Volume 131, Issue 5, Pages 1542-1552 (November 2006) Hydrogen Sulfide Is a Novel Prosecretory Neuromodulator in the Guinea-Pig and Human Colon  Rudolf Schicho, Dagmar Krueger, Florian Zeller, Claus Werner Hann Von Weyhern, Thomas Frieling, Hideo Kimura, Isao Ishii, Roberto De Giorgio, Barbara Campi, Michael Schemann  Gastroenterology  Volume 131, Issue 5, Pages 1542-1552 (November 2006) DOI: 10.1053/j.gastro.2006.08.035 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 The expression of CSE and CBS is shown in enteric neurons of guinea-pig colon, human colon, and myenteric interstitial cells of Cajal guinea-pig ileum. (A) In the guinea-pig submucous plexus, colocalization studies showed that CSE-IR is present in all HU-IR neurons. (B) Rabbit and mouse CSE antibodies label identical neurons. (C) CSE-IR and CBS-IR are found in the same neurons. (D) In human submucous plexus, CSE-IR is present in all NSE-IR neurons. (E) CSE-positive neurons entirely overlap with CBS-positive neurons in a human submucous ganglion. (F) Kit-IR ICC in guinea-pig myenteric plexus also are CSE-IR but not (G) CBS-IR. (H) Double-labeling with NSE shows neuronal expression of CSE in the myenteric plexus of the guinea-pig ileum. Scale bars = 100 μm. Gastroenterology 2006 131, 1542-1552DOI: (10.1053/j.gastro.2006.08.035) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 In Ussing chamber experiments, the H2S donors NaHS and L-cysteine induced chloride secretion in guinea-pig and human submucosa/mucosa preparations. (A) NaHS (200 μmol/L) and (B) L-cysteine (1 mmol/L) both evoked a transient increase of Isc; repeated applications (arrows) led to a decrease in secretory response and finally to full desensitization. (C) Concentration-dependent increase in Isc after NaHS application is shown in guinea pig (■, n = 14–19) and human colon (□, n = 4–15). (D) In the guinea pig, L-cysteine caused an increase in Isc that was reduced significantly by PAG (2 mmol/L), AOAA (1 mmol/L), and capsaicin desensitization (10 μmol/L) (all n = 9). Unlike the quite robust effect of NaHS, the magnitude of the secretory responses after L-cysteine application varied extensively between experiments. We therefore used L-cysteine–evoked responses of adjacent tissue from the same preparation as controls. The control values were set at 100%, and the responses to L-cysteine in the presence of PAG, AOAA, or capsaicin then were expressed as the percentage increase in Isc relative to the control values and tested in an unpaired design. ■, L-cysteine; , PAG + L-cysteine; , AOAA + L-cysteine; □, capsaicin + L-cysteine. (E) The neurotoxin TTX (0.5 μmol/L), the TRPV1 blocker capsazepine (10 μmol/L), and capsaicin desensitization (10 μmol/L) significantly reduced NaHS-induced Isc responses in guinea pig (n = 4–19) and human tissue (n = 4–15). □, NaHS alone; , TTX; , capsaicin; , capsazepine. *Significant differences for NaHS effects in TTX, capsaicin, and capsazepine vs NaHS alone or for effects of L-cysteine in PAG, AOAA, and capsaicin vs L-cysteine alone. Data are expressed as means ± standard error of mean or as medians and their percentiles (see Materials and Methods section). Gastroenterology 2006 131, 1542-1552DOI: (10.1053/j.gastro.2006.08.035) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Neuroimaging with the voltage-sensitive dye di-8-ANEPPS revealed that neurons in guinea-pig and human submucous ganglia were stimulated by NaHS. (A) Signals from 4 cells (1–4) of a guinea-pig submucous ganglion before and during perfusion of 500 μmol/L NaHS. In the presence of NaHS, cells 2 and 4 respond with action potential discharge whereas cells 1 and 3 remain quiet. Cell 2 shows one spontaneously occurring action potential during vehicle perfusion (Krebs solution). A fluorescent image of the ganglion was taken by the CCD camera and is shown on the right side (scale bar = 20 μm). Note that individual cells can be recognized by strong labeling of their cell membrane with di-8-ANEPPS. The relative change in fluorescence indicates relative changes in fluorescence. (B) Box plots show significant increase of action potential frequency in submucous ganglion cells of the guinea-pig and human colon during perfusion with 500 μmol/L NaHS. This response is reduced significantly by either capsaicin desensitization (10 μmol/L) or by application of the TRPV1 antagonist capsazepine (10 μmol/L), but not by the KATP channel blocker glibenclamide (10 μmol/L). *Significant differences between NaHS or glibenclamide + NaHS vs control. **Significant differences between capsaicin or capsazepine vs NaHS. □, Control; , NaHS 0.5 mmol/L; , NaHS 0.5 mmol/L in 10 μmol/L capsaicin; , NaHS 5 mmol/L in 10 μmol/L capsazepin; , NaHS 5 mmol/L in 10 μmol/L gibenclamide. Gastroenterology 2006 131, 1542-1552DOI: (10.1053/j.gastro.2006.08.035) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 Proposed model of action of H2S in the human and guinea-pig distal colon. H2S-induced secretion involves activation of TRPV1 (1, 2) on extrinsic afferent terminals that innervate enteric ganglia or terminate at the epithelial level. The result is an activation of enteric nerves and an increase in secretion. Gastroenterology 2006 131, 1542-1552DOI: (10.1053/j.gastro.2006.08.035) Copyright © 2006 AGA Institute Terms and Conditions