Sensitizing effects of lafutidine on CGRP-containing afferent nerves in the rat stomach

Abstract

1. Capsaicin sensitive afferent nerves play an important role in gastric mucosal defensive mechanisms. Capsaicin stimulates afferent nerves and enhances the release of calcitonin gene-related peptide (CGRP), which seems to be the predominant neurotransmitter of spinal afferents in the rat stomach, exerting many pharmacological effects by a direct mechanism or indirectly through second messengers such as nitric oxide (NO). 2. Lafutidine is a new type of anti-ulcer drug, possessing both an antisecretory effect, exerted via histamine H(2) receptor blockade, and gastroprotective activities. Studies with certain antagonists or chemical deafferentation techniques suggest the gastroprotective actions of lafutidine to be mediated by capsaicin sensitive afferent nerves, but this is an assumption based on indirect techniques. In order to explain the direct relation of lafutidine to afferent nerves, we conducted the following studies. 3. We determined CGRP and NO release from rat stomach and specific [(3)H]-resiniferatoxin (RTX) binding to gastric vanilloid receptor subtype 1 (VR1), which binds capsaicin, using EIA, a microdialysis system and a radioreceptor assay, respectively. 4. Lafutidine enhanced both CGRP and NO release from the rat stomach induced by a submaximal dose of capsaicin, but had no effect on specific [(3)H]-RTX and capsaicin binding to VR1. 5. In conclusion, our findings demonstrate that lafutidine modulates the activity of capsaicin sensitive afferent nerves in the rat stomach, which may be a key mechanism involved in its gastroprotective action.

Figure 1

Effect of capsaicin on CGRP release from isolated rat stomach. The stomach was incubated with capsaicin for 30 min and CGRP in the incubation medium was determined by EIA. Each column and vertical bar represents the mean and s.e.mean of three separate experiments. **P<0.01, ***P<0.001; significantly different from vehicle (Dunnett's multiple comparison test).

Figure 2

Effect of lafutidine on CGRP release from isolated rat stomach. The stomach was incubated with lafutidine for 30 min and CGRP in the incubation medium was determined by EIA. Capsaicin (10⁻⁶ M) was treated as a positive control. Each column and vertical bar represents the mean and s.e.mean of 10 separate experiments. ***P<0.001; significantly different from vehicle (Student's t-test).

Figure 3

Effect of lafutidine on capsaicin induced CGRP release from isolated rat stomach. The stomach was incubated with capsaicin for 30 min and CGRP in the incubation medium was determined by EIA. Lafutidine was added 10 min before capsaicin treatment. Each column and vertical bar represents the mean and s.e.mean of eight separate experiments. ###P<0.001; significantly different from capsaicin (Student's t-test). **P<0.01, ***P<0.001; significantly different from capsaicin (Dunnett's multiple comparison test).

Figure 4

Effect of capsazepine on CGRP release induced by capsaicin alone or combined with lafutidine from isolated rat stomach. The stomach was incubated with capsaicin for 30 min and CGRP in the incubation medium was determined by EIA. Lafutidine or capsazepine was added 10 min before capsaicin treatment. Each column and vertical bar represents the mean and s.e.mean of eight separate experiments. ***P<0.001; significantly different between two groups (Student's t-test).

Figure 5

Effect of capsaicin on NO production from stomachs of anesthetized rats. The perfused dialysates were collected from the stomach every 10 min and NO metabolites were determined with an automated NO detector HPLC system. (A) The sum of NO production over the basal level during 60 min period were calculated. (B) Changes in NO production in the dialysate. Capsaicin was intragastrically administered through a cannula in the forestomach. Each point or column and vertical bar represents the mean and s.e.mean of five separate experiments. (A), *P<0.05, ***P<0.001; significantly different from vehicle (Dunnett's multiple comparison test).

Figure 6

Effect of lafutidine on capsaicin-induced NO production from stomachs of anaesthetized rats. The perfused dialysates were collected from the stomach every 10 min and NO metabolites were determined with an automated NO detector HPLC system. (A) The sum of NO production over the basal level during 60 min period were calculated. (B) Changes in NO production in the dialysate. Capsaicin was intragastrically administered through a cannula in the forestomach and lafutidine was added 10 min before capsaicin treatment. Each point or column and vertical bar represents the mean and s.e.mean of 10 separate experiments. (A), **P<0.01; significantly different from capsaicin (Dunnett's multiple comparison test).

Figure 7

Effects of lafutidine, capsaicin and RTX on specific binding of [³H]-RTX to rat gastric mucosal membranes. Specific binding of [³H]-RTX was determined experimentally as the difference between total and nonspecific binding in parallel assays in the presence and absence of 1 μM RTX. One nM of [³H]-RTX was incubated with five doses of each drug. All data were run in duplicate and each point represents the mean value of eight separate experiments.