Immunohistochemical demonstration of the NK1 tachykinin receptor on muscle and epithelia in guinea pig intestine  Bridget R. Southwell, John B. Furness  Gastroenterology  Volume 120, Issue 5, Pages 1140-1151 (April 2001) DOI: 10.1053/gast.2001.23251 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 NK1-receptor IR is present on muscle. Distribution of NK1-receptor IR on muscle in rectum (A and B), distal colon (C and D), ileum (E and F), and duodenum (G). (A) Transverse section of rectum. NK1-receptor IR is visible on neurons in a myenteric ganglion (arrow). Spots of IR are also visible in the circular muscle in line with the muscle cells and as circles at the surfaces of longitudinal muscle cells. Single optical section, 1 μm thick. (B) NK1-receptor IR is present on the surface of longitudinal muscle cells in rectum (tissue incubated without SP and with Nic and TTX to inhibit the endogenous release of TKs). (C and D) Transverse section of distal colon incubated without SP. Single optical section (0.5 μm) showing NK1-receptor IR on surface of (C) circular muscle cells and (D) longitudinal muscle cells. (E) NK1-receptor IR in whole mount of circular muscle from ileum incubated without SP. Receptor outlines the circular muscle cells. (F) NK1-receptor IR on the surface of circular muscle cells in longitudinal section of ileum incubated without SP. (G) NK1-receptor IR on the surface of circular muscle cells in longitudinal section of duodenum incubated without SP. Scale bar: A = 100 μm; B = 20 μm; C–G = 10 μm. Gastroenterology 2001 120, 1140-1151DOI: (10.1053/gast.2001.23251) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 NK1 receptor on muscle is mobilized by SP. NK1 receptor on circular muscle in longitudinal sections of rectum. (A) Incubated without SP and with Nic and TTX to inhibit the endogenous release of TKs. NK1-receptor IR is located on the surfaces of circular muscle cells. (B) Incubated with 10−7 mol/L SP. NK1-receptor IR is clumped and the shape of the circular muscle cells is difficult to distinguish. (C) Incubated with 10−7 mol/L SP and 10−6 mol/L CP99994 (NK1-receptor antagonist). The NK1-receptor IR is on the surface of the muscle cells. Single optical section 0.5-μm thick. Scale bar = 10 μm. Gastroenterology 2001 120, 1140-1151DOI: (10.1053/gast.2001.23251) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Binding and uptake of Cy3 SP and distribution of NK1 receptor. Transverse section of duodenum (A and B) and rectum (C and D) incubated with Cy3 SP and warmed for 10 minutes. (A) A single optical section (0.5 μm) showing NK1-receptor IR on circular muscle (CM) cells and on neurons in the myenteric ganglion (MG). Receptor is present on surface and in cytoplasm of neurons and in the cytoplasm of muscle cells. (B) Cy3 SP in same optical section. Cy3 SP is taken up by neurons and is present in the cytoplasm of the circular muscle cells. Bright cells are red blood cells (nonspecific fluorescence). Scale = 20 μm. (C) NK1 receptor and (D) Cy3 SP in the same optical section (0.5 μm) of rectum incubated with 10−6 mol/L Cy3 SP. Boxes and arrows mark identical positions on each image. LM, longitudinal muscle. Scale = 10 μm. Gastroenterology 2001 120, 1140-1151DOI: (10.1053/gast.2001.23251) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Distribution of TK IR in nerve fibers and NK1-receptor IR in external muscle of the duodenum and colon. (A and B) Transverse section of duodenum incubated without SP (-SP). (A) A single optical section showing TK IR in nerve fibers in a myenteric ganglion (MG) and in nerve bundles in the circular muscle (CM). (B) NK1-receptor IR in the same optical section. NK1 receptor is present as speckles on the circular muscle cells and is bright on the ICC in the deep muscular plexus (DMP) and on epithelial cells in Brunner's glands (BG). (C and D) Transverse section of distal colon incubated without SP. (C) A single optical section showing TK IR in nerve fibers in a myenteric ganglion, and in nerve bundles in the circular and longitudinal (LM) muscle. (C) NK1-receptor IR in the same optical section. NK1 receptor is present on the surfaces of the longitudinal and circular muscle cells and on neurons in the myenteric ganglion. Optical sections taken with confocal microscope, all 0.5-μm thick. Scale bar: A, C = 20 μm. Gastroenterology 2001 120, 1140-1151DOI: (10.1053/gast.2001.23251) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 NK1-receptor distribution on mucosal cells in small intestine. (A) Transverse section of ileum incubated without SP. NK1-receptor IR is present on the surfaces of cells at the base of the crypts (long arrows) and on some cells at the lumenal surface of the mucosa (short arrow). MUC, mucosa; SMG, submucosal ganglia. (B and C) Transverse section of ileum incubated with Cy3 SP and warmed for 10 minutes. (B) NK1-receptor IR and (C) Cy3 SP are colocalized on the cell surface and in the cytoplasm of the crypt epithelial cells. Arrow indicates same location in both micrographs. (D and E) Whole mount of ileum incubated with Cy3 SP and warmed for 10 minutes. (D) NK1-receptor IR and (E) Cy3 SP are present on the surface of neurons in the submucosal ganglia (SMG) and on epithelial cells in the adjacent Brunner's gland (BG, arrows). The bright oval cells in C and E are red blood cells (nonspecific fluorescence). Scale bar: A = 50 μm; B, D = 20 μm. Gastroenterology 2001 120, 1140-1151DOI: (10.1053/gast.2001.23251) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 NK1-receptor distribution in the mucosa of large intestine. (A) Transverse section of distal colon incubated without SP. NK1-receptor IR is present on epithelial cells lining the glands (arrows). MUC, mucosa. (B–D) Transverse section of proximal colon incubated without SP. (B) There is very strong NK1-receptor IR on cells at the bases of glands (arrow). The muscularis mucosa (MM) is negative. (C) A single optical section at higher magnification showing lines of NK1-receptor IR in the cytoplasm of the gland cells. (D) The same optical section showing TK IR in nerve fibers (arrow) adjacent to the gland cells. Arrow marks the same location on C and D. (E) Higher magnification of gland cells from proximal colon incubated with SP. The bright NK1-receptor IR is granular and located in bands in the cytoplasm. Arrows indicate the cell membranes. Optical sections taken with confocal microscope. Scale bar: A, C = 20 μm; B = 100 μm; E = 10 μm. Gastroenterology 2001 120, 1140-1151DOI: (10.1053/gast.2001.23251) Copyright © 2001 American Gastroenterological Association Terms and Conditions