Colonic H₂S synthesis in CSE-deficient (CSE−/−) and CBS heterozygous (CBS+/−) mice.

<p>Panel A: In the presence of P5P, colonic tissue from CSE-deficient mice produced significantly less H₂S from L-cysteine than tisue from wild type mice (***p<0.001). Panel B: There was no difference in the ability of colonic tissue from CBS heterozygous mice to produce H₂S from L-cysteine in the presence P5P when compared to colonic tissue from wild type controls. Each bar represents the mean ± SEM of 4–7 mice.</p

H₂S synthesis by compartments of healthy and damaged colon.

<p>Panel A: The contributions of the mucosa and muscularis layers of the colon to H₂S synthesis via the α-ketoglutarate-dependent pathway. H₂S synthesis by the ulcerated mucosa was markedly increased compared to that of adjacent, non-ulcerated mucosaand that of the healthy mucosa (*p<0.05). Panel B: The contributions of the mucosa and muscularis layers of the colon to H₂S synthesis via the P5P-dependent pathways. H₂S production by muscularis and mucosa from sites of ulceration were significantly greater than that from healthy tissue or tissue from non-ulcerated sites (*p<0.05, ***p<0.001). Each bar represents the mean ± SEM of 4–6 rats.</p

Sources of H₂S synthesis in the healthy and inflamed rat colon.

<p>Panel A: In the presence of P5P and absence of α-ketoglutarate, colonic tissue from rats with colitis (3 days post-DNBS administration) synthesized significantly more H₂S than that from healthy rats (*p<0.05 vs. corresponding healthy group). Likewise, synthesis of H₂S by colonic tissue from rats with colitis was significantly greater than that from healthy rats when the assay was performed in the presence of α-ketoglutarate and the absence of P5P. For both healthy rats and rats with colitis, colonic H₂S synthesis was significantly greater via the α-ketoglutarate-dependent pathway than via the P5P-dependent pathways (^ψp<0.05). In the absence of both P5P and α-ketoglutarate there was no detectable H₂S production. Panel B: α-ketoglutarate-dependent H₂S synthesis by samples of colonic tissue from healthy rats and rats with colitis (3 days post-DNBS administration) was significantly inhibited by CHH (O-carboxymethyl-hydroxylamine hemihydrochloride) (10 µM) and by L-aspartate (1 mM) (***p<0.001 vs. the corresponding vehicle-treated group; (^ψp<0.05) vs. the corresponding healthy group). Each bar represents the mean ± SEM of 4–6 rats.</p

Major pathways of colonic hydrogen sulfide (H₂S) synthesis.

<p>Hydrogen sulfide can be produced from L-cysteine thorugh at least three enzymatic pathways. The pyridoxal-5′-phosphate (P5P)-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-Υ-lyase (CSE), can metabolize L-cysteine, resulting in the generation of H₂S. L-cysteine can also be converted to 3-mercaptopyruvate via the enzyme cysteine aminotransferase (CAT), the activity of which is depending upon the presence of α-ketoglutarate (α-KG). Mercaptopyruvate transferase (3MST), which is largely localized to mitochondria, can metabolize 3-mercaptopyruvate to generate H₂S.</p

H₂S synthesis by healthy colonic tissue occurs via multiple pathways.

<p><u>Panel A</u>: In the absence of pyridoxal-5′-phosphate (P5P), α-ketoglutarate increased the production of H₂S in by colonic tissue in a concentration-dependent manner (***p<0.001 vs. the group with no α-ketoglutarate added). <u>Panel B</u>: Maximal production of H₂S from L-cysteine by the healthy colon was observed in the presence of 3 mM P5P or 300 µM α-ketoglutarate. The maximal production of H₂S in the healthy colon in the presence of α-ketoglutarate was markedly higher than the maximal production of H₂S in the presence of P5P. <u>Panel C</u>: At a concentration of 1 mM, L-aspartate significantly inhibited H₂S synthesis by healthy colonic tissue. <u>Panel D</u>: CHH (O-carboxymethyl-hydroxylamine hemihydrochloride) concentration-dependently inhibited α-ketoglutarate-dependent colonic H₂S synthesis. The reactions shown in Panels C and D were carried out in the absence of pyridoxal-5′-phosphate and presence of α-ketoglutarate (100 µM). In the absence of both P5P and α-ketoglutarate there was no detectable H₂S production. Each bar represents the mean ± SEM of 4–6 rats (*p<0.05, **p<0.01, ***p<0.001 vs. the control group).</p

Increased naproxen-induced small intestinal damage in obese versus lean rats.

<p>Neither lean nor obese rats developed intestinal damage when given ATB-346. **p<0.01 versus the corresponding vehicle- and ATB-346-treated rats. n = 6 rats per group.</p

Co-administration of naproxen or celecoxib with omeprazole and/or low-dose aspirin results in marked exacerbation of small intestinal damage.

<p>In contrast, rats given a naproxen derivative (ATB-346 or NCX 429) did not develop significant intestinal injury when given alone or in combination with omeprazole, low-dose aspirin, or both. *p<0.05, **p<0.01 versus the corresponding group treated with NSAID alone (n≥6 per group). Aspirin and omeprazole, alone or given together, did not elicit significant intestinal damage.</p

Effects of NSAIDs in rats with adjuvant arthritis.

<p>Despite comparable suppression of paw swelling (panel A), gastric prostaglandin synthesis (panel B) and whole blood thromboxane synthesis (panel C), ATB-346 and NCX 429 did not cause significant gastric (panel D) or intestinal (panel E) damage. Celecoxib also did not cause significant GI damage. *p<0.05, **p<0.01 versus the naproxen-treated group. n = 8 per group.</p

Serum and biliary levels of naproxen are significantly reduced in rats given a naproxen derivative (ATB-346 or NCX 429) as compared to the levels observed in rats given an equimolar dose of naproxen itself.

<p>The test drugs were administered twice-daily for 2 days. *p<0.05, **p<0001 versus the naproxen-treated group. n = 4 per group.</p

Older (19 months of age) rats develop extensive gastric damage when given naproxen, but not when given equimolar doses of a hydrogen sulfide-releasing naproxen derivative (ATB-346) or a nitric oxide-releasing naproxen derivative (NCX 429).

<p>***p<0.001 versus the vehicle-treated group. n =  6 rats per group.</p