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

Abstract

The calcium ion dependence of soluble and particulate nitric oxyde synthase (NOS) activity from
Viviparus ater immunocytes was investigated. At a calcium ion concentration of 2 nM, the NOS activity measured by citrulline formation was 27.1 ± 2.2 and 9.3 ± 0.8 pmol/min/106 cell for soluble and particulate NOS, respectively. The increase in free calcium ion concentration to 300 nM increases enzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 pmol/min/106 cell, respectively. The 50 % activation of the calcium-dependent activity is 91 and 97 nM Ca2+ for soluble and particulate enzymes. Trifluoperazine, an inhibitor of the calmodulin-dependent enzyme, partially inhibits both activities. Soluble NOS is five times more sensitive than particulate NOS. The behaviour of both activities with three NOS inhibitors (7-nitroindazole, S-methylisothiourea sulphate, diphenyleneiodonium) is very similar, with IC50 values that are not significantly different. 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 nitric oxyde in V. ater immunocytes not only has a defensive role but also signalling relevance in crosstalking between immunocytes and other cells.