Exogenous asymmetric dimethylarginine (ADMA) in pathogenesis of ischemia-reperfusion-induced gastric lesions: interaction with protective nitric oxide (NO) and calcitonin gene-related peptide (CGRP)

Abstract

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide (NO) synthesis inhibitor and pro-inflammatory factor. We investigated the role of ADMA in rat gastric mucosa compromised through 30 min of gastric ischemia (I) and 3 h of reperfusion (R). These I/R animals were pretreated with ADMA with or without the combination of L-arginine, calcitonin gene-related peptide (CGRP) or a small dose of capsaicin, all of which are known to afford protection against gastric lesions, or with a farnesoid X receptor (FXR) agonist, GW 4064, to increase the metabolism of ADMA. In the second series, ADMA was administered to capsaicin-denervated rats. The area of gastric damage was measured with planimetry, gastric blood flow (GBF) was determined by H2-gas clearance, and plasma ADMA and CGRP levels were determined using ELISA and RIA. ADMA significantly increased I/R-induced gastric injury while significantly decreasing GBF, the luminal NO content, and the plasma level of CGRP. This effect of ADMA was significantly attenuated by pretreatment with CGRP, L-arginine, capsaicin, or a PGE2 analogue. In GW4064 pretreated animals, the I/R injury was significantly reduced and this effect was abolished by co-treatment with ADMA. I/R damage potentiated by ADMA was exacerbated in capsaicin-denervated animals with a further reduction of CGRP. Plasma levels of IL-10 were significantly decreased while malonylodialdehyde (MDA) and plasma TNF-α contents were significantly increased by ADMA. In conclusion, ADMA aggravates I/R-induced gastric lesions due to a decrease of GBF, which is mediated by a fall in NO and CGRP release, and the enhancement of lipid peroxidation and its pro-inflammatory properties.

Figure 1.

Mean lesion area, gastric blood flow (GBF) level, and gastric luminal NO concentration after application of vehicle (saline) or ADMA administered in graded doses (0.1–40 mg/kg i.g.) followed by exposure to ischemia/reperfusion. Results are mean ± S.E.M. of seven rats per group. Significant change (p < 0.05) as compared with the respective values in vehicle-control group is indicated by an asterisk. A cross and asterisk indicate significant change (p < 0.05) when compared to the values obtained in vehicle-controls and in those pretreated with ADMA (1 or 10 mg/kg i.g.).

Figure 2.

Mean lesion area and the changes in gastric blood flow (GBF) in vehicle and GW4064 (3 mg/kg i.p.) pretreated rats exposed to 3.5 h of ischemia/reperfusion in the absence and the presence of ADMA (20 mg/kg i.g.). Results are mean ± S.E.M of six rats per group. Significant change (p < 0.05) as compared with the respective values in vehicle-control group is indicated by an asterisk. A cross indicates a significant difference (p < 0.05) compared with the vehicle control treated group not treated with ADMA. A cross and an asterisk indicate a significant change (p < 0.05) compared to the values obtained in rats treated with GW4064 without ADMA.

Figure 3.

Mean lesion area and the changes in gastric blood flow (GBF) after application of a small “protective” dose of capsaicin (CAP, 0.25 mg/kg i.g.), l-arginine (l-Arg, 200 mg/kg i.g.) or CGRP (10 μg/kg s.c.). Each was applied alone and in combination with ADMA (20 mg/kg i.g.), followed by exposure to ischemia/reperfusion. Results are mean ± S.E.M of 6–8 rats per group. Significant change (p < 0.05) as compared with the respective values in the vehicle-control group, is indicated by an asterisk. A cross indicates a significant change (p < 0.05) compared to the values obtained in the vehicle-control group. An asterisk and a cross indicate a significant change (p < 0.05) as compared to the values obtained in rats treated with vehicle, capsaicin, l-arg, or CGRP without ADMA.

Figure 4.

Mean lesion area of ischemia/reperfusion-induced damage, the alterations in the gastric blood flow (GBF), and the plasma CGRP levels in rats pretreated with ADMA alone or ADMA given in combination with CGRP in rats with or without capsaicin-induced denervation. Results are mean ± S.E.M of six to eight rats per group. Significant change (p < 0.05) as compared with the respective values in vehicle (saline)-control group is indicated by asterisk. A cross and asterisk indicate significant change (p < 0.05) as compared with vehicle-control group and with rats treated with vehicle or ADMA alone (left panel). Significant difference (p < 0.05) between the respective values in the vehicle (saline)-control group and the vehicle-control with capsaicin denervation, is indicated by a cross. A double asterisk denotes the significant change (p < 0.05) as compared with the respective values in vehicle-controls treated with ADMA without capsaicin denervation. A cross and a hash indicate significant change (p < 0.05) compared to the values obtained in capsaicin-denervated rats treated with ADMA without concurrent treatment with CGRP.

Figure 5.

Plasma ADMA levels and MDA concentration in the gastric mucosa of intact rats, and those pretreated with vehicle (saline), l-arginine (200 mg/kg i.g.), CGRP (10 μg/kg s.c.), capsaicin (CAP) (applied in small protective dose (0.25 mg/kg i.g.)) or superoxide dismutase (SOD, 5000 U/rat i.p.) and exposed to 3.5 h of ischemia/reperfusion. Results are mean ± S.E.M of 7 rats per group. Significant change (p < 0.05) as compared with the intact group, is indicated by an asterisk. A cross indicates significant change (p < 0.05) as compared with vehicle-pretreated rats exposed to 3.5 h of I/R.

Figure 6.

Mean lesion area, the alterations in GBF, and the plasma concentrations of proinflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 in rats pretreated with ADMA alone or ADMA applied in combination with synthetic prostaglandin E₂ derivative, 16,16 dmPGE₂, and exposed 30 min later to gastric ischemia/reperfusion. Results are mean ± S.E.M of 8 rats per group. Significant change (p < 0.05) as compared with the respective values in the vehicle (saline)-control group, is indicated by an asterisk. A cross indicates the significant difference (p < 0.05) between the values obtained in vehicle-controls with and without ADMA administration. An asterisk and a cross indicate significant change (p < 0.05) as compared with the vehicle group in the presence of ADMA.