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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

RhoA Prenylation Inhibitor Produces Relaxation of Tonic Smooth Muscle of Internal Anal Sphincter

Division of Gastroenterology and Hepatology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania

Received January 2, 2007; accepted February 20, 2007.

	Abstract

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RhoA prenylation is a critical step for the translocation of RhoA to the membrane and its activation in response to agonist-induced sustained contraction of the smooth muscle. However, the effect and role of RhoA prenylation in the spontaneously tonic smooth muscle, such as internal anal sphincter (IAS), is not known. Present studies determined RhoA prenylation and its association with the basal tone in the IAS before and after the RhoA prenylation inhibitor, geranylgeranyl transferase inhibitor GGTI-297 [N-4-[2(R)-amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(L)-leucine,TFA]. Western blot analyses of cytosolic and membrane fractions determined the effects of RhoA prenylation inhibition on the cellular distribution of the RhoA. Additional studies were performed to determine the relationship between RhoA prenylation and Rho kinase (ROCK) activity. GGTI-297 decreased prenylation of RhoA, decreased ROCK activity, and caused a corresponding fall in the IAS tone. These inhibitory effects following RhoA prenylation blockade were demonstrated to be directly on the spontaneously contracted IAS smooth muscle cells. Western blot analysis revealed high levels of RhoA in the IAS smooth muscle cellular membrane in the basal state, and GGTI-297 shifted the RhoA localization to the cytosol. RhoA prenylation may play an important role in the translocation of RhoA to the smooth muscle cell membrane leading to its activation and for the maintenance of basal tone in the IAS.

Activation of Ser/Thr kinase Rho kinase (ROCK) by GTP·RhoA is a critical step in RhoA-ROCK-mediated Ca²⁺ sensitization of MLC₂₀ in the smooth muscle. RhoA cycles between a biologically inactive GDP-bound state and an active GTP-bound state. Thus, in resting cells, the Rho GDP dissociation inhibitor binds to GDP-RhoA and shifts GDP-RhoA from the membrane to the cytosol (Somlyo and Somlyo, 2003) (Fig. 1). With the agonist stimulation of G protein-coupled receptors, guanine nucleotide exchange factors convert GDP-RhoA to GTP-bound RhoA (active form of RhoA). GTP-bound RhoA associates with the plasma membrane via its prenylated tail and then activates ROCK (Gong et al., 1997).

View larger version (27K): [in this window] [in a new window]   Fig. 1. Schematics of the molecular changes for RhoA prenylation leading to RhoA/ROCK activation responsible for the basal tone in the IAS. GGTase I catalyzes the covalent attachment of the geranylgeranyl group from GGPP to RhoA (RhoA prenylation). RhoA prenylation plays a critical role in the translocation of RhoA to the membrane followed by the activation of RhoA/ROCK. GGTI-297 blocks RhoA prenylation via inhibiting transfer of geranylgeranyl group to RhoA via inhibition of GGTase I.

The purpose of the present study was to determine the role of RhoA prenylation in the activation of RhoA and in the maintenance of IAS tone. We determined changes in the IAS tone in the basal and stimulated (by U-46619) state, before and after prenylation inhibitor GGTI-297 (GGTase I-selective inhibitor). We also determined cellular distribution of RhoA and ROCK activity. For comparison, we used phasic rectal smooth muscle (RSM).

	Materials and Methods

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Tissue Preparation, Culture, and Drug Treatments. Sprague-Dawley rats (300–350 g) were killed by decapitation. The anal canal with an adjacent region of the rectum was quickly removed and transferred to oxygenated (95% O₂/5% CO₂) Krebs physiological solution of the following composition: 118.07 mM NaCl, 4.69 mM KCl, 2.52 mM CaCl₂, 1.16 mM MgSO₄, 1.01 mM NaH₂PO₄, 25 mM NaHCO₃, and 11.10 mM glucose (37°C). Circular smooth muscle strips (0.5 x 7 mm) of the IAS and the RSM were prepared as explained previously (Rattan and Chakder, 1992).

IAS and RSM strips were cultured in Leibovitz medium (L-15) (Bolz et al., 2000) with 5% penicillin-streptomycin, 50 µg/ml gentamicin, and 2 µg/ml amphotericin B at room temperature (RT) in a sterile environment in tissue culture hood. The strips were incubated with either GGTI-297 (0.01, 0.1, and 1 µM) or GGTI-297 plus 10 µM GGPP. Control set of strips were similarly incubated for 24 h in L-15 medium with the exception of GGTI-297 treatment. The viability of the tissues following 24-h incubation was evident from the development of tone in the IAS and robust responses to U-46619. U-46619 was used since it produces reproducible contraction both in the IAS and RSM.

Measurement of Isometric Tension. Smooth muscle strips prepared above were transferred to 2-ml muscle baths containing oxygenated Krebs physiological solution at 37°C. Isometric tension was measured via force transducers (model FT03; Grass Instruments, Quincy, MA) using the PowerLab/8SP data-acquisition system (AD Instruments, Colorado Springs, CO) using Chart 5 (AD Instruments) (Rattan et al., 2004, 2005; De Godoy and Rattan, 2005). Following 24-h culture, the IAS was characterized by the development of spontaneous tone and relaxation responses to electrical field stimulation. The RSM was included as the phasic tissue for the direct comparison for the role of RhoA prenylation in the phasic versus the tonic smooth muscle of the IAS. The changes in basal IAS tone were expressed as percentage of maximal relaxation by 10 mM EDTA, and increase in IAS tone was expressed as percentage of maximal contraction by 100 µM bethanechol at the end of each experiment (Biancani et al., 1985). 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.

Drug Responses. Concentration-response curves (CRCs) for U-46619 (1 nM to 10 µM) in the IAS smooth muscle strips pretreated for 24 h with GGTI-297 were obtained in a cumulative fashion. The corresponding controls were the responses of the smooth muscle strips incubated for 24 h under the above conditions except for the omission of the pretreatments with GGTI-297.

Immunoprecipitation of Prenylated Proteins and Western Blot Analysis. At the end of incubation tissues were put into liquid N₂ immediately and stored at –80°C. Later, the respective tissues were cut into small pieces, and homogenization buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM NaVO₄, 25 mM NaF) was added in a volume equal to 5 times the weight. The tissue homogenates were centrifuged (14,000 rpm) for 5 min. Protein concentration in the resultant supernatants was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard (Pierce Biotechnology, Inc., Rockford, IL).

Prenylated RhoA was immunoprecipitated using Roche Diagnostics immunoprecipitation kit (Protein G) (Fisher, Allentown, PA). Tissue lysate (200 µg) in 250-µl volume was precleared with 25 µl of protein G agarose beads. Precleared lysate was incubated with 1 µg of anti-farnesyl rabbit polyclonal antibody (Calbiochem, San Diego, CA) for 1 h (Baron et al., 2000). Then, 25 µl of protein G agarose beads was added and further incubated overnight to immobilize prenylated proteins. Agarose beads were centrifuged for 20 s at 10,000g, and supernatants were transferred to a fresh tube for a fraction containing unprenylated proteins. Agarose beads were washed repeatedly with wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate). Later, 50 µl of Lamellae sample buffer (LSB; with final concentrations of 62.5 mM Tris, 1% SDS, 15% glycerol, 0.005% bromphenol blue, and 2% -mercaptoethanol) was added to the beads and placed in a boiling water bath for 5 min. Likewise, 20 µg of proteins from unprenylated fraction and total tissue lysate were mixed with LSB. Protein samples were separated by 15% SDS-PAGE.

The separated proteins were electrophoretically transferred onto a nitrocellulose membrane (NCM) at 100 V for 1 h at 4°C. To block nonspecific antibody binding, the NCMs were soaked overnight at 4°C in Tris-buffered saline with Tween (TBS-T; composed of 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk. The membrane was then incubated with the RhoA primary antibody raised in rabbit 1:1000 diluted in TBS-T containing 1% milk for 1 h at RT. After washing with TBS-T three times (10 min each wash), the membranes were incubated with the horseradish peroxidase-conjugated bovine anti-rabbit secondary antibody (1: 10,000). The membranes were washed three times with TBS-T, and the corresponding bands were visualized with enhanced chemiluminescence substrate using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology Inc.) and Hyperfilm MP (Amersham Biosciences Corp., Piscataway, NJ). Different protein bands on X-ray films were scanned with a scanner (model SNAPSCAN 310; Agfa, Ridgefield Park, NJ), and their relative densities were determined by using Image-Pro Plus 4.0 software (Media Cybernetics, Inc., Silver Spring, MD).

Cytosolic and Particulate Fractionation. The IAS and RSM tissues (both control and incubated with GGTI-297) were homogenized in ice-cold homogenization buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 2 mM EDTA, 250 mM sucrose, and 1 mM dithiothreitol). The homogenates were centrifuged at 100,000g for 30 min at 4°C (Beckman L8–70 M Ultracentrifuge; Beckman Coulter, Fullerton, CA). The supernatants were then transferred to a fresh tube and used as the cytosolic fraction. The pellets were resuspended and homogenized in buffer containing 1% Triton X-100. The pellet extract was centrifuged at 800g for 10 min, and the supernatant was collected as the particulate fraction (Fujihara et al., 1997).

Protein extracts (20 µg) as prepared above were mixed with LSB and separated by 15% SDS-PAGE. The separated proteins were electrophoretically transferred onto an NCM, RhoA Western blots were performed as described before, and bands were captured on X-ray film. NCMs were then stripped of secondary and primary antibodies by incubating with Restore Western blot stripping buffer (Pierce Biotechnology Inc.) for 15 min at RT and then reprobed for -actin as described earlier (De Godoy and Rattan, 2005).

ROCK Activity Assay. At the end of 24-h incubations with GGTI-297, the tissues were put into liquid N₂ immediately and stored at –80°C. Later, the respective tissues were cut into small pieces and homogenized in homogenization buffer (in a volume equal to 5 times the weight). The composition of the homogenization buffer was: 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamide, and 0.3% (w/v) 2-mercaptoethanol. The homogenates were centrifuged (14,000 rpm) for 5 min, and supernatants were collected. Proteins (25 µg) in 10 µl of lysate were used for kinase assay.

The tissue lysates were mixed with 30 µM Long S6 kinase substrate peptide (Upstate, Lake Placid, NY). Kinase assays were initiated by the addition of 10 µCi of [-³²P]ATP (3000 Ci/mmol) (Amersham Biosciences Corp.) and 100 µM ATP, followed by incubation for 10 min at 30°C. [³²P]substrate peptide was absorbed onto P81 Whatman phosphocellulose discs (Fisher), and free radioactivity was removed by repeated washings with 75 mM phosphoric acid. The amount of radioactivity on the discs was measured by liquid scintillation. The results were expressed as percentage of the basal activity in the control IAS tissues (Murthy et al., 2003a,b).

Measurement of Smooth Muscle Cell Lengths. Smooth muscle cells (SMCs) were isolated from the IAS smooth muscle by sequential enzymatic digestion, filtration, and centrifugation as described previously (De Godoy and Rattan, 2005). The cells were cultured in 10-cm plates in DMEM containing 10% fetal bovine serum, 5% penicillin-streptomycin, 50 µg/ml gentamicin, and 2 µg/ml amphotericin B at 37°C with 5% CO₂ until they attained confluence. Cells were further incubated with GGTI-297 (0.01, 0.1, and 1 µM) for 24 h. Control cells were incubated with the vehicle solution only. Individual cell length (in the control state or with a test agent) was measured by computerized image microscopy as described before (De Godoy and Rattan, 2005).

Confocal Microscopy. The SMCs were cultured in DMEM with 10% fetal bovine serum, 5% penicillin-streptomycin, 50 µg/ml gentamicin, and 2 µg/ml amphotericin B on Lab-Tek II chamber slides (Nulge Nunc International, Naperville, IL) at 37°C and 5% CO₂ in an incubator with humidity. At confluence, culture medium was removed, and the cells were fixed in 4% paraformaldehyde solution in DPBS at RT for 15 min. SMCs were washed three times with DPBS and incubated overnight at RT in a humid environment with 1:100 dilution of RhoA primary antibody (raised in rabbit) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) in DPBS containing 0.2% Triton X-100 and 0.5% bovine serum albumin. SMCs were washed three times with DPBS and incubated with Texas red-conjugated anti-rabbit secondary antibody (1:200) (Santa Cruz) and fluorescein isothiocyanate-conjugated -actin monoclonal antibody (1:800) (Sigma Chemical Co., St. Louis, MO).) in DPBS with 0.3% Triton X-100 and 2% donkey serum for 1 h. SMCs were then washed three times with DPBS, and chambers were removed from slides.

The slides were then air-dried and coverslipped using Vectashield mounting medium (Vector Labs, Burlingame, CA). Florescence was analyzed with a Bio-Rad MRC 600 laser scanning confocal microscope (Zeiss Anxiovert 100, Overkochen, Germany). Texas Red was excited at 543 nm with a helium/neon laser, and fluorescein isothiocyanate was excited at 488 nm with an argon laser. The fluorophores were detected separately, and overlay images were generated automatically by the imaging software.

Data Analysis. Data are presented as means ± S.E. for the number of animals indicated. Western blot data were expressed as a ratio of relative densities in particulate over cytosolic fractions. One-way analysis of variance followed by Bonferroni post hoc test was used (p < 0.05) to calculate statistical significance for comparing more than two groups. CRCs were analyzed using two-way analysis of variance.

Drugs and Chemicals. GGTI-297 was purchased from Calbiochem. U-46619 was purchased from Bachem Bioscience Inc. (King of Prussia, PA). RhoA antibody was purchased from Santa Cruz Biotechnology Inc. -Actin antibody and GGPP were obtained from Sigma Chemical Co. L-15 and DMEM were from Fisher.

	Results

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Effect of GGTI-297 on the Basal Tone of the IAS. Twenty-four-hour pretreatment of the tissues with the prenylation inhibitor GGTI-297 caused a concentration-dependent decrease in the basal tone of the IAS. The prenylation inhibitor works by the inhibition of GGTase I and thus blocks the geranylgeranylation of RhoA. GGTI-297 (1 µM) caused 71 ± 4.6% inhibition in the basal IAS tone (*, p < 0.5; n = 4; Fig. 2A). Ten minutes of pretreatment of the tissues with the inhibitor, however, produced no change in the basal tone. The GGTase substrate GGPP (10 µM) caused significant reversal of the IAS tone inhibited by GGTI-297 (*, p < 0.05; n = 3; Fig. 2B). The results suggest RhoA prenylation is important in the basal tone of the IAS.

View larger version (26K): [in this window] [in a new window]   Fig. 2. A, smooth muscles pretreated with GGTI-297 for 24 h (but not 10 min) reveal a concentration-dependent and significant fall in the basal IAS tone (normalized as 100% in control group) (*, p < 0.05; n = 4). The control smooth muscle tissues underwent similar incubation as the pretreated group except that the inhibitor was omitted from the incubation medium. B, fall in the IAS tone caused by GGTI-297 is significantly reversed by GGPP (*, p < 0.05; n = 4).

Effect of GGTI-297 on the U-46619-Induced Contractions of the IAS. Thromboxane A2 analog U-46619 (known to activate RhoA/ROCK; Pang et al., 2005) caused a concentration-dependent increase in the IAS tone (EC_max = 59.9 ± 5.1%, EC₅₀ = 25 nM; n = 6). GGTI-297 (1 µM) significantly reduced the effect of U-46619 on IAS (EC_max = 29 ± 7.2%; EC₅₀ = 2.1 µM; n = 4; Fig. 3A). The inhibitory effect of GGTI-297 on U-46619 was significantly reversed by GGPP (10 µM) (n = 6; Fig. 3B). Data suggest that not only the basal tone but also the increase in the IAS tone by the agonist are mediated via RhoA prenylation.

View larger version (19K): [in this window] [in a new window]   Fig. 3. A, comparison of CRC with thromboxane A2 analog U-46619 (1 nM to 10 µM) in the IAS before and after 24-h pretreatment with 0.01, 0.1, and 1 µM GGTI-297. The RhoA prenylation inhibitor causes a significant rightward shift in the control U-46619 CRC (*, p < 0.05; n = 4). The control CRCs are obtained in the tissues that are incubated under the similar conditions as the pretreated ones with the exception that the inhibitor is omitted. B, inhibition of IAS contraction caused by U-46619 is significantly reversed by GGPP (*, p < 0.05; n = 4).

Effect of GGTI-297 on Actual RhoA Prenylation. To authenticate the inhibitory action of GGTI-297 on RhoA prenylation, we immunoprecipitated prenylated and unprenylated proteins using anti-farnesyl rabbit antibody (Baron et al., 2000). This was followed by the Western blot analysis using RhoA antibody. Anti-farnesyl antibody has been reported to recognize proteins that have been isoprenylated by farnesyltransferase or geranyltransferase. RhoA is geranylgeranylated, rather than farnesylated, by GGTase I (Solski et al., 2002).

Pretreatment of the IAS tissues for 24 h with selective GGTase I inhibitor GGTI-297 reduced the levels of prenylated RhoA. In contrast, the levels of unprenylated RhoA increased following pretreatment with GGTI-297 (Fig. 4A; n = 3). Data suggest the specificity of GGTI-297 (especially in the concentration that caused fall in the IAS tone) in inhibiting RhoA prenylation.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Western blots for the levels of prenylated, unprenylated, and total RhoA in the IAS smooth muscle before (0) and after GGTI-297 (A). B, quantitative data for the prenylated RhoA normalized over total RhoA. The respective tissue lysates (200 µg) were incubated with anti-farnesyl antibody. The prenylated proteins were immobilized and captured on protein G agarose beads. The proteins bound on beads were then denatured in sample buffer and separated by SDS-PAGE, followed by Western blot using RhoA antibody. Densities in control group are adjusted to 100%. Data show a significant decrease in the levels of prenylated RhoA by 0.1 and 1 µM GGTI-297 (*, p < 0.05; n = 3).

View larger version (44K): [in this window] [in a new window]   Fig. 5. RhoA levels in the particulate versus cytosolic fractions of the IAS smooth muscles before (0) and after GGTI-297 (A). Data are also shown with the RSM in the basal state. B, quantitative information on particulate/cytosolic ratios (adjusted to 100% in the control IAS group). Note concentration-dependent decrease (*, p < 0.05; n = 3) in the levels of RhoA in the particulate fractions of the IAS following pretreatment with GGTI-297.

View larger version (31K): [in this window] [in a new window]   Fig. 6. A, effect of GGTI-297 on the levels of RhoA in the particulate versus cytosolic fractions in the RSM. B, quantitative data to show the ratios of densities in particulate/cytosolic fractions of the RSM. Note in the RSM in contrast to the IAS, GGTI-297 causes no significant change in the levels of RhoA (*, p < 0.05; n = 3). Data in the RSM are normalized considering ratios in the control IAS as 100%.

Effect of GGTI-297 on ROCK Activity. The effects of RhoA prenylation inhibition by GGTI-297 on ROCK (the immediate target of RhoA) in the IAS were determined by directly monitoring ROCK activity. GGTI-297 in the concentrations of 0.1 and 1 µM significantly reduced ROCK activity by 34.8 ± 6.3 and 69.3 ± 2.7%, respectively (*, p < 0.05; Fig. 7).

View larger version (21K): [in this window] [in a new window]   Fig. 7. The RhoA prenylation inhibitor GGTI-297 reduces ROCK activity (an immediate downstream target of RhoA) in the IAS. ROCK activity in the control group is adjusted to 100%. GGTI-297 (0.1 and 1 µM) causes a significant decrease in the ROCK activity in the IAS (*, p < 0.05; n = 3). Decreases in the ROCK activity are comparable with the basal tone (see Fig. 1A).

View larger version (12K): [in this window] [in a new window]   Fig. 8. GGTI-297 (24-h pretreatment) causes concentration-dependent relaxation of the SMCs from the IAS (*, p < 0.05; n = 3). On the other hand, the RSM cells show no change (p > 0.05). Percent increase in the cell lengths (in the presence of different concentrations of the RhoA prenylation inhibitor) of the spontaneously contracted SMCs was calculated on the basis of original cell length.

View larger version (36K): [in this window] [in a new window]   Fig. 9. A, confocal microscopy reveals higher levels of RhoA toward the periphery of the SMCs from the IAS in the basal state (b). Pretreatment of the IAS SMC with GGTI-297 causes redistribution of RhoA and relaxation of the SMC (d). B, inhibitor on the other hand had no significant effect in the case of RSM SMC (b and d).

	Discussion

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Present studies show for the first time that inhibition of RhoA prenylation inactivates RhoA/ROCK, leading to the decrease in the basal tone in the IAS. Inhibition of RhoA geranylgeranylation by the GGTase I inhibitor GGTI-297 reduces IAS tone in the basal state as well as its increase following an agonist. GGTI-297-induced attenuation of the IAS tone correlates with the decreases in the RhoA prenylation, translocation of RhoA in the smooth muscle membrane, and in the ROCK activity.

RhoA/ROCK are known to be present in the cytosol of the SMCs, and during sustained phase of the smooth muscle contraction they translocate to the periphery of the cells for the activation of ROCK (Somlyo and Somlyo, 2003). Once activated, ROCK inhibits the myosin-binding subunit of myosin light-chain phosphatase, promoting an increase in the levels of phosphorylated-MLC₂₀ (Kimura et al., 1996; Uehata et al., 1997; Cao et al., 2002; Harnett et al., 2005). We speculate that the RhoA/ROCK-mediated increase in phosphorylated-MLC₂₀ is primarily responsible for the maintenance of the basal tone in the IAS (Rattan et al., 2006). This concept is substantiated by the agonist-induced sustained contraction of different smooth muscles (Ito et al., 2004).

Isoprenoids such as farnesylpyrophosphate and GGPPs are produced during the cholesterol biosynthesis pathway (Rikitake and Liao, 2005). These pyrophosphates play a significant role in the post-translational modification of Ras and Rho GTPase, respectively. Prenyltransferase GGTase I attaches the geranylgeranyl group from GGPP to the carboxyl-terminal cysteine of RhoA (RhoA prenylation). In agreement with this concept, as illustrated in Fig. 1, GGTI-297 inhibits RhoA geranylgeranylation by inhibiting prenyltransferase GGTase I (Sun et al., 1998; Allai et al., 2000). The GGTase I inhibitor GGTI-298 has been reported to attenuate agonist-induced contraction of vascular smooth muscles (Shiga et al., 2005). However, none of the earlier studies used truly tonic smooth muscles that remain contracted spontaneously.

Long incubation (24 h) with GGTI-297 for the inhibition of RhoA prenylation in the IAS is in agreement with the slow turnover of isoprenylation (Shiga et al., 2005). Following such regimen of pretreatment, GGTI-297 reduces IAS tone and its increase by U-46619. The inhibitory effects of GGTI-297 on IAS are reversible by the GGTase I substrate GGPP. We speculate the role of RhoA prenylation in the IAS tone in the basal state as well as its increase following U-46619. A decrease in IAS tone by the GGTase I inhibitor, GGTI-297, correlates directly with the decrease in the levels of prenylated RhoA.

Different G proteins and GTPases like the Ras, Rap, and Rab family of proteins may be isoprenylated via either farnesylation or geranylgeranylation (Van Aelst and D'Souza-Schorey, 1997). Present studies focused primarily on the role of isoprenylation of RhoA critically involved in the sustained contraction of different smooth muscles (Somlyo and Somlyo, 2003). Dependence of basal tone in the IAS smooth muscle on RhoA signal transduction pathway (Rattan et al., 2006) further supports this notion. Additionally, in contrast to RhoA, prenylation of some of the above-mentioned signal transduction proteins is dependent on farnesylation, which is not prone to inhibition by GGTI-297 (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998; Solski et al., 2002). Present data provide additional support by the significant correlation between the basal tone in the IAS as well as agonist-induced increase and the levels of RhoA prenylation before and after RhoA prenylation inhibitor. In addition, the appropriate substrate GGPP reverses these decreases in the IAS tone caused by the RhoA prenylation inhibitor.

In accordance with the above concept, we observed a significant shift in the levels of RhoA from the membrane to the cytosol in the presence of the GGTase I inhibitor. These data suggest dependence of basal tone on RhoA translocation, which in turn is dependent on RhoA prenylation. It is well known that activation and translocation of RhoA are important processes for the agonist induced Ca²⁺ sensitization and force generation (Gong et al., 1997; Somlyo and Somlyo, 2003). The prenylation status is critical for the translocation of RhoA to the membrane (Adamson et al., 1992). Interestingly, Gong et al. (1996) reported that prenylated GTP·RhoA_val14 Ca²⁺ sensitizes smooth muscle mildly permeabilized with -escin but not with Triton X-100. These data suggest the importance of intact membrane for RhoA translocation. Also, the authors determined that unprenylated GTP·RhoA_val14 fails to produce the above changes in force that are dependent on Ca²⁺ sensitization.

The IAS serves as the prototype of the tonic smooth muscle since it develops spontaneous tone in the absence of any stimulus, whereas RSM is primarily phasic smooth muscle (Rattan, 2005). The present studies show high levels of RhoA at membrane and low levels at the cytosol in the IAS, and vice versa in the RSM. These data suggest higher levels of RhoA prenylation and active RhoA are required in the IAS for maintaining higher activity of its downstream target ROCK. In the presence of the prenylation inhibitor, unprenylated RhoA fails to translocate to the membrane, causing a decrease in the basal tone in the IAS. Conversely, in the RSM, GGTI-297 causes no significant change in the location of RhoA. Consistent with this concept, earlier studies have shown the predominance of GDP dissociation inhibitor-bound RhoA in the phasic tissues (Somlyo and Somlyo, 2000).

The immediate and most important downstream target for RhoA (especially the prenylated form) in the smooth muscle is ROCK. Present data provide further evidence that the inhibitory effects of RhoA prenylation inhibitor in the IAS tone are mediated via ROCK inhibition. Active ROCK is important for the maintenance of IAS tone because the ROCK inhibitor Y 27632 almost completely abolishes IAS tone concomitant with the corresponding changes in ROCK activity (Rattan et al., 2006). In agreement with this concept, present studies show significant correlation for the reduction in ROCK activity and fall in IAS tone in the presence of the prenylation inhibitor.

Although NO is the major inhibitory neurotransmitter for the IAS relaxation (Rattan and Chakder, 1992; Rattan et al., 1992; Rattan, 2005; Terauchi et al., 2005), recent studies have shown that the IAS relaxation by ROCK inhibition is independent of NO production in the smooth muscle (Rattan et al., 2006). The effect of RhoA prenylation inhibitor on the IAS SMC in the present study further demonstrates direct relaxant effect of RhoA/ROCK inactivation on the SMC.

We conclude that RhoA prenylation is essential for membrane localization and downstream signal transduction of RhoA. The studies have important implications in the pathophysiology and on the new concepts in the therapeutic approaches in the rectoanal motility disorders characterized by the hyper- or hypomotility of the sphincteric smooth muscle.

	Footnotes

 
This study was supported by the National Institutes of Diabetes and Digestive and Kidney Diseases (Grant DK-35385) and by an institutional grant from Thomas Jefferson University (Philadelphia, PA).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.119339.

ABBREVIATIONS: IAS, internal anal sphincter; ROCK, Rho kinase; GDP-RhoA, GDP-bound RhoA (inactive form of RhoA); MLC, myosin light chain; GGTase I, geranylgeranyltransferase I; GGPP, geranylgeranyl pyrophosphate; U-46619, 9,11-dideoxy-11,9-methano-epoxy-PGF₂; GGTI-297, [N-4-[2(R)-amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(L)-leucine,TFA]; RSM, rectal smooth muscle; L-15, Leibovitz medium; RT, room temperature; CRC, concentration-response curve; LSB, Lamellae sample buffer; PAGE, polyacrylamide gel electrophoresis; NCM, nitrocellulose membrane; TBS-T, Tris-buffered saline with Tween; SMC, smooth muscle cell; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; Y27632, R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.

Address correspondence to: Dr. Satish Rattan, 901 College, Department of Medicine, Division of Gastroenterology and Hepatology, 1025 Walnut Street, Philadelphia, PA 19107. E-mail: satish.rattan{at}jefferson.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Adamson P, Paterson HF, and Hall A (1992) Intracellular localization of the P21 rho proteins. J Cell Biol 119: 617–627.[Abstract/Free Full Text]

Allai C, Favre G, Couderc B, Salicio S, Sixou S, Hamilton AD, Sebti SM, Lajoie-Mazenc I, and Pradines A (2000) Rho A prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription. J Biol Chem 275: 31001–31008.[Abstract/Free Full Text]

Baron R, Fourcade E, Lajoie-Mazenc I, Allai C, Couderc B, Barbaras R, Favre G, Faye J, and Pradines A (2000) RhoA prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidence in vivo by an anti-farnesyl cysteine antibody. Proc Natl Acad Sci USA 97: 11626–11631.[Abstract/Free Full Text]

Biancani P, Walsh JH, and Behar J (1985) Vasoactive intestinal polypeptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter. Gastroenterology 89: 867–874.[Medline]

Bolz S-S, Piperhoff S, de Wit C, and Pohl U (2000) Intact endothelial and smooth muscle function in small resistance arteries after 48 h in vessel culture. Am J Physiol 279: H1434–H1439.

Cao W, Harnett KM, Behar J, and Biancani P (2002) PGF₂-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle. Am J Physiol 283: G282–G291.

Culver PJ and Rattan S (1986) Genesis of anal canal pressures in the opossum. Am J Physiol 251: G765–G771.[Medline]

De Godoy MAF and Rattan S (2005) Autocrine regulation of internal anal sphincter tone by renin-angiotensin system: comparison with phasic smooth muscle. Am J Physiol 289: G1164–G1175.

Epstein WW, Lever D, Leining LM, Bruenger E, and Rilling HC (1991) Quantitation of prenylcysteines by a selective cleavage reaction. Proc Natl Acad Sci USA 88: 9668–9670.[Abstract/Free Full Text]

Fujihara H, Walker LA, Gong MC, Lemichez E, Bouquet P, Somlyo AV, and Somlyo AP (1997) Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B. Mol Biol Cell 8: 2437–2447.[Abstract/Free Full Text]

Gong MC, Fujihara H, Somlyo AV, and Somlyo AP (1997) Translocation of rhoA associated with Ca²⁺ sensitization of rabbit smooth muscle. J Biol Chem 272: 10704–10709.[Abstract/Free Full Text]

Gong MC, Iizuka K, Nixon G, Browne JP, Hall A, Eccleston JF, Sugai M, Kobayashi S, Somlyo AV, and Somlyo AP (1996) Role of guanine nucleotide-binding proteins ras-family or trimeric proteins or both in Ca²⁺ sensitization of smooth muscle. Proc Natl Acad Sci USA 93: 1340–1345.[Abstract/Free Full Text]

Hall A (1998) Rho GTPases and the actin cytoskeleton. Science (Wash DC) 279: 509–514.[Abstract/Free Full Text]

Harnett KM, Cao W, and Biancani P (2005) Signal-transduction pathways that regulate smooth muscle function: signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles. Am J Physiol 288: G407–G416.

Ito M, Nakano T, Erdodi F, and Hartshorne DJ (2004) Myosin phosphatase: structure, regulation and function. Mol Cell Biochem 259: 197–209.[CrossRef][Medline]

Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, et al. (1996) Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science (Wash DC) 273: 245–248.[Abstract]

Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275.[Free Full Text]

Madoff RD and Fleshman JW (2004) American gastroenterological association technical review on the diagnosis and treatment of hemorrhoids. Gastroenterology 126: 1463–1473.[CrossRef][Medline]

McCallion K and Gardiner KR (2002) Progress in the understanding and treatment of chronic anal fissure. Postgrad Med J 77: 753–758.[CrossRef]

Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, and Makhlouf GM (2003a) Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via G_i3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC₂₀ (20 kDa regulatory light chain myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit and protein kinase C/CPI-17 pathway. Biochem J 374: 145–155.[CrossRef][Medline]

Murthy KS, Zhou H, Grider JR, and Makhlouf GM (2003b) Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of rhoA. Am J Physiol 284: G1006–G1016.

Pang H, Guo ZG, Su W, Eto M, and Gong MC (2005) RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells. Am J Physiol 289: C352–C360.[CrossRef]

Rattan S (2005) The internal anal sphincter: regulation of smooth muscle tone and relaxation. Neurogastroenterol Motil 17: 50–59.[CrossRef][Medline]

Rattan S, Al Haj R, and De Godoy MAF (2004) Mechanism of internal anal sphincter relaxation by CORM-1, authentic CO, and NANC nerve stimulation. Am J Physiol 287: G605–G611.

Rattan S and Chakder S (1992) Role of nitric oxide as a mediator of internal anal sphincter relaxation. Am J Physiol 262: G107–G112.[Medline]

Rattan S, De Godoy MAF, and Patel CA (2006) Rho kinase as a novel molecular therapeutic target for hypertensive internal anal sphincter. Gastroenterology 131: 108–116.[CrossRef][Medline]

Rattan S, Regan RF, Patel CA, and De Godoy MAF (2005) Nitric oxide not carbon monoxide mediates nonadrenergic noncholinergic relaxation in the murine internal anal sphincter. Gastroenterology 129: 1954–1966.[CrossRef][Medline]

Rattan S, Sarkar A, and Chakder S (1992) Nitric oxide pathway in rectoanal inhibitory reflex of opossum internal anal sphincter. Gastroenterology 103: 43–50.[Medline]

Rikitake Y and Liao JK (2005) Rho GTPases, statins, and nitric oxide. Circ Res 97: 1232–1235.[Abstract/Free Full Text]

Schiller LR (2002) Fecal incontinence, in Sleisenger & Fordrtran's Gastrointestinal and Liver Disease (Feldman M ed) pp 164–174, W.B. Saunders Co., Philadelphia, PA.

Shiga N, Hirano K, Hirano M, Nishimura J, Nawata H, and Kanaide H (2005) Long-term inhibition of RhoA attenuates vascular contractility by enhancing endothelial NO production in an intact rabbit mesenteric artery. Circ Res 96: 1014–1021.[Abstract/Free Full Text]

Solski PA, Helms W, Keely PJ, Su L, and Der CJ (2002) RhoA biological activity is dependent on prenylation but independent of specific isoprenoid modification. Cell Growth Differ 13: 363–373.[Abstract/Free Full Text]

Somlyo AP and Somlyo AV (2000) Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol (Lond) 522: 177–185.[Abstract/Free Full Text]

Somlyo AP and Somlyo AV (2003) Ca²⁺ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83: 1325–1358.[Abstract/Free Full Text]

Sun J, Qian Y, Hamilton AD, and Sebti SM (1998) Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth the nude mouse xenografts. Oncogene 16: 1467–1473.[CrossRef][Medline]

Terauchi A, Kobayashi D, and Mashimo H (2005) Distinct roles of constitutive nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation. Am J Physiol 289: G291–G299.

Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, et al. (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature (Lond) 389: 990–994.[CrossRef][Medline]

Van Aelst L and D'Souza-Schorey C (1997) RhoGTPases and signaling network. Genes Dev 11: 2295–2322.[Free Full Text]

Vanderwinden J-M, De Laet M-H, Schiffmann SN, Mailleux P, Lowenstein CJ, Snyder SH, and Vanderhaeghen J-J (1993) Nitric oxide synthase distribution in the enteric nervous system of Hirschsprung's disease. Gastroenterology 105: 969–973.[Medline]

Yokoyama K and Gelb MH (1993) Purification of a mammalian protein geranylgeranyltransferase: formation and catalytic properties of an enzyme-geranylgeranyl pyrophosphate complex. J Biol Chem 268: 4055–4060.[Abstract/Free Full Text]

Yokoyama K, Goodwin GW, Ghomashchi F, Glomset JA, and Gelb MH (1991) A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity. Proc Natl Acad Sci USA 88: 5302–5306.[Abstract/Free Full Text]

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