Volume 141, Issue 2, Pages 565-575.e4 (August 2011) Transient Receptor Potential Ankyrin 1 Is Expressed by Inhibitory Motoneurons of the Mouse Intestine  Daniel P. Poole, Juan Carlos Pelayo, Fiore Cattaruzza, Yien–Ming Kuo, Gregory Gai, Jonathon V. Chiu, Romke Bron, John B. Furness, Eileen F. Grady, Nigel W. Bunnett  Gastroenterology  Volume 141, Issue 2, Pages 565-575.e4 (August 2011) DOI: 10.1053/j.gastro.2011.04.049 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 (A) TRPA1 (227 base pairs) was amplified in muscularis externa-myenteric plexus and mucosa-submucosa from all regions. RT, reverse transcriptase. (B) Localization of TRPA1-IR in antrum, duodenum (Duo), proximal colon (PC), and distal colon (DC). Inset is a higher-magnification image showing TRPA1-IR in epithelial cells of the proximal colon. ec, epithelial cells; mp, myenteric plexus; lm, longitudinal muscle; cm, circular muscle. Preadsorption abolished TRPA1-IR in intestine. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Localization of TRPA1-IR and PGP9.5-IR in whole mounts of myenteric ganglia of the (A) duodenum, (B) cecum, and (C) proximal colon. In the duodenum, TRPA1-IR was localized to PGP9.5-IR fibers but was absent from the soma (arrowheads with asterisks). In the cecum and proximal colon, TRPA1-IR was detected in a subset of PGP9.5-IR neurons. Neurons positive for both markers (arrowheads) or for PGP9.5-IR only (arrowheads with asterisks) are indicated. (D) Preadsorption abolished TRPA1-IR in myenteric neurons in proximal colon, but the preadsorbed antibody gave a particulate pattern of nonspecific staining of nonneuronal tissues. Scale = 50 μm. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Localization of TRPA1-IR and NOS-IR in myenteric plexus whole mounts. TRPA1-IR colocalized with NOS-IR in neurons of the (A) cecum, (B) proximal colon, and (C) distal colon (arrowheads). Neurons positive for only TRPA1-IR or NOS-IR are indicated by arrowheads with asterisks, and TRPA1-IR in proximal processes is indicated with an asterisk. Scale = 50 μm. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 TRPA1 signaling to cultured myenteric neurons. AITC (100 μmol/L) increased [Ca2+]i in a subset of neurons from (A) trpa1+/+ but not (B) trpa1−/− mice. (C) Vehicle had no effect on [Ca2+]i in neurons from trpa1+/+ mice. The relative proportion of neurons responsive to AITC (100 μmol/L) and CMA (10 or 100 μmol/L) and the magnitude of the response to AITC (100 μM) are summarized in D and E. F shows colocalization of TRPA1-IR, PGP9.5-IR, and 4′,6-diamidino-2-phenylindole (DAPI) in cultured neurons (arrowheads), indicating intense labeling of the proximal process (asterisks). Inset is a high-power magnification image showing prominent intracellular localization. Scale = 20 μm. (n = 3–10 independent experiments). *P < .05, ***P < .001 to vehicle or trpa1−/− mice. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Effects of TRPA1 agonists on contractility of isolated tissues. (A–C) CMA (100 μmol/L) caused a rapid, prolonged inhibition of phasic contractions of colon from trpa1+/+ but not trpa1−/− mice (A and B). Vehicle had no effect in trpa1+/+ mice. Tetrodotoxin or l-NAME attenuated effects of CMA in trpa1+/+ mice (C). Representative traces in A, pooled data in B and C. (D and E) CMA (100 μmol/L) did not affect contractility of the gastric antrum, duodenum, or ileum. Representative traces in D and pooled data in E. (n ≥ 6 mice per group). **P < .01, ***P < .001 to vehicle or trpa1−/− mice. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Effects of TRPA1 deletion or AITC on (A and B) GE, (C and D) SIT, and (E) colonic transit. Summarized data are shown in A and C, and scatter plots of values for individual mice are shown in B, D, and E. There was no difference in GE or SIT between vehicle-treated trpa1+/+ and trpa1−/− mice, and AITC (low, 0.0125 or high, 0.25 μmole/mouse) had no effect in either strain. Intracolonic AITC (0.5%, 100 μL) significantly delayed colonic bead expulsion in trpa1+/+ mice but not trpa1−/− mice. No significant difference in colonic transit was detected in vehicle-treated trpa1+/+ and trpa1−/− mice (n ≥ 6 mice per group). *P < .05. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Colocalization of TRPA1-IR and PGP9.5-IR in whole mounts of submucosal ganglia of the (A) cecum, (B) proximal colon, and (C) distal colon. TRPA1-IR was present in the cytoplasm of the majority of PGP9.5-IR submucosal neurons (arrowheads). Intense labeling of the axon hillock and proximal processes was also evident (asterisks). PGP9.5-IR neurons that are TRPA1-IR negative are indicated by arrowheads with an asterisk. Scale = 20 μm. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Localization of TRPA1-IR in myenteric plexus whole mounts and cell lines using a previously characterized antibody.13 (A) TRPA1-IR (left panels) was colocalized with NOS-IR (middle panels), a marker of inhibitory motor neurons and descending interneurons in a subset of neurons of the proximal colon (arrowheads). Examples of neurons positive for only TRPA1-IR are indicated by arrowheads with an asterisk. (B) TRPA1-IR colocalized with HA-IR in HEK-FLP TREX cells stably expressing rat TRPA1 with a hemagglutinin (HA) epitope tag (TRPA1-HA). TRPA1-IR was detected in TRPA1-HA–expressing cells and was colocalized with HA-IR. (C) Neither TRPA1-IR nor HA-IR was detected in cells expressing vector control (VC) without TRPA1 insert. Scale = 50 μm (whole mounts) and 20 μm (cells). Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Localization of TRPA1-IR and choline acetyltransferase (ChAT)-IR in myenteric plexus whole mounts. TRPA1-IR colocalized with ChAT-IR in neurons of the (A) cecum, (B) proximal colon, and (C) distal colon (arrowheads). Examples of neurons positive for only TRPA1-IR or ChAT-IR are indicated by arrowheads with an asterisk. Intense TRPA1-IR in proximal processes is indicated by asterisks. Scale = 50 μm. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Localization of TRPA1-IR and neurofilament medium (NFM)-IR in myenteric plexus whole mounts. The intrinsic primary afferent neurons are medium to large multipolar (Dogiel type II) neurons (arrowheads). TRPA1-IR was colocalized with NFM-IR in a subset of neurons of the (A) cecum, (B) proximal colon, and (C) distal colon (arrowheads). Examples of neurons positive only for TRPA1-IR are indicated by arrowheads with an asterisk. Prominent TRPA1-IR in NFM-IR proximal processes of intrinsic primary afferent neurons is marked with asterisks. Scale = 50 μm. Gastroenterology 2011 141, 565-575.e4DOI: (10.1053/j.gastro.2011.04.049) Copyright © 2011 AGA Institute Terms and Conditions