Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization to localize receptors in the rat gastrointestinal tract

Abstract

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.

Fig. 1.

Confocal photomicrographs showing localization of NK1-R, NK2-R, and NK3-R in cell lines. The top horizontal panels show KNRK-NK1-R cells, the second horizontal panels show CHO-NK2-R cells, and the third horizontal panels show KNRK-NK3-R cells. The bottom horizontal panels show transfected cell lines expressing NK1-R (left), NK2-R (middle), and NK3-R (right). Cells were incubated with NK1-R antiserum (#94168, left vertical panels), NK2-R antiserum (#94179,middle vertical panels), and NK3-R antiserum (#94192,right vertical panels), followed by Texas Red or FITC-conjugated secondary antibodies. The bottom horizontal panels show transfected cells that were incubated with antisera preabsorbed with the peptides used for immunization. Thearrows indicate that the antisera stain the plasma membrane of cells expressing the appropriate receptor and do not cross-react with cells expressing the other receptors. Thearrows in the bottom horizontal panelsindicate that staining was abolished by preabsorption with the receptor fragment used for immunization. Scale bar, 20 μm.

Fig. 2.

Western blot analysis usingNK1-R antiserum (#94168), NK2-R antiserum (#94179), and NK3-R antiserum (#94192). Each lane contains 10 μg of protein. Lane 1, Transfected cells expressing NK1-R, NK2-R, or NK3-R. Lane 2, Transfected cells expressing NK1-R, NK2-R, or NK3-R, with antisera preabsorbed with receptor fragment. Lane 3, Nontransfected cells. Thearrows indicate the major bands that were detected in transfected cells.

Fig. 3.

Confocal photomicrographs showing localization of NK1-R (A–E) and NK3-R (F) in tissue sections and whole mounts. Tissues were incubated with NK1-R antiserum #94168 or NK3-R antiserum AP951.A, Section of antrum, showing localization of NK1-R in myenteric neurons. B, Section of jejunum, showing localization of NK1-R in myenteric neurons and interstitial cells of Cajal. C, Whole mount of the myenteric plexus of the ileum. D, Whole mount of interstitial cells of Cajal of the ileum. The arrows indicate prominent staining of the plasma membrane and endosomes. E, The sum of five optical sections of jejunum, collected at 0.36 μm intervals, showing localization of NK1-R in myenteric neurons and in interstitial cells of Cajal. F, An adjacent section to E (8 μm apart), the sum of five optical sections showing localization of NK3-R. The same neurons expressing the NK1-R express the NK3-R, whereas the interstitial cells are unstained.B–E show NK1-R-immunoreactive endosomes.mp, Myenteric plexus; cm, circular muscle. Scale bar (shown in F):A–D, 20 μm; E,F, 13 μm.

Fig. 4.

Confocal photomicrographs showing localization of NK2-R in tissue sections and whole mounts. Tissues were incubated with NK2-R antiserum #94179. A, Section of circular and longitudinal muscle layers of the duodenum. B, Serial section from A in which the antiserum is preabsorbed with 1 μm of the receptor fragment that was used for immunization.C, Section of circular and longitudinal muscle layers of the fundus. D, Section of circular muscle layer of the colon. E, Section of muscularis mucosa and circular muscle layer of the colon. The muscularis mucosa is identified byarrows. F, The sum of 10 optical sections, collected at 0.54 μm intervals, of a whole mount of the submucosal plexus of the ileum showing a stained nerve ending.G, Section of circular muscle of the fundus. Thearrows indicate prominent staining of the plasma membrane. The arrowhead indicates endosomes.H, Image of the same optical section shown inG, showing staining with Cell Tracker CM-DiI to outline the cell surface. mp, Myenteric plexus;cm, circular muscle; lm, longitudinal muscle; mm, muscularis mucosa. Scale bar (shown inH): A–E, 20 μm;F, 10 μm; G, H, 5 μm.

Fig. 5.

Confocal photomicrographs showing localization of NK3-R in tissue sections and whole mounts. Tissues were incubated with NK3-R antiserum #94192 in A,B, D, and E, or AP951 inC, F, G, andH. A, Section of duodenum, showing localization of NK3-R in myenteric neurons. B, Serial section from A in which the antiserum is preabsorbed with 1 μm of the receptor fragment that was used for immunization. C, Section of fundus, showing localization of NK3-R in myenteric neurons. D, Section of jejunum, showing localization of NK3-R in myenteric neurons. E, Section of ileum, showing localization of NK3-R in submucosal neurons.F, Section of colon, showing localization of NK3-R in myenteric neurons. G, Section of colon, showing localization of NK3-R in submucosal neurons. H, Whole mount of the myenteric plexus of the ileum showing NK3-R at the plasma membrane and in endosomes. The arrows indicate prominent staining of the plasma membrane and endosomes. mp, Myenteric plexus; sp, submucosal plexus;cm, circular muscle; lm, longitudinal muscle; muc, mucosa. Scale bar (shown inH), 20 μm.

Fig. 6.

Confocal photomicrographs showing localization of neurokinin receptors (red, arrows) and tachykinins (green, arrowheads).A–C, NK1-R and tachykinins in whole mounts of the ileal myenteric plexus (A,B) and interstitial cells of Cajal (C). D, E, NK2-R and tachykinins in the gastric antrum. F,G, NK3-R and tachykinins in whole mounts of the ileal myenteric plexus. Cells expressing the neurokinin receptors (arrows) are closely associated with tachykinin containing nerve fibers (arrowheads). Not all neurons within a plexus express a given neurokinin receptor (asterisks), but all neurons are surrounded by tachykinin-containing fibers. Scale bar (shown inF): A, C,E–G, 20 μm; B, 10 μm;D, 30 μm.