CHARACTERIZATION OF ANTISERA SPECIFIC TO NK1, NK2, AND NK3 NEUROKININRECEPTORS AND THEIR UTILIZATION TO LOCALIZE RECEPTORS IN THE RAT GASTROINTESTINAL-TRACT

Understanding the physiological role of tachykinins requires precise c ellular and subcellular localization of their receptors. We raised ant isera 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 a nd confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the p lasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the pept ides 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. Therefo re, the antisera are specific and do not cross-react with other neurok inin receptors. We examined the distribution of the neurokinin recepto rs in the gastrointestinal tract of the rat. NK1-R was detected in mye nteric 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. Rece ptors were detected at the plasma membrane and in endosomes. Cells exp ressing the receptors were closely associated with tachykinin-containi ng nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by ta chykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate t he effects of tachykinins on interstitial and smooth muscle cells, res pectively.