| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
HORMONES AND SIGNALING
Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University, Philadelphia, Pennsylvania
Submitted 22 September 2006 ; accepted in final form 21 January 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
smooth muscle tone; RhoA/ROCK; tonic smooth muscle
RhoA/Rho kinase (ROCK) are critical for the Ca2+ sensitization of 20-kDa myosin regulatory light chain (MLC20) in smooth muscles. Inactive RhoA in the cytoplasm remains as RhoA-GDP complexed with Rho guanine nucleotide dissociation inhibitor (GDI) (23). Guanine nucleotide exchange factors catalyze the exchange of GDP-RhoA-GDI to active RhoA-GTP that associates with plasma membrane. RhoA-GTP binding to Rho binding domain of ROCK leads to autophosphorylation and activation of ROCK (4, 10). Activated ROCK inhibits myosin light chain phosphatase (MLCP) (30).
MLCP causes dephosphorylation of MLC20. MLCP present in the smooth muscle is a heterotrimeric enzyme that consists of a catalytic 38-kDa type 1 protein phosphatase 
isoform (PP1c) and two regulatory subunits, a 110-kDa myosin phosphatase target subunit 1 (MYPT1) and a 20-kDa small regulatory subunit (M20). ROCK-mediated phosphorylation of MYPT1 at the threonine-696 (Thr696) residue is associated with inhibition of MLCP and force (14, 30). ROCK also inhibits the catalytic subunit of MLCP via phosphorylation of CPI-17, an endogenous inhibitory protein of the catalytic subunit of MLCP. Phosphorylated CPI-17 at threonine-38 (Thr38) residue is 
7,000-fold more potent than nonphosphorylated CPI-17 (7). Both ROCK and protein kinase C (PKC) are capable of phosphorylating CPI-17 at Thr38 residue (7, 16, 18). Although the relative contribution of ROCK vs. PKC in the IAS smooth muscle cells (SMC) is not known, we speculate that ROCK-activated inhibition of MLCP in the SMC is primarily responsible for the maintenance of basal tone in the IAS.
In the present study using freshly isolated SMC from the IAS, we determined cellular distribution of RhoA/ROCK, levels of RhoA-GTP and RhoA-RhoGDI complex formation, and SMC relaxation following C3 exoenzyme (RhoA inhibitor). We also determined the effects of Y 27632 (ROCK inhibitor), Gö 6850 (PKC inhibitor), C3 exoenzyme (RhoA inhibitor), active ROCK-II, and active PKC on the cell lengths of SMC. In addition, we performed Western immunoblottings to determine the levels of RhoA, ROCK-II (primary isoform of ROCK in smooth muscle cells), MYPT1, and MLC20 in rat SMC isolated from IAS vs. rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 x 7 mm) of the IAS and RSM were prepared (26). Likewise, the smooth muscle strips of the ASM were prepared (6). The experimental protocol 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.
SMC were isolated from IAS, RSM, and ASM by sequential enzymatic digestion, filtration, and centrifugation as described previously (6). Briefly, the smooth muscle tissues were cut into 0.2 x 0.2-mm blocks and incubated in KPS containing 0.1% collagenase and 0.01% trypsin inhibitor. The partly digested tissues were washed, and SMC were allowed to disperse spontaneously for 30 min. Cells were harvested by filtration through 500-µm Nitex mesh and centrifuged twice at 350 g for 10 min. SMC were incubated in 10-cm plates in DMEM containing 5% penicillin-streptomycin, 50 µg/ml gentamycin, and 2 µg/ml amphotericin B at 37°C with 5% CO2 for 24 h.
Later, DMEM was removed and homogenization buffer (1% SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, pH7.4) was added to the SMC. The mixture was homogenized with a homogenizer 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. (20) using BSA as a standard (Pierce, Rockford, IL).
Measurement of cell lengths. At the end of incubation period, culture medium was removed from the plates and SMC were incubated for 5–10 min with 1 ml of 0.05% trypsin-EDTA. SMC were then centrifuged and washed twice with fresh KPS and then resuspended in KPS, and 104 SMC suspended in 100 µl of KPS were treated with 20 µl of a solution containing test agents. The reaction was stopped at 5–10 min by the addition of acrolein (1% final concentration). Lengths of the individual cells were measured before and after the test agent by computerized image microscopy. The average length of cells in the control state or with a test agent was obtained from 50 cells encountered randomly in successive microscopic fields. The experiments were repeated in at least three animals. Changes in SMC lengths were measured following incubation with C3 exoenzyme (1 to 10 µg/ml), Y 27632 (0.1 to 10 µM), Gö 6850 (10 µM), 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; 100 µM), active ROCK-II (3 to 30 nM), active PKC (30 nM), and bethanechol (100 µM).
Permeabilization of SMC for the introduction of C3 exoenzyme and ROCK-II and PKC. SMC were permeabilized by the method previously used in our laboratory with a few modifications (8). Briefly, SMC were suspended in KPS following 24-h culture as described earlier followed by the centrifugation. KPS was then replaced with cytosolic buffer (20 mM NaCl, 100 mM KCl, 5 mM MgSO4, 0.96 mM NaH2PO4, 25 mM NaHCO3, 1 mM EGTA, 0.48 mM CaCl2, and 1% BSA) containing saponin (75 µg/ml) for 3 min. At the end of incubation, saponin-containing cytosolic buffer was replaced with fresh cytosolic buffer with omission of saponin. The procedure was repeated twice to remove the saponin completely. C3 exoenzyme (1 to 10 µg/ml) was then added to the SMC for 30 min. Control SMC received similar treatments except for the omission of C3 exoenzyme. In some SMC, active ROCK-II (3 to 30 nM) or active PKC (30 nM) was added for 10 min. SMC lengths were measured as described earlier.
Assay for GTP-bound active RhoA.
IAS, RSM, and ASM SMC in the basal state and following C3 exoenzyme (1, 5, or 10 µg/ml) treatments were assayed for GTP-bound RhoA. GTP-bound RhoA was assayed by using Rhotekin (Rho binding domain). The GST-tagged fusion protein corresponding to residues 7–89 of mouse Rhotekin bound to glutathione-agarose (Upstate, Lake Placid, NY) was used to measure selectively active GTP-bound RhoA (22). SMC were lysed in ice-cold lysis buffer (composed of 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 1 mM EDTA, and 2% glycerol) on ice. SMC lysates (100 µg of protein) were incubated with glutathione-agarose slurry of Rhotekin at 4°C for 45 min. The beads were washed three times with the washing buffer (composed of 50 mM Tris·HCl, pH 7.2; 150 mM NaCl; 10 mM MgCl2; 0.1 mM PMSF, and 1% Triton X-100). GTP-bound RhoA was solubilized in Laemmli sample buffer (LSB; with final concentrations 62.5 mM Tris, 1% SDS, 15% glycerol, and 0.005% bromophenol blue, and 2% 
-mercaptoethanol) and analyzed by 15% SDS-PAGE followed by Western blot analysis and chemiluminescence.
Coimmunoprecipitation of RhoA-RhoGDI complexes. SMC in the basal state and following C3 exoenzyme treatments were used for the coimmunoprecipitation studies. SMC were homogenized on ice in lysis buffer (composed of 50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM NaVO4, 25 mM NaF). The lysates were centrifuged (14,000 rpm) for 5 min and supernatants were collected. Protein concentration in resultant supernatants was determined as described above. RhoA-RhoGDI complexes were immunoprecipitated using Roche Diagnostics immunoprecipitation kit (Protein G) (Fisher), and 200 µg of lysate in 250 µl were precleared with 25 µl protein G agarose beads. Precleared lysate was incubated with 1 µg of RhoA rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h (9). Then 25 µl of protein G agarose beads were added and further incubated for overnight to immobilize protein complexes. Agarose beads were centrifuged for 20 s at 10,000 g and washed repeatedly with wash buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate). Later, 50 µl of LSB were added to the beads and placed in a boiling water bath for 5 min. Protein samples were separated by 15% SDS-PAGE and Western blot was performed using RhoGDI antibody.
Confocal microscopy.
SMC were allowed to attach to the bottom of the Lab-Tek II chamber slides (Nulge Nunc International, Naperville, IL) in DMEM with 10% fetal bovine serum, 5% penicillin-streptomycin, 50 µg/ml gentamycin, and 2 µg/ml amphotericin B on at 37°C and 5% CO2 in an incubator with humidity for 24 h. Later, culture medium was removed and SMC were fixed in 4% paraformaldehyde solution in Dulbecco's phosphate-buffered saline (DPBS) at room temperature for 15 min. SMC were washed three times with DPBS and incubated overnight at room temperature in a humid environment with 1:100 dilution of RhoA or ROCK-II primary antibody (raised in rabbit) (Santa Cruz Biotechnology) in DPBS containing 0.2% Triton X-100 and 0.5% BSA. SMC were washed three times with DPBS and incubated with Texas red (TR)-conjugated anti-rabbit secondary antibody (1:200) (Santa Cruz) and fluorescein isothiocyanate (FITC)-conjugated 
-actin monoclonal antibody (1:800) (Sigma) in DPBS with 0.3% Triton X-100 and 2% donkey serum for 1 h. SMC were then washed three times with DPBS and chambers were removed from slides. The slides were air dried and coverslipped with 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). TR was excited at 543 nm with a helium-neon laser and FITC was excited at 488 nm with an argon laser. The fluorophores were detected separately and overlay images were generated automatically by the imaging software.
Cytosolic and particulate fraction collections. SMC were homogenized in ice-cold homogenization buffer (10 mmol/l Tris·HCl, pH 7.5, 5 mmol/l MgCl2, 2 mmol/l EDTA, 250 mmol/l sucrose, and 1 mmol/l dithiothreitol). The homogenates were centrifuged at 100,000 g for 30 min at 4°C (Beckman L8–70M Ultracentrifuge, Fullerton, CA). The supernatants were then transferred to a fresh tube and used as the cytosolic fraction. Pellets containing membrane proteins were resuspended in homogenization buffer containing 1% Triton X-100 and homogenized with a homogenizer. The extract was centrifuged at 800 g for 10 min, and the supernatant was collected as the particulate fraction (9). Protein samples were mixed with LSB and analyzed by 15% SDS-PAGE and chemiluminescence.
Western blot analysis.
Twenty micrograms of proteins in 20 µl of lysate were mixed with 2 x LSB (with final concentrations 62.5 mM Tris, 1% SDS, 15% glycerol, and 0.005% bromophenol blue, and 2% -mercaptoethanol) and placed in a boiling water bath for 5 min. Protein samples were separated by SDS-PAGE (7.5% gel for ROCK-II, MYPT1, and pThr696-MYPT1; 15% gel for RhoA, RhoGDI, MLC20, and pThr18/Ser19-MLC20).
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, RhoGDI, MLC20, and pThr18/Ser19-MLC20 at 100 V for 1 h at 4°C. To block nonspecific antibody binding, the membranes 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 membranes were then incubated with the specific primary antibodies diluted in TBS-T containing 1% milk (1:1,000 for RhoA, RhoGDI, MYPT1, pThr696-MYPT1, MLC20, and pThr18/Ser19-MLC20; 1:20,000 for -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-conjugated secondary antibody (bovine anti-rabbit 1:10,000 for RhoA, RhoGDI, ROCK-II, MYPT1, pThr696-MYPT1, and pThr18/Ser19-MLC20; bovine anti-mouse 1:5,000 for MLC20 and 1:20,000 for -actin). The membranes were washed three times with TBS-T and the corresponding bands were visualized with enhanced chemiluminescence substrate by use of the SuperSignal West Pico Chemiluminescent Substrate (Pierce) and Hyperfilm MP (Amersham Bioscience, Piscataway, NJ). The membranes were stripped of secondary and primary antibodies by incubation with Restore Western blot stripping buffer (Pierce) for 15 min at room temperature. The membranes were reprobed with -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, Silver Spring, MD).
Chemicals and drugs.
-Actin, FITC-conjugated -actin and MLC20 antibodies, C3 exoenzyme, and 8-Br-cGMP were from Sigma (St. Louis, MO); RhoA, RhoGDI, ROCK-II, MYPT1, pThr696-MYPT1, pThr18/Ser19-MLC20, horseradish peroxidase-conjugated secondary antibodies, and TR-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. Y 27632 was purchased from Biomol (Plymouth Meeting, PA). Gö 6850 (bisindolylmaleimide I) was from Tocris Bioscience (Ellisville, MO). Active ROCK-II and PKC were from Upstate.
Data analysis.
Results are expressed as mean densities ± SE from three independent experiments. For the Western blot analysis, mean density and area for the band were calculated by using Image-Pro Plus 4.0 software (Media Cybernetics). Mean density was multiplied with the area to calculate relative density. The relative density for the protein of interest was normalized over -actin densities. For the comparison of ASM, RSM, and IAS SMC the relative densities for IAS were adjusted to 1. One-way ANOVA followed by a Bonferroni post hoc test was used (P < 0.05) to calculate statistical significance.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
Y 27632 produced significantly greater relaxation of the IAS SMC vs. that by Gö 6850 (31.7 ± 3.2 and 16.2 ± 2.3%, respectively) (n = 4; Fig. 3A). The combined effect of Y 27632 and Gö 6850 was not significantly different from that of Y 27632 alone (32.4 ± 3.1%) (P > 0.05; n = 4). PKG activator 8-Br-cGMP (100 µM) produced relaxation of the IAS SMC (32.9 ± 4.1%) that was not significantly different from that by Y 27632 (P > 0.05; n = 4, Fig. 3A).
|
Distribution of RhoA and ROCK-II in the SMC of the IAS vs. the RSM. To determine the cellular distribution of RhoA/ROCK, we performed confocal microscopy studies. The data revealed significantly higher levels of RhoA and ROCK-II toward the periphery of the IAS SMC compared with those of the RSM SMC (Fig. 4, A and B). These findings suggest that RhoA/ROCK are in active form in the basal state of the IAS SMC.
|
|
Expression and cellular distribution of RhoA in SMC of the IAS. The expression of the signal transduction components related to RhoA/ROCK pathway was examined in the cellular protein extracts, by Western blot analysis. Figure 6 illustrates the representative Western blots of RhoA in SMC from functionally diverse tissues.
|
The expression of protein levels in the SMC isolated from different tissues were normalized using -actin. The latter did not differ significantly among the three different SMC types. The RhoA protein expression was the highest in the tonic SMC of the IAS followed by RSM and the least in the ASM (n = 3; Fig. 6B).
Cellular distribution of ROCK-II in the SMC isolated from IAS vs. the ASM and RSM, in the basal state. Cellular distribution of ROCK-II in the basal state was similar to that of the RhoA, higher levels in the membrane and lower in the cytosol in the tonic IAS. The SMC from the RSM and ASM on the other hand demonstrated higher levels of ROCK-II in the cytosolic fraction (n = 3; Fig. 7A). Western blot analysis of total ROCK-II revealed highest levels in the IAS SMC, followed by the RSM and ASM (n = 3; Fig. 7B).
|
|
Levels of phospho-MYPT1 and MLC20 followed a trend similar to those of RhoA and ROCK-II, highest in the IAS followed by the RSM and ASM (n = 3; Fig. 8, C and D). The primary sites of MLC20 phosphorylation are Ser19 and Thr18 (17).
Effects of C3 exoenzyme on RhoA/ROCK activity. Effects of C3 exoenzyme on RhoA activity in the IAS SMC were analyzed via RhoA-GTP levels and RhoA-RhoGDI complex formation. C3 exoenzyme caused increase in the amount of RhoGDI in the complexes pulled down with RhoA antibody. RhoA-GTP levels were reduced in the SMC pretreated with C3 exoenzyme compared with control SMC (n = 3, Fig. 9, A and B). ROCK is the immediate downstream target of RhoA. Activity of ROCK leads to decrease in the phosphorylation of MYPT1 at Thr696 residue (pThr696-MYPT1). C3 exoenzyme concentration dependently reduced the levels of pThr696-MYPT1 (n = 3, Fig. 9C).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
The role of RhoA/ROCK in the SMC was examined by the use of RhoA and ROCK inactivation as well as by the direct introduction of active ROCK-II into the SMC. Kureishi et al. (19) have reported elevation in the levels of p-MLC20 and sustained contraction, independent of the Ca2+-calmodulin-dependent myosin light chain kinase pathway, upon introduction of active ROCK into the Triton X-100-permeabilized phasic smooth muscle. Membrane-associated RhoA-GTP activates its downstream target ROCK (30). Activated ROCK inhibits MLCP and PP1c by the increased phosphorylation of MYPT1 (pThr696-MYPT1) and CPI-17 (pThr38-CPI-17), respectively (31). Inhibition of MLCP reduces MLC20 dephosphorylation, resulting in sustained contraction of the smooth muscle. This concept is supported by the higher levels of total RhoA/ROCK in the tonic SMC of the IAS. Also introduction of active ROCK-II causes further shortening of the IAS SMC.
Studies further demonstrate that actual levels of RhoA-GTP are higher in the IAS SMC and the lowest in the purely phasic SMC. In the resting phase of the SMC a majority of RhoA may be trapped in the cytosol by RhoGDI. RhoGDI captures the prenylated COOH terminus of RhoA in its hydrophobic cavity and inhibits nucleotide exchange from GDP to GTP (23). In addition, a high concentration of GDI may extract RhoA-GTP translocated to membrane and reduce agonist-induced smooth muscle contractions (11). Our studies reveal the highest levels of RhoA-RhoGDI complex formation in phasic SMC and the lowest in tonic IAS SMC. We speculate that such complex formation prevents the formation of RhoA-GTP and in turn the activation of ROCK. In addition, data show distinctly higher levels of RhoA/ROCK at the membrane than the cytosol in the IAS SMC vs. ASM and RSM. Confocal microscopy combined with immunocytochemical staining for RhoA and ROCK-II in the IAS further supports this notion.
Studies further demonstrate lowest levels of MYPT1 in the IAS SMC, moderate in RSM and the highest in the purely phasic ASM SMC, and the trend was reversed in the case of pThr696-MYPT1. We speculate that higher levels of unphosphorylated MYPT1 unleash MLCP in phasic SMC, resulting in low levels of p-MLC20. Conversely, in the IAS SMC higher levels of phospho-MYPT1 (as a result of RhoA/ROCK upregulation) restrict MLCP, resulting in high levels of p-MLC20. ROCK may also inhibit MLCP activity via phosphorylation of CPI-17 (18). It is of interest that CPI-17 by itself inhibits the catalytic subunit of MLCP, but with a lower potency than pThr38-CPI-17 (7). Our preliminary studies reveal higher levels of CPI-17 as well as pThr38-CPI-17 in the IAS SMC vs. those of the ASM. These findings suggest that spontaneous contractions in the IAS SMC are also regulated in part by RhoA/ROCK activation-induced MLCP inhibition via CPI-17.
Further evidence in favor of the RhoA/ROCK pathway in the spontaneous contractions of IAS SMC comes from the use of the RhoA inhibitor C3 exoenzyme. C3 exoenzyme is an ADP-ribose transferase that ribosylates the Asn-41 residue of RhoA, causing inactivation of RhoA and inhibition of Ca2+ sensitization (9). C3 exoenzyme is highly specific and extensively used for inhibiting the RhoA/ROCK pathway upstream (30). ROCK inhibitor Y 27632 reduces pThr696-MYPT1 and completely abolishes the basal tone in the IAS smooth muscle (24). Inhibition of RhoA/ROCK may unleash MLCP, causing rapid dephosphorylation of MLC20, preventing spontaneous contraction in the IAS SMC. In agreement with this concept, C3 exoenzyme causes concentration-dependent increase in the RhoA-RhoGDI binding and decrease in the levels of RhoA-GTP. This results in reduced levels of pThr696-MYPT1 and relaxation of spontaneously contracted IAS SMC that is comparable to the relaxation by ROCK inhibitor (Y 27632). On the other hand, introduction of active ROCK-II into IAS SMC restores spontaneous contractions. In sharp contrast to tonic IAS SMC, RhoA/ROCK inhibitors are relatively ineffective in cells from phasic smooth muscles of RSM and ASM.
Relaxation of the IAS SMC by ROCK inhibitor is significantly greater compared with the PKC inhibitor. Direct introduction of active PKC similar to the earlier studies causes contraction of the IAS SMC (2). The ROCK inhibitor attenuates PKC-induced contraction in the IAS SMC whereas ROCK-II-mediated contraction of the SMC is not affected by the PKC inhibitor. Interestingly, in the vascular smooth muscle it has been shown that contraction caused by PKC is mediated primarily via ROCK activation (15). In addition, Y 27632 blocks active PKC-induced increase in the pThr38-CPI-17 (data not shown). Present results in the SMC suggest that both ROCK and PKC are working on the same signaling pathway but the former may play a larger and direct role in basal IAS tone. The effects of PKC on the other hand, at least in part may be dependent on ROCK activation.
Smooth muscles of the gut represent functionally diverse organ systems characterized either with true tone, e.g., the sphincters, or with the phasic contractions of nonsphincteric regions. Woodsome et al. (32) 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. Systematic studies to compare the levels of RhoA/ROCK and related intracellular machinery in functionally diverse SMC of gut (ranging from truly tonic to the phasic types) in the basal state have not been reported before the present study.
In summary, upregulation of RhoA/ROCK in the SMC play a significant role in the maintenance of spontaneous tone in the IAS and PKC in part mediates its effects via ROCK. Whether this concept also holds for the other sphincteric regions of the GI tract remains to be determined. These data provide important insights into the role of RhoA/ROCK in the pathophysiology and therapeutic approaches in the rectoanal motility disorders especially affected with hypertensive IAS.
|
FOOTNOTES |
|---|
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 |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
implication of the plecktrin homology domain in ROK function using region-specific antibodies. J Biol Chem 277: 12680–12688, 2002.This article has been cited by other articles:
|
|
 |
|
  M. A. F. de Godoy, N. Rattan, and S. Rattan COX-1 vs. COX-2 as a determinant of basal tone in the internal anal sphincter Am J Physiol Gastrointest Liver Physiol, February 1, 2009; 296(2): G219 - G225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. McDonnell, R. Hamilton, M. Fong, S. M. Ward, and K. D. Keef Functional evidence for purinergic inhibitory neuromuscular transmission in the mouse internal anal sphincter Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G1041 - G1051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rattan and C. A. Patel Selectivity of ROCK inhibitors in the spontaneously tonic smooth muscle Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G687 - G693. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
