Neurokinin₃ receptors couple to the activation of neuronal nitric-oxide synthase in stably transfected Chinese hamster ovary cells

Several physiological effects induced by activation of neurokinin₃ (NK₃) receptors are mediated by the production of nitric oxide (NO). We investigated 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 cell line was assayed directly, by measuring the formation of L- citrulline, another product of NOS, as well as indirectly, by measuring the production of cGMP in cultured rat fetal lung fibroblasts (RFL-6 cells). MePhe⁷-neurokinin B (NKB) stimulation of L-[³H]citrulline production was concentration-dependent and yielded a two-site model for the concentration- response relationship. The production of L-citrulline in response to two other tachykinins, substance P or neurokinin A, revealed only a one-site nature of the response. The production of cGMP in response to MePhe⁷-NKB had an EC₅₀ value that corresponded to the high-potency component of MePhe⁷- NKB-induced production of L-[³H]citrulline. Agonist-induced calcium signaling was also concentration-dependent, and the acute increase in the production of cGMP by MePhe⁷-NKB (0.1 nM) was dependent on the release of calcium from intracellular stores. Results of this study provide the first direct evidence that NK₃ receptors couple to the generation of NO within the same cell.