No abstract

The effects of a lectin (AaL) from seeds of Araucaria angustifolia were investigated in the model of rat paw edema. In vivo anti-and pro-inflammatory activities, role of sugar residues, inflammatory mediators and systemic toxicity were assessed. Intravenous injection of AaL (0.1-1 mg/kg) dose-dependently inhibited the dextran-induced increase in edema and vascular permeability, which were prevented by association of the lectin with its binding sugar N-acetyl-glucosamine (Glyc-Nac). AaL also significantly inhibited edema induced by serotonin (18%) and compound 48/80 (33%), but not edema induced by histamine. In contrast, when applied by the s.c. route, AaL evoked a paw edema that peaked 1 h later and was partially prevented by association with Glyc-Nac (59%) or by prior i.v. administration of the lectin itself (38.8%). This AaL edematogenic activity was significantly inhibited by pentoxifylline (44.4%) or dexamethasone (51%) and also by depletion of rat paw mast cells (45.6%), but not by L-N-nitro-arginine methyl ester or indomethacin, excluding involvement of nitric oxide and prostaglandins. Treatment of animals with a single anti-inflammatory dose of AaL (1 mg/kg, i.v.) for 7 days did not affect rat corporal mass, liver, kidney, spleen or stomach wet weight, blood leukocyte count, and urea, creatinine or serum transaminase activity. Systemic toxicity was apparent only at much higher doses (LD50=88.3 mg/kg) than those required for the anti-inflammatory effect. Summarizing, AaL exerts anti-and pro-edematogenic actions via interaction with its specific lectin domain. These actions may share a common pathway involving either activation or inhibition of inflammatory mediators from resident mast cells.

PAL is a glucose/mannose-specific lectin isolated from Pisum arvense seeds. Previously, we demonstrated the capacity of other leguminous lectins to induce oedema formation and neutrophil stimulation. To investigate the potential pro-inflammatory activity of PAL, we have studied its ability to induce neutrophil migration into peritoneal cavities of rats and neutrophil chemotaxis in-vitro. The role of resident cells and sugar residues on PAL activity was analysed. PAL or saline (control) were administered intraperitoneally to rats, and total and differential leucocyte (macrophages, neutrophils and mast cells) counts were performed. The role of resident cells on the PAL effect was evaluated using three strategies: reducing the total resident cell population by lavage of rat cavities with saline; increasing macrophage population by treating animals with thioglycolate; and depleting mast cell population by subchronic treatment of rats with compound 48/80. PAL induced in-vitro and in-vivo neutrophil migration. In-vivo, PAL (50, 100, 200 and 300 μg) significantly (P < 0.05) and dose-dependently increased neutrophil migration by 600, 740, 900 and 940%, respectively, showing maximal effect 4 h after injection. PAL induced mononuclear cell migration. The neutrophil stimulatory effect of PAL was potentiated in animals treated with both thioglycolate and compound 48/80. The indirect lectin chemotactic effect was shown in rats injected with supernatant from cultured macrophages stimulated by PAL. In conclusion, PAL was shown to exhibit in-vivo and in-vitro proinflammatory activity. The in-vivo effect seemed to occur by a dual mechanism that was independent, but also dependent, on resident cells.

1 We have investigated the inhibitory effects of blockers of volume-activated (Cl vol ) and calciumactivated (Cl Ca ) chloride channels on hypotonic solution (HS)-induced contractions of rat trachea, comparing their effects with those of the voltage-dependent calcium channel (VDCC) blocker nifedpine. 2 HS elicited large, stable contractions that were partially dependent on the cellular chloride gradient; a reduction to 41.4577.71% of the control response was obtained when extracellular chloride was removed. In addition, HS-induced responses were reduced to 26.875.6% of the control by 1 mM nifedipine, and abolished under calcium-free conditions, indicating a substantial requirement for extracellular calcium entry, principally via VDCCs. 3 The established Cl vol blockers tamoxifen (p10 mM) and 4,4 0 -diisothiocyanatostilbene-2,2 0 -disulphonic acid (1-100 mM), at concentrations previously reported to inhibit Cl vol in smooth muscle, did not significantly inhibit HS-induced contractions. 4 In contrast, the recognized Cl Ca blocker niflumic acid (NFA; 1-100 mM) produced a reversible, concentration-dependent inhibition of HS responses, with a reduction to 36.676.4% of control contractions at the highest concentration. The mixed Cl vol and Cl Ca blocker, 5-nitro 2-(3-phenylpropylamine) benzoic acid (NPPB; 10-100 mM) also elicited concentration-related inhibition of HS-induced contractions, producing a decrease to 35.9711.3% of the control at 100 mM. 5 Our results show that HS induces reversible, chloride-dependent contractions of rat isolated trachea that were inhibited by NFA and NPPB, while exhibiting little sensitivity to recognized blockers of Cl vol . The data support the possibility that opening of calcium-activated chloride channels under hypotonic conditions in respiratory smooth muscle may ultimately lead to VDCC-mediated calcium entry and contraction.

The vasorelaxant effect of the lectin of Pisum arvense (PAL) seeds was investigated in rat aorta. PAL (10-100 µg/ml) was applied on aorta rings, with or without endothelium, pre-contracted with phenylephrine (Phe; 0.1 µM). Participation of endothelium derived relaxant factors was evaluated incubating the tissue with indomethacin (10 µM), L-nitro arginine methyl ester (L-NAME, 100 µM) and tetraethylammonium (TEA, 5 mM) before addition of PAL. The role of the lectin domain was investigated by addition of PAL into tissue in presence of glucose (3x 10⁻⁵ M), or N-acetyl Dglucosamine (GlcNAc; 3 x 10⁻⁴ M). The importance of extracellular calcium (Ca²⁺e) or interaction with muscarinic receptors in the relaxant effect was evaluated by addition of PAL into aorta rings containing calcium free solution (OCa) and atropine (1 µ M), respectively. PAL induced concentration-dependent relaxation in endothelized aorta (IC50 =58.38 ± 1.87 µg/ml), which was reversed by L-NAME and glucose. The lectin effect was totally inhibited when the preparation was inserted in OCa, but not in presence of atropine. Summarizing, our data showed a relaxant effect of PAL in isolated rat aorta rings in presence of endothelium, suggestive of interaction between the lectin carbohydrate binding sites with specific receptors located in vascular endothelial cells leading to nitric oxide synthase activation. This effect seems to require Ca²⁺e but is independent on muscarinic receptors interaction.