Two-way analysis of variance (two-way ANOVA) with Bonferroni post-hoc test was used to compare the concentration-response curves. One-way ANOVA with Dunnett’s Multiple Comparison post-hoc test was used for single-concentration venom assays and Western Blot experiments. The level of significance was set at P < 0.05 and statistical analysis was performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). Lasiodora sp. venom (0.06-64 μg/ml) induced a concentration-dependent relaxation in aortic rings containing a functional endothelium pre-contracted with phenylephrine ( Fig. 1A). The IC50 value

for Lasiodora sp. venom was 6.6 ± 1.8 μg/ml (n = 5). The effect observed at the maximum venom concentration was 88.9 ± 2.4% relaxation. To investigate the possible role of vascular endothelium in the vasorelaxation induced by the venom, a single concentration (8 μg/ml) was used in endothelium denuded aortic rings pre-contracted selleck chemical with phenylephrine. In contrast to the previous result, the venom did not induce any significant relaxant effect in aortic rings without functional endothelium (n = 5, P < 0.01; Fig.

1B). This result showed that the relaxant effect provoked Selleckchem Ibrutinib by the crude venom depends on the presence of a functional endothelium. To investigate the possible role of prostanoids in the relaxant effect induced by the crude venom, the vessels were pre-incubated with indomethacin (10 μM), a nonselective inhibitor of cyclo-oxygenase. Indomethacin was not able to modify the vasorelaxation induced by 8 μg/ml venom (n = 5; Fig. 1B). On the other hand, when the aortic rings were pre-incubated with the NO synthase (NOS) inhibitor L-NAME (300 μM), the vasodilator effect induced by the venom was abolished (n = 5, P < 0.01; Fig. 1B). Rat aortic rings were incubated with 16 μg/ml Lasiodora sp. venom or 0.1 μM acetylcholine (positive control) during different time intervals:

PRKACG 0, 5, 15 and 30 min. We analysed the phosphorylation state of a serine (Ser1177) site of eNOS by Western blot. Results show that Lasiodora sp. venom significantly increased the level of Ser1177 phosphorylation, the activation site of eNOS, after 15 and 30 min of incubation (P < 0.05; Fig. 2). Acetylcholine (0.1 μM, positive control) stimulated Ser1177-eNOS phosphorylation at 5, 15 and 30 min. The expression of total eNOS was not altered after both treatments ( Fig. 2). After venom filtration using Vivaspin centrifugal tubes, pharmacological screenings in aortic rings showed that the filtrate from 3 kDa cutoff tubes concentrated the vasoactive fraction (data not shown). Subsequently, the filtrate from 3 kDa tube was fractionated by reversed-phase chromatography (Fig. 3A). All fractions derived from the first step on HPLC were tested in isolated rat aorta and the results revealed that only fraction 2 (Fig. 3A, black arrow) induced relaxation. Additionally, UV spectra showed that fraction 2 had absorbance peaks at 214 and 254 nm (data not shown).