Protective role of PI3-kinase-Akt-eNOS signalling pathway in intestinal injury associated with splanchnic artery occlusion shock

Abstract

Background and purpose: Endothelial NO synthase (eNOS) is a dynamic enzyme tightly controlled by co- and post-translational lipid modifications, phosphorylation and regulated by protein-protein interactions. Here we have pharmacologically modulated the activation of eNOS, at different post-translational levels, to assess the role of eNOS-derived NO and of these regulatory mechanisms in intestinal injury associated with splanchnic artery occlusion (SAO) shock.

Experimental approach: SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk for 45 min followed by 30 min of reperfusion. During ischemia, 15 min prior to reperfusion, mice were given geldanamycin, an inhibitor of hsp90 recruitment to eNOS, or LY-294002 an inhibitor of phosphatidylinositol 3-kinase (PI3K), an enzyme that initiates Akt-catalysed phosphorylation of eNOS on Ser1179. After 30 min of reperfusion, samples of ileum were taken for histological examination or for biochemical studies.

Key results: Either LY-294002 or geldanamycin reversed the increased activation of eNOS and Akt observed following SAO shock. These molecular effects were mirrored in vivo by an exacerbation of the intestinal damage. Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity, and with an increased expression of the adhesion proteins: ICAM-I, VCAM, P-selectin and E-selectin.

Conclusions and implications: Overall these results suggest that activation of the Akt pathway in ischemic regions of reperfused ileum is a protective event, triggered in order to protect the intestinal tissue from damage induced by ischaemia/reperfusion through a fine tuning of the endothelial NO pathway.

Figures

Figure 1

(a) eNOS expression was increased during reperfusion returning to basal levels within 120 min. (b) During ischemia, eNOS expression was unaltered but its activation was increased as assessed by the decreased expression of CAV-1 and the increased phosphorylation on eNOS Ser1179. Following reperfusion, eNOS was also significantly phosphorylated on Ser1179. The increased activation of eNOS was also confirmed by a decrease in CAV-1 expression during reperfusion. ***P<0.001 vs sham (S). The blot shown is representative of three different experiments.

Figure 2

(a) Akt expression was increased during reperfusion (30 min) and its phosphorylation significantly enhanced. When mice were pretreated with LY-294002 (30 mg kg−1) or geldanamycin (GA; 1 mg kg−1), Akt upregulation was reversed; S, sham; IR, ischemia-reperfusion; **P<0.01 vs vehicle (V). (b) Similarly, administration of LY-294002 (30 mg kg−1) or GA (1 mg kg−1) restored CAV-1 expression, but prevented the increase in eNOS expression and phosphorylation **P<0.01 vs sham (S). The blots are representative of three different experiments.

Figure 3

Histological evaluation of damage to mouse ileum after reperfusion and SAO. Hematoxylin/eosin staining of sections of ileum from sham-treated mice (a). In (b–d), sections are from mice subjected to SAO shock, pretreated with (b), vehicle; (c) LY-294002 (30 mg kg−1); (d) geldanamycin (GA; 1 mg kg−1). (e) Histological damage score following treatment with vehicle (V), LY-294002 (10–100 mg kg−1) or GA (1 mg kg−1) 15 min before reperfusion *P<0.05; **P<0.01 vs vehicle.

Figure 4

MPO activity in homogenates of ileum from mice subjected to SAO following treatment with vehicle (V), LY-294002 or geldanamycin (GA). All values are expressed as mean±s.e.m. *P<0.001 vs sham #P<0.001 vs I/R vehicle.

Figure 5

(A) Expression of E-selectin, P-selectin, VCAM and ICAM-1 in ileum obtained by sham mice or subjected to SAO following treatment with vehicle, LY-294002 (30 mg kg−1) or geldanamycin (GA) (1 mg kg−1). All values are expressed as mean±s.e.m. *P<0.001 vs sham #P<0.001 vs I/R vehicle. (B) Immunohistochemical localization of ICAM (panel 1), VCAM (panel 2), P-selectin (panel 3) and E-selectin (panel 4) following treatment with: (a) vehicle, (b) sham, (c) LY-294002 30 mg kg−1 and (d) GA (1 mg kg−1).