Differential effects of luminal arginine and glutamine on metalloproteinase production in the postischemic gut

Abstract

Background: Matrix metalloproteinases (MMPs) are a group of endopeptidases induced under inflammatory conditions in the intestine which possess the capacity to degrade components of the extracellular matrix. We have previously demonstrated that MMP-2 expression correlates with increased inducible nitric oxide synthase (iNOS) production in the stomach and that iNOS is upregulated in the postischemic gut by the luminal nutrient arginine and repressed by luminal glutamine. We therefore hypothesized that arginine would enhance expression of MMP-2 in the postischemic gut.

Methods: Jejunal sacs were created in rats at laparotomy and filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 minutes of superior mesenteric artery occlusion (SMAO) and 6 hours of reperfusion and compared with shams. Jejunum was harvested, and membrane type-1 matrix metalloproteinase (MT1-MMP), MMP-2, and iNOS protein expression was determined by Western analysis and MMP-9 production by gelatin zymography.

Results: MMP-2, MT1-MMP, MMP-9, and iNOS were all increased after SMAO compared with shams. Arginine maintained while glutamine inhibited the increase in iNOS, MT1-MMP, and MMP-2 expression in the postischemic gut. Pretreatment of the arginine group with a selective iNOS inhibitor blunted the induction of MMP-2 in the postischemic gut. There was no differential modulation of MMP-9 by the luminal nutrients.

Conclusions: The arginine-induced upregulation of iNOS may contribute to increased activity of MT1-MMP and MMP-2. The mechanism for this differential regulation by arginine warrants further investigation.

Figure 1

Effect of ischemia–reperfusion (IR) and luminal nutrients in jejunal production of inducible nitric oxide synthase (iNOS). Jejunal gut sacs were created and filled with 60 mM of arginine (Arg), glutamine (Glut), or magnesium sulfate as an iso-osmotic control and subjected to IR as described in the Materials and Methods section. Representative immunoblot and densitometric analysis demonstrates significantly increased iNOS production in the jejunal gut sacs after IR in the magnesium sulfate and Arg groups as compared with sham controls. There was no significant increase in iNOS production in the Glut group. *P < .05 vs corresponding sham (ANOVA)

Figure 2

Effect of ischemia–reperfusion (IR) and luminal nutrients on jejunal production of matrix metalloproteinase-2 (MMP-2). Jejunal gut sacs were created and filled with 60 mM of arginine (Arg), glutamine (Glu), or magnesium sulfate as an osmotic control and subjected to IR as described in the Materials and Methods section. Representative immunoblot and densitometric analysis demonstrates significantly increased MMP-2 production in the Arg IR group as compared with sham controls. *P < .05 vs arginine sham (ANOVA).

Figure 3

Effect of ischemia–reperfusion (IR) and luminal nutrients on jejunal production of membrane type-1 matrix metalloproteinase (MT1-MMP). Jejunal gut sacs were created and filled with 60 mM of arginine (Arg), glutamine (Glut), or magnesium sulfate as an osmotic control and subjected to IR as described in the Materials and Methods section. Representative immunoblot and densitometric analysis demonstrates significantly increased MT1-MMP production after IR in magnesium sulfate as well as the Arg group as compared with sham controls, while Glut attenuated the increase in MT1-MMP after IR. *P < .05 vs sham (ANOVA).

Figure 4

Effect of ischemia–reperfusion (IR) and luminal nutrients on jejunal production of matrix metalloproteinase-9 (MMP-9). Jejunal gut sacs were created and filled with 60 mM of arginine (Arg), glutamine (Glut), or magnesium sulfate as an osmotic control and subjected to IR as described in the Materials and Methods section. MMP-9 production was assessed by gelatin zymography. Areas of increased MMP-9 activity are detected as areas of increased lucency in the gel at 92 kDa. Representative zymography demonstrates increased MMP-9 activity after IR, which was not modulated by luminal nutrients. The increase in activity was significant when compared with magnesium sulfate controls. There was no increase in MMP-9 activity upon the addition of luminal nutrients in the absence of IR (data not shown). *P < .05 vs magnesium sulfate (ANOVA).

Figure 5

Effect of the selective inducible nitric oxide synthase (iNOS) inhibitor 1400w on matrix metalloproteinase-2 (MMP-2) production in arginine filled gut sacs after ischemia–reperfusion (IR). Jejunal gut sacs were created and filled with arginine (Arg) and 1400w or vehicle as described in the Materials and Methods section. MMP-2 production was assessed by Western immunoblot. MMP-2 was significantly increased in the Arg gut sacs after IR. This effect was inhibited by the addition of the selective iNOS inhibitor 1400w. *P < .05 vs sham vehicle; #P < .05 vs IR vehicle (ANOVA).

Figure 6

Effect of the selective inducible nitric oxide synthase (iNOS) inhibitor 1400w on membrane type-1 matrix metalloproteinase (MT1-MMP) production in arginine filled gut sacs after ischemia–reperfusion (IR). Jejunal gut sacs were created and filled with arginine and 1400w or vehicle as described in the Materials and Methods section. MT1-MMP production was assessed by Western immunoblot. MT1-MMP was significantly increased in the Arg gut sacs after IR. This effect was inhibited by the addition of the selective iNOS inhibitor 1400w. *P < .05 vs sham vehicle; #P < .05 vs IR vehicle (ANOVA).