Nitrite in saliva increases gastric mucosal blood flow and mucus thickness (original) (raw)

Animal and tissue preparation. All experiments were approved by the Uppsala University Ethical Committee for Animal Experiments and the Local Ethics Committee for Human Research at the Karolinska Institute. Male Sprague-Dawley rats (B&K Universal AB, Sollentuna, Sweden) weighing 165–280 g were kept in standard conditions of temperature (21–22°C) and illumination (12 h light/12 h darkness). The rats were kept in wide, mesh-bottomed cages with free access to pelleted food and tap water. Before the experiment they were fasted for 18–20 hours with free access to water. Anesthesia was conducted with 120 mg/kg thiobutabarbital sodium (Inactin; Sigma-Aldrich, St. Louis, Missouri, USA) given intraperitoneally and a PE-200 cannula was inserted into the trachea to facilitate spontaneous breathing. Core temperature was kept at 37–38°C by a heating pad regulated with a rectal thermistor. The right femoral artery was catheterized with a PE-50 cannula containing heparin (12.5 IU/ml) dissolved in 0.9% saline, and connected to a pressure transducer for continuous measurement of systemic blood pressure. The right femoral vein was cannulated for drug administration and continuous infusion of a modified Ringer solution (25 mM NaHCO3, 120 mM NaCl, 2.5 mM KCl, and 0.75 mM CaCl2) at a rate of 1 ml/h. In one group of animals, the left femoral vein was also cannulated for administration of N-nitro-L-arginine (L-NNA).

The gastric preparation for blood flow and mucus thickness measurements has been described previously in detail (15, 30). In brief, the abdomen was opened through a midline incision and the stomach was gently exteriorized. The forestomach was opened along the greater curvature and the rat was placed on its left side on a Lucite microscope stage. The corpus of the stomach was everted through the incision and loosely draped over a truncated cone with the luminal side up. A mucosal chamber with a hole in the bottom was placed over the stomach, exposing approximately 1.2 cm2 of the mucosal surface, and the junction was sealed with silicone grease. The mucosal chamber was filled with unbuffered 0.9% saline (5 ml) and kept at 37–38°C by warm water perfusing the bottom of the chamber. The saline was changed every 10 minutes and pH was measured. The animals were allowed to recover for at least 1 hour after surgical preparation until systemic blood pressure and gastric mucosal blood flow were stabilized.

Since basal acid secretion is very low or absent in fasted, anesthetized animals, pentagastrin was given to stimulate secretion. Pentagastrin (40 μg/kg/h intravenously) was given as a continuous infusion throughout the experiments, starting at least 30 minutes before the experiments. The time needed to achieve the desired acidity in the chamber (pH 2) was very long and highly variable (70–180 minutes). This is mainly due to the relatively large chamber volume (5 ml) in relation to the small acid-producing area exposed (approximately 10%) and the variability in acid secretion rate between the animals. Therefore, to make it possible to perform dose-response experiments and comparative studies in the same rat, chamber pH was set with exogenous acid in most experiments.

Blood flow. Laser-Doppler flowmetry (PeriFlux 4001 Master and Periflux PF3; Perimed AB, Stockholm, Sweden) was used for mucosal blood flow measurements. The laser light (wavelength 635 nm, helium neon laser) is guided to the tissue by an optical fiber, and back-scattered light is detected by a pair of fibers with a separation of 0.5 mm. The nature of the Doppler shift from an illuminated tissue depends on the velocity and number of moving red blood cells (31). The accuracy of the Laser-Doppler flowmetry technique for gastrointestinal applications was described earlier (32, 33). The laser probe was fixed to a micromanipulator and kept at a distance of 0.5–1 mm from the gastric mucosa in the chamber solution. The recorded blood flow was considered to be mainly mucosal, since the amount of back-scattered light decreases exponentially with the distance from the probe and since a majority of the total blood flow in the gastric wall is mucosal. Blood flow was determined as a voltage signal and expressed as perfusion units, and was continuously recorded throughout the experiment. Blood flow was then expressed as percent of baseline values. Mean blood flow response was calculated from the area under the curve during a 10-minute period.

Mucus thickness. Mucus thickness was measured using micropipettes connected to a micromanipulator (Leitz GmbH & Co., Oberkochen, Germany) (15, 34). The micropipettes were pulled to a tip diameter of 1–3 μm with a pipette puller (pp-83; Narishige Scientific Instrument Laboratories, Tokyo, Japan). To prevent mucus from adhering to the glass, the tip was dipped into a silicone solution (Wacker Silicone, Wacker-Chemie GmbH, Munich, Germany) and dried at 100°C for 30 minutes. The luminal surface of the mucus gel was visualized with carbon particles (extra pure activated charcoal; Merck Inc., Darmstadt, Germany). The epithelial cell surface was visible through the microscope. The micropipette was pushed into the mucus gel at an angle (a) of 25–35° to the epithelial cell surface, and the distance (D) traveled by the micropipette from the luminal surface of the mucus gel to the epithelial cell surface was measured with a digimatic indicator (IDC Series 543; Mitutoyo Corp., Tokyo, Japan) connected to the micromanipulator. Mucus gel thickness (T) was then calculated from the formula T = D(sin a). A mean value from four to five measurements at different locations was used as one observation. Removal of the outer loosely adherent mucus layer was performed by gentle suction with a thin catheter coupled to a syringe. The inner firmly adherent mucus layer remained and the thickness of this layer was immediately measured.

Collection of human saliva. To obtain saliva with different nitrite content, saliva was collected from overnight fasting volunteers before and 1 hour after ingestion of sodium nitrate (0.1 mmol/kg). Previous studies have shown that salivary nitrite/nitrate is greatly increased 1 hour after a nitrate load (35). The saliva was mixed, centrifuged at 400 g for 10 minutes, and stored at –20°C until immediately before use. Nitrite and nitrate content of saliva was measured with the chemiluminescence method described below.

Headspace NO. To determine the relative rate of NO formation from saliva and sodium nitrite we used an in vitro model. A plastic cup (100 ml) was placed over the mucosal chamber to collect headspace NO. The same concentrations and pH as in the in vivo experiments were used for sodium nitrite and saliva. For the sodium nitrite experiments, the chamber was filled with 5 ml isotonic HCl (pH 2), and nitrite (100 mM) was added through a hole in the base of the cup to a final concentration of 0.1, 0.5, 1, and 5 mM. For the saliva experiments, the chamber was filled with 2.5 ml saliva, after which 2.5 ml HCl (32 mM) was added to reach pH 2. In addition, we studied NO formation from saliva at pH 5.5. Headspace NO was measured through a sample tube connected to a chemiluminescence analyzing system (Aerocrine AB, Stockholm, Sweden). Calibration with cylinder gas (10 ppm NO; AGA AB, Lidingö, Sweden) was performed before each experiment. The sample flow rate was 100 ml/min, and peak NO concentration was measured after adding the compounds. The 8-mm hole in the base of the cup secured circulation of air during measurements.

Nitrite, nitrate, and S-nitrosothiols. For measurements of nitrite, nitrate, and S-nitrosothiols, we used a chemiluminescence method as described in detail by Feelisch et al. (36). Human saliva was collected before and after ingestion of sodium nitrate as described above. After centrifugation at 400 g for 10 minutes, saliva was mixed (1:1) with isotonic HCl to reach pH 2 or pH 5.5. The solutions were kept at room temperature for 15 minutes before analysis.

In vivo experimental protocol. The effects of topical administration of a variety of substances were tested in several groups of rats as described below. When the substances were coadministered with acid, an isotonic HCl (pH 2) was used. The total volume in the mucosal chamber was 5 ml in all experiments. All rats were given pentagastrin to secure a standardized endogenous acid secretion. Before each intervention a baseline mucosal blood flow was obtained during a 10-minute period to which each response was compared. The effects of each intervention were followed for 10 minutes. Mucosal blood flow and mean arterial blood pressure (MAP) were continuously registered during the experiments, and mucosal vascular resistance was calculated as the ratio of MAP to mucosal blood flow.

Effects of saliva on mucosal blood flow. Rat stomachs were treated topically with human saliva collected after a nitrate load (n = 6) or with fasting human saliva (n = 5). Saliva (2.5 ml) and HCl were mixed in the mucosal chamber. In all animals, the response to sodium nitrite (1 mM) in pH 2 HCl was also tested. Between each intervention the mucosal chamber was emptied and replaced with saline.

Dose-dependent effects of sodium nitrite on mucosal blood flow. Mucosal blood flow was measured in 7–13 animals after increasing doses of sodium nitrite. The mucosal chamber was filled with HCl (pH 2), and sodium nitrite (100 mM stock solution) was immediately added to a final concentration of 0.1, 0.5, 1, and 5 mM, respectively. Nitrite (1 mM) was also administered to six separate rats in an experiment in which chamber pH was allowed to drop to pH 2 spontaneously without addition of exogenous acid. Eight separate animals were exposed to HCl without addition of nitrite. Six additional animals were pretreated with an intravenous infusion of L-NNA (10 mg/kg bolus followed by 3 mg/kg/h throughout the experiments). After 30 minutes, when the blood flow was stable, the effects of 1 mM of sodium nitrite in HCl were studied.

Effects of guanylyl cyclase inhibition. In six rats, mucosal blood flow responses to acidified sodium nitrite (1 mM or 5 mM) and the NO donor S-nitroso-N-acetyl-penicillamine (SNAP; 0.3 mM, pH 5.5) were studied before and after 30 minutes’ exposure in the mucosal chamber to the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 mM) dissolved in 5% DMSO. In three additional rats, acidified nitrite and SNAP were tested before and after 5% DMSO alone.

Effects of a COX inhibitor. In four rats, the effects of sodium nitrite (1 mM, pH 2) on gastric mucosal blood flow were studied after pretreatment with the COX inhibitor indomethacin (3 mg/kg given intravenously).

Effects of an NO donor. In four rats, the gastric mucosal blood flow response to the NO donor diethylenetriamine NONOate (DETA/NO) (1 mM, pH 5.5) was studied.

After all experiments intravital microscopy was performed to detect any mucosal injury.

Effects of saliva and sodium nitrite on mucus thickness. Rats (n = 13) were divided into three groups in which nitrite-rich saliva (pH 2, n = 3), sodium nitrite (1 mM, pH 2, n = 5), or pH 2 HCl alone (n = 5) was applied for a total of 60 minutes. During this period the solutions in the chamber were replaced every 15 minutes to maintain nitrite concentration throughout the experiment. To standardize diffusion distance to the mucosa and to allow measurements of total mucus increase, the outer loosely adherent mucus layer was removed before intervention and the remaining inner firmly adherent mucus layer was measured. After the 60-minute study period, total mucus thickness and the inner firmly adherent mucus layer were measured again.

Intragastric NO generation. In 14 additional rats we measured intragastric NO levels after pretreatment with sodium nitrate. Fasting rats were given either sodium nitrate (0.1 mmol/kg, n = 7) or the same amount of NaCl (control, n = 7) in 1 ml distilled water intragastrically. After 2 hours, the rats were anesthetized and a laparotomy was performed. A thin needle was inserted intragastrically via the stomach wall and the stomach was inflated with 4 ml of NO-free air. External clamps prevented passage of air into the esophagus or duodenum. The dilution of gastric gas was necessary to achieve a satisfactory gas volume for further analysis. After 15 seconds the air was aspirated and immediately injected into a rapid-response chemiluminescence NO analyzer (Aerocrine AB).

Chemicals. The following chemicals and drugs were used: thiobutabarbital sodium (Inactin, Sigma-Aldrich), pentagastrin (Cambridge Laboratories Ltd., Wallsend, United Kingdom), 1 M HCl (Titrisol; Merck Inc.), ODQ (Tocris Cookson Inc., Ballwin, Missouri, USA), heparin (LEO Pharma, Ballerup, Denmark), SNAP, DETA/NO, L-NNA, DMSO, sodium nitrite, and sodium nitrate (Sigma-Aldrich).

Statistics. Differences between groups of animals were evaluated by one-way ANOVA, followed by the Fisher protected least significant difference test. For comparison within groups, we used ANOVA for repeated measures followed by the Fisher protected least significant difference test. All statistical calculations were performed with a data analysis software system (STATISTICA version 6; StatSoft Inc., Tulsa, Oklahoma, USA). All data are presented as mean ± SEM. A P value of less than 0.05 was considered significant.