Anatomy of the external anal sphincter in man

The anatomy of the external anal sphincter was studied in 20 adult human cadavers of both sexes by dissection and histological examination. The external anal sphincter is not naturally divided into layers or laminae. The lowermost muscle bundles differ from the main part in being completely surrounded by thick fibrous tissue septa derived form the longitudinal anal coat. The perineal branch of S4 supplies the posterior third, the inferior haemorrhoidal nerves supply the anterior two thirds. All fibres of the sphincter retain their skeletal attachment to the anococcygeal ligament to the coccyx. Some fibres cross the median plane to be continuous with the transversus perinei of the opposite side. A cleft was displayed separating the lower border of the levator ani muscle from the upper border of the external anal sphincter. The muscle bundles of the sphincter are arranged circumferentially in one continuous circle and not in loops. Fibres from the anterior border of the levator ani muscle extend to cover and blend with the outer surface of the external sphincter in their way to the anococcygeal ligament.     

Function of the anal sphincters following pull-through operations without anal eversion

It has been investigated the function of the anal sphincters following Bacon type pull-through operation. Our data have shown a significant decrease of the anal resting pressure after pull-through excision signifying an impairment in the internal sphincter function. But the tonic external sphincter contraction induced by stimulation can maintain continence.     

In vitro relaxation of rabbit and human internal anal sphincter by rutaecarpine, an alkaloid isolated from Evodia rutaecarpa

Rutaecarpine, a compound extracted from the Chinese medicinal herb Evodia rutaecarpa, has been shown to possess relaxing action on vascular smooth muscle from rat thoracic aorta. The internal anal sphincter is a specialized smooth muscle regulating important anorectal physiology. To investigate the effect and underlying mechanisms of rutaecarpine on internal anal sphincter, muscle strips from rabbit internal anal sphincter were used. The results showed that rutaecarpine (1 x 10(-10) M to 1 x 10(-4) M) produced a concentration-dependent muscular relaxation effect in our preparations, which were precontracted with acetylcholine. This muscular relaxation effect was not affected by treatment with L-N(G)-nitro-arginine methyl ester (a nitric oxide synthase inhibitor), methylene blue (a guanylate cyclase inhibitor), N-ethylmaleimide (an adenylate cyclase inhibitor), or by removal of the mucosa and submucosa tissue. Pretreatment with nifedipine (a calcium channel blocker) or extracellular Ca+2 removal by ethylenediaminetetraacetic acid (EDTA) greatly attenuated the relaxation effect, suggesting that calcium ion might be involved. In experiments using strips from human internal anal sphincter, an even more prominent relaxation effect was shown. It is thus concluded that rutaecarpine caused relaxation on internal anal sphincter from rabbits and human subjects. The relaxation action was not related to NO-cGMP pathway, instead calcium ion might play an important role and shed insight into clinical implications for those anorectal disorders with hyperactive anal tone.     

Mesenchymal stem cells can improve anal pressures after anal sphincter injury

Objective.Fecal incontinence reduces the quality of life of many women but has no long-term cure. Research on mesenchymal stem cell (MSC)-based therapies has shown promising results. The primary aim of this study was to evaluate functional recovery after treatment with MSCs in two animal models of anal sphincter injury.Methods.Seventy virgin female rats received a sphincterotomy (SP) to model episiotomy, a pudendal nerve crush (PNC) to model the nerve injuries of childbirth, a sham SP, or a sham PNC. Anal sphincter pressures and electromyography (EMG) were recorded after injury but before treatment and 10 days after injury. Twenty-four hours after injury, each animal received either 0.2 ml saline or 2 million MSCs labelled with green fluorescing protein (GFP) suspended in 0.2 ml saline, either intravenously (IV) into the tail vein or intramuscularly (IM) into the anal sphincter.Results.MSCs delivered IV after SP resulted in a significant increase in resting anal sphincter pressure and peak pressure, as well as anal sphincter EMG amplitude and frequency 10 days after injury. MSCs delivered IM after SP resulted in a significant increase in resting anal sphincter pressure and anal sphincter EMG frequency but not amplitude. There was no improvement in anal sphincter pressure or EMG with in animals receiving MSCs after PNC. GFP-labelled cells were not found near the external anal sphincter in MSC-treated animals after SP.Conclusion.MSC treatment resulted in significant improvement in anal pressures after SP but not after PNC, suggesting that MSCs could be utilized to facilitate recovery after anal sphincter injury.     

The gonyautoxin 2/3 epimers reduces anal tone when injected in the anal sphincter of healthy adults

The primary clinical symptom of Paralytic Shellfish Poisoning is acute paralytic illness produced by paralyzing toxins. Paralytic shellfish poison is formed by a mixture of phycotoxins and their toxicity is due to its reversible binding to a receptor site on the voltage-gated sodium channel on excitable cells, thus blocking neuronal transmission. We studied the effect of the gonyautoxin 2/3 epimers by local infiltration in the anal internal sphincter of healthy voluntary adults in order to reduce anal tone. The toxin was injected after prior clinical evaluation, anoscopy and anorectal manometry. Post injection clinical examination, electromyography and anorectal manometry were performed. Resting and voluntary contraction pressures were measured and the anorectal inhibitory and anocortical reflexes were tested by manometry. Blood and urine samples were obtained from each participant, and hemogram, basic metabolic panel, and urinalysis were done both before and one week after the injection. This study shows, for the first time, that gonyautoxin 2/3 reduces the anal tone by relaxing the anal sphincters in 100 % of the participants. Manometric recordings showed a significant decrease in anal maximal voluntary contraction pressure after the toxin injection, dropping to 55.2+/-6.2 % and 47.0+/-6.8% (Mean Value+/-Std.Dev.) of the baseline values at 2 minutes and at 24 hours respectively after the injection. Post-injection electromyography showed that activity of the muscle was abolished. We conclude that local administration of gonyautoxin 2/3 to the anal sphincter produces immediate relaxation and a statistically significant decrease in the anal tone (p <0.001).     

Function of the anal sphincters following anterior resections of rectum

The purpose of the present work is to evaluate the function of the anal sphincter following anterior resections of the rectum. Our data have shown that the anorectal reflex did not depend on the presence of the rectal mucosa. The external anal sphincter contraction involves a reflex which is initiated by stretch receptors in the levator ani muscle. The internal anal sphincter relaxation is likely a local reflex involving nerve pathways not yet completely defined.     

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK- and PKC-selective inhibitors Y 27632 and G? 6850 (10(-8) to 10(-4) M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-α, MYPT1, CPI-17, and MLC(20) in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC(20), before and after Y 27632 and G? 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment.     

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.     

Third degree obstetric anal sphincter tears: risk factors and outcome of primary repair.

OBJECTIVES--To determine (i) risk factors in the development of third degree obstetric tears and (ii) the success of primary sphincter repair. DESIGN--(i) Retrospective analysis of obstetric variables in 50 women who had sustained a third degree tear, compared with the remaining 8553 vaginal deliveries during the same period. (ii) Women who had sustained a third degree tear and had primary sphincter repair and control subjects were interviewed and investigated with anal endosonography, anal manometry, and pudendal nerve terminal motor latency measurements. SETTING--Antenatal clinic in teaching hospital in inner London. SUBJECTS--(i) All women (n = 8603) who delivered vaginally over a 31 month period. (ii) 34 women who sustained a third degree tear and 88 matched controls. MAIN OUTCOME MEASURES--Obstetric risk factors, defecatory symptoms, sonographic sphincter defects, and pudendal nerve damage. RESULTS--(i) Factors significantly associated with development of a third degree tear were: forceps delivery (50% v 7% in controls; P = 0.00001), primiparous delivery (85% v 43%; P = 0.00001), birth weight > 4 kg (P = 0.00002), and occipito-posterior position at delivery (P = 0.003). No third degree tear occurred during 351 vacuum extractions. Eleven of 25 (44%) women who were delivered without instruments and had a third degree tear did so despite a posterolateral episiotomy. (ii) Anal incontinence or faecal urgency was present in 16 women with tears and 11 controls (47% v 13%; P = 0.00001). Sonographic sphincter defects were identified in 29 with tears and 29 controls (85% v 33%; P = 0.00001). Every symptomatic patient had persistent combined internal and external sphincter defects, and these were associated with significantly lower anal pressures. Pudendal nerve terminal motor latency measurements were not significantly different. CONCLUSIONS--Vacuum extraction is associated with fewer third degree tears than forceps delivery. An episiotomy does not always prevent a third degree tear. Primary repair is inadequate in most women who sustain third degree tears, most having residual sphincter defects and about half experiencing anal incontinence, which is caused by persistent mechanical sphincter disruption rather than pudendal nerve damage. Attention should be directed towards preventive obstetric practice and surgical techniques of repair.     

Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca(2+) concentration ([Ca(2+)](i)) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca(2+) mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca(2+)](i) resembled that induced by the Ca(2+) channel blocker nifedipine and were reversed specifically by the Ca(2+) channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca(2+) influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.     

Development of a three-dimensional physiological model of the internal anal sphincter bioengineered in vitro from isolated smooth muscle cells

Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. The object of this study was to develop a three-dimensional (3-D) physiological model of the IAS bioengineered in vitro from isolated smooth muscle cells. Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5-mm-diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. We found that 1) the bioengineered IAS rings generated a spontaneous basal tone, 2) stimulation with 8-bromo-cAMP (8-Br-cAMP) caused a sustained decrease in the basal tone (relaxation) that was calcium-independent, 3) upon stimulation with ACh, bioengineered IAS rings showed a calcium- and concentration-dependent peak contraction at 30 s that was sustained for 4 min, 4) addition of 8-Br-cAMP induced rapid relaxation of ACh-induced contraction and force generation of IAS rings, and 5) bioengineered sphincter rings show striking functional differences when compared with bioengineered rings made from isolated colonic smooth muscle cells. This is the first report of a 3-D in vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction.     

Overview of mathematical computer models of striated sphincter muscles

The present paper compares current mathematical striated sphincter models. Current models are subdivided in four categories: (1) simple models, (2) implementations of the urethral resistance relation, (3) models with realistic muscle dynamics and (4) finite element models. In our research group a neural network model, representing Onuf's nucleus, the spinal motor nucleus that innervates the external urethral and anal sphincters, was developed. A realistic sphincter model is needed to test the neural network. To decide whether or not a model is applicable in our research two requirements should be fulfilled: (1) the presence of realistic muscle dynamics preferably by implementation of a Huxley type muscle model and (2) the model should consist of more than one muscle unit to form a more dimensional model. Reviewing the literature, if a myogenic sphincter is modelled, mainly the Hill-equation is applied. Moreover, single muscle unit models are published. In general a multi-unit muscle model of the sphincter is lacking, prohibiting the study of the inherent properties of sphincter muscles, which could give information on the realistic behaviour of elements in circular muscles. It is concluded that the functionality of current sphincter models is limited for our purpose.     

Investigation of anal motor characteristics of the sensorimotor response (SMR) using 3-D anorectal pressure topography

Desire to defecate is associated with a unique anal contractile response, the sensorimotor response (SMR). However, the precise muscle(s) involved is not known. We aimed to examine the role of external and internal anal sphincter and the puborectalis muscle in the genesis of SMR. Anorectal 3-D pressure topography was performed in 10 healthy subjects during graded rectal balloon distention using a novel high-definition manometry system consisting of a probe with 256 pressure sensors arranged circumferentially. The anal pressure changes before, during, and after the onset of SMR were measured at every millimeter along the length of anal canal and in 3-D by dividing the anal canal into 4 × 2.1-mm grids. Pressures were assessed in the longitudinal and anterior-posterior axis. Anal ultrasound was performed to assess puborectalis morphology. 3-D topography demonstrated that rectal distention produced an SMR coinciding with desire to defecate and predominantly induced by contraction of puborectalis. Anal ultrasound showed that the puborectalis was located at mean distance of 3.5 cm from anal verge, which corresponded with peak pressure difference between the anterior and posterior vectors observed at 3.4 cm with 3-D topography (r = 0.77). The highest absolute and percentage increases in pressure during SMR were seen in the superior-posterior portion of anal canal, reaffirming the role of puborectalis. The SMR anal pressure profile showed a peak pressure at 1.6 cm from anal verge in the anterior and posterior vectors and distinct increase in pressure only posteriorly at 3.2 cm corresponding to puborectalis. We concluded that SMR is primarily induced by the activation and contraction of the puborectalis muscle in response to a sensation of a desire to defecate.     

Length-tension relationship of the external anal sphincter muscle: implications for the anal canal function

The length at which a muscle operates in vivo (operational length) and the length at which it generates maximal force (optimal length) may be quite different. We studied active and passive length-tension characteristics of external anal sphincter (EAS) in vivo and in vitro to determine the optimal and operational length of rabbit EAS. For the in vitro studies, rings of EAS (n = 4) were prepared and studied in a muscle bath under isometric conditions. For in vivo studies, female rabbits (n = 19) were anesthetized and anal canal pressure was recorded by use of a sleeve sensor placed in the custom-designed catheter holders of 4.5-, 6-, and 9-mm diameters. Measurements were obtained at rest and during EAS electrical stimulation. Sarcomere length of EAS muscle was measured by laser diffraction technique with no probe and three probes in the anal canal. In vitro studies revealed 2,054 mN/cm(2) active tension at optimal length. In vivo studies revealed a probe size-dependent increase in anal canal pressure and tension. Maximal increase in anal canal tension with stimulation was recorded with the 9-mm probe. Increases in anal canal tension with increase in probe size were completely abolished by pancuronium bromide. EAS muscle sarcomere length without and with 9-mm probe in the anal canal were 2.11 +/- 0.08 and 2.99 +/- 0.07 microm, respectively. Optimal sarcomere length, based on the thin filament length measured by thin filament analysis, is 2.44 +/- 0.10 microm. These data show that the operational length of EAS is significantly shorter than its optimal length. Our findings provide insight into EAS function and we propose the possibility of increasing anal canal pressure by surgical manipulation of the EAS sarcomere length.     

Dilatation and closing anal reflexes. Description and clinical significance of new reflexes: preliminary report.

The present communication describes new reflexes which are called 'dilatation and closing anal reflexes', and discusses their clinical significance. The study comprised 21 healthy volunteers and 15 incontinent patients (7 with partial fecal incontinence and 8 with urinary stress incontinence). The technique comprised the introduction into the rectal neck of a balloon-tipped catheter. The balloon was inflated with air in increments of 10 ml up to 50 ml, and the EMG response of the external and urethral sphincters to balloon inflation and deflation was recorded. A new device called 'switch inflation' apparatus was used to inflate the balloon simultaneously with switching of the EMG apparatus. Rapid rectal neck inflation and deflation evoked external anal and urethral sphincter contraction. Slow and gradual inflation or deflation did not initiate the response. The anesthetized external anal sphincter did not respond to the stimulus, while the saline-infiltrated sphincter responded. The latency of the reflexes was recorded. In fecal incontinent patients, the external anal sphincter, on rapid rectal neck inflation or deflation, showed lower EMG activity and longer latency than in normal volunteers; the external urethral sphincter responded as in normal volunteers. In urinary stress incontinent patients, the external anal sphincter responded normally for both rectal neck inflation and deflation. The external urethral sphincter showed lower EMG activity and prolonged latency than normal on rectal neck inflation; it did not respond to deflation. The dilatation and closing reflexes seem to play a role in fecal and urinary continence as well as in fecal sampling. Detectable changes in latency or amplitude of the evoked response indicate a defect in the reflex pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells

Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10(-7) M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10(-6) M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10(-6) M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined.     

Fluorescence histochemical study on the noradrenergic control to the anterior column of the spinal lumbosacral segments of the rat and dog,with special reference to motoneurons innervating the perineal striated muscles (Onuf's nucleus)

Summary The organization of noradrenergic fibers in the lumbosacral anterior column of rats and dogs was examined in detail using a modification of a highly sensitive glyoxylic acid fluorescence histochemical method. In both rat and dog, there were greater concentrations of fluorescing noradrenergic fibers around the motoneurons innervating the perineal striated muscles (Onuf's nucleus) than around other motoneuronal groups. The preferential accumulation of noradrenergic fibers in Onuf's nucleus may indicate that the noradrenergic neuron system in the spinal cord of rodents and carnivores is closely related to the functional peculiarities of the perineal striated muscles, including the external anal and urethral sphincter muscles.     

Fluorescence histochemical study on the noradrenergic control to the anterior column of the spinal lumbosacral segments of the rat and dog, with special reference to motoneurons innervating the perineal striated muscles (Onuf's nucleus)

The organization of noradrenergic fibers in the lumbosacral anterior column of rats and dogs was examined in detail using a modification of a highly sensitive glyoxylic acid fluorescence histochemical method. In both rat and dog, there were greater concentrations of fluorescing noradrenergic fibers around the motoneurons innervating the perineal striated muscles (Onuf's nucleus) than around other motoneuronal groups. The preferential accumulation of noradrenergic fibers in Onuf's nucleus may indicate that the noradrenergic neuron system in the spinal cord of rodents and carnivores is closely related to the functional peculiarities of the perineal striated muscles, including the external anal and urethral sphincter muscles.     

Role of nitric oxide in beta3-adrenoceptor activation on basal tone of internal anal sphincter

Effects of activation of beta3-adrenoceptor (beta3-AR) have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a selective beta3-AR agonist, on the basal smooth muscle tone and direct release of nitric oxide (NO) by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a beta3-AR antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a nonselective NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) and by putative inhibitor of eNOS l-N5-(1-iminoethyl)-ornithine dihydrochloride (l-NIO). Inhibitors of iNOS [N-(3-aminomethyl)benzyl acetamide 2HCl] and nNOS [1-[2-(trifluoromethylphenyl)imidazole]] had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by l-NNA and l-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by beta3-AR activation, which is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal-transduction pathways including NOS activation may explain the characteristic IAS relaxation by beta3-AR activation. The studies may have therapeutic implications in anorectal motility disorders.     

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

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 microM; renin inhibitor) and captopril (1 microM; 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.