Nitrite in saliva increases gastric mucosal blood flow and mucus thickness

Abstract

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to NO and related compounds, which have potential biological activity. We used an in vivo rat model as a bioassay to test effects of human saliva on gastric mucosal blood flow and mucus thickness. Gastric mucosal blood flow and mucus thickness were measured after topical administration of human saliva in HCl. The saliva was collected either after fasting (low in nitrite) or after ingestion of sodium nitrate (high in nitrite). In additional experiments, saliva was exchanged for sodium nitrite at different doses. Mucosal blood flow was increased after luminal application of nitrite-rich saliva, whereas fasting saliva had no effects. Also, mucus thickness increased in response to nitrite-rich saliva. The effects of nitrite-rich saliva were similar to those of topically applied sodium nitrite. Nitrite-mediated effects were associated with generation of NO and S-nitrosothiols. In addition, pretreatment with an inhibitor of guanylyl cyclase markedly inhibited nitrite-mediated effects on blood flow. We conclude that nitrite-containing human saliva given luminally increases gastric mucosal blood flow and mucus thickness in the rat. These effects are likely mediated through nonenzymatic generation of NO via activation of guanylyl cyclase. This supports a gastroprotective role of salivary nitrate/nitrite.

Figure 1

Dynamic changes in gastric mucosal vascular resistance (resistance), MAP, and gastric mucosal blood flow following topical application of human nitrite-rich saliva or sodium nitrite (1 mM) at pH 2 to the rat gastric mucosa. Values are expressed as mean ± SEM (n = 6).

Figure 2

Changes in mean gastric mucosal blood flow during 10 minutes after topical application of human saliva or sodium nitrite to the rat gastric mucosa at pH 2. Nitrite levels were 23 μM in the experiments with fasting saliva (n = 5) and 1.1 mM after nitrate load (n = 6). In both groups the response to 1 mM sodium nitrite (NaNO₂, pH 2) was also studied. *P < 0.05 compared with baseline.

Figure 3

Effects of sodium nitrite (0.1–5 mM in HCl, pH 2) on gastric mucosal vascular resistance, MAP, and gastric mucosal blood flow. Values are expressed as mean ± SEM (n = 7–13).

Figure 4

Maximum mucosal blood flow responses (upper panel, n = 7–13 rats) after topical application of human saliva or sodium nitrite (0.1–5 mM) to the gastric mucosa in relation to generation of NO (lower graphs, n = 5). Saliva or sodium nitrite solutions were mixed with HCl to pH 2. Final nitrite levels were 0.23 μM in experiments with fasting saliva and 1.1 mM when using saliva collected after a nitrate load. Values are expressed as mean ± SEM. *P < 0.01 compared with baseline.

Figure 5

Effects of topical administration of sodium nitrite (1 mM, pH 2) and SNAP (0.3 mM) on rat gastric mucosal blood flow before and after topical pretreatment with the guanylyl cyclase inhibitor ODQ (1 mM, n = 6). Data are presented as mean ± SEM. *P < 0.05.

Figure 6

Thickness of the firmly adherent gastric mucus layer following 60 minutes of exposure to human saliva or sodium nitrite (1 mM). All experiments were performed at pH 2; in the control group the mucosa was exposed to acid alone. *P < 0.05 compared with control.

Figure 7

Generation of NO (filled circles) and S-nitrosothiols (open circles) from saliva mixed with HCl to pH 5.5 or pH 2. Saliva was collected from healthy volunteers after ingestion of nitrate (0.1 mmol/kg); nitrite content was 0.9 mM. Data are expressed as mean ± SEM of three experiments.