches and elicited a twofold increase (P = 0.004) in whole cell conductance in amphotericin B-permeabilized membrane patches, changes that were inhibited by the specific IK(Ca) channel blockers TRAM-34 (100 nM) and clotrimazole (CLT; 10 μM). In colonic sheets apically permeabilized with nystatin, DNP elicited a twofold increase (P = 0.005) in G(S), which was largely inhibited by the serosal addition of 50 μM CLT. We conclude that, in intestinal epithelia, chemical hypoxia increases G(S) through a mechanism involving basolateral IK(Ca) channel activation. Basolateral IK(Ca) channel inhibition may prevent or limit increased intestinal permeability during liver surgery.

Fig. 1.

Chemical hypoxia activates basolateral intermediate-conductance (IK_Ca) channels. A: representative recording showing channel activation by 100 μM dinitrophenol (DNP) and 5 mM deoxyglucose (DG) [command voltage = 100 mV; channel openings indicated by current transitions between closed channel current (C) and open state (O₁)]. B: summary of effect of DNP and DG, and subsequent washout (Wash), on channel activity [channel open probability (NP_o)] (n = 10, *P = 0.04 compared with control).

Fig. 2.

Chemical hypoxia increases whole cell conductance. A: representative experiment showing effect of 100 μM DNP and 5 mM DG, and subsequent washout, on whole cell currents. B: current (I)-voltage (V) relationship using data from A, showing that DNP and DG increased whole cell conductance (I/V) and hyperpolarized the cell membrane. C: summary of effect of DNP and DG, and subsequent washout, on whole cell conductance (n = 7; *P = 0.005 compared with control, **P = 0.05 compared with DNP and DG).

Fig. 3.

Representative data showing time course of stimulation of whole cell conductance by 100 μM DNP and 5 mM DG and subsequent inhibitory effect of 10 μM clotrimazole (CLT).

Fig. 4.

1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) inhibits increase in whole cell conductance induced by chemical hypoxia. A: representative experiment showing effect of 100 μM DNP and 5 mM DG, and subsequent addition of 100 nM TRAM-34, on whole cell currents. B: current-voltage relationship using data from A, showing that DNP and DG increased whole cell conductance and hyperpolarized the cell membrane, whereas these effects were completely inhibited by TRAM-34. C: summary of effect of DNP and DG, and subsequent addition of 100 nM TRAM-34, on whole cell conductance (n = 6; *P = 0.03 compared with control, **P = 0.01 compared with DNP and DG alone).

Fig. 5.

CLT inhibits increase in whole cell conductance induced by chemical hypoxia. A: representative experiment showing effect of 100 μM DNP and 5 mM DG, and subsequent addition of 10 μM CLT, on whole cell currents. B: current-voltage relationship using data from A, showing that DNP and DG increased whole cell conductance and hyperpolarized the cell membrane, whereas these effects were completely inhibited by CLT. C: summary of effect of DNP and DG, and subsequent addition of 10 μM CLT, on whole cell conductance (n = 7; *P = 0.02 compared with control, **P = 0.006 compared with DNP and DG alone).

Fig. 6.

Chemical hypoxia increases paracellular conductance (G_S). A: representative experiment showing increases in transepithelial voltage (V_T) after nystain-induced apical membrane permeabilization in control and paired DNP-treated colon. B: plots of total tissue conductance (G_T) against short-circuit current (I_sc; corrected for tissue area) obtained from A, the y-intercepts providing estimates of G_S. C: summary of effect of DNP on G_S (n = 6 pairs of tissue, *P = 0.005 compared with control).

Fig. 7.

CLT inhibits increase in paracellular conductance (G_S) induced by chemical hypoxia. A: representative experiment showing increases in V_T after nystain-induced apical membrane permeabilization in control and paired DNP-treated colon in the presence of 50 μM CLT. B: plots of G_T against I_sc (corrected for tissue area) obtained from A, the y-intercepts providing estimates of G_S. CLT inhibited the DNP-induced increase in G_S shown in Fig. 6. C: summary showing that CLT largely prevented the increase in G_S induced by DNP (n = 5 pairs of tissue, P = 0.22 compared with control).