Nitric Oxide Not Carbon Monoxide Mediates Nonadrenergic Noncholinergic Relaxation in the Murine Internal Anal Sphincter  Satish Rattan, Raymond F. Regan, Chirag A. Patel, Márcio A.F. De Godoy  Gastroenterology  Volume 129, Issue 6, Pages 1954-1966 (December 2005) DOI: 10.1053/j.gastro.2005.08.050 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Effect of inhibitory neurotransmitter candidates CO, NO, and VIP on the basal IAS tone in (A) wild-type and (B) HO-2−/− mice. Note significant differences in the potencies of different inhibitors in the IAS VIP > NO > CO. HO-2 deletion has no significant effect on the effects of these inhibitors. Data represent the mean ± SE of 5–7 independent determinations. Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Genotyping of wild-type vs HO-2−/− mice for HO-2. Genotypes were identified by PCR using genomic DNA isolated from tail clippings. PCR products were separated via 1.5% agarose gel electrophoresis. Ethidium bromide-stained DNA bands were visualized under UV source and photographed. PCR from a wild-type animal (HO-2+/+) shows a single band at 460 bp and PCR from a knockout (HO-2−/−) animal shows a single band at ∼550 bp. The heterozygous animals (HO-2+/−) produced 2 distinct bands. Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Immunoblots of HO-1, HO-2, nNOS, and VIP in the IAS smooth muscle of wild-type (WT) and HO-2−/− mice. (A) The actual blots for the specific presence of the respective enzyme proteins in IAS smooth muscle. (B) The quantitative densitometric analyses of the blots. Note selective deletion of HO-2 protein in the HO-2−/− group of mice. In addition, in such mice, there is a significant decrease in HO-1 protein (*P < .05; data represent mean ± SE of 3 independent determinations). Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 (A) Immunohistochemistry for α-actin showing general view of the IAS smooth muscle layers and adjacent structures. (B) Detailed view of the IAS smooth muscle area focusing on the area within the square in A. The IAS smooth muscle layers are immunoreactive (IR) for α-actin (green). The myenteric plexus IR for NF-L is shown in red. Bar in panel A = 100 μm/L; original magnification, ×10 power. Bar in panel B = 20 μm/L. Immunohistochemistry for NF-L (green) and HO-2 (red) in IAS smooth muscle of (C) wild-type (WT) and (D) HO-2−/− mice. Note the presence of IR for NF-L and HO-2 in the mp of WT IAS, whereas the mp of HO-2−/− IAS lack HO-2-IR. mu, mucosa; mm. muscularis mucosa; sm, submucosa; cm, circular smooth muscle layer; mp, myenteric plexus; lm, longitudinal smooth muscle layer. Bar in panel C = 20 μm/L for panels C and D. Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Immunohistochemistry for NF-L (green) and nNOS (red) in IAS smooth muscle of (A) wild-type (WT) and (B) HO-2−/− mice. Note the presence of NF-L and nNOS-IR throughout the mp of WT and HO-2−/− IAS. Bars in panels A and B = 20 μm/L. Immunohistochemistry for NF-L (green) and VIP (red) in IAS smooth muscle of (C) wild-type (WT) and (D) HO-2−/− mice. cm, circular smooth muscle layer; mp, myenteric plexus; lm, longitudinal smooth muscle layer. Bar in panel C = 20 μm/L for panels C and D. Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Influence of ODQ on the IAS relaxation by CO (A), NO (B), VIP (C), and EFS (D). Note that the decrease in the IAS tone by all of the maneuvers in wild-type (HO-2+/+) (+/+) vs HO-2−/− (−/−) mice are similar. In addition, ODQ causes significant attenuation of the IAS relaxation by different maneuvers (*P < .05; n = 5–7 determinations) that was similar in wild-type and HO-2−/− mice. Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effect of SnPP IX (A), L-NNA (B), and VIP10–28 (C) on the IAS relaxation caused by EFS. Note, among different manipulations, only L-NNA and VIP10–28 caused significant attenuation of the IAS relaxation by EFS (*P < .05; n = 5–7 determinations). This attenuation is similar both in the wild-type (HO-2+/+) (+/+) and HO-2−/− (−/−) mice. An inset in panel C shows the linear regression for calculation of VIP10–28 IC50 in inhibiting VIP-induced relaxation in the murine IAS. Data represent the mean ± SEM of 4 independent determinations. The interception of the linear regression with the “x” axis provides the IC50 value of 4.3 μmol/L (indicated by the arrow). CR, concentration ratio. Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Comparison of EFS-induced relaxation of the IAS in wild-type (A) vs nNOS−/− mice (B). Data show that, in wild-type mice, responses to NANC relaxation by EFS are not modified by SnPP IX (P > .05) but are significantly attenuated by L-NNA (*P < .05; n = 8). Conversely, in the smooth muscle strips from nNOS−/− animals, neither L-NNA nor SnPP IX have a significant effect on EFS-induced relaxation in the IAS (P > .05; n = 8 determinations). Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Effect of L-NNA on the fall in the IAS tone by CO (A), NO (B), and VIP (C) in the wild-type mice. L-NNA attenuates IAS relaxation by VIP (*P < .05; n = 5–7 determinations) but not that by CO and NO (P > .05; n = 5–7 determinations). Gastroenterology 2005 129, 1954-1966DOI: (10.1053/j.gastro.2005.08.050) Copyright © 2005 American Gastroenterological Association Terms and Conditions