Calcium-dependent nitric oxide synthase activity in rat thymocytes.
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 IC50 of 1.2 microM and 19.4 microM, 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+]i inside 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.
Cruz
M T
MT
Faculdade de Farmácia, Universidade de Coimbra, Portugal.
Carmo
A
A
Carvalho
A P
AP
Lopes
M C
MC
D000818:Animals; D001120:Arginine / Q000378:metabolism; D018801:CD2 Antigens / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019335:Nitroarginine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D013950:Thymus Gland / Q000166:cytology* / Q000201:enzymology / Q000276:immunology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D018801:CD2 Antigens; D019335:Nitroarginine; D019323:omega-N-Methylarginine; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D002118:Calcium