FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1964438177> ?p ?o ?g. }

• W1964438177 endingPage "1409" @default.
• W1964438177 startingPage "1401" @default.
• W1964438177 abstract "Background & Aims: The present studies evaluated the role of H-ras and its implications in the RhoA/Rho kinase (ROCK) pathway in regulating basal tone in the internal anal sphincter (IAS). Methods: Studies were performed in the IAS from the wild-type (H-ras+/+) and knock-out (H-ras−/−) mice. The basal tone of smooth muscle strips was measured by isometric force transducers. Length of smooth muscle cells (SMC) isolated from the IAS in the basal state was determined by phase contrast microscopy. Experiments were repeated in the presence of Y 27632, a ROCK inhibitor. Involvement of the RhoA/ROCK machinery was analyzed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Reversal of H-ras knock-out effect was evaluated by transfection of SMCs with the constitutively activated (G12V) mutant. Results: Basal tone of the H-ras−/− IAS was significantly higher and resistant to relaxation by Y 27632, compared with the H-ras+/+ IAS. Similarly, the length of SMCs from H-ras−/− IAS was significantly shorter. Y 27632 eliminated this difference. RhoA immunoreactivity shifted from cytoplasm to plasma membrane in H-ras−/− SMCs, a change typically associated with contraction. Further, SMCs from H-ras−/− mice exhibited higher levels of the contractile proteins ROCK II, phosphorylated-MYPT1 and phosphorylated-MLC20. Transfection with the G12V mutant increased the length of H-ras−/− cells. Conversely, the dominant negative H-ras (S17N) mutant decreased the length of H-ras+/+ cells. Conclusions: H-ras negatively regulates basal tone in the IAS by inhibiting RhoA/Rho-kinase machinery. Studies may have significant relevance in the pathophysiology and therapy of certain anorectal motility disorders associated with the IAS dysfunction. Background & Aims: The present studies evaluated the role of H-ras and its implications in the RhoA/Rho kinase (ROCK) pathway in regulating basal tone in the internal anal sphincter (IAS). Methods: Studies were performed in the IAS from the wild-type (H-ras+/+) and knock-out (H-ras−/−) mice. The basal tone of smooth muscle strips was measured by isometric force transducers. Length of smooth muscle cells (SMC) isolated from the IAS in the basal state was determined by phase contrast microscopy. Experiments were repeated in the presence of Y 27632, a ROCK inhibitor. Involvement of the RhoA/ROCK machinery was analyzed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Reversal of H-ras knock-out effect was evaluated by transfection of SMCs with the constitutively activated (G12V) mutant. Results: Basal tone of the H-ras−/− IAS was significantly higher and resistant to relaxation by Y 27632, compared with the H-ras+/+ IAS. Similarly, the length of SMCs from H-ras−/− IAS was significantly shorter. Y 27632 eliminated this difference. RhoA immunoreactivity shifted from cytoplasm to plasma membrane in H-ras−/− SMCs, a change typically associated with contraction. Further, SMCs from H-ras−/− mice exhibited higher levels of the contractile proteins ROCK II, phosphorylated-MYPT1 and phosphorylated-MLC20. Transfection with the G12V mutant increased the length of H-ras−/− cells. Conversely, the dominant negative H-ras (S17N) mutant decreased the length of H-ras+/+ cells. Conclusions: H-ras negatively regulates basal tone in the IAS by inhibiting RhoA/Rho-kinase machinery. Studies may have significant relevance in the pathophysiology and therapy of certain anorectal motility disorders associated with the IAS dysfunction. Basal tone in the internal anal sphincter (IAS) plays a major role in rectoanal continence. The IAS that comprises an inner ring of smooth muscle maintains spontaneous tone in the basal state, which is primarily myogenic in nature.1Culver P.J. Rattan S. Genesis of anal canal pressures in the opossum.Am J Physiol Gastrointest Liver Physiol. 1986; 251: G765-G771PubMed Google Scholar A decrease in basal tone may result in anal incontinence.2Schiller L.R. Fecal incontinence.in: Feldman M. Sleisenger & Fordrtran’s gastrointestinal and liver disease. 7 ed. W.B. Saunders Co, Philadelphia, PA2002: 164-174Google Scholar H-ras belongs to the ras family of small GTPases, which comprises H-ras, K-ras, and N-ras Ras homologs that regulate a wide spectrum of cellular processes by switching between the inactive GDP- and the active GTP-bound states. When bound to GTP, these proteins translocate from the cytoplasm to the plasma membrane and interact with effector molecules leading to changes in cellular activities.3Rebollo A. Martinez-A C. Ras proteins: recent advances and new functions.Blood. 1999; 94: 2971-2980Crossref PubMed Google Scholar, 4Prior I.A. Hancock J.F. Compartmentalization of Ras proteins.J Cell Sci. 2001; 114: 1603-1608Crossref PubMed Google Scholar, 5Fiordalisi J.J. Holly S.P. Johnson II, R.L. Parise L.V. Cox A.D. A distinct class of dominant negative Ras mutants.J Biol Chem. 2002; 277: 10813-10823Crossref PubMed Scopus (40) Google Scholar, 6Niv H. Gutman O. Kloog Y. Henis Y.I. Activated K-Ras and H-Ras display different interactions with saturable sites at the surface of live cells.J Biol Chem. 2002; 157: 865-872Google Scholar, 7Prior I.A. Muncke C. Parton R.G. Hancock J.F. Direct visualization of Ras proteins in spatially distinct cell surface microdomains.J Biol Chem. 2003; 160: 165-170Google Scholar H-ras down-regulates expression of genes encoding smooth muscle contractile proteins such as α-actin, caldesmon 1, myosin heavy chains, and tropomyosin 1.8Kaplan-Albuquerque N. Garat C. Desseva C. Jones P.L. Nemenoff R.A. Platelet-derived growth factor-BB-mediated activation of Akt suppresses smooth muscle-specific gene expression through inhibition of mitogen-activated protein kinase and redistribution of serum response factor.J Biol Chem. 2003; 278: 39830-39838Crossref PubMed Scopus (54) Google Scholar, 9Kaplan-Albuquerque N. Bogaert Y.E. Van Putten V. Weiser-Evans M.C. Nemenoff R.A. Patterns of gene expression differentially regulated by platelet-derived growth factor and hypertrophic stimuli in vascular smooth muscle cells: markers for phenotypic modulation and response to injury.J Biol Chem. 2005; 280: 19966-19976Crossref PubMed Scopus (40) Google Scholar In addition, H-ras inhibits RhoA-GTP formation and coupling to RhoA/Rho kinase (ROCK) by arresting the target molecules to the cytoplasm preventing signaling. The mechanism seems to involve the protein p21, which forms a physical complex with the target protein and inhibits its activity.4Prior I.A. Hancock J.F. Compartmentalization of Ras proteins.J Cell Sci. 2001; 114: 1603-1608Crossref PubMed Google Scholar, 5Fiordalisi J.J. Holly S.P. Johnson II, R.L. Parise L.V. Cox A.D. A distinct class of dominant negative Ras mutants.J Biol Chem. 2002; 277: 10813-10823Crossref PubMed Scopus (40) Google Scholar, 6Niv H. Gutman O. Kloog Y. Henis Y.I. Activated K-Ras and H-Ras display different interactions with saturable sites at the surface of live cells.J Biol Chem. 2002; 157: 865-872Google Scholar, 7Prior I.A. Muncke C. Parton R.G. Hancock J.F. Direct visualization of Ras proteins in spatially distinct cell surface microdomains.J Biol Chem. 2003; 160: 165-170Google Scholar, 10Pawlak G. Helfman D.M. Post-transcriptional down-regulation of ROCK/Rho-Kinase through an MEK-dependent pathway leads to cytoskeleton disruption in Ras-transformed fibroblasts.Mol Biol Cell. 2002; 13: 336-347Crossref PubMed Scopus (78) Google Scholar, 11Lee S. Helfman D.M. Cytoplasmic p21cip1 is involved in ras-induced inhibition of the ROCK/LIMK/Cofilin pathway.J Biol Chem. 2004; 279: 1885-1891Crossref PubMed Scopus (142) Google Scholar Importantly, maintenance of IAS basal tone is critically dependent on the RhoA/ROCK signaling pathway.12Rattan S. Puri R.N. Fan Y.-P. Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II.Exp Biol Med. 2003; 228: 972-981Crossref PubMed Scopus (37) Google Scholar, 13Rattan S. De Godoy M.A.F. Patel C.A. Rho kinase as a novel molecular therapeutic target for hypertensive internal anal sphincter.Gastroenterology. 2006; 131: 108-116Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar There are reports of upregulation of H-ras14Arber N. Shapira I. Ratan J. Stern B. Hibshoosh H. Moshkowitz M. Gammon M. Fabian I. Halpern Z. Activation of c-K-ras mutations in human gastrointestinal tumors.Gastroenterology. 2000; 118: 1045-1050Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar in certain forms of colorectal cancer, and associated rectoanal incontinence with lower intraluminal pressures in the IAS.15van Duijvendijk P. Slors F. Taat C.W. Heisterkamp S.H. Obertop H. Boeckxstaens G.E. A prospective evaluation of anorectal function after total mesorectal excision in patients with a rectal carcinoma.Surgery. 2003; 133: 56-65Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Together, present studies using multipronged approaches suggest that H-ras modulates the development of basal tone in smooth muscle by regulating the RhoA/ROCK pathway. Here, the role of H-ras in regulating basal tone of the IAS through RhoA/ROCK signaling was explored in wild-type (H-ras+/+) mice and mice from which H-ras expression was eliminated (H-ras knock-out mice; H-ras−/−). H-ras+/− mice16Ise K. Nakamura K. Nakao K. Shimizu S. Harada H. Ichise T. Miyoshi J. Gondo Y. Ishikawa T. Aiba A. Katsuki M. Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis.Oncogene. 2000; 19: 2951-2956Crossref PubMed Scopus (114) Google Scholar were bred in the facility of Animal Resources of Thomas Jefferson University. Genomic DNA was extracted from mouse-tail biopsies as previously described.17Laird P.W. Zijderveld A. Linders K. Rudnicki M.A. Jaenisch R. Berns A. Simplified mammalian DNA isolation procedure.Nucleic Acids Res. 1991; 19: 4293Crossref PubMed Scopus (1300) Google Scholar Tail biopsies were incubated at 55°C in lysis buffer (100 mmol/L Tris-HCl [pH 8.0] 5 mmol/L EDTA, 0.2% sodium dodecyl sulfate [SDS], 200 mmol/L NaCl, 200 μg of proteinase K/mL) overnight. DNA was precipitated using 100% ethanol, washed in 75% ethanol, and resuspended in 50 μL of distilled water. Routine genotyping of DNA isolated from mouse-tail biopsies sacs was performed by polymerase chain reaction (PCR). The primers for H-ras were H-ras A (5′-CCG CAA TTT ATG CTG CCG AAT CTC-3′), H-ras B (5′-GCT CCC TAT TTG TGT TGG TTT TGC-3′), and H-ras Neo-PGK promoter (5′-TCC TGC CGA GAA AGT ATC CA-3′) from the Kimmel Cancer Center (Philadelphia, PA). Oligonucleotides were used in a 25-μL Promega 2× Master Mix (Promega, Madison, WI). Cycling conditions were 94°C for 2 minutes, followed by 30 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, followed by an elongation cycle of 72°C for 7 minutes, using a Perkin-Elmer Thermal Cycler (Perkin-Elmer Life and Analytical Sciences, Inc., Waltham, MA). Amplified products were analyzed by electrophoresis in 1.5% agarose gel containing ethidium-bromide. H-ras+/+ and H-ras−/− mice (∼30 g) were sacrificed by decapitation and the anal canal with an adjacent region of the rectum was quickly removed and transferred to oxygenated (95% O2/5% CO2) Krebs’ physiological solution (KPS) of the following composition (in mmol/L): 118.07 mmol/L NaCl, 4.69 mmol/L KCl, 2.52 mmol/L CaCl2, 1.16 mmol/L MgSO4, 1.01 mmol/L NaH2PO4, 25 mmol/L NaHCO3, and 11.10 mmol/L glucose at 37°C. Extraneous tissues connected to the IAS were removed carefully by sharp dissection. The anal canal was then opened and pinned flat with the mucosal side up on a dissecting tray containing oxygenated KPS. The mucosa was removed carefully using sharp dissection. Circular smooth muscle strips (∼1 mm × 5 mm) of the IAS (identified as a thickened circular smooth muscle situated at the lowermost part of the anal canal) were prepared. The experimental protocol of the study was approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University, and was in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. Isolated smooth muscle strips were transferred to 2-mL muscle baths containing oxygenated KPS at 37°C. One end of the muscle strip was anchored to the bottom of the muscle bath while the other end was connected to a force transducer (model FT03; Grass Instruments, Quincy, MA). Isometric tension was measured by the PowerLab/8SP data acquisition system (AD Instruments, Colorado Springs, CO) and recorded using Chart 4.1.2 (AD Instruments). Each smooth muscle strip was initially stretched to a tension of 0.5 g followed by 90 minutes of equilibration. During this period the smooth muscle strips were replenished with fresh KPS every 20 minutes. Only the smooth muscle strips that developed spontaneous tone and relaxed in response to electrical field stimulation were used in this study. The fall in IAS tone by Y 27632 was calculated on the basis of percent maximal relaxation induced by EDTA (50 mmol/L) at the end of each experiment.18Biancani P. Walsh J.H. Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter.Gastroenterology. 1985; 89: 867-874PubMed Google Scholar, 19Cao W. Harnett K.M. Behar J. Biancani P. PGF2α-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G282-G291Crossref PubMed Scopus (27) Google Scholar Concentration–response curves for Y 27632 (10 nmol/L–100 μmol/L) were obtained in IAS smooth muscle by adding the Rho-kinase inhibitor to muscle baths in a cumulative fashion. Time courses (minutes) for relaxation by Y 27632 (10 μmol/L) and recovery of basal tone after washing out the Rho-kinase inhibitor were quantified in the IAS smooth muscle strips from H-ras+/+ vs. H-ras−/− mice. SMCs from the circular smooth muscle layer of the mouse IAS were isolated.20Rattan S. Chakder S. Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation.Am J Physiol Gastrointest Liver Physiol. 1993; 265: G799-G804PubMed Google Scholar, 21Kuemmerle J.F. Murthy K.S. Coupling of the insulin-like growth factor-1 receptor tyrosine kinase to Gi2 in human intestinal smooth muscle.J Biol Chem. 2001; 276: 7187-7194Crossref PubMed Scopus (78) Google Scholar Briefly, the IAS smooth muscle was cut into small pieces (1–2-mm cubes) and incubated in oxygenated KPS containing 0.1% collagenase and 0.01% soybean trypsin inhibitor at 37°C for 2 successive 1-hour periods. After incubation, the mixture was filtered through a 500-μm Nitex mesh. The tissue trapped on the mesh was rinsed with 25 mL (5 × 5 mL) of collagenase-free KPS. The tissue was incubated at 37°C and dispersion of the cells (0–1 hour) was monitored periodically by examining a 10-μL aliquot of the mixture under the microscope. SMCs were harvested by filtration through the Nitex mesh. The filtrate containing the cells was centrifuged at 350 × g for 10 minutes at room temperature. The cells in the pellet were resuspended in oxygenated KPS (at 37°C) at a cell density of 3 × 104Prior I.A. Hancock J.F. Compartmentalization of Ras proteins.J Cell Sci. 2001; 114: 1603-1608Crossref PubMed Google Scholar cells/mL. Individual cell lengths were measured by micrometry using phase contrast microscopy22Murthy K.S. Makhlouf G.M. Differential regulation of phospholipase A2 (PLA2)-dependent Ca2+ signaling in smooth muscle by cAMP- and cGMP-dependent protein kinases—inhibitory phosphorylation of PLA2 by cyclic nucleotide-dependent protein kinases.J Biol Chem. 1998; 273: 34519-34526Crossref PubMed Scopus (57) Google Scholar on a custom-assembled microscope (Olympus, Tokyo, Japan), closed-circuit video camera (model Pulnix MC-7; PULNIX America, Inc., Sunnyvale, CA), and PC computer. Images of cells were stored digitally and cell lengths measured with Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD). To determine the effect of Y 27632 on cell lengths, SMCs were first treated with Y 27632 (10 μmol/L) for 5 minutes, and then fixed with acrolein (final concentration 1%) and transferred onto chrome alum-coated glass slides (Fisher Scientific, Pittsburg, PA). The lengths of each group of cells were measured and results were expressed as the mean ± SE. Studies were performed with tissues isolated from 5 to 6 animals of each group of mice (H-ras+/+ and H-ras−/−). pcDNA3.1-eYFP containing the human wild-type (wt), constitutively activated (G12V), or dominant negative (S17N) H-ras coding sequences were generously provided by Dr. Thomas Schmidt from the Leiden Institute of Physics (Leiden, The Netherlands). Dr. Lily Dong (University of Texas Health Science Center at San Antonio, TX) provided the Empty pcDNA3.1-eYFP. Plasmid DNA (50 ng) was transfected and amplified in Escherichia coli strains cultivated in Luria-Bertani (LB) Agar, Miller (10 ng tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar in 1000 mL of distilled water) and LB Broth, Miller medium (10 ng tryptone, 5 g yeast extract, and 10 g NaCl in 1000 mL of distilled water) at 37°C in a humid incubator. Plasmids were purified using the plasmid purifying kit QIAfilter Plasmid Midi Kit from Qiagen (Valencia, CA), and the final concentration was determined by spectrophotometry at 260 nm. The quality of plasmids was evaluated by running a PCR agarose gel (0.8%). The expected size for the circular plasmids was about 3300 bp. The procedures were performed as described previously.23Lommerse P.H. Snaar-Jagalska B.E. Spaink H.P. Schmidt T. Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation.J Cell Sci. 2005; 118: 1799-1809Crossref PubMed Scopus (103) Google Scholar Murine IAS cells were isolated as described above and transferred into 60 × 15 mm dishes (BD Biosciences, Bedford, MA) containing 3 mL of DMEM medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), gentamycin (100 μg/mL), and amphotericin B (2 μg/mL), and filtered in a 0.22-μm mesh (Millipore, Billerica, MA). DMEM aliquots (0.25 mL) were mixed with 6 μg of plasmid and 12 μL of lipofectamine (Qiagen Inc.) and left undisturbed at room temperature for 15 minutes. Subsequently, the plasmid-containing solution was poured into the DMEM medium containing cells and incubated in a 5% CO2 humidified atmosphere at 37°C for 40 hours. The cells attached to the culture plate were resuspended using 1.5 g/L trypsin-EDTA, centrifuged at 350×g for 10 minutes and resuspended in oxygenated KPS at 37°C. Cells were fixed using acrolein (final concentration 1%) and transferred onto chrome alum-coated glass slides for cell length measurements under phase-contrast microscopy. Cell viability was evaluated by the ability to exclude trypan blue (0.2%). To check transfection efficiency, cells were transferred to Lab-Tek II chamber slides (Nulge, Nunc International, Naperville, IL) and fixed with 4% paraformaldehyde solution in Dulbecco’s phosphate-buffered saline at room temperature for 15 minutes, washed with Dulbecco’s phosphate-buffered saline, air dried, and coverslips were mounted on the slides using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Yellow fluorescence was evaluated with a Bio-Rad MRC 600 laser scanning confocal microscope (Zeiss Anxiovert 100; Zeiss, Overkochen, Germany) at 514 nm at the Bioimaging Facility of the Kimmel Cancer Center (Thomas Jefferson University, Philadelphia, PA). Western blot studies were performed to determine the relative distribution of p-MLC20, pThr696-MYPT1, RhoA, ROCK I, and ROCK II.24Fan Y.-P. Puri R.N. Rattan S. Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action.Am J Physiol Gastrointest Liver Physiol. 2002; 282: G461-G469Crossref PubMed Scopus (24) Google Scholar The IAS smooth muscle tissues isolated from both H-ras+/+ and H-ras−/− mice was cut in small pieces and rapidly homogenized in 5 volumes of boiling lysis buffer (1% SDS, 1.0 mmol/L sodium orthovanadate, and 10 mmol/L Tris, pH 7.4) and then microwaved for 10 seconds. Homogenates were centrifuged (16,000×g; 4°C) for 15 minutes and the protein contents in supernatants were quantified25Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar using BSA as standard. In the case of RhoA, samples were centrifuged at 100,000 × g and immunoprecipitated using RhoA polyclonal antibody and protein G agarose before Western blots for signal intensification. Supernatant and cytosolic precipitates were considered the cytosolic and particulate fractions of cell lysates.26Murthy K.S. Zhou H. Grider J.R. Makhlouf G.M. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of rhoA.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G1006-G1016Crossref PubMed Scopus (117) Google Scholar, 27Harnett K.M. Cao W. Biancani P. Signal-transduction pathways that regulate smooth muscle function Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G407-G416Crossref PubMed Scopus (75) Google Scholar The above protein samples were mixed with 2× sample buffer (125 mmol/L Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, and 2% β-mercaptoethanol) and placed in boiling water for 3 minutes. Proteins in the sample (20 μL containing 40 μg protein) were separated on 7.5% SDS polyacrylamide gels. Following this, proteins were transferred to a nitrocellulose membrane (NCM) by electrophoresis at 4°C. To block nonspecific antibody binding, the NCM was soaked overnight at 4°C in Tris-buffered saline containing Tween (TBS-T; 20 mmol/L Tris pH 7.6, 137 mmol/L NaCl, 0.1% Tween-20) and 1% BSA. The NCM was then incubated with specific primary antibodies (1:200 for RhoA and p-MLC20; 1:500 for ROCK I, ROCK II; 1:1,000 for pThr696-MYPT1) for 1 hour at room temperature. After washing with TBS-T, NCMs were incubated with horseradish peroxidase-labeled secondary antibody (1:10,000) for 1 hour at room temperature. The corresponding bands were visualized with the SuperSignal, West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and Hyperfilm MP (Amersham Bioscience, Corp, Piscataway, NJ). At this stage, NCMs were stripped of secondary and primary antibodies by incubating with Restore Western Blot Stripping Buffer (Pierce, Rockford, IL) for 5 minutes at room temperature. NCMs were soaked again overnight at 4°C in TBS-T and immunoblots for α-actin were obtained using specific primary (1:10,000) and secondary (1:20,000) antibodies as described previously. Specifically for p-MLC20 and pThr696-MYPT1, immunoblots for unphosphorylated myosin light chain (MLC)20 and MYPT1 were obtained instead of α-actin. Bands corresponding to different proteins on X-ray films were scanned (SnapScn.310; Agfa, Ridgefield Park, NJ) and the respective areas and optical densities determined by using Image-Pro Plus 4.0 software (Media Cybernetics). Integrated Optical Densities (IODs) were calculated for protein bands and results expressed as the ratio of IODs for protein bands relative to IODs for their respective α-actin or unphosphorylated MLC20 and MYPT1 bands. Immunocytochemistry of isolated cells was performed by the indirect immunofluorescence method.28Battish R. Cao G.Y. Lynn R.B. Chakder S. Rattan S. Heme oxygenase-2 distribution in anorectum: colocalization with neuronal nitric oxide synthase.Am J Physiol Gastrointest Liver Physiol. 2000; 278: G148-G155PubMed Google Scholar To determine the localization of RhoA, IAS cells from H-ras+/+ and H-ras−/− mice were isolated as described previously and fixed with ice-cold 4% paraformaldehyde and 0.2% picric acid in PBS, pH 7.4 for 10 minutes, and then washed in PBS. Cells were rinsed with PBS and incubated in a mixture of 1:200 H-ras antiserum raised in rabbit and 1:200 RhoA antiserum raised in mouse diluted in PBS containing 0.5% BSA and 0.2% Triton X-100 overnight at room temperature in a humid chamber. Cells were then rinsed with PBS and incubated in a mixture of secondary antibodies raised in donkey. H-ras was labeled with secondary antibody conjugated to FITC (at 1:100) in a solution of 2% normal donkey serum and 0.3% Triton X-100 in PBS, whereas RhoA was labeled with secondary antibody conjugated to Texas red (TxR) (at 1:200) in the same solution. The slides were incubated for 60 minutes at room temperature, rinsed with PBS, air dried, and coverslipped with Vectashield (Vector Labs). Cells were observed with fluorescence microscopy using the appropriate filters and photographed by a Leica DC350F Digital Camera (Leica Microsystems Digital Imaging, Cambridge, UK). Some cells were immunostained as described previously but with the primary antibodies removed (controls). Y 27632 was a generous gift from Yoshitomi Pharmaceutical Industries (Osaka, Japan). α-Actin antibody was purchased from Sigma-Aldrich Chemicals (St. Louis, MO). ROCK I and ROCK II antibodies were purchased from BD Biosciences (San Jose, CA). All other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Results are expressed as means ± SE for the number of animals indicated. Y 27632 concentration–response curves were fitted using a nonlinear interactive fitting program (GraphPad Prism 3.00, Graph Pad Software Incorporated, San Diego, CA). Potencies and maximum response were expressed as the pIC50 (negative logarithm of the molar concentration of Y 27632 producing 50% of the maximum inhibitory response) and Imax (maximum inhibition elicited by Y 27632), respectively. Statistical significance was determined by paired or unpaired Student t-test as appropriate. One-way analysis of variance (ANOVA) followed by the Tukey’s Multiple Comparison post hoc test were used to determine the existence of statistical significance among the means of SMC lengths isolated from H-ras+/+ and H-ras−/− strips measured in the basal state and after treatment with Y 27632. A P value <.05 was considered statistically significant. Breeding between heterozygous (H-ras+/−) mice produced offsprings with wild-type (H-ras+/+), and heterozygous (H-ras+/−) and homozygous (H-ras−/−; H-ras knockout) mutant genotypes (Figure 1A), which did not exhibit differences in anatomy, physiology, or fertility, confirming that H-ras is not required for normal development in mice.29Esteban L.M. Vicario-Abejon C. Fernandez-Salguero P. Fernandez-Medarde A. Swaminathan N. Yiennger K. Lopez E. Malumbres M. McKay R. Ward J.M. Pellicer A. Santos E. Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensibility of both loci and mouse growth and development.Mol Cell Biol. 2001; 21: 1444-1452Crossref PubMed Scopus (248) Google Scholar IAS strips from H-ras+/+ mice developed lower spontaneous basal tone (0.033 ± 0.005 g/mg; n = 5) compared with H-ras−/− mice (0.048 ± 0.004 g/mg; n = 7; P < .05; Figure 1B). The Rho kinase inhibitor Y 27632, which induced concentration-dependent relaxation in the IAS, was more potent in relaxing the IAS of H-ras+/+, compared with H-ras−/− mice (Figure 1C). Maximum relaxation (88.6% ± 5.0%) was reached at 10 μmol/L of Y 27632 in the IAS of H-ras+/+ mice. In contrast, maximum relaxation (77.7% ± 6.0%) was achieved at 30 μM of Y 27632 in the IAS from H-ras−/− mice. The corresponding pIC50 values for relaxation by Y 27632 in the IAS from H-ras+/+ and H-ras−/− mice were 5.93 ± 0.08 and 5.55 ± 0.06, respectively (Figure 1C; P < .05). The fall in basal IAS tone by Y 27632 was time-dependent and maximal at 21 minutes and 30 minutes in IAS from H-ras+/+ and H-ras−/− mice, respectively (Figure 1D and E). Further, recovery of basal tone in H-ras+/+ and H-ras−/− mice following removal of Y 27632 was 76.53% ± 8.92% and 91.93% ± 7.86%, respectively (Figure 1D and E). SMCs from H-ras+/+ and H-ras−/− IAS strips were isolated and their cell lengths measured. The mean length of IAS SMCs isolated from H-ras+/+ mice was 42.5 ± 3.9 μmol/L (Figure 2), as described previously.30Bitar K.N. Aging and neural control of the GI tract: V Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G1-G7Crossref PubMed Scopus (22) Google Scholar In contrast, the mean length of SMCs isolated from H-ras−/− mice was 15.1% ± 0.94% shorter than the H-ras+/+ group, reflecting increased basal shortening (Figure 2; P < .05). Y 27632 (10 μmol/L) completely reversed (P < .05) basal contraction of IAS SMCs and eliminated the differences in length between cells from H-ras+/+ and H-ras−/− mice (Figure 2B). Enhanced basal shortening in IAS SMC cells from which H-ras expression was eliminated (Figure 3A1, A3), was associated with increased translocation of RhoA from the cytoplasm to the plasma membrane (Figure 3A2, A4, and B). Further, RhoA translocation was associated with a specific increase in the Rho kinase isoform ROCK II, a principal mediator of sustained contraction of smooth muscle,13Rattan S. De Godoy M.A.F. Patel C.A. Rho kinase as a novel molecular therapeutic target for hypertensive internal anal sphincter.Gastroenterology. 2006; 131: 108-116Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar but not in ROCK I (Figure 3C). In addition, activation of RhoA/Rho kinase signaling following elimination of H-ras expression resulted in increased phosphorylation on threonine 696 of MYPT1 (Figure 3D), the canonical post-translational modification of this regulatory subunit that inhibits myosin light-chain phosphatase, promoting sustained contr" @default.
• W1964438177 created "2016-06-24" @default.
• W1964438177 creator A5013279293 @default.
• W1964438177 creator A5040582134 @default.
• W1964438177 creator A5062664304 @default.
• W1964438177 creator A5064742753 @default.
• W1964438177 creator A5079512176 @default.
• W1964438177 creator A5089696151 @default.
• W1964438177 date "2007-04-01" @default.
• W1964438177 modified "2023-09-26" @default.
• W1964438177 title "H-ras Inhibits RhoA/ROCK Leading to a Decrease in the Basal Tone in the Internal Anal Sphincter" @default.
• W1964438177 cites W1520019314 @default.
• W1964438177 cites W1580959101 @default.
• W1964438177 cites W1775749144 @default.
• W1964438177 cites W1852817901 @default.
• W1964438177 cites W1868092669 @default.
• W1964438177 cites W1964158918 @default.
• W1964438177 cites W1964860761 @default.
• W1964438177 cites W1972591285 @default.
• W1964438177 cites W1988699822 @default.
• W1964438177 cites W1996965585 @default.
• W1964438177 cites W2014102108 @default.
• W1964438177 cites W2031692128 @default.
• W1964438177 cites W2034036293 @default.
• W1964438177 cites W2037181830 @default.
• W1964438177 cites W2038982544 @default.
• W1964438177 cites W2048684437 @default.
• W1964438177 cites W2052022100 @default.
• W1964438177 cites W2066602623 @default.
• W1964438177 cites W2083822658 @default.
• W1964438177 cites W2089742414 @default.
• W1964438177 cites W2090442581 @default.
• W1964438177 cites W2097906793 @default.
• W1964438177 cites W2101707171 @default.
• W1964438177 cites W2123450550 @default.
• W1964438177 cites W2134674740 @default.
• W1964438177 cites W2136731748 @default.
• W1964438177 cites W2144804934 @default.
• W1964438177 cites W2276321447 @default.
• W1964438177 cites W2292368642 @default.
• W1964438177 cites W2325577633 @default.
• W1964438177 cites W81352928 @default.
• W1964438177 cites W99794425 @default.
• W1964438177 doi "https://doi.org/10.1053/j.gastro.2007.01.043" @default.
• W1964438177 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17408635" @default.
• W1964438177 hasPublicationYear "2007" @default.
• W1964438177 type Work @default.
• W1964438177 sameAs 1964438177 @default.
• W1964438177 citedByCount "9" @default.
• W1964438177 countsByYear W19644381772013 @default.
• W1964438177 countsByYear W19644381772020 @default.
• W1964438177 countsByYear W19644381772021 @default.
• W1964438177 countsByYear W19644381772022 @default.
• W1964438177 crossrefType "journal-article" @default.
• W1964438177 hasAuthorship W1964438177A5013279293 @default.
• W1964438177 hasAuthorship W1964438177A5040582134 @default.
• W1964438177 hasAuthorship W1964438177A5062664304 @default.
• W1964438177 hasAuthorship W1964438177A5064742753 @default.
• W1964438177 hasAuthorship W1964438177A5079512176 @default.
• W1964438177 hasAuthorship W1964438177A5089696151 @default.
• W1964438177 hasBestOaLocation W19644381771 @default.
• W1964438177 hasConcept C124952713 @default.
• W1964438177 hasConcept C126322002 @default.
• W1964438177 hasConcept C127313418 @default.
• W1964438177 hasConcept C142362112 @default.
• W1964438177 hasConcept C2777093181 @default.
• W1964438177 hasConcept C2778024521 @default.
• W1964438177 hasConcept C2779306644 @default.
• W1964438177 hasConcept C2780035688 @default.
• W1964438177 hasConcept C2780583480 @default.
• W1964438177 hasConcept C2781074409 @default.
• W1964438177 hasConcept C2781102422 @default.
• W1964438177 hasConcept C62478195 @default.
• W1964438177 hasConcept C71924100 @default.
• W1964438177 hasConcept C86803240 @default.
• W1964438177 hasConcept C95444343 @default.
• W1964438177 hasConceptScore W1964438177C124952713 @default.
• W1964438177 hasConceptScore W1964438177C126322002 @default.
• W1964438177 hasConceptScore W1964438177C127313418 @default.
• W1964438177 hasConceptScore W1964438177C142362112 @default.
• W1964438177 hasConceptScore W1964438177C2777093181 @default.
• W1964438177 hasConceptScore W1964438177C2778024521 @default.
• W1964438177 hasConceptScore W1964438177C2779306644 @default.
• W1964438177 hasConceptScore W1964438177C2780035688 @default.
• W1964438177 hasConceptScore W1964438177C2780583480 @default.
• W1964438177 hasConceptScore W1964438177C2781074409 @default.
• W1964438177 hasConceptScore W1964438177C2781102422 @default.
• W1964438177 hasConceptScore W1964438177C62478195 @default.
• W1964438177 hasConceptScore W1964438177C71924100 @default.
• W1964438177 hasConceptScore W1964438177C86803240 @default.
• W1964438177 hasConceptScore W1964438177C95444343 @default.
• W1964438177 hasIssue "4" @default.
• W1964438177 hasLocation W19644381771 @default.
• W1964438177 hasLocation W19644381772 @default.
• W1964438177 hasOpenAccess W1964438177 @default.
• W1964438177 hasPrimaryLocation W19644381771 @default.
• W1964438177 hasRelatedWork W1964438177 @default.
• W1964438177 hasRelatedWork W2053302426 @default.

• next