Volume 117, Issue 5, Pages 1136-1146 (November 1999) Expression of cholecystokinin a receptors in neurons innervating the rat stomach and intestine  Catia Sternini, Helen Wong, Thong Pham, Roberto de Giorgio, Laurence J. Miller, Susan M. Kuntz, Joseph R. Reeve, John H. Walsh, Helen E. Raybould  Gastroenterology  Volume 117, Issue 5, Pages 1136-1146 (November 1999) DOI: 10.1016/S0016-5085(99)70399-9 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Structure of the CCK-A receptor and the sequence chosen for antibody production. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Western blot of membranes prepared from CHO–CCK-A receptor–transfected cells (CHO-CCKR) and from non–receptor-expressing parent CHO cells (CHO) incubated with CCK-A receptor affinity-purified antiserum (1:2000). The CCK-A receptor antiserum labels a broad band at approximately 85–95 kilodaltons in CCK-A receptor–expressing CHO cells but not in parent CHO cells not expressing the receptor. Molecular size markers (Mr × 10−3) are shown on the left. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Confocal images of (A and B) CHO–CCK-A receptor–transfected cells and (C and D) nontransfected CHO cells incubated with the CCK-A receptor antiserum. Note the punctate staining confined to the cell surface (A, arrow) in transfected cells and the lack of immunostaining in nontransfected cells (C and D). B and D show cells exposed to CCK stimulation. CCK-A receptor staining is concentrated in a perinuclear position (B, arrow), indicating that the receptors were internalized. Bar = 20 μm. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 CCK-A receptor immunoreactivity in the (A) mucosa and (C) myenteric plexus of the stomach. CCK-A receptor–immunoreactive fibers innervating the gastric mucosa (A) run parallel to the epithelial cells from the base of the mucosa to the luminal aspect. In the myenteric plexus (C), CCK-A receptor immunoreactivity is localized to myenteric neurons and to thin, beaded fibers (arrows). (B and D) Lack of CCK-A receptor immunostaining in the mucosa and myenteric plexus of sections of the stomach incubated with the CCK-A receptor antibody preadsorbed with 10 μmol/L synthetic peptide. Bars = 50 μm; lm, longitudinal muscle; mp, myenteric plexus; cm, circular muscle. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 CCK-A receptor immunoreactivity in (A) a Dogiel type I neuron and (B) varicose fibers forming a dense network surrounding unstained cell bodies in the myenteric plexus of the ileum. (C) CCK-A receptor–immunoreactive fibers in the circular muscle layer of the proximal duodenum. Bar = 100 μm. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Confocal microscopic images of CCK-A receptor immunoreactivity in varicose fibers in the (A) myenteric plexus and (B) interconnecting strand of the ileum. *(A) Myenteric neuron encircled by CCK-A receptor–immunoreactive beaded processes. Bar = 50 μm; mp, myenteric plexus; is, interconnecting strand. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Confocal microscopic images showing the cytoplasmic localization of CCK-A receptor immunoreactivity in myenteric neurons of the (A and B) stomach and (C and D) ileum. Confocal images were taken near the cell surface (A and C) and through the center of the cell (B and D). Bars = 25 μm; cm, circular muscle. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 CCK-A receptor–immunoreactive fibers innervating the gastric mucosa from a (A) control rat and (B) vagotomized rat. Note the marked decrease in fiber density in the vagotomized rat. Bar = 50 μm. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Confocal microscopic images showing the CCK-A receptor (green) and VIP (red) immunoreactivities and their colocalization (orange) in a myenteric neuron and in fibers in the mucosa of the stomach. Bars = 25 μm; mp, myenteric plexus; m, mucosa. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 10 Colocalization of CCK-A receptor and SP immunoreactivities in neuron and varicose processes (arrows) of the myenteric plexus of the stomach. Light microscopic images. Bar = 100 μm; mp, myenteric plexus; lm, longitudinal muscle; cm, circular muscle. Gastroenterology 1999 117, 1136-1146DOI: (10.1016/S0016-5085(99)70399-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions