Volume 130, Issue 1, Pages 150-164 (January 2006) GLP-2 Receptor Localizes to Enteric Neurons and Endocrine Cells Expressing Vasoactive Peptides and Mediates Increased Blood Flow  Xinfu Guan, Heidi E. Karpen, John Stephens, John T. Bukowski, Sanyong Niu, Guangcheng Zhang, Barbara Stoll, Milton J. Finegold, Jens J. Holst, Darry L. Hadsell, Buford L. Nichols, Douglas G. Burrin  Gastroenterology  Volume 130, Issue 1, Pages 150-164 (January 2006) DOI: 10.1053/j.gastro.2005.11.005 Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 1 Expression of GLP-2R mRNA determined by real-time qRT-PCR of laser capture microdissected porcine jejunal tissue samples. Panel A: Morphologically defined villus epithelium (a–d) and myenteric plexus (e–h) were procured by laser capture microdissection (LCM) from frozen jejunum tissue sections. Shown are representative images of before LCM (a and e), after laser shot (b and f), tissue residue after LCM (c and g), and captured subtissue (d and h). On panel B is 1 representative result of pGLP-2R mRNA expression by real-time qRT-PCR from 3 experiments, in which 50 ng total RNA from intact jejunum extract and a much lower, undetectable amount of total RNA from the LCM samples (water-negative control) were used in each reaction, indicating that the GLP-2R mRNA was expressed in both villus epithelium and myenteric plexus because their threshold cycles (Ct) were significantly different from the negative control value. From the PCR reaction tubes, 5% of the intact jejunum and 100% of the LCM (or water) of the amplicons were loaded, run, and visualized by 3% agarose gel electrophoresis (on panel D), appearing at a predicted size (76 bp). Note that the primers and probe for the porcine glp2r gene specifically recognized the mRNA but not the genomic DNA (panel C); no band appeared in the porcine intestinal RNA sample when either the reverse transcription (RT) was omitted or in the porcine genomic DNA sample. Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 2 Expression of GLP-2R mRNA was visualized in paraffin sections by fluorescence in situ hybridization with tyramide signal amplification using in vitro reverse transcription-generated pGLP-2R cRNA probes (A). The mRNA of GLP-2R was expressed in villus epithelium and submucosal/myenteric plexus of the porcine jejunum. Positive-stained cells (in red, indicated by arrows) are shown in epithelial noncolumnar cells (A, b) and myenteric plexus (A, d), and negative controls are shown in jejunal villi (A, a) and submucosal/myenteric plexus (A, c). Nuclei (DNA) were counterstained by TOPRO-3 (in blue). In addition, expression of GLP-2R mRNA was detected in frozen sections by an alkaline phosphatase-based chromogen reaction using PCR-generated pGLP-2R DNA probes (B). The mRNA of GLP-2R is expressed in distinct epithelial cells (in blue, B, b), submucosal plexus (in blue, B, c), and myenteric plexus (in blue, B, d); the negative control is shown (B, a). Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 3 A human GLP-2R antibody validated by coimmunoprecipitation Western blotting and double immunostaining. Specific bands (at 66 and 73 kilodaltons) were detected by Western blotting (panel A) using a rabbit anti-N-terminal hGLP-2R antibody in either whole cell lysate or coimmunoprecipitated (with anti-c-myc IgG) proteins from hGLP-2R-transfected COS cells but not from nontransfected COS cells. In panel A, lanes 1 and 2 were loaded with cell lysate samples from the nontransfected cells and transfected cells, respectively, and lanes 3 and 4 with coimmunoprecipitated proteins samples from the nontransfected cells and transfected cells, respectively. COS cells transfected with hGLP-2R DNA were positively immunostained by the hGLP-2R antibody (in panel B). With nuclei (DNA) counterstained by TOPRO-3 (in blue), COS cells were double immunostained with mouse anti-c-myc antibody (in red; a, d, g, and j), with the rabbit hGLP-2R antibody (in green; b and k), with the rabbit hGLP-2R antibody but preabsorbed with its control peptide (in green; h), or no hGLP-2R antibody (in green; e). Confocal images (a–c) are from nontransfected cells and confocal images (d–l) from hGLP-2R-transfected cells. Note that the myc-tagged hGLP-2R protein was coimmunostained (in yellow; l) by these 2 antibodies. Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 4 The GLP-2R protein localized to both enteroendocrine cells (EC) and enteric neurons (EN) of the porcine jejunum by double immunostaining. The pGLP-2R protein was expressed in EC scattered in both villus epithelium (A; in yellow, indicated by arrows) and crypt epithelium (E; in yellow, indicated by arrows) and submucosal EN (I; in yellow, indicated by arrows). Note that not all EC or EN (in red, indicated by arrowheads) were GLP-2R immunoreactive. The GLP-2R protein was immunostained by the hGLP-2R antibody (in green; B, F, and J); the EC were labeled by antichromogranin A (Chro A) antibody (in red; C and G); and myenteric EN were labeled by anti-HuC/HuD antibody (in red; K). The pGLP-2R protein was colocalized with the EC-specific marker (Chro A, in yellow; D and H) and the EN-specific marker (HuC/HuD, in yellow; L). Nuclei (DNA) were counterstained by TOPRO-3 (in blue). White squares (in A and E) are enlarged for details (as shown in B–D and F–H, respectively). Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 5 The GLP-2R protein localized to VIP-positive enteric neurons (EN) and eNOS-expressing EN in the whole-mount submucosal preparation of the porcine jejunum by double immunostaining. The GLP-2R protein was immunostained by the hGLP-2R antibody (in green; A, D, and G). The enteric neurons were immunostained by antibodies against a neuron-specific marker (PGP 9.5, in red; B), a specific vasoneurotransmitter (VIP, in red; E), or eNOS protein (eNOS, in red; H). The GLP-2R protein was coexpressed (in yellow, indicated by arrows) with PGP 9.5 (C), VIP (F), and eNOS (I). Note that representative images are shown of enteric neurons within the ganglia of the submucosal plexus of the neonatal porcine jejunum. Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 6 The GLP-2R protein localized to 5-HT-containing enteroendocrine cells (EC) of the human jejunum by double immunostaining. The hGLP-2R protein was expressed in EC (in yellow, indicated by arrows) scattered in villus epithelium (A) and crypt epithelium (E) and expressed in 5-HT-containing EC (I). Note also that not all EC (in red, indicated by arrowheads) were GLP-2R immunoreactive. hGLP-2R protein was immunostained by the hGLP-2R antibody (in green; B, F, and J); the EC were labeled by anti-chromogranin A (Chro A) antibody (in red; C and G); and 5-HT-containing EC were labeled by anti-5-HT antibody (in red; K). hGLP-2R protein was coexpressed with the EC-specific marker (Chro A, in yellow; D and H), specifically with a 5-HT-containing subset of EC (5-HT, in yellow; L). Nuclei (DNA) were counterstained by TOPRO-3 (in blue). White squares (in A, E, and I) are enlarged for details (as shown in B–D, F–H, and J–L; respectively). Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 7 The GLP-2R protein localized to VIP-positive enteric neurons (EN) and eNOS-expressing EN of the human jejunum by double immunostaining. hGLP-2R protein was expressed in EN (in yellow; A) and coexpressed with VIP (in yellow; E) and eNOS (in yellow; I). However, not all hGLP-2R immunoreactivity was VIP positive (in green, indicated by arrows; E), and not all PGP 9.5 immunoreactivity was eNOS positive (in green; indicated by an arrow; M). hGLP-2R protein was immunostained by the hGLP-2R antibody (in green; B, F, and J). The EN were labeled by a general neuron marker (PGP 9.5; C and O) and a specific neurotransmitter (VIP; G). A subset of cells was immunostained by mouse anti-eNOS antibody (in red; K and N). hGLP-2R protein was coexpressed with the neuronal marker (PGP 9.5, in yellow; D), the neurotransmitter (VIP, in yellow; H), and the eNOS protein (in yellow; I) that was colocalized with the neuronal marker (PGP 9.5, in yellow; P). Nuclei (DNA) were counterstained by TOPRO-3 (in blue). White squares (in A, E, I, and M) are enlarged for details (as shown in B–D, F–H, J–L, and N–P; respectively). Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 8 The GLP-2R protein localized to eNOS-expressing enteric neurons (EN) of the human jejunum by triple immunostaining. The hGLP-2R, eNOS, and PGP 9.5 (a neuron marker) were immunostained by their antibodies, respectively, in green (C), red (D), and blue (E); and nuclei (DNA) were counterstained by DAPI (in yellow). The hGLP-2R protein was coexpressed in submucosal eNOS-expressing EN (in white, indicated by arrows in A and F). The confocal images at a lower power were shown for morphologic reference (A, no nuclei counterstaining; B, with nuclei counterstaining). Note that a white square (in B) is enlarged for details (as shown in C–F). Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 9 GLP-2-mediated vascular actions in endogenously GLP-2-deficient neonatal pigs. Intravenous infusion of GLP-2 acutely increased SMA blood flow in a dose-dependent manner (A). Furthermore, GLP-2-stimulated SMA flow rate was positively correlated with arterial GLP-2 concentrations (B). In contrast, there was no correlation between arterial blood pressure and arterial GLP-2 concentrations (C). Finally, infusion of GLP-2 acutely increased jejunal expression of eNOS (mRNA and protein) and activation of the eNOS protein (by phosphorylation at Ser1177). Both blood flow and blood pressure were quantified by area under curve during the first 2-h of GLP-2 infusion period and further normalized by their saline baselines (A–C). Asterisk and double asterisk indicate P < .05 and P < .01, respectively, when compared with saline baseline (A) or with zero time (D). Gastroenterology 2006 130, 150-164DOI: (10.1053/j.gastro.2005.11.005) Copyright © 2006 American Gastroenterological Association Terms and Conditions