Internal Anal Sphincter Tone Research Articles

STUDY ON EFFECTIVENESS OF LATERAL ANAL SPHINCTEROTOMY AS AN OUT PATIENT PROCEDURE USING DIATHERMY UNDER LOCAL ANAESTHESIA FOR CHRONIC ANAL FISSURE

Anal fissure is one of the most common surgical disorders of the anal canal, ranking next to hemorrhoids and chronic constipation. The incidence of anal fissure has been increasing since the last two decades due to irregular foot habits as well as improper modern lifestyle habits. The prevalence of anal fissure in general population is probably much more higher than that is seen in clinical practice. This is because ,most of the patients do not seek medical attention or resort to some form of native treatment. In early stages, it can be managed by increasing intake of dietary fibres, stool softeners and Sitz baths .But chronic fissure in ano needs surgical treatment, as the underlying pathology is increased internal anal sphincter tone. Lateral anal sphincterotomy is the procedure of choice, which is usually performed under regional anaesthesia. This procedure can also be performed under local anaesthesia as a day care procedure with similar results. This study aims to determine the feasibility and efficacy of lateral anal sphincterotomy for chronic anal fissure under local anaesthesia using diathermy as an outpatient procedure.

Safety and effectiveness of saving sphincter procedure in the treatment of chronic anal fissure in female patients

IntroductionLateral internal sphincterotomy (LIS) is still the approach of choice for the treatment of chronic anal fissure (CAF) regardless to the internal anal sphincter tone but it is burdened by high risk post-operative faecal incontinence (FI). In female patient there are some anatomical and functional differences of the sphinteric system which make them more at risk of FI and vaginal birth could cause sphinteric lesions affecting the anal continence function. The aim of our study is to evaluate the results of saving sphincter procedure as treatment for female patients affected by CAF.MethodsWe studied 110 female patients affected by CAF undergone fissurectomy and anoplasty with V–Y cutaneous flap advancement associating pharmacological sphincterotomy in patients with hypertonic IAS. The follow up was at least for 2 years. The goals were patient’s complete healing, the evaluation of FI, recurrence rate and manometry parameters.ResultsAll wounds healed within 40 days after surgery. We recorded 8 cases of recurrences 6 healed with medical therapy and 2 with dilatation. We recorded 2 “de novo” temporary and low grade post-operative cases of FI. Post-operative value of MRP were unmodified in patient with normotonic IAS but significantly lower at 12 months follow up as compared with the pre-operative ones in patients with hypertonic IAS; after 24 months from surgery MRP values were within the normal range.ConclusionThe fissurectomy and anoplasty with V–Y cutaneous flap alone or in association with a pharmacological sphincterotomy in patients with hypertonic IAS may represent an effective approach for the treatment of CAF in female patients.

A prospective clinical study of fissure in ano and analysis of various treatment modalities

Background: Fissure-in-ano is a very common problem across the world. The rationale of treating this condition is to reduce the internal anal sphincter tone, thereby relieving the spasm and improving the circulation. This study aims to analyse the various modalities of treatment given for fissure in ano and observe the presentations and tailor the treatment needs of the patient. Methods: This study was done prospectively on 165 patients with fissure in ano in Apollo BGS hospitals Mysore, and patients were treated with Diltiazem gel and Left lateral sphincterotomy when and as needed. Results: 165 patients were prospectively studied, treated with conservative management with Diltiazem gel and left lateral sphincterotomy when and as needed. 134 patients (81.2%) improved and fissure healed and 31 patients (18.8%), whose fissure was not healed were subjected to Left lateral sphincterotomy. Mean time taken for fissure to heal with conservative management was 5.26 weeks and with surgery was 3.41 weeks. Conclusion: Topical Diltiazem gel therapy should be advocated as the first line of treatment and surgery should be reserved for patients with relapse and therapeutic failure of prior pharmacological treatment.

Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations [SCaOs] and asynchronized Ca2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

Fissurectomy and anoplasty in posterior normotensive chronic anal fissure.

Purpose:Anal Fissure (AF) is the second most frequent proctological disease in Italy. Chronic AF (CAF) most common located at the posterior anal commissure (CAPF). CAPF are thought to be associated with hypertonic internal anal sphincter (IAS) but manometric findings showed that a normotonic IAS is present in the 20-40%. Sphincterotomy is often recommended as treatment of choice for CAF independently from IAS tone; nevertheless, this approach appears less logical for CAF with normotonic IAS, as in those cases there’s a higher risk of post-operative anal incontinence. The aim of this study is to evaluate the results of fissurectomy and anoplasty with V-Y cutaneous advancement flap, as treatment for patients suffering from CAPF without hypertonic IAS.Methods:We enrolled 30 patients affected by CAPF without IAS hypertonia. All patients were followed up for 2 years after the surgical procedure, with evaluation of anal continence, recurrence rate and maximum resting pressure, maximum squeeze pressure, ultraslow wave activity.Results:All patients healed within 40 days after surgery. We didn’t observe any “de novo” post-operative anal incontinence cases. We reported 2 cases of recurrences, within 18 months from surgery, all healed after conservative therapy. We didn’t record statistically significant differences in pre- and post-operative manometry findings.Conclusion:At 2 years after the surgical procedure we achieved good results, these evidences shows that sphincter preserving procedures are more suitable for CAPF without hypertonic IAS.

Surgical Sphincter Saving Approach and Topical Nifedipine for Chronic Anal Fissure with Hypertonic Internal Anal Sphincter.

The role of augmented internal anal sphincter (IAS) tone in the genesis of posterior chronic anal fissure (CAPF) is still unknown. Lateral internal sphincterotomy is the most employed surgical procedure, nevertheless it is burdened by high risk post-operative anal incontinence. The aim of our study is to evaluate results of sphincter saving procedure with post-operative pharmacological sphincterotomy for patients affected by CAPF with IAS hypertonia. Methods: We enrolled 30 patients, undergone fissurectomy and anoplasty with V-Y cutaneous flap advancement; all patients received topical administration of nifedipine 0.3% and lidocaine 1.5% ointment-based therapy before and for 15 days after surgery. The primary goal was patient's complete healing and the evaluation of incontinence and recurrence rate; the secondary goal included the evaluation of manometry parameters, symptom relief and complications related to nifedipine and lidocaine administration. Results: All wounds healed within 40 days after surgery. We didn't observe any de novo postoperative anal incontinence case. We reported 2 cases of recurrences, healed after conservative therapy. We didn't report any local complications related to the administration of the ointment therapy; with whom all patients reported a good compliance. Conclusions: Fissurectomy and anoplasty with V-Y cutaneous advancement flap and topical administration of nifedipine and lidocaine, is an effective treatment for CAPF with IAS hypertonia.

BDNF augments rat internal anal sphincter smooth muscle tone via RhoA/ROCK signaling and nonadrenergic noncholinergic relaxation via increased NO release.

Presently, there are no studies examining the neuromodulatory effects of brain-derived neurotropic factor (BDNF) on the basal internal anal sphincter (IAS) tone and nonadrenergic noncholinergic (NANC) relaxation. To examine this, we determined the neuromuscular effects of BDNF on basal IAS smooth muscle tone and the smooth muscle cells (SMCs) and the effects of NANC nerve stimulation before and after high-affinity receptor tyrosine kinase receptor B (TrkB) antagonist K252a. We also investigated the mechanisms underlying BDNF-augmented increase in the IAS tone and NANC relaxation. We found that BDNF-increased IAS tone and SMC contractility were TTX resistant and attenuated by K252a. TrkB-specific agonist 7,8-dihydroxyflavone, similar to BDNF, also produced a concentration-dependent increase in the basal tone, whereas TrkB inhibitors K252a and ANA-12 produced a decrease in the tone. In addition, BDNF produced leftward shifts in the concentration-response curves with U46619 and ANG II (but not with bethanechol and K+ depolarization), and these shifts were reversed by K252a. Effects of Y27632 and Western blot data indicated that the BDNF-induced increase in IAS tone was mediated via RhoA/ROCK. BDNF-augmented NANC relaxation by electrical field stimulation was found to be mediated via the nitric oxide (NO)/soluble guanylate cyclase (sGC) pathway rather than via increased sensitivity to NO. In conclusion, the net effect of BDNF was that it caused an increase in the basal IAS tone via RhoA/ROCK signaling. BDNF also augmented NANC relaxation via NO/sGC. These findings may have relevance to the role of BDNF in the pathophysiology and therapeutic targeting of the IAS-associated rectoanal motility disorders.NEW & NOTEWORTHY These studies for the first time to our knowledge demonstrate that increased levels of brain-derived neurotrophic factor (BDNF; conceivably released from smooth muscle cells and/or the enteric neurons) has two major effects. First, BDNF augments the internal anal sphincter (IAS) tone via tyrosine kinase receptor B/thromboxane A2-receptor, angiotensin II receptor type 1/RhoA/ROCK signaling; and second, it increases nonadrenergic noncholinergic relaxation via nitric oxide/soluble guanylate cyclase. These studies may have relevance in therapeutic targeting in the anorectal motility disorders associated with the IAS.

Downregulation of thromboxane A2 and angiotensin II type 1 receptors associated with aging-related decrease in internal anal sphincter tone

Aging-associated decrease in internal anal sphincter (IAS) tone (AADI) is a major contributor in the rectoanal incontinence (RI). To determine the pathogenesis of AADI, we investigated the effect of aging on GPCR activation and related downstream signaling. We particularly investigated two GPCRs that characterize IAS smooth muscle cells (SMCs): thromboxane A2 and angiotensin II type 1. Two groups of Fischer 344 rats (6-month-old [young group] and 26-month-old [old group]) were employed to determine the GPCR function by isometric contraction, the expressions of GPCRs, and their downstream regulatory signaling proteins (regulator of G-protein signaling 2, RGS2; GPCR Kinase 5, GRK5; and β-arrestin, Arrb2) using RT-PCR, qPCR, and western blot analyses. We used reversible biotinylation to monitor the GPCR trafficking using SMCs. Aging selectively attenuated thromboxane A2 and Ang II-induced IAS contraction. RT-PCR, qPCR, and WB data revealed a significant decrease in the expressions of the GPCRs and increase in the expression of RGS2, GRK5, and Arrb2. The increased GPCR internalization and decreased recycling under aging were validated by reversible biotinylation. We conclude that downregulation of GPCR, accompanied by upregulation of regulatory proteins, plays an important role in receptor desensitization and may be important underlying mechanisms of RI in certain aging patients.

Sphincter-sparing treatment of anal fissure

Surgical treatment of the anal fissure is associated with unreasonably high risks of delayed development of fecal incontinence to gas or liquid stool. Standardized sphincter-preserving therapy, based on the pharmacological reduction of increased internal anal sphincter tone (chemical sphincterotomy) allows to improve significantly the results of the non-surgical approach of treating one of the most common pathology in proctological practice. Our work presents a retrospective analysis of the treatment of 295 patients with anal fissure treated with diltiazem ointment, nifedipine ointment, nitroglycerin ointment and botulinum toxin A. Significant improvement or disappearance of complaints was noted in 84% of patients. The use of botulinum toxin A was successfull in 10 out of 11 patients without the need of surgical intervention. High efficiency (91% of patients) of the sphincter-preserving approach with a significant decrease in the need for aggressive surgical manipulation allows to decrease sphincterotomy rate and reduces the risk of delayed fecal incontinence.

In vivo magnetofection: a novel approach for targeted topical delivery of nucleic acids for rectoanal motility disorders.

In these studies, we developed a novel approach of in vivo magnetofection for localized delivery of nucleic acids such as micro-RNA-139-5p (miR-139-5p; which is known to target Rho kinase2) to the circular smooth muscle layer of the internal anal sphincter (IAS). The IAS tone is known to play a major role in the rectoanal continence via activation of RhoA-associated kinase (RhoA/ROCK2). These studies established an optimized protocol for efficient gene delivery using an assembly of equal volumes of in vivo PolyMag and miR139-5p or anti-miR-139-5p (100 nM each) injected in the circular smooth muscle layer in the pinpointed areas of the rat perianal region and then incubated for 20 min under magnetic field. Magnetofection efficiency was confirmed and analyzed by confocal microscopy of FITC-tagged siRNA. Using physiological and biochemical approaches, we investigated the effects of miR-139-5p and anti-miR-139-5p on basal intraluminal IAS pressure (IASP), fecal pellet count, IAS tone, agonist-induced contraction, contraction-relaxation kinetics, and RhoA/ROCK2 signaling. Present studies demonstrate that magnetofection-mediated miR-139-5p delivery significantly decreased RhoA/ROCK2, p-MYPT1, and p-MLC20 signaling, leading to decreases in the basal IASP and IAS tone and in rates of contraction and relaxation associated with increase in fecal pellet output. Interestingly, anti-miR-139-5p transfection had opposite effects on these parameters. Collectively, these data demonstrate that magnetofection is a promising novel method of in vivo gene delivery and of nucleotides to the internal anal sphincter for the site-directed and targeted therapy for rectoanal motility disorders. NEW & NOTEWORTHY These studies for the first time demonstrate the success of topical in vivo magnetofection (MF) of nucleic acids using perianal injections. To demonstrate its effectiveness, we used FITC-tagged siRNA via immunofluorescence microcopy and functional and biochemical evidence using miR-139-5p (which is known to target ROCK2). In conclusion, MF allows safe, convenient, efficient, and targeted delivery of oligonucleotides such as siRNAs and microRNAs. These studies have direct therapeutic implications in rectoanal motility disorders especially associated with IAS.

Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone

The present study focused on the role of microRNA-139-5p (miRNA-139-5p) in the regulation of basal tone in internal anal sphincter (IAS). Applying genome-wide miRNA microarrays on the phenotypically distinct smooth muscle cells (SMCs) within the rat anorectrum, we identified miRNA-139-5p as differentially expressed RNA repressor with highest expression in the purely phasic smooth muscle of anococcygeus (ASM) vs. the truly tonic smooth muscle of IAS. This pattern of miRNA-139-5p expression, previously shown to target ROCK2, was validated by target prediction using ingenuity pathway (IPA) and by qPCR analyses. Immunoblotting, immunocytochemistry (ICC), and functional assays using IAS tissues and cells subjected to overexpression/knockdown of miRNA-139-5p confirmed the inverse relationship between miRNA-139-5p and ROCK2 expressions/IAS tone. Overexpression of miRNA-139-5p caused a decrease, while knockdown by anti-miRNA-139-5p caused an increase in the IAS tone; these tissue contractile responses were confirmed by single-cell contraction using magnetic twisting cytometry (MTC). These findings suggest miRNA-139-5p is capable of significantly influencing the phenotypic tonicity in smooth muscle via ROCK2: a lack of tone in ASM may be associated with the suppression of ROCK2 by high expression of miRNA-139-5p, whereas basal IAS tone may be associated with the persistence of ROCK2 due to low expression of miRNA-139-5p.

AB285. SPR-12 Evidence supporting a pivotal role for intramuscular interstitial cells of Cajal in the generation of pacemaker activity, phasic contractions and tone in the internal anal sphincter

ObjectiveA fundamental property of the internal anal sphincter (IAS) is its ability to generate tone. However, the mechanism underlying tone generation is still controversial. In other GI regions, interstitial cells of Cajal (ICC) have been demonstrated to be the pacemaker cells that generate slow waves (SWs). We have shown that SWs are also present in the IAS along with a population of intramuscular ICC (ICC-IM). However, the identity of the cell that generates SWs in the IAS is still uncertain. We hypothesize that SWs are generated by ICC-IM via a rise in intracellular calcium, activation of calcium-activated chloride channels (ANO1) and voltage-dependent L-type calcium channels (CavL). We further propose that SWs play a critical role in tone development in the IAS through the generation and summation of phasic contractile activity.MethodsCell specific protein expression was examined with immunohistochemistry while gene expression was determined with qPCR on whole muscles and cells isolated with FACS. An inducible Cre/loxP technique was used to express a genetically-encoded Ca2+ biosensor (GCaMP3) in a cell-specific manner. Calcium transients were imaged from the IAS of mice expressing GCaMP3 in ICC (Kit-GCaMP3) and smooth muscle cells (SMC; smMHC-GCaMP3) with an Olympus DSU.ResultsDual labeling immunohistochemistry revealed ANO1 expression in ICC but not SMC while gene expression of ANO1 (Ano1) was 26× greater in FACS-sorted ICC than in sorted SMC. In contrast, gene expression of CavL (Cacna1c) was only 2× greater in SMC than in ICC. Calcium transients were visualized in Kit-GCaMP3+ cells in the circular muscle layer of the IAS. Kit-GCaMP3+ cells exhibited rhythmic whole cell Ca2+ transients that occurred at the same frequency as the SWs we have previously recorded from this muscle. The activity of adjacent Kit-GCaMP3+ cells (<200 µm) was synchronized suggesting coupling between ICC-IM. Rhythmic calcium transients in Kit-GCaMP3+ cells were abolished by removal of extracellular calcium, by the CavL blocker nicardipine (1 µM) and the ANO1 blocker CaCCinh-A01 (3–10 µM). SWs and phasic contractions were also abolished by these blockers. Like SWs, Ca2+ transients in both smMHC-GCaMP3+ and Kit-GCaMP3+ cells were greatest in frequency at the distal extremity of the IAS and declined in the proximal direction. However, when the distal edge of the IAS was removed, both SWs and Ca2+ transients persisted in the remaining muscle although there was a reduction in frequency.ConclusionsThis study provides the first direct evidence that ICC-IM are the pacemaker cells of the IAS. The properties of Ca2+ transients in Kit-GCaMP3+ and smMHC-GCaMP3+ cells are commensurate with the electrical and contractile properties of this muscle. The ability of nicardipine and CaCCinh-A01 to abolish tone, SWs and rhythmic calcium transients in Kit-GCaMP3+ cells suggests a causal relationship between these events. The importance of the distal edge of the IAS in regulating phasic contractions in this muscle is underscored by the decline in both SW and Ca2+ transient frequency and amplitude in the proximal direction indicating that like the heart there is a dominant site of pacemaker activity in the IAS. The observation that the greatest frequency of these events is at the distal edge suggests an important function in ensuring fecal continence.Funding Source(s)NIH DK078736

The molecular basis of the genesis of basal tone in internal anal sphincter.

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca2+ channels (VDCCs) or TMEM16A Ca2+-activated Cl− channels significantly changes global cytosolic Ca2+ concentration ([Ca2+]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca2+]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca2+]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.

Bimodal effect of oxidative stress in internal anal sphincter smooth muscle.

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 p(Thr18/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.

Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans

The extracellular signal that triggers activation of rho-associated kinase (RhoA/ROCK), the major molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone, is not known. Using human IAS tissues, we identified the presence of the biosynthetic machineries for angiotensin II (ANG II), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α). These end products of the renin-angiotensin system (RAS) (ANG II) and arachidonic acid (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were studied. A multipronged approach utilizing immunocytochemistry, Western blot analyses, and force measurements was implemented. Additionally, in a systematic analysis of the effects of respective inhibitors along different steps of biosynthesis and those of antagonists, their end products were evaluated either individually or in combination. To further describe the molecular mechanism for the IAS tone via these pathways, we monitored RhoA/ROCK activation and its signal transduction cascade. Data showed characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS compared with the RSM. Additionally, specific inhibition of the arachidonic acid (AA) pathway caused ~80% decrease in the IAS tone, whereas that of RAS lead to ~20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products TXA2, PGF2α, and ANG II, respectively), provide extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS.

Sa1362 Rectal Hyposensitivity and Slow Colonic Transit Are More Common in Patients With Outlet Obstruction by Balloon Expulsion Testing: A Retrospective Cohort Analysis

G A A b st ra ct s along different steps of biosynthesis of these end products, and their antagonists on the basal IAS tone. These agents were given either individually or in certain combinations. Molecular mechanisms for the IAS tone via these pathways were tracked down by monitoring RhoA/ROCK signal transduction cascade. Activation of RhoA/ROCK was monitored using translocation of these molecular proteins in particulate fractions. Results: U46619, a stable analog of TXA2, PGF2α and Ang II produced concentration (10-9 to 10-5 M)-dependent increase in the IAS tone. Interestingly, maximal increase in the IAS tone by U46619 and PGF2α was 98.5 ± 1.5% and 65.0 ± 6.0% maximal respectively, while increase in the IAS tone by Ang II was limited to 19.1 ± 2.8%. These increases in IAS tone were significantly higher than those in the RSM (p < 0.05; n = 6 to 8). Data further showed characteristically higher expression of biosynthetic enzymatic machineries of RAS and AA pathways in the IAS as compared with the RSM. Additionally, specific inhibition of AA pathway caused ~80% decrease in the IAS tone, while that of RAS produced only ~20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in IAS tone via activation of RhoA/ROCK. Conclusion: Activation of arachidonic acid (AA) and renin-angiotensin system (RAS) pathways both increase basal tone in the internal anal sphincter (IAS). The AA pathway is more potent activator of the IAS tone than the RAS pathway. Both the RAS and AA pathways lead to synthesis and release the end products (TXA2, PGF2α, and Ang II, respectively), which in turn activate their respective G-protein-coupled receptors TPR, FPR, and AT1-R. This activation provides extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS. These studies were supported by the NIH grant (RO1DK035385).

Heme oxygenase-1 upregulation modulates tone and fibroelastic properties of internal anal sphincter.

A compromise in the internal anal sphincter (IAS) tone and fibroelastic properties (FEP) plays an important role in rectoanal incontinence. Herein, we examined the effects of heme oxygenase (HO)-1 upregulation on these IAS characteristics in young rats. We determined the effect of HO-1 upregulator hemin on HO-1 mRNA and protein expressions and on basal IAS tone and its FEP before and after HO-1 inhibitor tin protoporphyrin IX. For FEP, we determined the kinetics of the IAS smooth muscle responses, by the velocities of relaxation, and recovery of the IAS tone following 0 Ca(2+) and electrical field stimulation. To characterize the underlying signal transduction for these changes, we determined the effects of hemin on RhoA-associated kinase (RhoA)/Rho kinase (ROCK) II, myosin-binding subunit of myosin light chain phosphatase 1, fibronectin, and elastin expression levels. Hemin increased HO-1 mRNA and protein similar to the increases in the basal tone, and in the FEP of the IAS. Underlying mechanisms in the IAS characteristics are associated with increases in the genetic and translational expressions of RhoA/ROCKII, and elastin. Fibronectin expression levels on the other hand were found to be decreased following HO-1 upregulation. The results of our study show that the hemin/HO-1 system regulates the tone and FEP of IAS. The hemin/HO-1 system thus provides a potential target for the development of new interventions aimed at treatment of gastrointestinal motility disorders, specifically the age-related IAS dysfunction.

Aging-associated oxidative stress leads to decrease in IAS tone via RhoA/ROCK downregulation

Internal anal sphincter (IAS) tone plays an important role in rectoanal incontinence (RI). IAS tone may be compromised during aging, leading to RI in certain patients. We examined the influence of oxidative stress in the aging-associated decrease in IAS tone (AADI). Using adult (4-6 mo old) and aging (24-30 mo old) rats, we determined the effect of oxidative stress on IAS tone and the regulatory RhoA/ROCK signal transduction cascade. We determined the effect of the oxidative stress inducer LY83583, which produces superoxide anions (O2 (·-)), on basal and stimulated IAS tone before and after treatment of intact smooth muscle strips and smooth muscle cells with the O2 (·-) scavenger SOD. Our data showed that AADI was associated with a decrease in RhoA/ROCK expression at the transcriptional and translational levels. Oxidative stress with a LY83583-mediated decrease in IAS tone and relaxation of IAS smooth muscle cells was associated with a decrease in RhoA/ROCK signal transduction, which was reversible by SOD. In addition, LY83583 caused a significant decrease in IAS contraction produced by the RhoA activator and a known RhoA/ROCK agonist, U46619, that was also reversible by SOD. The inhibitory effects of LY83583 and the ROCK inhibitor Y27632 on the U46619-induced increase in IAS tone were similar. We conclude that an increase in oxidative stress plays an important role in AADI in the elderly and may be one of the underlying mechanisms of RI in certain aging patients.

Interstitial Cells of Cajal Modulate the Tone of the Human Internal Anal Sphincter In Vitro

Interstitial cells of Cajal, expressing the proto-oncogene c-kit, have been shown to regulate the spontaneous activity of the gastrointestinal tract. They have been described in the human internal anal sphincter; however, their function is still unclear. We examined the effects of the c-kit tyrosine kinase inhibitor imatinib mesylate on sphincter strips to investigate the function of the interstitial cells. This was a case series study. This was a single-center study conducted at the University of Oxford. Internal anal sphincter strips were collected from 10 patients undergoing abdominoperineal resection or proctectomy and mounted in organ bath. Responses to electrical field stimulation and chemical agents were monitored in the absence of drugs and after the administration of increasing doses of imatinib mesylate. Immunohistochemistry was performed to identify interstitial cells. The role of the interstitial cells in the internal anal sphincter was assessed. Imatinib mesylate significantly reduced the tone and the spontaneous activity of the strips. In the absence of drugs, the tone generated was 147.7 ± 33.0 mg/mg of tissue. Administration of ≥5 μM of imatinib mesylate caused a dose-dependent reduction in the tone. Strips exhibited spontaneous activity characterized by intermittent low-amplitude contractions superimposed on basal tone (135.6 ± 4.6 contractions in 10 minutes). Imatinib mesylate significantly reduced the number of contractions at concentration >5 μM. No differences were observed in the responses to electrical field stimulation, carbachol, or phenylephrine. Immunohistochemistry showed c-kit-positive cells. This study was limited by the relatively small number of patients enrolled and thus the difficulty of finding human tissue for laboratory studies. Our results suggest that the interstitial cells modulate the tone and the spontaneous activity of the internal anal sphincter. This provides a foundation for new approaches to preclinical and clinical research. Moreover, these cells may represent a target for drugs inhibiting the c-kit receptor and provide a new approach for treating anorectal diseases.

A double‐blind, placebo‐controlled, randomised, parallel‐group, dose‐escalating, repeat dose study in healthy volunteers to evaluate the safety, tolerability, pharmacodynamic effects and pharmacokinetics of the once daily rectal application of NRL001 suppositories for 14 days

The 1R,2S stereoisomer of methoxamine hydrochloride, NRL001, is a highly selective α1-adrenoceptor agonist being developed for the local treatment of non-structural faecal incontinence caused by weak internal anal sphincter tone. This study investigated the steady state pharmacokinetics (PK) and safety of 2 g rectal suppositories containing NRL001 in different strengths (7.5, 10, 12.5 or 15 mg). Healthy volunteers aged 18-45 years received 14 daily doses of NRL001 2 g suppositories or matching placebo. In each dose group nine participants received NRL001 and three received placebo. Blood samples to determine NRL001 concentrations were taken on Days 1, 7 and 14. Cardiovascular parameters were collected via electrocardiograms, Holter monitoring (three lead Holter monitor) and vital signs. Forty-eight volunteers were enrolled; 43 completed the study and were included in the PK analysis population. AUC and Cmax broadly increased with increasing dose, Tmax generally occurred between 4.0 and 5.0 h. Although the data did not appear strongly dose proportional, dose proportionality analysis did not provide evidence against dose proportionality as the log(dose) coefficients were not significantly < 1. NRL001 did not accumulate over time for any dose. Increasing NRL001 concentrations were related to changes in vital sign variables, most notably decreased heart rate. The most commonly reported adverse events (AEs) in the active treatment groups were paraesthesia and piloerection. Treatment with NRL001 was generally well tolerated over 14 days once daily dosing and plasma NRL001 did not accumulate over time. Treatment was associated with changes in vital sign variables, most notably decreased heart rate. AEs commonly reported with NRL001 treatment were events indicative of a systemic α-adrenergic effect.