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Background: 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., Objective: We examined the effects of the c-kit tyrosine kinase inhibitor imatinib mesylate on sphincter strips to investigate the function of the interstitial cells., Design: This was a case series study., Settigs: This was a single-center study conducted at the University of Oxford., Patients: 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., Main Outcome Measures: The role of the interstitial cells in the internal anal sphincter was assessed., Results: 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., Limitations: This study was limited by the relatively small number of patients enrolled and thus the difficulty of finding human tissue for laboratory studies., Conclusions: 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.

Background: Neoadjuvant chemoradiotherapy is recommended in the treatment of locally advanced rectal cancer. Studies have suggested that chemoradiotherapy adversely affects anorectal function. However, the functional implication and the underlying neuromyogenic changes involved in radiation-induced damage are poorly understood., Objective: This study evaluated the functional changes following chemoradiotherapy on the internal anal sphincter., Design and Patients: This article describes an in vitro study on the internal anal sphincter collected from patients undergoing abdominoperineal resection or proctectomy. Five patients were treated by surgery alone (control group), and 6 received preoperative chemoradiotherapy (treatment group). Sphincter strips were mounted in organ bath, and the responses to electrical field stimulation and drugs were monitored., Settings: The study was performed at the University of Oxford., Main Outcome Measures: The end points of this study were to investigate whether chemoradiotherapy has any significant effects on internal anal sphincter function and, subsequently, to establish the type of injury induced., Results: Chemoradiotherapy strips developed similar tone, but significantly lower spontaneous activity (p = 0.001) than controls. Electrical field stimulation induced relaxation, followed by contraction. At 50 Hz, electrical field stimulation produced 25.6 ± 4.9% (mean ± SE) of maximum relaxation followed by a contraction of 5.5 ± 0.9% of basal tone in chemoradiotherapy strips i9n comparison with 47.0 ± 6.2% (p = 0.009) and 17.7 ± 4.0% (p = 0.007) in controls. Relaxation was significantly attenuated by N-nitro-L-arginine. Significant differences were found in responses to carbachol (p = 0.018) and phenylephrine (p = 0.022), but not to sodium nitroprusside., Limitations: This work was limited by the relatively small number of patients enrolled, because of the difficulty of finding human tissue for laboratory studies, and the lack of long-term results., Conclusions: Chemoradiotherapy significantly impairs internal anal sphincter function and intrinsic nerves seem more susceptible than smooth muscle. The exclusion of anal canal from the radiation field is recommended, when oncologically safe.

Purpose: In vitro data suggest that nitric oxide is an important inhibitory neurotransmitter in the internal anal sphincter, and morphologic evidence implies that it mediates the rectoanal inhibitory reflex. This study examined the anatomy, physiology, and pharmacology of the internal sphincter in control and neuronal nitric oxide synthase knockout mice., Methods: Neuronal nitric oxide synthase, nicotinamide adenosine triphosphate dinucleotide phosphate diaphorase histochemistry, and PGP 9.5 immunohistochemistry were compared between knockout and sibling control mice. Anorectal manometry was performed with a balloon-tipped water-perfused catheter. In vitro studies were performed on both whole internal anal sphincter rings and strips., Results: Staining of the myenteric plexus and nerves traversing the internal anal sphincter in sibling control mice demonstrated the presence of neuronal nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase at these sites. These markers were absent in knockout mice. Maximum anal resting pressure was similar in control and knockout mice (15.6 +/- 2.6 cm H(2)O (n = 4) vs. 14.0 +/- 2.3 cm H(2)O (n = 7)). The rectoanal inhibitory reflex was present in all control mice (n = 4) but in only four of seven knockout mice. Field stimulation with parameters designed to activate inhibitory nerves produced relaxation of internal sphincter tissue from both control and knockout mice, which was partially attenuated in control mice only by pretreatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine. Further inhibition of nerve-induced relaxation in control mice was achieved with antagonists of vasoactive intestinal peptide, adenosine triphosphate, and heme oxygenase., Conclusions: Although in the normal mouse, nitric oxide is an inhibitory neurotransmitter in the internal sphincter, other transmitters may play a role in the rectoanal inhibitory reflex. These other inhibitory neurotransmitters can apparently compensate for the absence of nitric oxide synthase in knockout mice to maintain approximately normal function.

Purpose: Pharmacologic treatments are gaining widespread acceptance as first-line therapy for anal fissure. However, existing treatments have limited clinical usefulness because of side effects and incomplete healing rates., Methods: Fresh human surgical resection specimens containing internal anal sphincter and rectal circular muscle were collected. Strips of smooth muscle were cut from each muscle group and mounted in a superfusion organ bath. The effects of increasing concentrations of phosphodiesterase inhibitors were evaluated., Results: All phosphodiesterase inhibitors tested caused a dose-dependent reduction in the tone of the internal anal sphincter, with potencies as follows: vinpocentine (phosphodiesterase-1 inhibitor; 50 percent maximum inhibition concentration = 0.87 +/- 0.10 microM), erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (phosphodiesterase-2 inhibitor; 32 +/- 4.8 microM), trequinsin (phosphodiesterase-3 inhibitor; 0.28 +/- 0.041 microM), rolipram (phosphodiesterase-4 inhibitor; 63 +/- 9 microM), zaprinast (phosphodiesterase-1,5,6,9,11 inhibitor; 3 +/- 0.69 microM), and dipyridamole (phosphodiesterase-5,6,8,10,11 inhibitor; 5.5 +/- 2 microM). Although all inhibitors were also effective on rectal circular muscle strips, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, trequinsin, and rolipram were at least an order of magnitude more potent in this tissue than in the internal anal sphincter., Conclusions: There are several functionally important phosphodiesterases in the internal anal sphincter and rectal circular muscle. Both adenosine 3', 5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate appear to be important in the myogenic tone of the internal anal sphincter, and this study provides further evidence of the sphincteric specialization of this tissue. Phosphodiesterase inhibitors might represent a new therapy for the treatment of anal fissure.

Introduction: Glyceryl trinitrate reduces anal resting pressure and aids the healing of anal fissures. However, some patients develop tachyphylaxis and the fissure fails to heal, suggesting that other agents are needed. This study assesses the effects of nifedipine (a calcium channel antagonist) in modulating resting tone and agonist-induced contractions in human internal anal sphincter (IAS) and rectal circular muscle., Methods: Smooth muscle strips from the IAS and rectal circular muscle from ten patients undergoing surgical resection were mounted for isometric tension recording in a superfusion organ bath. The effects of noradrenaline and carbachol were assessed in the presence of various perfusates., Results: LAS strips developed tone and spontaneous activity. Noradrenaline produced dose-dependent contractions. In calcium-free Krebs solution, tone and activity were abolished and no contractions were elicited in response to noradrenaline. Nifedipine also abolished tone and spontaneous activity, but contractions to noradrenaline were only slightly attenuated. In contrast, rectal smooth muscle strips developed spontaneous activity but no resting tone and contracted in response to carbachol. In calcium-free Krebs solution, the spontaneous activity and carbachol contractions were abolished. Addition of nifedipine to the perfusate abolished spontaneous activity and greatly reduced contractions., Discussion: These data suggest that spontaneous activity and resting tone are dependent on extracellular calcium and flux across the cells. Agonist-induced contraction in the IAS is attributable mainly to the release of calcium from intracellular stores, whereas rectal circular smooth muscle depends principally on extracellular calcium entering the cell for contraction. The attenuation of contractions in both tissues and the abolition of resting tone in the IAS suggest that nifedipine may be useful in the management of patients with anorectal disorders.

Purpose: This study was designed to investigate the role of nitric oxide in neurogenic relaxation of the longitudinal layer of human rectal smooth muscle., Methods: Tissue was obtained from the mid rectum of patients undergoing anterior resection for carcinoma. Adjacent strips of longitudinal muscle were dissected and mounted in organ baths for isometric tension recording. In preliminary experiments to determine the response of strips to cholinergic, adrenergic, and potential excitatory agonists, strips were superfused with standard Krebs solution (37 +/- 0.5 degrees C; pH, 7.4 +/- 0.05). Investigation of inhibitory, nonadrenergic noncholinergic responses required the addition of 3 x 10(-6) M histamine to induce reproducible and stable tension for five-minute "test" periods, during which electrical field stimulation (EFS) and additional drugs were applied. In these experiments, strips were superfused with Krebs solution that contained atropine sulfate (3 x 10(-6) M) and guanethidine (3 x 10(-6) M)., Results: The response to cholinergic and adrenergic agonists was typical of nonsphincter specialized gastrointestinal smooth muscle. EFS elicited frequency-dependent, neurogenic (tetrodotoxin-sensitive) relaxations of precontracted strips, which were reduced in dose-dependent fashion by addition of N omega-nitro-L-arginine and restored by addition of 3 x 10(-4) M L-arginine but not by D-arginine. Addition of exogenous nitric oxide (sodium nitroprusside) mimicked the relaxant response induced by EFS., Conclusion: Smooth muscle from the longitudinal layer of human rectum receives an intrinsic inhibitory innervation mediated by nitric oxide.

Purpose: This study was designed to investigate whether nitric oxide mediates inhibitory innervation in human rectal circular smooth muscle., Methods: Tissue was obtained from the midrectum of patients undergoing anterior resection for carcinoma. Adjacent strips of circular muscle were dissected and mounted in superfusion organ baths for isometric tension recording and initially loaded with 1 g of weight. Strips were continuously bathed with standard Krebs solution (37 degrees C, bubbled with 97 percent O2/3 percent CO2) containing 3 X X 10(-6) M guanethidine and 3 X 10(-6) M atropine sulfate to block adrenergic and muscarinic cholinergic neurotransmission. After equilibration, strips had no intrinsic tone, and reproducible and stable tension was, therefore, induced by the addition of 3 X 10 M histamine for five-minute "test" periods, during which electrical field stimulation (EFS) and additional drugs were applied., Results: EFS elicited frequency-dependent, neurogenic (tetrodotoxin-sensitive) relaxations of precontracted strips. Addition of N-w-nitro-L-arginine, a powerful competitive inhibitor of nitric oxide synthase, reduced the relaxant response to EFS in a dose-dependent fashion, and effect reversed by addition of s X 10(-4) M L-arginine but not by D-arginine. Addition of exogenous nitric oxide (sodium nitroprusside) mimicked the relaxant response induced by EFT., Conclusions: Human rectal circular smooth muscle receives an intrinsic inhibitory innervation mediated by nitric oxide. The presennce of a residual response following blockade of the enzyme nitric oxide synthase suggests the involvement of additional neurotransmitters.

Purpose: Following the demonstration that a novel neurotransmitter, nitric oxide (NO), is released during neurogenic relaxation of the internal anal sphincter in vitro, it has been suggested that NO could mediate the rectoanal inhibitory reflex in vivo. The aim of this study was to establish whether the distribution of NO-producing nerves in the anorectum is consistent with this proposed role., Methods: NO is synthesized in neurons which contain the enzyme nitric oxide synthase and their presence in the anorectum was determined in tissue obtained from nine abdominoperineal and three anterior resection specimens in patients undergoing surgery for rectal carcinoma. Cryostat sections were stained for nitric oxide synthase immunoreactivity, pan-neuronal/axonal immunoreactivity, and NADPH diaphorase activity., Results: Nitric oxide synthase immunoreactivity is present in a subpopulation of neurons in rectal myenteric ganglia which also contain NADPH diaphorase activity. Use of the latter histochemical technique enabled the structure and distribution of nitric oxide synthase containing neurons to be determined in whole-mount preparations. Individual neurons have Dogiel type 1 morphology and are present throughout the rectal myenteric plexus. In the distal rectum, positively stained axons enter shunt fascicles which descend into the anal canal, where they ramify into and throughout the internal anal sphincter. Within the sphincter, positively stained nerves lie in close proximity to smooth muscle cells., Conclusion: These results are consistent with the hypothesis that NO is the neurotransmitter that mediates the rectoanal inhibitory reflex.