Volume 132, Issue 4, Pages 1401-1409 (April 2007) H-ras Inhibits RhoA/ROCK Leading to a Decrease in the Basal Tone in the Internal Anal Sphincter  Márcio A.F. de Godoy, Chirag A. Patel, Scott A. Waldman, Motoya Katsuki, Raymond F. Regan, Satish Rattan  Gastroenterology  Volume 132, Issue 4, Pages 1401-1409 (April 2007) DOI: 10.1053/j.gastro.2007.01.043 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 H-ras regulates the basal tone of the internal anal sphincter (IAS) via a Rho-kinase-dependent mechanism. (A) PCR products of genomic DNA from mice demonstrating wild-type (+/+; 336 bp), knock-out (−/−; 434 bp), and heterozygous (+/−; both 336 bp and 434 bp) H-ras mice. (B) Data show significantly higher basal IAS tone (mean ± SEM) in the H-ras−/− vs. H-ras+/+ mice (*P < .05 by unpaired Student’s t-test; n = 5–7 independent determinations). (C) Data show Rho kinase inhibitor Y 27632 is significantly more potent in causing IAS smooth muscle relaxation (mean ± SEM) in the H-ras+/+ vs. H-ras−/− mice (*P < .05 by unpaired Student’s t-test; n = 4–5). (D) Time course of the effect of Y 27632 (10 μmol/L) on the basal IAS tone in H-ras+/+ and H-ras−/− mice. Data show that in response to Y 27632 the kinetics of the fall in the IAS tone was slower while its recovery following relaxation was faster in the H-ras−/− mice vs. the wild-type (mean ± SEM of 4–5 independent determinations). (E) Representative tracings to show differences in the kinetics of Y 27632 effects in H-ras+/+ vs. H-ras−/− mice. Gastroenterology 2007 132, 1401-1409DOI: (10.1053/j.gastro.2007.01.043) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 H-ras regulates the length of smooth muscle cells isolated from the IAS. (A) Representative images of smooth muscle cells (SMCs) isolated from H-ras+/+ and H-ras−/− mice (panels 1 and 2, respectively). Cells are stained for α-actin and visualized with FITC. (B) Data show significantly reduced basal length of the SMC from H-ras−/− vs. H-ras+/+ mice (*P < .05; mean ± SE of 5–6 animals). In addition, Y 27632 causes significant relaxation of the SMC in both H-ras+/+ and H-ras−/− mice (**P < .05; n = 5–6 animals), and it is noteworthy that Y 27632 eliminated the differences in the cell lengths between the 2 strains of mice (P > .05; n = 5–6 animals). Results were analyzed using 1-way analysis of variance followed by the Newman-Keuls post hoc test. Gastroenterology 2007 132, 1401-1409DOI: (10.1053/j.gastro.2007.01.043) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 H-ras regulates contractile elements in SMCs isolated from the IAS. (A) SMCs isolated from IAS of H-ras+/+ and H-ras−/− mice were subjected to immunofluorescent staining for H-ras (FITC) or RhoA (TR) as described in the Materials and Methods section. (Panels 1, 3) H-ras immunofluorescence (FITC) in H-ras+/+ (1) and H-ras−/− (3) cells. (Panels 2, 4) RhoA immunofluorescence (TR) in H-ras+/+ (2) and H-ras−/− (4) cells. (B) Distribution of RhoA in cytosolic and particulate fractions of IAS smooth muscle strips isolated from H-ras+/+ and H-ras−/− mice. Data show significantly higher levels of RhoA in the particulate fractions of the IAS smooth muscles taken from H-ras−/− vs. H-ras+/+ (*P < .05; n = 3). (C–E) Expression of ROCK I, ROCK II, and α-actin (C), pThr696-MYPT1, and MYPT1 (D), and pthr18/ser19-MLC20, and MLC20 (E) in IAS smooth muscles isolated from H-ras+/+ and H-ras−/− mice. Graphs illustrate the relative densities (RhoA IOD/α-actin, pthr696-MYPT1/MYPT1 or pthr18/ser19-MLC20/MLC20 IODs) of immunoblots. Data show significantly higher levels of ROCK II, pThr696-MYPT1 and pthr18/ser19-MLC20 in the H-ras−/− vs. H-ras+/+ mice (*P < .05; n = 3–4). Gastroenterology 2007 132, 1401-1409DOI: (10.1053/j.gastro.2007.01.043) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 IAS SMCs isolated from H-ras+/+ (A) and H-ras−/− (B) mice were transfected with pcDNA(+)3.1 eYFP H-ras 17N (dominant negative) and pcDNA3.1(+) eYFP H-ras G12V (constitutively activated), respectively. Empty pcDNA(+)3.1 eYFP was used as control. Only trypan-blue negative cells were used. Transfection efficiency was confirmed by visualization of yellow fluorescence by confocal microscopy at 514 nm. Data show that dominant negative construct causes significant decrease in the lengths of the SMC in H-ras+/+ mice and increase with constitutively active construct in the case of H-ras−/− (*P < .05; mean ± SE of 5–7 animals). Gastroenterology 2007 132, 1401-1409DOI: (10.1053/j.gastro.2007.01.043) Copyright © 2007 AGA Institute Terms and Conditions