Hydrogen sulfide preconditioning or neutrophil depletion attenuates ischemia-reperfusion-induced mitochondrial dysfunction in rat small intestine

Abstract

The objectives of this study were to determine whether neutrophil depletion with anti-neutrophil serum (ANS) or preconditioning with the hydrogen sulfide (H(2)S) donor NaHS (NaHS-PC) 24 h prior to ischemia-reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium-activated, large conductance potassium (BK(Ca)) channels were involved in this protective effect. I/R was induced by 45-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in rats preconditioned with NaHS (NaHS-PC) or a BK(Ca) channel activator (NS-1619-PC) 24 h earlier or treated with ANS. Mitochondrial function was assessed by measuring mitochondrial membrane potential, mitochondrial dehydrogenase function, and cytochrome c release. Mucosal myeloperoxidase (MPO) and TNF-α levels were also determined, as measures of postischemic inflammation. BK(Ca) expression in intestinal mucosa was detected by immunohistochemistry and Western blotting. I/R induced mitochondrial dysfunction and increased tissue MPO and TNF-α levels. Although mitochondrial dysfunction was attenuated by NaHS-PC or NS-1619-PC, the postischemic increases in mucosal MPO and TNF-α levels were not. The protective effect of NaHS-PC or NS-1619-PC on postischemic mitochondrial function was abolished by coincident treatment with BK(Ca) channel inhibitors. ANS prevented the I/R-induced increase in tissue MPO levels and reversed mitochondrial dysfunction. These data indicate that neutrophils play an essential role in I/R-induced mucosal mitochondrial dysfunction. In addition, NaHS-PC prevents postischemic mitochondrial dysfunction (but not inflammation) by a BK(Ca) channel-dependent mechanism.

Fig. 1.

Expression of calcium-activated, large conductance potassium (BK_Ca) channel in rat intestinal enterocytes. Top: Western blot of total membrane and crude mitochondrial fractions from isolated mouse ileal enterocytes (see methods), each probed for the α-subunit of BK_Ca channel, mitochondrial porin (VDAC, outer membrane marker for mitochondria), and E-cadherin (plasma membrane protein used as marker for the total membrane fraction). Bottom, A: immunostaining of BK_Ca α-subunit protein in enterocytes of rat ileal mucosa. Bottom, B: negative controls were performed by using BK_Ca-α primary antibody that had been preabsorbed by use of a control peptide. Scale bar = 20 μm.

Fig. 2.

Reductions in mitochondrial membrane potential (top) and respiratory activity (bottom) induced by ischemia-reperfusion (I/R) are prevented by preconditioning with the hydrogen sulfide donor NaHS (NaHS-PC+I/R) or the BK_Ca channel activator NS-1619 (NS-1619+I/R) 24 h earlier. The effects of NaHS-PC to preserve postischemic mitochondrial function are prevented by coincident administration of the BK_Ca channel inhibitor paxilline (Pax+NaHS-PC+I/R) or penitrem A (penitrem A+NaHS-PC+I/R) with NaHS 24 h prior to I/R. Data are means ± SE for 6 rats per treatment group. *P < 0.05 compared with sham, NaHS-PC+I/R and NS-1619+I/R groups.

Fig. 3.

I/R induced release of cytochrome c into the cytosol of mucosal epithelial cells relative to sham (nonischemic) intestine. This disruption in postischemic mitochondrial membrane integrity was prevented by preconditioning with either the hydrogen sulfide (H₂S) donor NaHS (NaHS-PC+I/R) or the BK_Ca channel activator NS-1619 (NS-1619+I/R) 24 h prior to induction of I/R. Coincident treatment with paxilline, a BK_Ca channel inhibitor, with NaHS 24 h prior to I/R (Pax+NaHS-PC+I/R) abrogated the protective actions of this H₂S donor on postischemic mitochondrial membrane integrity. Top: representative Western blot showing comparison of signals from cytochrome c vs. β-actin. Bottom: densitometric analysis of full data set from 6 rats per treatment group. *P < 0.05 compared with sham, NaHS-PC+I/R, and NS-1619+I/R groups.

Fig. 4.

Increase in ileal mucosal myeloperoxidase (MPO) activity induced by I/R relative to sham (nonischemic) controls is prevented by neutrophil depletion by use of anti-neutrophil serum (ANS). Data are means ± SE for 6 rats per treatment group. *P < 0.001 compared with sham and ANS+I/R groups.

Fig. 5.

Neutrophil depletion with ANS prevents the reductions in mitochondrial membrane potential (top) and respiratory activity (bottom) induced by I/R relative to sham (nonischemic) controls. Data represents means ± SE for 6 rats per group. *P < 0.01 compared with sham group, #P < 0.05 compared with ANS+I/R group. JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; MTT-FZ, formazan derivative of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide.

Fig. 6.

ANS+I/R treatment prevented the postischemic release of cytochrome c into enterocyte cytosol induced by I/R compared with sham (nonischemic) preparations. Top: representative Western blot from full data set. Bottom: densitometric analysis of aggregate data. Data are means ± SE from 6 rats per group. *P < 0.05 compared with sham and ANS+I/R groups.

Fig. 7.

I/R induced an increase in intestinal MPO activity relative to sham (nonischemic) controls, an effect that was not attenuated by preconditioning with the H₂S donor NaHS (NaHS-PC+I/R) or the BK_Ca activator NS-1619 (NS-1619+I/R) 24 h prior to I/R. Coincident administration of the BK_Ca inhibitor paxilline with NaHS 24 h prior to I/R (Pax+NaHS+I/R) was associated with mucosal MPO levels that were significantly elevated relative to sham but were not statistically different from I/R. Data are means ± SE for 6 rats per group. *P < 0.05 compared with sham. &P < 0.001 compared with sham.

Fig. 8.

Ileal mucosal tumor necrosis factor-α (TNF-α) levels were increased following I/R. This postischemic increase in mucosal TNF-α was not affected by preconditioning with the H₂S donor NaHS (NaHS-PC+I/R) or the BK_Ca channel activator NS-1619 (NS-1619+I/R) nor by coincident administration of the BK_Ca channel inhibitor paxilline with NaHS (Pax+NaHS+I/R) 24 h prior to I/R. Data are means ± SE for 6 rats per group. *P < 0.001 compared with sham, &P < 0.05 compared with sham.

Fig. 9.

Summary diagram illustrating the role of endothelial and mucosal BK_Ca channels as mediators of the effects of antecedent treatment with NaHS or NS-1619 (NaHS or NS-1619 Preconditioning) to limit I/R-induced leukocyte rolling and adhesion and preserve mucosal mitochondrial function. Neutrophil depletion with ANS prior to the onset of I/R also preserved mucosal mitochondrial function. Since I/R-induced neutrophil infiltration (assessed using tissue MPO activity as a marker) was prevented by ANS treatment, but not by administration of NaHS or NS-1619, our results suggest that infiltrating neutrophils may not be activated to produce collateral injury in the postischemic intestine of animals preconditioned with NaHS or NS-1619. Although the postischemic increase in TNF-α was not attenuated by NaHS or NS-1619 preconditioning, multiple chemotactic agents are released and act in concert to promote neutrophil in the interstitial space after intestinal I/R. It is possible that release of at least some of these neutrophil activators may be abrogated by preconditioning, thereby limiting activation of extravasated leukocytes.