P8 Hydrogen sulfide production during colonic inflammation occurs primarily via a pyridoxal-5′-phosphate (P5P)-independent pathway

Abstract

Hydrogen sulfide (H 2 S) is an important endogenous anti-inflammatory mediator, which has been shown to promote resolution of colitis and healing of ulcers. H 2 S synthesis was believed to occur primarily via two P5P-dependent enzymes (cystathionine- β -synthase (CBS) and cystathionine- γ -lyase (CSE)). In the present study, we examined the possible contribution of a 3 rd pathway for H 2 S synthesis, in the healthy and inflamed colon, which involves the enzymes cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). Methods The capacity of tissue to produce H 2 S was measured from homogenized tissue in the presence of exogenous substrate and/or inhibitors using a zinc-trapping assay. Production of H 2 S via the CAT-3MST pathway was explored using the substrate α -ketoglutarate and the CAT inhibitors l -aspartate and O -carboxymethyl-hydroxylamine hemihydrochloride (CHH). Colitis was induced in rats by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Rats were euthanized at different time points up to 28 days post-DNBS for measurement of H 2 S synthesis. To assess the contributions of different colonic layers to total colonic H 2 S synthesis, healthy and inflamed colonic sections collected on day 3 after DNBS-instillation were dissected to separate mucosal and smooth muscle layers. Diseased colonic sections were further dissected at the edges of the ulcer yielding distinct ulcerated mucosal and smooth muscle layers. Results Colonic H 2 S synthesis via the CAT-3MST pathway was 5-fold greater than that produced via the previously studied pathways (CBS and CSE) (225.2 ± 58.9 nmol/g/h vs. 41.8 ± 21 nmol/g/h respectively; p 2 S synthesis via CAT-3MST markedly increased (>4-fold) compared to controls (963.6 ± 115.0 nmol/g/h vs. 225.2 ± 58.9 nmol/g/h, respectively; p 2 S synthesis were observed when inflammation was most robust (3 days post DNBS) and were suppressed by both l -aspartate and CHH (1132.3 ± 87.7 nmol/g/h control vs. 424.2 ± 153.4 nmol/g/h; p p 2 S synthesis decreased to control levels. Furthermore, the ability of the ulcerated mucosa to produce H 2 S via the CAT-3MST pathway was markedly increased when compared to smooth muscle layers (both healthy and ulcerated) and the mucosa from healthy colons. The capacity of the ulcerated mucosa to produce H 2 S via the CBS/CSE pathways also increased significantly when compared to smooth muscle sections (both healthy and ulcerated) and healthy mucosal layers. Conclusions These data suggest that in both the healthy and inflamed colon CAT and 3MST represent the primary pathway for H 2 S synthesis. The increased production of H 2 S via the CAT-3MST enzymes appears to be occurring at the site of mucosal ulceration. Interestingly, the CBS/CSE pathways also make important contributions to H 2 S synthesis at the ulcerated mucosa. Further understanding of the pathways for H 2 S production in the colon can present new opportunities for modulating colonic H 2 S production, which may have important implications in the development of novel therapies for inflammatory bowel disease.