Effect of BaP1, a metalloproteinase isolated from Bothrops asper snake venom, on the release of NO by elicited macrophages.

¹Fernandes,C.M; ¹Zuliani, JP; ²Leanza, EC; ²Bevilacqua, E; ³Gutiérrez, JM; ¹Teixeira; CFP.

1 Lab. Farmacologia, Instituto Butantan, Brazil, 2 Departamento de Histologia – ICB USP, 3 Instituto Clodomiro Picado, Universidad de Costa Rica, Costa Rica.

Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms and are involved in local and systemic toxic effects of these venoms. BaP1, a 22,7 kDa metalloproteinase with weak hemorrhagic activity, was purified from the venom of Bothrops asper. In this study, we evaluated the effects of BaP1 in the release of nitric oxide (NO) by peritoneal macrophages (Mfs). Mfs of swiss male mice were obtained 96h after intraperitoneal injection of thioglycollate and incubated with various concentrations of BaP1 (6.25 – 200mg/ml) or RPMI (control). Viability of Mfs was assayed by Trypan Blue. NO production and mRNA expression of iNOS was measured by Griess Method and RT-PCR, respectively. BaP1 was not citotoxic to elicited Mfs up to 3h. This metalloproteinase (6,25 – 50mg/ml) significantly increased the release of NO from Mfs, maximum at concentration of 12.5mg/ml (16 ± 1,3; control: 7 ± 1 mMNO^-₂). This effect was inhibited in the presence of 1 mM L-NAME and aminoguanidine and abrogated by incubation of BaP1 with 2mM EDTA. BaP1 increased mRNA expression of iNOS (1,2 UD; control: 0,45 UD) 1h after incubation. These findings demonstrate the ability of BaP1 to activate elicited Mfs to produce NO. This effect depends on the activity of inducible NO synthase, and  BaP1-induced increase on this enzyme gene expression appears to be the primary mechanism. In addition, the enzymatic activity of BaP1 is relevant for induction of NO release.

Financial Support: FAPESP (03/08529-9); CNPq

Correspondence to: cristinaf@butantan.gov.br