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Volume 16, Number 21, Issue of November 1, 1996 pp. 6975-6986
Copyright ©1996 Society for NeuroscienceCharacterization of Antisera Specific to NK1, NK2, and NK3 Neurokinin Receptors and their Utilization to Localize Receptors in the Rat Gastrointestinal Tract
Received March 18, 1996; revised July 12, 1996; accepted Aug. 16, 1996.
Eileen F. Grady1, Peter Baluk2, 5, Stephan Böhm1, Patrick D. Gamp1, Helen Wong6, Donald G. Payan3, John Ansel7, Andrea L. Portbury8, John B. Furness8, Donald M. McDonald2, 5, and Nigel W. Bunnett1, 4 Departments of 1 Surgery, 2 Anatomy, 3 Medicine, and 4 Physiology, and the 5 Cardiovascular Research Institute, University of California, San Francisco, California 94143; 6 CURE VA/University of California Los Angeles Gastroenteric Biology Center, Los Angeles, California 90095; 7 Department of Dermatology, Emory University, Atlanta, Georgia 30322; and 8 Department of Anatomy and Cell Biology, University of Melbourne, Parkville Victoria 3052, Australia
Understanding the physiological role of tachykinins requires precise cellular and subcellular localization of their receptors. We raised antisera by immunizing rabbits with peptides corresponding to portions of the intracellular tails of the rat neurokinin 1, 2, and 3 receptors (NK1-R, NK2-R, NK3-R). Receptors were localized by immunofluorescence and confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the plasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the peptides used for immunization. Nontransfected cells were unstained. Each antiserum only stained cells transfected with the appropriate receptor and did not stain cells transfected with the other receptors. Therefore, the antisera are specific and do not cross-react with other neurokinin receptors. We examined the distribution of the neurokinin receptors in the gastrointestinal tract of the rat. NK1-R was detected in myenteric and submucosal neurons and in interstitial cells of Cajal. NK2-R was localized to circular and longitudinal muscle cells and to nerve endings in the plexuses. NK3-R was detected in numerous myenteric and submucosal neurons. Some neurons expressed both NK1-R and NK3-R. Receptors were detected at the plasma membrane and in endosomes. Cells expressing the receptors were closely associated with tachykinin-containing nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by tachykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate the effects of tachykinins on interstitial and smooth muscle cells, respectively.
Key words: tachykinins; neurokinin receptors; receptor antisera; enteric neurons; interstitial cells of Cajal; smooth muscle
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