Heme oxygenase activity in the internal anal sphincter: Effects of nonadrenergic, noncholinergic nerve stimulation  Sushanta Chakder, Gao-Yuan Cao, Richard B. Lynn, Satish Rattan  Gastroenterology  Volume 118, Issue 3, Pages 477-486 (March 2000) DOI: 10.1016/S0016-5085(00)70253-8 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Relationship between HO activity as measured by the release of free 55Fe2+ and concentrations of IAS smooth muscle tissue extract protein. The amount of released 55Fe2+ was a direct measure of the levels of HO activity of the IAS tissue extracts in the incubates. HO activity of the IAS extracts increased linearly with increasing protein concentration up to 5 mg/mL. Note a steep linear relationship between the amount of 55Fe2+ and the protein concentrations from 0.04 to 2 mg/mL. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 HO activity of the IAS tissue extracts shown as free 55Fe2+ released as a function of the time of incubation. When 100 μg of tissue protein was incubated with a fixed amount of radiolabeled hemin (see Materials and Methods), HO activity increased with increasing time of incubation and plateaued at 20 minutes. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Increase in HO activity (2) and the corresponding percent fall in IAS tension (▨) in response to neural stimulation with EFS (0.25–5.0 Hz). The increase in HO activity of the IAS was significant at 2 and 5 Hz of EFS (*P < 0.05; n = 4). There was a significant correlation between the degree of IAS smooth muscle relaxation and increase in HO activity, especially at frequencies up to 2 Hz. *Values significantly different from basal. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Increase in HO activity (2) and the corresponding percent fall in IAS smooth muscle tension (▨) by different concentrations of DMPP. DMPP caused an increase in HO activity and relaxation of the IAS smooth muscle. In the lower concentrations of DMPP, there was a significant correlation between the 2 parameters. *Values significantly different from basal (P < 0.05; n = 4). Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Increase in HO activity (2) and the corresponding percent fall in IAS smooth muscle tension (▨) by different concentrations of VIP. VIP (especially in the lower concentrations) caused a concentration-dependent increase in HO activity and relaxation of the IAS smooth muscle. *Values significantly different from basal (P < 0.05; n = 4). Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of the HO inhibitor SnPP IX on the increase in HO activity (2) and the corresponding percent fall in IAS smooth muscle tension (▨) by (A) EFS (5 Hz) and (B) DMPP (1 × 10−5 mol/L). EFS and DMPP caused a significant increase in HO activity and relaxation of the IAS, which were significantly reduced by SnPP IX (1 × 10−4 mol/L) (n = 4). *Values significantly higher than basal. **The fall in the IAS tension and increase in HO activity caused by 5 Hz of EFS and 1 × 10−5 mol/L DMPP were significantly attenuated by SnPP IX. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of the HO inhibitor SnPP IX on the increase in HO activity (2) and the corresponding percent fall in IAS smooth muscle tension (▨) by (A) EFS (5 Hz) and (B) DMPP (1 × 10−5 mol/L). EFS and DMPP caused a significant increase in HO activity and relaxation of the IAS, which were significantly reduced by SnPP IX (1 × 10−4 mol/L) (n = 4). *Values significantly higher than basal. **The fall in the IAS tension and increase in HO activity caused by 5 Hz of EFS and 1 × 10−5 mol/L DMPP were significantly attenuated by SnPP IX. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Autoradiographs showing HO-1 and HO-2 protein bands in the opossum IAS and rectal tissue extracts. Interestingly, the expression of HO-2 protein was higher both in the IAS and rectum than that of HO-1. There was no significant difference in HO-1 levels between the rectum and IAS. (B) Quantitative analysis showing the relative expression of HO-1 and HO-2 protein bands using densitometric analysis. The data show that the relative expression of HO-1 in the IAS and rectum were similar (P > 0.05). The relative expression of HO-2, on the other hand, in both the tissues was found to be higher than that of HO-1 (*P < 0.05; n = 4). Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Autoradiographs showing HO-1 and HO-2 protein bands in the opossum IAS and rectal tissue extracts. Interestingly, the expression of HO-2 protein was higher both in the IAS and rectum than that of HO-1. There was no significant difference in HO-1 levels between the rectum and IAS. (B) Quantitative analysis showing the relative expression of HO-1 and HO-2 protein bands using densitometric analysis. The data show that the relative expression of HO-1 in the IAS and rectum were similar (P > 0.05). The relative expression of HO-2, on the other hand, in both the tissues was found to be higher than that of HO-1 (*P < 0.05; n = 4). Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Localization of (A) HO-2 and (B) HO-1 immunoreactivities in the opossum IAS. Both HO-1 and HO-2 immunoreactivities were present in the ganglia of the myenteric neurons. However, HO-1 immunoreactivity was relatively light compared with HO-2, and there were occasional HO-2 immunoreactive structures in the circular muscle layers of the IAS. cm, circular muscle; mp, myenteric plexus; lm, longitudinal muscle. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Localization of (A) HO-2 and (B) HO-1 immunoreactivities in the opossum IAS. Both HO-1 and HO-2 immunoreactivities were present in the ganglia of the myenteric neurons. However, HO-1 immunoreactivity was relatively light compared with HO-2, and there were occasional HO-2 immunoreactive structures in the circular muscle layers of the IAS. cm, circular muscle; mp, myenteric plexus; lm, longitudinal muscle. Gastroenterology 2000 118, 477-486DOI: (10.1016/S0016-5085(00)70253-8) Copyright © 2000 American Gastroenterological Association Terms and Conditions