Neurokinin(3) receptors couple to the activation of neuronal nitric-oxide synthase in stably transfected Chinese hamster ovary cells

Several physiological effects induced by activation of neurokinin(3) (NK3) receptors are mediated by the production of nitric oxide (NO). We investiga ted the intracellular coupling of NK3 receptors to NO synthase (NOS) using a Chinese hamster ovary cell line that was stably transfected with both the NK3 receptor and type I (neuronal) NOS. NOS activity in the transfected ce ll line was assayed directly, by measuring the formation of L-citrulline, a nother product of NOS, as well as indirectly, by measuring the production o f cGMP in cultured rat fetal lung fibroblasts (RFL-6 cells). MePhe(7)-neuro kinin B (NKB) stimulation of L-[H-3]citrulline production was concentration -dependent and yielded a two-site model for the concentration-response rela tionship. The production of L- citrulline in response to two other tachykin ins, substance P or neurokinin A, revealed only a one-site nature of the re sponse. The production of cGMP in response to MePhe(7)-NKB had an EC50 valu e that corresponded to the high-potency component of MePhe(7)-NKB-induced p roduction of L-[H-3]citrulline. Agonist-induced calcium signaling was also concentration-dependent, and the acute increase in the production of cGMP b y MePhe(7)-NKB (0.1 nM) was dependent on the release of calcium from intrac ellular stores. Results of this study provide the first direct evidence tha t NK3 receptors couple to the generation of NO within the same cell.