Tu2003 Rho Kinase is the Major Molecular Pathway for the Basal Tone in Intact Human Internal Anal Sphincter (IAS) Smooth Muscle

2012 ◽  
Vol 142 (5) ◽  
pp. S-898
Author(s):  
Satish C. Rattan ◽  
Jagmohan Singh
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Rho Kinase ◽  
Molecular Pathway ◽  
Basal Tone

Autocrine regulation of internal anal sphincter tone by renin-angiotensin system: comparison with phasic smooth muscle

2005 ◽  
Vol 289 (6) ◽  
pp. G1164-G1175 ◽  
Author(s):  
Márcio A. F. De Godoy ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Renin Angiotensin System ◽  
Control Mechanisms ◽  
Ang Ii ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Autocrine Regulation ◽  
Basal Tone

The myogenic control mechanisms that govern the basal tone in the internal anal sphincter (IAS) are not known. The present studies determined the autocrine regulation of ANG II in the IAS. The studies were performed in the freshly isolated smooth muscle cells (SMC) of the IAS. We determined the presence of ANG II precursor angiotensinogen (Angen), and the enzymes that convert it into ANG II, using functional, molecular biology, and immunocytochemical studies in rats. ANG II levels in the SMC were determined using ELISA. The IAS SMC generate ANG II at a rate severalfold higher than those from the adjoining smooth muscle of rectum (RSM). RT-PCR data show that IAS exclusively expresses significant higher levels of renin, Angen, and angiotensin-converting enzyme (ACE). These data were confirmed using Western blot analyses and immunocytochemistry. In the IAS SMC, H-77 (10 μM; renin inhibitor) and captopril (1 μM; ACE inhibitor) decreased the basal as well as Angen-increased levels of ANG II. The following functional data corroborate the role of renin-angiotensin system (RAS) in the IAS tone. Angen produced concentration-dependent shortening of the IAS SMC that was inhibited by H-77 and captopril. In addition, H-77 or captopril caused a concentration-dependent fall in the IAS tone vs. nontonic tissues. Basal tone in IAS is partially under the autocrine control of cellular RAS evident by the expression of mRNA coding Angen, renin, and ACE and translation to the respective proteins in the SMC.

scholarly journals Successful implantation of physiologically functional bioengineered mouse internal anal sphincter

2010 ◽  
Vol 299 (2) ◽  
pp. G430-G439 ◽  
Author(s):  
Shreya Raghavan ◽  
Eiichi A. Miyasaka ◽  
Mohamed Hashish ◽  
Sita Somara ◽  
Robert R. Gilmont ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Intracellular Signaling ◽  
Internal Anal Sphincter ◽  
Muscle Cells ◽  
Nitric Oxide Donor ◽  
Proof Of Concept ◽  
Smooth Muscle Tissue ◽  
Basal Tone

We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.

Role of phospholipase A2 (group I secreted) in the genesis of basal tone in the internal anal sphincter smooth muscle

2007 ◽  
Vol 293 (5) ◽  
pp. G979-G986 ◽  
Author(s):  
Márcio A. F. de Godoy ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Critical Role ◽  
Smooth Muscles ◽  
Electrical Field Stimulation ◽  
Group I ◽  
Basal Tone ◽  
Bromoenol Lactone

The role of phospholipase A2 (PLA2) in the genesis of basal tone in the internal anal sphincter (IAS) is not known. We determined the effects of PLA2 and inhibitors on the basal tone and intraluminal pressures (IASP) in the rat IAS vs. rectal smooth muscles (RSM). In addition, we determined the correlations between the IAS tone, PLA2 levels, and the actual enzymatic activity. Inhibition of PLA2 by 4-bromophenacyl bromide (universal inhibitor of PLA2) and MJ33 [selective inhibitor of secreted isoform of PLA2 (sPLA2)] caused concentration-dependent decrease in the IAS tone and in the IASP. Maximal decreases in the IAS tone and IASP by 4-bromophenacyl bromide and MJ33 were 58.8 ± 6.9 and 51.5 ± 6.3%, and 66.7 ± 5.1 and 79.8 ± 8.2%, respectively. The sPLA2 inhibitors were ∼100 times more potent in decreasing the IASP than the mean blood pressure. Conversely, the selective inhibitors of the cytosolic and calcium-independent PLA2 arachidonyl trifluoromethyl ketone and bromoenol lactone, respectively, produced no significant effect. The IAS had characteristically higher levels of sPLA2 activity (26.5 ± 4.9 μmol·min−1·ml−1) vs. the RSM (3.2 ± 0.4 μmol·min−1·ml−1), and higher levels of sPLA2 as shown by Western blot and RT-PCR. Interestingly, administration of sPLA2 transformed RSM into the tonic smooth muscle like that of the IAS: it developed basal tone and relaxed in response to the electrical field stimulation. From the present data, we conclude that sPLA2 plays a critical role in the genesis of tone in the IAS. PLA2 inhibitors may provide potential therapeutic target for treating anorectal motility disorders.

scholarly journals COX-1 vs. COX-2 as a determinant of basal tone in the internal anal sphincter

2009 ◽  
Vol 296 (2) ◽  
pp. G219-G225 ◽  
Author(s):  
Márcio A. F. de Godoy ◽  
Neeru Rattan ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Tone ◽  
Smooth Muscles ◽  
Wild Type ◽  
Selective Inhibitors ◽  
Basal Tone ◽  
Cox 2 ◽  
Cox 1

Prostanoids, produced endogenously via cyclooxygenases (COXs), have been implicated in the sustained contraction of different smooth muscles. The two major types of COXs are COX-1 and COX-2. The COX subtype involved in the basal state of the internal anal sphincter (IAS) smooth muscle tone is not known. To identify the COX subtype, we examined the effect of COX-1- and COX-2-selective inhibitors, SC-560 and rofecoxib, respectively, on basal tone in the rat IAS. We also determined the effect of selective deletion of COX-1 and COX-2 genes (COX-1−/− and COX-2−/− mice) on basal tone in murine IAS. Our data show that SC-560 causes significantly more efficacious and potent concentration-dependent decreases in IAS tone than rofecoxib. In support of these data, significantly higher levels of COX-1 than COX-2 mRNA were found in the IAS. In addition, higher levels of COX-1 mRNA and protein were expressed in rat IAS than rectal smooth muscle. In wild-type mice, IAS tone was decreased 41.4 ± 3.4% (mean ± SE) by SC-560 (1 × 10−5 M) and 5.4 ± 2.2% by rofecoxib ( P < 0.05, n = 5). Basal tone was 0.172 ± 0.021 mN//mg in the IAS from wild-type mice and significantly less (0.080 ± 0.015 mN/mg) in the IAS from COX-1−/− mice ( P < 0.05, n = 5). However, basal tone in COX-2−/− mice was not significantly different from that in wild-type mice. We conclude that COX-1-related products contribute significantly to IAS tone.

Mechanism of action of cholera toxin on the opossum internal anal sphincter smooth muscle

1999 ◽  
Vol 277 (1) ◽  
pp. G152-G160 ◽  
Author(s):  
Ya-Ping Fan ◽  
Sushanta Chakder ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Cholera Toxin ◽  
Anal Sphincter ◽  
Mechanism Of Action ◽  
Internal Anal Sphincter ◽  
Inhibitory Action ◽  
Kinase Inhibitor ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Basal Tone

Cholera toxin (CTX), an activator of Gsprotein, is an important pharmacological tool in G protein research. The effect and the mechanism of action of CTX in the gastrointestinal smooth muscle, including the internal anal sphincter (IAS), are not known. The present investigation was carried out to examine the effects of CTX on the signal transduction associated with the adenylate cyclase (AC) pathway on the basal tone of the IAS smooth muscle. CTX caused a prompt and dose-dependent fall in the basal tone of the IAS that was not affected by the neurotoxins TTX and ω-conotoxin or the nitric oxide synthase inhibitor NG-nitro-l-arginine. The cyclooxygenase inhibitor indomethacin, cAMP-dependent protein kinase inhibitor Rp-8-bromoadenosine 3′,5′ cyclic monophosphorothioate inhibited CTX-induced IAS smooth muscle relaxation. Furthermore, CTX caused a concentration-dependent relaxation of the isolated smooth muscle cells (SMC) of the IAS, which was blocked by Gsα antibody (Gsα-Ab). The IAS smooth muscle relaxation was accompanied with an increase in the GTPase activity that was also specifically blocked by Gsα-Ab. We conclude that a major part of the inhibitory action of CTX in the IAS is via the direct response of the SMC that is linked with Gsprotein to the AC pathway. A part of the inhibitory action of CTX on the smooth muscle occurs via the activation of cyclooxygenase pathway. The relative contribution of such actions of CTX in the smooth muscle in the gastrointestinal motility disturbances following cholera infection remains to be determined.

Cellular regulation of basal tone in internal anal sphincter smooth muscle by RhoA/ROCK

2007 ◽  
Vol 292 (6) ◽  
pp. G1747-G1756 ◽  
Author(s):  
Chirag A. Patel ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Light Chain ◽  
Internal Anal Sphincter ◽  
Critical Role ◽  
Active Form ◽  
Rock Inhibitor ◽  
Basal Tone ◽  
C3 Exoenzyme ◽  
Guanine Nucleotide Dissociation Inhibitor

Sustained contractions of smooth muscle cells (SMC) maintain basal tone in the internal anal sphincter (IAS). To examine the molecular bases for the myogenic tone in the IAS, the present studies focused on the role of RhoA/ROCK in the SMC isolated from the IAS vs. the adjoining phasic tissues of the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM) of rat. We also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP, RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex formation, levels of pThr696-MYPT1, and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ROCK were higher at the cell membrane in the IAS SMC compared with those from the RSM and ASM. C3 exoenzyme (RhoA inhibitor) and Y 27632 (ROCK inhibitor) caused a concentration-dependent relaxation of the IAS SMC. In addition, active ROCK-II (primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3 exoenzyme increased RhoA-RhoGDI binding and reduced the levels of RhoA-GTP and pThr696-MYPT1. ROCK inhibitor attenuated PKC-induced contractions in IAS SMC. Conversely, a PKC inhibitor (Gö 6850, which causes partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, active form of RhoA (RhoA-GTP), ROCK-II, 20-kDa myosin regulatory light chain (MLC20), phospho-MYPT1, and phospho-MLC20 in the IAS vs. RSM and ASM SMC. However, the trend was the reverse with the levels of inactive RhoA (GDP-RhoA-RhoGDI complex) and MYPT1. We conclude that RhoA/ROCK play a critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light chain phosphatase.

scholarly journals Bimodal effect of oxidative stress in internal anal sphincter smooth muscle

2015 ◽  
Vol 309 (5) ◽  
pp. G292-G300 ◽  
Author(s):  
Jagmohan Singh ◽  
Sumit Kumar ◽  
Satish Rattan
Keyword(s):  
Oxidative Stress ◽  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Rho Kinase ◽  
Inhibitory Effects ◽  
Ly 83583 ◽  
Nanc Relaxation ◽  
Rock Activity ◽  
Nnos Inhibition

Changes in oxidative stress may affect basal tone and relaxation of the internal anal sphincter (IAS) smooth muscle in aging. We examined this issue by investigating the effects of the oxidative stress inducer 6-anilino-5,8-quinolinedione (LY-83583) in basal as well as U-46619-stimulated tone, and nonadrenergic, noncholinergic (NANC) relaxation in rat IAS. LY-83583, which works via generation of reactive oxygen species in living cells, produced a bimodal effect in IAS tone: lower concentrations (0.1 nM to 10 μM) produced a concentration-dependent increase, while higher concentrations (50–100 μM) produced a decrease in IAS tone. An increase in IAS tone by lower concentrations was associated with an increase in RhoA/Rho kinase (ROCK) activity. This was evident by the increase in RhoA/ROCK in the particulate fractions, in ROCK activity, and in the levels of phosphorylated (p) Thr696-myosin phosphatase target subunit 1 and pThr18/Ser19-20-kDa myosin light chain. Conversely, higher concentrations of LY-83583 produced inhibitory effects on RhoA/ROCK. Interestingly, both the excitatory and inhibitory effects of LY-83583 in the IAS were reversed by superoxide dismutase. The excitatory effects of LY-83583 were found to resemble those with neuronal nitric oxide synthase (nNOS) inhibition by l-NNA, since it produced a significant increase in the IAS tone and attenuated NANC relaxation. These effects of LY-83583 and l-NNA were reversible by l-arginine. This suggests the role of nNOS inhibition and RhoA/ROCK activation in the increase in IAS tone by LY-83583. These data have important implications in the pathophysiology and therapeutic targeting of rectoanal disorders, especially associated with IAS dysfunction.

Sign in / Sign up

Export Citation Format

Share Document