Vol. 293, Issue 2, 559-568, May 2000

Neurokinin₃ Receptors Couple to the Activation of Neuronal Nitric-Oxide Synthase in Stably Transfected Chinese Hamster Ovary Cells¹

David R. Linden, Melissa J. Chell, Esam E. El-Fakahany and Virginia S. Seybold

Department of Neuroscience (D.R.L., V.S.S.) and Division of Neuroscience Research in Psychiatry (M.J.C., E.E.E.), University of Minnesota, Minneapolis, Minnesota

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.