Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C26
Pepsinogen is nitrated in the stomach in vivo: a dual function for dietary nitrate with functional and physiological consequences
B. Rocha1, B. Gago2, R. Barbosa3, R. Radi4, J. Laranjinha5
1. Center for Neurosciences and Cell Biology, Coimbra, Portugal. 2. Center for Neuroscience and Cell Biology, Coimbra, Portugal. 3. Center for Neurosciences and Cell Biology, Coimbra, Portugal. 4. Center for Free Radical and Biomedical Research, Montevideo, Uruguay. 5. Center for Neurosciences and Cell Biology, Coimbra, Portugal.
Photomicrographs of the spatial distribution of nitrotyrosine labeling in the rat gastric mucosa. (A) Nitrotyrosine immunoreactivity was detected within glandular cells (arrowhead) and (B) scattered through the lamina propria where polymorphonuclear cells were identified (asterisk). Transversely orientated gastric glands (GG) and the respective lumen (GL) are represented in figure (A’) by H&E staining (original magnification X200).
Impact of dietary nitrite on pepsinogen nitration in healthy and ulcerated stomachs in vivo. (A) Pepsinogen is shown to be nitrated under physiological conditions (lane 4), but nitration yields increase under inflammatory states (lane 2). The exposure of stomachs with active gastric ulcers to physiological concentrations of nitrite further increases pepsinogen nitration (lane 1). Dietary nitrite decreases the levels of nitrated pepsinogen in healthy stomachs (lane 3). Pepsinogen was immunoprecipitated by an anti-pepsinogen polyclonal antibody and nitration was detected by western blot using an anti-nitrotyrosine antibody. The densitometric analysis of the data is depicted in figure (B). Values are means
Dietary nitrate may be a novel regulator of physiological functions in the gastrointestinal tract, due to its stepwisely reduction to nitrite and nitric oxide (NO). While NO is engaged in gastroprotection, nitrite generates nitrogen oxides able to nitrate tyrosine residues of endogenous proteins. This work studies the impact of nitrite dietary supplementation in the nitration status of the healthy and ulcerated rat gastric mucosa. In addition, we tracked nitration of specific gastric mediators in vivo, namely pepsinogen and evaluated the impact of this post-translational modification on protein (pepsin) function. Wistar rats (n=4, per group) were used to monitor gastric nitration in vivo. The rats were divided in two groups: a healthy (untreated) group and another with acute gastric inflammation (induced by diclofenac - 30 mg/Kg). Ulceration developed for 4 hours, after which the animals were killed. Animals from both groups were further fed (or not) with nitrite 1.38 mg/Kg. All drugs were given by oral gavage. Protein tyrosine nitration was evaluated by immunohistochemistry and immunoprecipitation. Nitrotyrosine (NT) labeling was detected in the deep mucosa of untreated rats suggesting that nitration is a physiological event in the stomach. NT yields increased in the stomach of rats with acute gastric ulceration (p < 0.01) and were further enhanced when they were subsequently fed with nitrite (p < 0.01). NT staining was located within the lamina propria and blood vessels but also in cells of the oxyntic glands, where an intense cytoplasmatic staining suggests nitration of specific gastric mediators stored in cytoplasmatic vesicles, such as pepsinogen (fig. 1). Pepsinogen nitration occurs under basal conditions but increases under gastric ulceration. In this later case, nitrite further enhanced nitration yields (p < 0.05)(fig.2) but nitrite-fed rats showed reduced levels of both, overall and pepsinogen nitration in respect to untreated rats. Pepsinogen nitration has implications to the proteolytic function of the derived pepsin as its activity is c.a. 5 times lower than the one derived from non nitrated pepsinogen (p < 0.001). We demonstrate that pepsinogen is nitrated under physiological conditions in the stomach and that nitration yields increase under acute ulceration. Moreover, nitrite increases the nitration yields under inflammatory events but, in the healthy stomach, it decreases the efficiency of nitration, thus having a dual role depending on the health and disease status. We also provide evidence that pepsinogen nitration impairs the proteolytic function of the derived pepsin, thereby impacting on protein function. Expectedly, an inefficient protease would impair the digestion of dietary proteins but, on the other hand, would also prevent the breakdown of endogenous proteins (mucins, collagen) vital for gastric integrity, thus preventing peptic ulcer disease.
Where applicable, experiments conform with Society ethical requirements