Role of prostaglandins, nitric oxide, sensory nerves and gastrin in acceleration of ulcer healing by melatonin and its precursor, L‐tryptophan

Melatonin, a major hormone of pineal gland, was recently shown to attenuate acute gastric lesions induced by strong irritants because of the scavenging of free radicals but its role in ulcer healing has been little investigated. In this study we compared the effects of intragastric (i.g.) administration of melatonin and its precursor, L‐tryptophan, with or without concurrent treatment with luzindole, a selective antagonist of melatonin MT2 receptors, on healing of chronic gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm2). The involvement of endogenous prostaglandins (PG), nitric oxide (NO) and sensory nerves in ulcer healing action of melatonin and L‐tryptophan was studied in rats treated with indomethacin and NG‐nitro‐L‐arginine (L‐NNA) to suppress, respectively, cyclo‐oxygenases (COX) and NO synthases or in those with functionally deactivated sensory nerves with capsaicin. The influence of melatonin on gastric secretion during ulcer healing was tested in separate group of rats with gastric ulcer equipped with gastric fistulas (GF). At day 8 and 15 upon the ulcer induction, the area of gastric ulcers was measured by planimetry, the mucosal blood flow (GBF) was determined by H2‐gas clearance technique and gastric luminal NO2–/NO3– levels was assessed by Griess reaction. Plasma melatonin and gastrin levels were measured by specific radioimmunoassay (RIA). Biopsy mucosal samples were taken for expression of constitutive NO‐synthase (cNOS) and inducible NOS (iNOS) by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Melatonin (2.5–20 mg/kg‐d i.g.) and L‐tryptophan (25–100 mg/kg‐d i.g.) dose‐dependently accelerated ulcer healing, the dose inhibiting by 50% (ED50) of ulcer area being 10 and 115 mg/kg, respectively. This inhibitory effect of melatonin (10 mg/kg‐d i.g.) and L‐tryptophan (100 mg/kg‐d i.g.) on ulcer healing was accompanied by a significant rise in the GBF at ulcer margin and an increase of plasma melatonin, luminal NO2–/NO3– and plasma gastrin levels. Gastric acid and pepsin outputs were significantly inhibited during the ulcer healing in melatonin‐treated gastric mucosa as compared with those in vehicle‐treated animals. Luzindole abolished completely the healing effects of melatonin and L‐tryptophan and attenuated significantly the rise in plasma gastrin evoked by the hormone and its precursor. Indomethacin (5 mg/kg‐d i.p), that blocked PG biosynthesis by 90% or L‐NAME (20 mg/kg i.v), inhibitor of NOS, that suppressed luminal NO release, attenuated significantly melatonin and L‐tryptophan‐induced acceleration of ulcer healing and accompanying rise in GBF at ulcer margin and luminal NO release. The melatonin‐induced acceleration of ulcer healing, hyperemia at ulcer margin and increase in the release of NO were enhanced when L‐arginine but not D‐arginine was added to L‐NAME. The ulcer healing and the GBF effects of melatonin and L‐tryptophan were significantly impaired in rats with capsaicin‐induced denervation of sensory nerves and both, ulcer healing and the hyperemia at ulcer margin were restored in these rats by addition of exogenous CGRP to melatonin and L‐tryptophan. Expression of cNOS mRNA was detected by RT‐PCR in the intact gastric mucosa as well as at the edge of gastric ulcers treated with both, vehicle and melatonin, while iNOS mRNA that was undetectable in the intact gastric mucosa, appeared during ulcer healing and especially this was strongly up‐regulated in the melatonin‐treated gastric mucosa. We conclude that (1) exogenous melatonin and that derived from its precursor, L‐tryptophan, accelerate ulcer healing probably via interaction with MT2 receptors; (2) this ulcer healing action is caused by an enhancement by melatonin of the microcirculation at the ulcer margin possibly mediated by COX‐derived PG and NO because of overexpression of iNOS and (3) gastrin, which exhibits trophic activity in the gastric mucosa and calcitonin gene related peptide (CGRP), released from sensory nerves, may also contribute to the ulcer healing action of melatonin.