Abstract
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 ofl-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 ofl-[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.
Footnotes
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Send reprint requests to: Dr. Virginia S. Seybold, Department of Neuroscience, 6-145 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455. E-mail: ginger{at}med.umn.edu
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↵1 This work was supported by grants from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (Grants NS17702 to V.S.S. and NS25743 to E.E.E.). D.R.L. was supported by a grant from the National Institute on Drug Abuse, National Institutes of Health (Grant T32-DA07234).
- Abbreviations:
- NK
- neurokinin
- NO
- nitric oxide
- nNOS
- neuronal NO synthase
- CHO
- Chinese hamster ovary
- RFL
- rat fetal lung
- IP3
- inositol-1,4,5-trisphosphate
- BAPTA-AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- cPTIO
- 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
- PKC
- protein kinase C
- Received October 26, 1999.
- Accepted January 17, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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