CALCIUM-DEPENDENT NITRIC-OXIDE SYNTHASE ACTIVITY IN RAT THYMOCYTES

We examined the conversion of L-[H-3]arginine to L-[H-3] 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 -[H-3]citrulline formation by about 97%. The L-[H-3]citrulline formati on was completely inhibited by the NO synthase inhibitors, N-G-nitro-L -arginine and N-G-monomethyl-1-arsnine, with values for IC50 of 1.2 mu M and 19.4 mu M, respectively. In intact thymocytes, the L-[H-3]-citr ulline formation was dependent on the intracellular Ca2+ ([Ca2+](i)) c oncentration. Increasing the extracellular free-Ca2+ concentration up to 1.5 mM, was accompanied by an increase in [Ca2+](i) inside the thym ocytes and there was a parallel increase in the intracellular L-[H-3]c itrulline formation, which reached a maximal value of 371.2 nM of [Ca2 +];. Addition of N-G-nitro-L-arsnine to the medium, completely inhibit ed the formation of L-[H-3]citrulline. The immunolabeling study reveal ed that 15% of the thymocytes isolated from rat thymus constitutively expressed the endothelial isoform of NO synthase. (C) 1998 Academic Pr ess.