Rho Kinase as a Novel Molecular Therapeutic Target for Hypertensive Internal Anal Sphincter  Satish Rattan, Marcio A.F. De Godoy, Chirag A. Patel  Gastroenterology  Volume 131, Issue 1, Pages 108-116 (July 2006) DOI: 10.1053/j.gastro.2006.03.043 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Effect of Y-27632 in the IAS versus the RSM. Considering the median effective concentration values, ROK inhibitor was 30 times more potent in the IAS versus the RSM (*P < .05; n = 5). Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Typical tracings of the effect of Y-27632 in the (A) IAS and (B) RSM. The ROK inhibitor causes a concentration-dependent decrease in the IAS tone and phasic contractions of the RSM. However, the ROK inhibitor was severalfold more sensitive in the IAS versus the RSM. Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 In vivo studies show that in the lower dose range, Y-27632 causes a significant decrease in the IASP (*P < .05; n = 4) without significant effect on the MSBP. Considering the median effective concentration values, Y-27632 was ∼200 times more potent in causing a decrease in the IASP versus the MSBP. Maximal decrease in the IASP was in reference to the pressures in the rectum. Conversely, maximal decrease in the MSBP was in reference to a possible decrease of 60 mm Hg; in the data shown, 200 nmol/kg of Y-27632 caused a decrease of ∼30 mm Hg. Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 (A) The neurohumoral inhibitors, including the NOS inhibitor l-NNA, have no significant effect on the IAS relaxation caused by the ROK inhibitor (P > .05; n = 5–7). (B) The ROK inhibitor causes concentration-dependent and significant relaxation of the SMCs from the IAS (*P < .05; n = 4) but no significant effect in the RSM SMCs. Percent increase in the cell length (in the presence of different concentrations of Y-27632) of the spontaneously contracted SMCs was calculated on the bases of original cell length. As pointed out in Materials and Methods, in each case average length of cells was obtained from 50 cells in a random manner. Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 (A) Comparison of ROK activity in the IAS versus the RSM reveals higher levels of the activity in the IAS (*P < .05; n = 4). (B) Relationship between the decrease in the IAS tone and in the ROK activity following different concentrations of ROK inhibitor. The comparison reveals significant correlation between the 2 events (r2 = 0.95). (C) Western blot analysis demonstrates higher levels of ROCK-II protein expression in the IAS versus the RSM (*P < .05; n = 4). Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Effect of Y-27632 (10 μmol/L) on free intracellular Ca2+ levels and PKC activity in the IAS. Data show that the ROK inhibitor has no significant effect on the levels of free intracellular Ca2+ and PKC activity (P > .05; n = 4). Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Confocal microscopy showing the immunocytochemical localization of ROCK-II in the SMCs of the IAS. Data show higher levels of ROCK-II toward the periphery of the SMCs isolated from the IAS in the basal state (upper right panel). Pretreatment of the IAS SMCs with Y-27632 causes redistribution of ROCK-II and relaxation of the SMCs (lower right panel). Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 Effect of Y-27632 on the basal tone in the control and hypertensive IAS. The ROK inhibitor causes relaxation of the IAS; in the submaximal concentrations, it causes significantly higher relaxation in the hypertensive IAS (*P < .05; n = 5). Gastroenterology 2006 131, 108-116DOI: (10.1053/j.gastro.2006.03.043) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions