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Am J Physiol Gastrointest Liver Physiol 291: G830-G837, 2006. First published June 8, 2006; doi:10.1152/ajpgi.00130.2006
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HORMONES AND SIGNALING

Spontaneously tonic smooth muscle has characteristically higher levels of RhoA/ROK compared with the phasic smooth muscle

Chirag A. Patel and Satish Rattan

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

Submitted 22 March 2006 ; accepted in final form 27 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The internal anal sphincter (IAS) tone is important for the rectoanal continence. The RhoA/Rho kinase (ROK) pathway has been associated with the agonist-induced sustained contraction of the smooth muscle, but its role in the spontaneously tonic smooth muscle is not known. Present studies compared expression of different components of the RhoA/ROK pathway between the IAS (a truly tonic SM), the rectal smooth muscle (RSM) (a mixture of phasic and tonic), and anococcygeus smooth muscle (ASM) (a purely phasic SM) of rat. RT-PCR and Western blot analyses were performed to determine RhoA, ROCK-II, CPI-17, MYPT1, and myosin light-chain 20 (MLC20). Phosphorylated CPI-17 at threonine-38 residue (pThr38-CPI-17), MYPT1 at threonine-696 residue (pThr696-MYPT1), and MLC20 at threonine-18/serine-19 residues (pThr18/Ser19-MLC20) were also determined in the basal state and after pretreatment with the ROK inhibitor Y 27632. In addition, we compared the effect of Y 27632 on the basal isometric tension and ROK activity in the IAS vs. the RSM. Our data show the highest levels of RhoA, ROCK-II, CPI-17, MLC20, and of phospho-MYPT1, -CPI-17, and -MLC20 in the IAS followed by in the RSM and ASM. Conversely, MYPT1 levels were lowest in the IAS and highest in the ASM. In the IAS, Y 27632 caused a concentration-dependent decrease in the basal tone, levels of phospho-MYPT1, -CPI-17, and -MLC20, and ROK activity. We conclude that RhoA/ROK plays a critical role in the basal tone in the IAS by the inhibition of MLC phosphatase via the phosphorylation of MYPT1 and CPI-17.

internal anal sphincter; catalytic subunit of myosin light-chain phosphatase; regulatory targeting subunit of myosin light-chain phosphatase; spontaneous tone; endogenous inhibitory protein of PPIC{delta}

SMOOTH MUSCLES have been broadly classified into tonic and phasic types based on their electrophysiological and mechanical characteristics (9, 33). This classification has been made largely on the bases of agonist-induced contraction of the smooth muscles, an initial contraction being the "phasic" and the latter component of sustained contraction being the "tonic." In this regard, RhoA/Rho kinase (ROK) play an important role in the agonist-induced sustained contraction (21, 28, 35, 36). However, there are no such data on the truly tonic smooth muscles that remain contracted in the basal state (without the addition of any agonist). Classic examples of truly tonic smooth muscles are the lower esophageal sphincter and the internal anal sphincter (IAS) (4, 37). The myogenic tone in the IAS plays a pivotal role in anorectal continence (4, 24, 31).

Phosphorylation of smooth muscle 20-kDa myosin regulatory light chain (pThr18/Ser19-MLC20) and the resultant force is determined by the opposing actions of myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP) (10). Activation of ROK inhibits MLCP (35), which shifts the MLCK/MLCP ratio, resulting in sustained higher levels of pThr18/Ser19-MLC20.

Activation of Ser/Thr kinase ROK by RhoA-GTP is perhaps the most important step in RhoA/ROK-mediated Ca2+ sensitization of MLC20 in the smooth muscle. RhoA-GTP binding to Rho binding domain of ROK leads to autophosphorylation and activation of ROK (3).

MLCP dephosphorylates pThr18/Ser19-MLC20. MLCP is a heterotrimeric enzyme that consists of a catalytic 38-kDa protein phosphatase 1c (catalytic subunit targeting of MLCP) (PP1c), an associated 110- to 130-kDa regulatory subunit of MLCP or myosin-binding subunit of MLCP or myasin-targeting subunit (MYPT1), and a tightly bound 20-kDa subunit (M20) of unknown function. ROK inhibits MLCP primarily by phosphorylation of MYPT1 at the threonine-696 (Thr696) residue (8, 13).

The endogenous inhibitory protein of catalytic subunit of MLCP (CPI-17) (17-kDa peptide) is another potential mediator of Ca2+ sensitization and modulator of MLCP activity. Phosphorylated CPI-17 at Thr-38 residue (pThr38-CPI-17) enhances its potency for inhibiting MLCP. Thiophosphorylated CPI-17 inhibits PP1c ~7,000-fold more efficiently than the nonphosphorylated form (7). CPI-17 was initially recognized as a PKC substrate (7). However, ROK can also lead to pThr38-CPI-17 (15, 16).

ROK modulates smooth muscle contraction independently from Ca2+-calmodulin dependent MLCK (17). Previously, Richards et al. (30) reported radio-labeled ATP incorporation into the MYPT1 with agonist-induced Ca2+ sensitization in adult gizzard preparation. Similarly, other investigators (15, 22, 38) reported functional significance of increased MYPT1 and CPI-17 phosphorylation during agonist-induced force in portal vein, vas deferens, and femoral artery. But the functional status of these phosphoproteins has never been studied in the spontaneously tonic smooth muscle such as the IAS.

The present study uses RT-PCR and immunoblotting techniques to determine the levels of RhoA, ROCK-II (primary isoform of Rho kinase involved in the smooth muscle contraction), CPI-17, MYPT1, and MLC20 in rat IAS vs. the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM). We also determined the levels of pThr38-CPI-17, pThr696-MYPT1, pThr18/Ser19-MLC20, and ROK activity and the basal tone before and after pretreatment with ROK inhibitor Y 27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Male Sprague-Dawley rats (300–350 g) were killed by decapitation, and ASM and the anal canal with an adjacent region of the RSM was quickly removed and transferred to oxygenated (95% O2-5% CO2) Krebs physiological solution (KPS) of the following composition (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose (37°C). Extraneous adventitious blood vessels and skeletal muscle 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 by sharp dissection. Circular smooth muscle strips (~1 x 7 mm) of the IAS (identified as a thickened circular smooth muscle situated at the lowermost part of the anal canal), RSM, and ASM were prepared (26). The experimental protocols of the study were 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.

Measurement of tone and isometric tension. The smooth muscle strips were transferred to 2-ml muscle baths containing oxygenated KPS at 37°C. One end of the strips was anchored at the bottom of the muscle bath, whereas 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 (ADInstruments, Castle Hill, Australia) and recorded using Chart 4.1.2 (AD Instruments). Each smooth muscle strip was initially stretched to a tension of 0.7 g. The muscle strips were then given at least 1 h to equilibrate during which they were washed with KPS every 20 min. For ASM, only smooth muscle strips that responded by contraction to electrical field stimulation (EFS) were used. For IAS, only smooth muscle strips that developed spontaneous tone and responded by relaxation to EFS were used in this study (5, 25, 29). The RSM were characterized by the presence of a low grade tone with the superimposed phasic contractions. After the equilibration period, the smooth muscle strips were treated with different concentrations of Y 27632 (10–9 to 10–5 M).

RNA isolation and RT-PCR analysis. Total RNA was collected from IAS, RSM, and ASM tissues using TRI Reagent, and concentrations were measured as described previously (23). One microgram of RNA was reverse transcribed using the sensi-script RT kit (Qiagen, Valencia, CA), and RhoA, ROCK-II, CPI-17, MYPT1, MLC20, and beta-actin cDNA was amplified using gene specific primers described in Table 1. PCR reactions were carried out with Takara Ex Taq DNA polymerase (Fisher), using an Eppendorf Mastercycler personal (Fisher). The PCR cycle consisted of 94°C for 5 min, 94°C for 30 s (denaturation phase), 60°C for 30 s (annealing phase), and 72°C for 1 min (elongation phase); repeated for 30 cycles; 72°C for 5 min. For beta-actin amplification, annealing temperature was set at 56°C. Amplified DNA was run on 2% agarose gel containing ethidium bromide.


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  		Table 1. Primers used in RT-PCRs for amplification of mRNA in the IAS, RSM, and ASM

 
Tissue lysate preparation and Western blot analyses. While the isometric tension was monitored, the IAS, ASM, and RSM strips were quick-frozen in the basal state and after pretreatment with different concentrations of Y 27632. To freeze the tissues, first the tissue chambers were rapidly lowered, exposing the tissues, and a Wollenberger clamp precooled in liquid N2 was used to snap-freeze the tissues. The frozen tissues were placed in a fresh tube, submerged in liquid N2, and stored at –80°C (27). Later, the respective tissues were cut into small pieces, and homogenization buffer (1% SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, pH 7.4) was added to the tissues in a volume equal to five times the weight. The mixture was homogenized on ice. The homogenates were centrifuged (14,000 rpm) for 5 min, and supernatants were collected. Protein concentration in resultant supernatants was determined by the method of Lowry et al. (18) using BSA as a standard (Pierce). Twenty micrograms of protein in 20 µl of lysates were mixed with 2x Laemmli sample buffer (LSB; with final concentrations of 62.5 mM Tris, 1% SDS, 15% glycerol, 0.005% Bromophenol blue, and 2% beta-mercaptoethanol) and placed in a boiling water bath for 5 min. Protein in the samples were separated by SDS-polyacrylamide gel. (7.5% gel for ROCK-II, MYPT1, and pThr696-MYPT1; 15% gel for RhoA, MLC20, pThr18/Ser19-MLC20, CPI-17, and pThr38-CPI-17).

The separated proteins were electrophoretically transferred onto either a nitrocellulose membrane for ROCK-II, MYPT1, and pThr696-MYPT1 or a polyvinylidene difluoride membrane for RhoA, MLC20, pThr18/Ser19-MLC20, CPI-17, and pThr38-CPI-17 at 100 V for 1 h at 4°C. To block nonspecific antibody binding, the membrane was 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 specific primary antibodies diluted in TBS-T containing 1% milk (1:1,000 for RhoA, ROCK-II, MYPT1, pThr696-MYPT1, MLC20, pThr18/Ser19-MLC20, CPI-17, and pThr38-CPI-17; 1:20,000 for {alpha}-actin) for 1 h at room temperature. After being washed with TBS-T three times (10 min each wash), the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (bovine anti-rabbit 1:10,000 for ROCK-II, MYPT1, and pThr696-MYPT1; bovine anti-goat 1:5,000 for CPI-17 and pThr38-CPI-17; bovine anti-mouse 1:5,000 for RhoA, MLC20, and pThr18/Ser19-MLC20, and 1:20,000 for {alpha}-actin). 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, Rockford, IL) and Hyperfilm MP (Amersham Bioscience, Piscataway, NJ). The membranes were stripped of secondary and primary antibodies by incubating with Restore Western blot stripping buffer (Pierce) for 15 min at room temperature. The membranes were then reprobed with {alpha}-actin antibody. Bands corresponding to different proteins 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).

Tissue lysate preparation for kinase activity assay. While isometric tension was monitored, the smooth muscle strips were quick-frozen in the basal state and following the treatment with different concentrations of Y 27632. The tissues were quickly frozen in liquid N2 and stored at –80°C. Later, the respective tissues were cut into small pieces, and homogenization buffer (50 mM Tris·HCl, pH 7.5; 5 mM EDTA; 10 mM EGTA; 1 mM phenylmethylsulfonyl fluoride; 10 mM benzamide; 0.3% wt/vol 2-mercaptoethanol) was added to the tissues in a volume equal to 5 times the weight. After homogenization, the homogenates were centrifuged (14,000 rpm) for 5 min, and supernatants were collected. Protein concentration in resultant supernatants was determined as described above. Twenty-five micrograms of protein in 10 µl of lysate were used for the kinase assay.

ROK activity assay. ROK activity was determined by immunokinase assay in tissue lysates. 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 [{gamma}-32P]ATP (3,000 Ci/mmol) (Amersham Biosciences) 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 in the basal state and as percentage decrease in activity following Y 27632 as explained before (21, 39)

Chemicals and drugs. Y 27632 was purchased from Biomol (Plymouth Meeting, PA). The following antibodies were used in this study: {alpha}-actin and MLC20 antibodies were from Sigma (St. Louis, MO); RhoA, ROCK-II, MYPT1, pThr696-MYPT1, CPI-17, pThr38-CPI-17, pThr18/Ser19-MLC20, and HRP-conjugated mouse, goat, and rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Data analysis. Values are means ± SE from at least three independent experiments. For the comparison of ASM, RSM, and IAS, the relative densities for IAS were normalized to 1. The changes in phosphorylation were normalized to basal phosphorylation, and basal phosphorylation was plotted as 100%. One-way ANOVA followed by a Bonferroni post hoc test was used (P < 0.05) to calculate statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of RhoA and ROCK-II in the IAS vs. the ASM and RSM in the basal state. The expression of levels of the signal transduction components related to the RhoA/ROK pathway were examined in the tissue mRNA and tissue protein extracts, using RT-PCR and Western blot analyses, respectively. mRNA and protein extracts were obtained from the circular smooth muscle layers of the IAS vs. the RSM and ASM. Figure 1 illustrates the representative RT-PCR and Western blots of RhoA and ROCK-II. The expressions of mRNA transcripts and protein levels in different tissues were normalized using beta-actin and {alpha}-actin, respectively. The expression levels of beta-actin and {alpha}-actin did not differ significantly among the three different smooth muscle types.


Figure 1
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  		Fig. 1. A and B: RT-PCR analyses for RhoA and ROCK-II (primary isoform of Rho kinase involved in the smooth muscle contraction), respectively, of the anococcygeus smooth muscle (ASM), rectal smooth muscle (RSM), and internal anal sphincter (IAS) in the basal state. C and D: Western blot analyses for the relative expression of RhoA and ROCK-II, respectively, in the ASM, RSM, and the IAS in the basal state. The relative expressions of RhoA or ROCK-II mRNA transcripts are calculated in relation to the density of beta-actin and those of the related proteins are calculated in relation to {alpha}-actin. Data show expression of RhoA and ROCK-II in the IAS is highest in the IAS (*P < 0.05) followed by that in the RSM and ASM.

 
The RhoA and ROCK-II mRNA and protein expression were the highest in the tonic smooth muscle of the IAS followed by RSM, with the least in the ASM (n = 3; Fig. 1, A-D).

RT-PCR and immunoblots for MYPT1 and pThr696-MYPT1 in the IAS vs. the ASM and RSM in the basal state. MLCP activity was evaluated via expression of MYPT1, pThr696-MYPT1, CPI-17, and pThr38-CPI-17.

In contrast to RhoA and ROCK-II, the pattern of expression of MYPT1 mRNA transcripts and protein levels was the reverse. The highest levels of MYPT1 were found in the phasic ASM, and the lowest levels were found in the tonic smooth muscle of the IAS, whereas these levels in the RSM lied in between the IAS and ASM (Fig. 2, A and B).


Figure 2
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  		Fig. 2. A: relative expression of MYPT1 [regulatory subunit of myosin light-chain phosphatase (MLCP)] mRNA using RT-PCR. B and C: Western blot (WB) analyses of MYPT1 and of phospho-MYPT1 (pThr696-MYPT1), respectively. The data show the lowest expressions of mRNA transcripts and the proteins of MYPT1 in the IAS (*P < 0.05) followed by that in the RSM and highest in the ASM. The expression of pThr696-MYPT1 on the other hand follows the trend opposite to that of MTPT1 but similar to that with RhoA and ROCK-II, i.e., higher levels in the IAS (*P < 0.05) compared with the other tissues.

 
Conversely, levels of pThr696-MYPT1 were found to follow the pattern similar to that of RhoA/ROCK-II, highest in the IAS followed by the RSM and ASM (Fig. 2C).

RT-PCR and immunoblots for CPI-17 and pThr38-CPI-17 in the IAS vs. the RSM and ASM. CPI-17 mRNA was amplified using gene specific primers. CPI-17 protein expression was determined using anti-CPI-17 antibody, and phospho-CPI-17 was determined using anti-pThr38-CPI-17 antibody. CPI-17 mRNA transcripts and protein levels of unphosphorylated and pThr38-CPI-17 levels were found to be highest in the IAS > RSM > ASM (mRNA levels were normalized over beta-actin, and protein levels were normalized over {alpha}-actin) (n = 3; Fig. 3, A-C). Relative levels of pThr38-CPI-17 (expressed as the ratios of unphosphorylated protein) in the ASM and RSM were 0.15 ± 0.12 and 0.78 ± 0.16, respectively (data adjusted to 1).


Figure 3
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  		Fig. 3. Relative expression of the endogenous inhibitory protein of the catalytic subunit of MLCP (CPI-17) mRNA transcripts (A) and Western blot analyses of CPI-17 (B) and pThr38-CPI-17 (C) in the ASM, RSM, and IAS in the basal state. Levels of CPI-17 and pThr38-CPI-17 are highest in the IAS (*P < 0.05) followed by those in the RSM, with the lowest in the ASM.

 
RT-PCR and immunoblots for MLC20 and pThr18/Ser19-MLC20 in the IAS vs. the RSM and ASM in the basal state. Levels of MLC20 mRNA transcripts (Fig. 4A) and protein levels of MLC20 and pThr18/Ser19-MLC20 (Fig. 4, B and C) were found to be the highest in the tonic smooth muscle of the IAS followed by in the RSM. These levels were found to be the lowest in the purely phasic smooth muscle of the ASM (n = 3; Fig. 4, A-C). These data were normalized with {alpha}-actin. Ratios of pThr18/Ser19-MLC20 vs. MLC20 were 0.14 ± 0.05 (ASM) and 0.81 ± 0.13 (RSM) (data adjusted to 1).


Figure 4
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  		Fig. 4. Relative expression of myosin light-chain 20 (MLC20) mRNA (A) and protein expression of MLC20 (B) and of pThr18/Ser19-MLC20 (C) in the ASM, RSM, and the IAS in the basal state. The trend of these levels was as follows: IAS > RSM > ASM.

 
Influence of ROK inhibitor Y 27632 on the levels of pThr696-MYPT1 and pThr38-CPI-17 in the basal state. To determine the trafficking of RhoA/ROK, we followed two immediate primary downstream targets of ROK, pThr696-MYPT1 and pThr38-CPI-17, before and after pretreatment with different concentrations of Y 27632. Levels of pThr696-MYPT1 following 10–9, 10–8, 10–7, 10–6, and 10–5 M were 94 ± 3%, 69 ± 4%, 31 ± 2%, 17 ± 3%, and 2 ± 2%, respectively, considering basal levels of pThr696-MYPT1 to be 100% (Fig. 5A). Interestingly, a similar pattern of expression was observed in the case of pThr38-CPI-17 and pThr18/Ser19-MLC20 before and after the pretreatment of the preparations with different concentrations of Y 27632 (Fig. 5, B and C, respectively). Further analysis revealed a close relationship between the changes in the phosphoproteins and decrease in the basal tone of the IAS in the presence of different concentrations of Y 27632.


Figure 5
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  		Fig. 5. Effect of ROK inhibitor Y 27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide] (10–9 to 10–5 M) on pThr696-MYPT1 (A), pThr38-CPI-17 (B), and pThr18/Ser19-MLC20 (C) in the IAS smooth muscle. Y 27632 causes concentration-dependent decrease in these phosphoproteins in the IAS (the basal levels were considered as 100%).

 
ROK activity in the IAS, RSM, and ASM in the basal state and in the presence of Y 27632. Actual activity of ROK was the highest in the IAS followed by that in the RSM and the ASM in their basal states (Fig. 6A). Basal ROK activity was significantly lower in the RSM and ASM, 44 ± 7% and 21 ± 1%, respectively, compared with the IAS considered to be 100% (*P < 0.05; n = 3; Fig. 6A).


Figure 6
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  		Fig. 6. A: comparison of the basal activities of RhoA/Rho kinase in the IAS vs. ASM and RSM. Data show the highest levels of ROK activity in the IAS (considered as 100%). Percent decrease in the basal tension (B) vs. ROK activity (C) of the ASM, RSM, and IAS after different concentrations of Y 27632. Data show that at all concentrations of Y 27632 examined, the ROK inhibitor produced a significantly greater decrease in the basal tone and ROK activity (*P < 0.05) in the IAS compared with the other tissues and a close relationship between the decreases in tension and ROK activity.

 
ROK inhibitor was more potent in decreasing tonic activity of IAS than the phasic activity of RSM. Y 27632 in the concentrations of 0.1, 1, and 10 µM decreased the tonic activity of the IAS by 20.9 ± 3%, 50 ± 2%, and 81.2 ± 2%, respectively. In the case of RSM, in the presence of above concentrations of Y 27632, the respective decrease in the RSM activity was 1% ± 1, 9 ± 5%, and 40 ± 4%, respectively (*P < 0.05; n = 3; Fig. 6B). The corresponding decreases in the ROK activity with Y 27632 in the IAS were 36.7 ± 2.0%, 54.2 ± 2.2%, and 75.3 ± 1.0%, respectively (Fig. 6C). In the case of RSM, the corresponding decreases in the ROK activity were only 1 ± 1%, 13 ± 2%, and 13 ± 4%, respectively. Interestingly, the ASM, on the other hand, showed no change in ROK activity following incubation with Y 27632 (Fig. 6C).

Note that the basal levels of the tone and the effects of Y 27632 in the ASM were almost nonexistent because of the relative lack of the tone and ROK activity in the basal state in this purely phasic smooth muscle.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Present studies report characteristically higher levels of RhoA/ROK and the related signal transduction machinery in the truly tonic vs. the phasic smooth muscle. The IAS serves as the prototype of the tonic smooth muscle, since it develops spontaneous tone in the absence of any stimulus. The phasic smooth muscle on the other hand is the ASM that develops no tone and exhibits phasic contraction only in response to EFS (5). The RSM is primarily phasic smooth muscle with an admixture of a low grade tonic activity. The comparison of these tissues provides important clues on the molecular mechanisms for the basal tone in the IAS. Data are consistent with the concept that RhoA/ROK components (responsible for the inhibition of MLCP causing sustained elevated levels of p-MLC20), are highest in the IAS, moderate in the RSM, and lowest in the ASM.

The immediate target of RhoA is ROK, and the most common isoform of ROK involved in the smooth muscle contraction is ROCK-II (also called ROK{alpha}) (8, 34). Activation of ROK inhibits MLCP primarily by the increase in the pThr696-MYPT1 and partly via pThr38-CPI-17 (8, 13, 15). Inhibition of MLCP causes sustained increase in the pThr18/Ser19-MLC20 by minimizing its dephosphorylation. There are ample data in support of this concept for the sustained contraction in different smooth muscles in response to agonists (35). However, presently, there are no such data in the spontaneously tonic smooth muscles such as those of gastrointestinal sphincters (1, 2, 4, 24). It is in this regard that the present studies offer novel information on the role of RhoA/ROK in the spontaneous tone of the IAS. Following the concept detailed in Fig. 7, low levels of RhoA/ROK may unleash MLCP, causing rapid dephosphorylation of p-MLC20, preventing the development of any basal tone, and causing rapid fade of stimulated contraction in the phasic smooth muscles such as the ASM or the esophageal body (5, 11).


Figure 7
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  		Fig. 7. Working model to explain the role of RhoA/ROK in the basal tone of the IAS. The model suggests that the basal tone in the IAS is primarily via the inhibition of dephosphorylation of p-MLC20 via the inactivation of MLCP. Inactivation of MLCP is under the regulation of RhoA/ROK primarily by the phosphorylation of MYPT1 and partly by CPI-17. The role of the 20-kDa subunit of MLCP (M20), however, is not known. PP1c{delta}, protein phosphatase 1c (catalytic subunit of MLCP); CaM/MLCK, calmodulin/myosin light-chain kinase.

 
Our studies show that the IAS is characterized with the molecular apparatus designed to provide sustained levels of p-MLC20 responsible for the basal tone. Accordingly, higher levels of RhoA/ROK and related downstream signal transduction proteins inhibit MLCP. This concept is supported by the higher levels of RhoA and ROCK-II in the tonic smooth muscle of the IAS, at the messenger level using RT-PCR, and at the translational level via Western blot analysis. Conversely, the lowest levels are present in the truly phasic smooth muscle the ASM.

Additional data show the lowest levels of MYPT1 in the IAS smooth muscle and the highest in the purely phasic smooth muscle. This is evident both at the mRNA transcripts levels and at the translational levels. In addition, the data show distinctly higher levels of pThr696-MYPT1 in the IAS, with the reverse to be the case in the ASM. We speculate the lower levels of MYPT1 minimize the dephosphorylation of pThr18/Ser19-MLC20. On the other hand, higher levels of pThr696-MYPT1 maintain elevated levels of the p-MLC20 by the inhibition of MLCP.

Although the primary target of ROK for the inhibition of MLCP is MYPT1, a part of the mechanism resides in the phosphorylation of CPI-17 (15, 16). In addition, CPI-17, even in the unphosphorylated state, is considered to be endogenous inhibitor of catalytic subunit of MLCP, however, with a considerably lower potency than in its phosphorylated form (6, 7, 32). Our studies reveal higher levels of CPI-17 as well as pThr38-CPI-17 in the IAS, moderate levels in the RSM, and the lowest levels in the ASM. It is of interest that Woodsome et al. (38) also reported lower levels of MYPT1 and higher levels of CPI-17 in tonic smooth muscle of the femoral artery in contrast to the phasic smooth muscle of the vas deferens. However, the relationship between the functional status and the levels of these different signal transduction proteins in the tonic vs. phasic smooth muscle in their basal state has not been determined before the present studies.

pThr18/Ser19-MLC20 plays a pivotal role in the smooth muscle contraction, whether in the initial phase or in the sustained component (12, 19, 20). Higher levels of Ca2+CM/MLCK have been shown to be characteristic of the phasic smooth muscle of the esophageal body by Szymanski et al., (37). The authors suggest that increased Ca2+CM and MLCK contents allow a rapid and high amplitude contraction of the smooth muscle followed by the complete relaxation. In the present studies we did not examine differences in the Ca2+CM/MLCK in different smooth muscles. However, significantly higher levels of pThr18/Ser19-MLC20 maintained throughout the recording of the basal tone in the IAS suggest lower rates of dephosphorylation regulated by the inhibition of MLCP either via ROK/MYPT1 and CPI-17/MYPT1.

To further establish the functional significance of the RhoA/ROK pathway in the IAS tone, we investigated the effect of the selective inhibitor of ROK, Y 27632. The inhibitor causes a concentration-dependent fall in the IAS tone and in the pThr696-MYPT1, pThr38-CPI-17, and pThr18/Ser19-MLC20. The highest correlation between the decrease in the IAS tone and in the pThr696-MYPT1 suggests that phosphorylation of MYPT1 is perhaps the most important mechanism for the inhibition of MLCP.

Direct evidence for the role of ROK in the IAS tone, however, comes from the direct correlation between the decreases in the basal IAS tone and in the actual ROK activity in the presence of different concentrations of the ROK inhibitor. These data suggest that basal tone in the IAS is regulated by the RhoA/ROK-mediated MLCP inhibition primarily via direct phosphorylation of MYPT1 and in part via CPI-17 phosphorylation. Relative contribution by the ROK/MYPT1 and CPI-17 in the inhibition of MLCP for the resultant tone in the IAS is not known. Also not known is the extent and nature of any interaction between these two pathways. The present studies were performed in the smooth muscles; however, our preliminary data suggest a similar trend for the distinct molecular mechanisms in the smooth muscle cells isolated from the tonic vs. phasic tissues (data not shown).

The effects of Y 27632 are considered to be selective because of the lower concentrations of the ROK inhibitor used (14) to selectively decrease the above parameters in the IAS. In addition, the studies demonstrate a gradient in the ROK activity in the functionally diverse smooth muscle tissues being tonic on one end and phasic on the other end of the spectrum. The highest levels of ROK activity are present in the IAS, moderate levels are present in the RSM, and the lowest levels are present in the ASM.

In conclusion, RhoA/ROK play a significant role in the maintenance of the basal tone in the IAS, and perhaps in the other smooth muscle sphincters. This study may provide insights into the role of RhoA/ROK in the pathophysiology and therapeutic approaches in the rectoanal motility disorders specifically involving hypertensive IAS. The notion may also apply to the motility disorders characterized by the hypertonicity of the other smooth muscle regions of the gastrointestinal tract.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and by an institutional grant from Thomas Jefferson University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Rattan, Dept. of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson Univ., 1025 Walnut St., Rm. 901, Philadelphia, PA 19107 (e-mail: Satish.Rattan{at}Jefferson.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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