Kazuo Nishiyama

Role of endogenous substance P in ethanol-induced mucosal damage in the rat stomach.

To determine the role of endogenous substance P in ethanol-induced mucosal damage, two experiments were performed. In the first experiment, the stomachs of anesthetized rats were doubly cannulated and gastric damage was induced with 5 ml of 30% ethanol in the gastric lumen. The damage was ameliorated by pretreatment with capsaicin (0.16 and 1.6 mM) and spantide (100 mg/kg, i.v.). In the second experiment, the gastric mucosa of these rats was perfused with physiological saline containing pepstatin (10 μl/ml). Endogenous substance P (SP) in the perfusate was measured by enzyme immunoassay (EIA). The peak SP levels were increased by capsaicin (0.16–1.6 mM) in a concentration-dependent manner. Perfusion with 50% ethanol for 5 min increased the SP levels approximately threefold. Perfusion with 1.6 mM capsaicin, followed by 50% ethanol, reduced the injured area to about one-quarter of the original injured area. The peak SP levels during perfusion with 50% ethanol after pretreatment with 1.6 mM capsaicin did not differe from those observed after vehicle pretreatment (control). The area under the curve for SP release during 50% ethanol perfusion after vehicle perfusion was not reduced by previous perfusion with 1.6 mM capsaicin followed by 50% ethanol, indicating that the prevention of ethanol-induced injury by capsaicin may be due to excess amounts of different neuropeptides released simultaneously.

Direct observation of microcirculation of the basal region of rat gastric mucosa

We modified and improved techniques for the intravital microscopic observation of the rat gastric microcirculation. The stomach of anesthetized rats was cut along the greater curvature, and the posterior wall of the glandular stomach was fixed in a chamber with the serosal side up and perfused with warmed Tyrodes solution. A portion of the muscularis externa was resected with the serosa to make an observation window. Vascular casts were studied histologically after the injection of Monastral blue B gelatin solution. Vascular casts revealed that most of the microvasculature observed in the window was not located in the submucosa, but in the basal part of the mucosa. Microscopic observation showed that the basal mucosal arterioles branched to form the mucosal capillaries, and the collecting venules from the mucosal surface were seen in cross-sections to drain into the venules located in the basal mucosa, without penetrating the muscularis mucosae. Topical application of acetylcholine (0.03–10μM) to the window dilated the arterioles, and topical application of epinephrine (0.03–3μM) constricted them dose-dependently without affecting the collecting venules and the venules. This method made possible the direct observation of the microvasculature in the basal mucosa of the stomach, in which common microvessel characteristics were shown.

Suppression of myoelectrical activity of gastric smooth muscle by endogenous gastric prostaglandin E2

The myoelectrical activity of the gastric smooth muscle, recorded by a bipolar electrode placed at the gastric antrum in rats, could be recorded when the stomach was distended with 5 ml of physiological saline. This activity may be induced by a mucosal reflex and was inhibited both by atropine and by pirenzepine. Replacement of physiological saline with solutions of NaCl suppressed this myoelectrical activity. This suppression was dependent on the concentrations of NaCl in the solutions, from 0.3 to 1.0 M. Pretreatment with indomethacin (10 mg/kg, intravenous) completely prevented this suppression induced by different concentrations of NaCl solutions. Intragastric administration of 1.0 M NaCl in solution caused an increase in the levels of PGE2 in the gastric lumen. Intragastric administration of OU-1308, a synthetic derivative of PGE1, also suppressed the myoelectrical activity in a dose-dependent manner. It is concluded that the suppression of gastric myoelectrical activity by hyperosmolar NaCl may be attributable to the generation of endogenous PGE2 in the stomach.

Possible role of endogenous prostaglandins against ethanol injury in rat stomach

Exposure of the gastric mucosa for 3 min to 30% intragasric ethanol induced mucosal injury that appeared dark red n anesthetized rats. Microscopic observation of the microvasculature in the injured area after exsanguination of rats and living rats confirmed that there was congestion of the blood flow in the collecting venules and capillaries. Hemor-chage was observed about 30 min after application of ethanol. Monastral blue dye was deposited along the postcapilary venules only at the border of the congestion area. A selective 5-lipoxygenase inhibitor, AA-861, inhibited the gastric lesion, and immunoreactive leukotriene C4 (LTC4) was detected in the gastric wall. Previous exposure of the gastric mucosa to 1.0 M NaCl solution inhibited the ethanol-induced mucosal injury markedly. NaCl solutions 0.5–1.0 M) also suppressed the electrical activity of smooth muscle and the suppression was released by indomethacin. Having observed that PGE2 generation in the stomach was induced dose-dependently by intragastric NaCl solutions (0.5–1.0 M), we propose the hypothesis that Peptide LTs released by intragastric ethanol constrict the Collecting venules or lamina muscularis mucosae and induce congestion of these venules. whereas the PGE2 released by intragastric NaCl solution releases the congestion Of the collecting venules directly or through suppression of contraction of the lamina muscularis.

Interference in the radioimmunoassay of gastric prostaglandins by cimetidine, a histamic H2 blocker

The present study was undertaken to assess whether cimetidine alters the levels of endogenous prostaglandin (PG) E2 and 6-keto-PGF1 alpha in the rat stomach. When cimetidine was mixed in vitro with a suspension of dextran-coated charcoal, to which [3H]-PGE2 had been adsorbed, levels of free [3H]-PGE2 were increased by cimetidine at concentrations above 1.0 mM, because of preferable adsorption of this blocker to the charcoal. Dextran-coated charcoal is used for the separation of antibody-bound [3H]-PGs from the free labelled compounds in the radioimmunoassay of PGs. Addition of cimetidine to standard solutions of PGE2 shifted the PGE2 calibration curve upward. Thus, when PGs in cimetidine-containing samples were quantitated by reference to the normal calibration curve, the assessed levels of PG were lower than the actual levels. Removal of cimetidine from the assay samples was successfully achieved by use of SEP-PAK C18 columns. Rats were injected intraperitoneally with 100 mg/kg of cimetidine only once or with 20 mg/kg twice daily for 7 days. Using this cleaning method, we found that both the basal levels of PGE2 and 6-keto-PGF1 alpha and those levels increased by intragastric administration of 1.0 M NaCl solution did not differ between rats treated with cimetidine and those treated with vehicle. It can be, therefore, concluded that cimetidine does not affect the gastric PG production.