Calcium-Dependent Nitric Oxide Synthase Activity in Rat Thymocytes

Abstract

We examined the conversion of L-[3H]arginine to L-[3H]citrulline in lysate from rat thymocytes, which was dependent on Ca2+and cofactors (FAD, BH4, NADPH). Removal of Ca2+of the medium, reduced the total L-[3H]citrulline formation by about 97%. The L-[3H]citrulline formation was completely inhibited by the NO synthase inhibitors, NG-nitro-L-arginine and NG-monomethyl-L-arginine, with values for IC50of 1.2 [mu]M and 19.4 [mu]M, respectively. In intact thymocytes, the L-[3H]citrulline formation was dependent on the intracellular Ca2+([Ca2+]i) concentration. Increasing the extracellular free-Ca2+concentration up to 1.5 mM, was accompanied by an increase in [Ca2+]iinside the thymocytes and there was a parallel increase in the intracellular L-[3H]citrulline formation, which reached a maximal value of 371.2 nM of [Ca2+]i. Addition of NG-nitro-L-arginine to the medium, completely inhibited the formation of L-[3H]citrulline. The immunolabeling study revealed that 15% of the thymocytes isolated from rat thymus constitutively expressed the endothelial isoform of NO synthase.