PARENT SESSION
4K Cyanobacterial toxins: health and environmental hazards
9:00 AM to 7:00 PM, Wednesday, 09 May 2001

(W/EH110) In vitro effects of microcystins on gill Na⁺, K⁺-ATPase of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae).

Vinagre, Tânia¹, Yunes, J.¹, Bianchini, Adalto¹, Monserrat, José¹, ¹

ABSTRACT- Microcystins are inhibitors of PP1 and PP2A phosphatases. Recent evidences suggest that microcystins can inhibit enzymes involved in osmoregulation of aquatic animals, such as Na⁺, K⁺-ATPase (NKA). The enzymatic cycle includes a phosphorylation and a dephosphorylation step. The latter is usually measured by the phosphatase K⁺ dependent (PKD) activity. In this study we evaluated the in vitro effects of an aqueous extract of the cyanobacteria Microcystis aeruginosa on the posterior gills NKA and PKD activities of the hyperosmoregulating crab Chasmagnathus granulata. Forty seven crabs were employed (12.26±0.52 g). NKA and FKD activity was determined by measuring, respectively, the phosphorus released from ATP (4mM) and the p-nitrophenyl phosphate (5mM) hydrolisis, in both presence and absence of ouabain (1mM). Gill homogenates were incubated with different microcystin concentrations (determined by ELISA), for 1h. IC₅₀ values were estimated by probit analysis. IC₅₀ (95% confidence intervals) for the different enzymes was estimated as follows: (a) NKA = 0.46 g/L (0.40-0.52 g/L); (b) total ATPase activity (Mg²⁺dependent) = 0.92 g/L (0.78-1.10 g/L); (c) FKD = 1.31 g/l (1.14-1.51 g/L); and (d) total phosphatase activity (Mg²⁺dependent) = 1.68 g/L (1.44-1.99 g/L). Considering that NKA is phosphorylated in an aspartate residue, our results suggest that microcystins are also aspartate phosphatases inhibitors. The difference in IC₅₀ values between NKA and total ATPase can be explained considering the existence of other ATPases which could also be inhibited by microcystin. The similar IC₅₀ estimated for FKD and total phosphatase activity suggests that FKD is the main phosphatase in gills. In vitro results suggest that microcystin can also have a possible in vivo effect on crab osmoregulation.

Key words: Na⁺, K⁺-ATPase , phosphatase K⁺ dependent , microcystin, osmoregulation