Background: L-arginine is the substrate for the nitric oxide synthase (NOS) pathway that is essential for GI wound healing. Inhibition of the inducible NOS isoform impairs anastomotic bursting pressure.L-arginine is also the substrate for the enzyme arginase which metabolizes L-arginine to ornithine and urea. This pathway generates polyamines and proline both known to interact in cell proliferation and collagen synthesis. Arginase exists in two isoforms: AI and AII. The goal of this study was to investigate the L-arginine metabolism in colon anastomosis in respect to arginase expression and activity.
Methods: Lewis-rats underwent laparotomy. The left sided colon was dissected, divided and subsequently reanastomosed using 6-0 prolene. The laparotomy was closed with a running suture. Sham operation was performed in controls. On day 2,5,7,14 and 28 after surgery the anastomosis was excised. The edge 5 mm above and below the suture line was considered "anastomosis"(ANAST). The 5 mm above and below these regions were harvested and considered proximal/distal colon (PDC).The tissue was homogenized in lysis buffer for arginase activity which was measured by newly formed urea (nmol urea/min/mg protein, mean±SEM) or for western blotting. RNA was extracted for RT-PCR of AI and AII and GAPDH. Comparision was made between ANAST and PDC using student's t-test, statistical significance was reached when p<0.05.
Results: Arginase activity was significantly higher at the anastomosis compared to sham controls and PDC (see table). Protein expression as assessed by western blotting detected the AI isoform at all days in ANAST and PDCs. RNA-Expression of AI was only seen on day 2. There was no expression of AII isoform neither by RT-PCR nor by western blotting.
Conclusion: Arginase activity is significantly increased during anasotomic healing probably by a combination of transcriptional and post-translational mechanism.
Arginase activity of colon anastomosis
sham day 2 day 5 day 7 day 14 day 28
PDC 1.75±0.1 1.60±0.3 2.31±0.2 1.40±0.1 1.58±0.1 2.70±0.5
ANAST 1.75±0.1 7.96±0.9 8.68±1.7 7.11±1.4 5.35±1.2 3.21±0.1
p<0.05 ANAST vs. PDC