Nitric oxide synthase activity in molluscan hemocytes

The hemocytes of the freshwater snail Viviparus ater have nitric oxide synthase (NOS) activity, as demonstrated by [H-3]citrulline and nitrite + nitrate formation. The enzyme is NADPH dependent and is competitively inhibited by the mammalian NOS inhibitor N-G-monomethyl-L-arginine (K-i = 4.7 mu M). The K-m for L-arginine is 2.5 mu M. 70% of the total activity is observed at very low free Ca2+ concentration (3 nM). LPS treatment increased total NOS activity 2.4 fold. The activity is partly present in the non-soluble fraction of hemocytes (24% and 8% in non-stimulated and LPS-stimulated snails, respectively). An antiserum to the C-terminal synthetic pentadecapeptide of the rat cerebellar NOS inhibited the enzyme activity in a concentration-dependent manner. This is the first biochemical demonstration of the existance of NOS activity in molluscan hemocytes, the cells responsible for defence mechanisms.