Calcium/calmodulin dependence of nitric oxide synthase from Viviparus aeter immunocytes

The calcium ion dependence of soluble and particulate NOS activity from Viviparus ater hemocytes has been investigated. At 2 nM calcium ion concentration the NOS activity measured by citrulline formation is 27.1 ± 2.2 and 9.3 ± 0.8 pmol.min-1.106cell-1 for soluble and particulate NOS respectively. The increase of free calcium ion concentration up to 300 nM increases the enzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 respectively. The 50% activation of the calcium dependent activity is 91 and 97 nM Ca2+ for soluble and particulate enzyme. Trifluoperazine, an inhibitor of calmodulin dependent enzyme, partially inhibits both activities. The soluble NOS is five time more sensible than particulate NOS. The behaviour of both activities to three NOS inhibitors (7-nitroindazole, S-methylisothiourea sulfate, diphenyleneiodonium) is very similar with not significant differences in IC50 values. The calcium ion dependence of NOS activities in a range of free calcium ion variations which are transiently observed in receptor stimulated cells suggests that NO in V. ater hemocytes has not only a defensive role but also a signalling relevance in cross-talking between hemocytes and other cells.