Botulinum toxin injection inhibits myogenic tone and sympathetic nerve function in the porcine internal anal sphincter

Objective Botulinum toxin is an effective treatment for anal fissure, though there is a lack of agreement over the optimal site for its injection. This reflects our current ignorance of its mechanism, and whether it has any action on the nerves of the internal anal sphincter (IAS). This study set out to resolve this issue through use of a pig model. Materials and methods Eight pigs were studied in pairs: one of each pair received a botulinum toxin injection into the anal sphincter, whilst the other acted as its control. Manometry was performed every two weeks under anaesthesia. Pigs were slaughtered at between four and six weeks after injection and the properties of the IAS compared in vitro. Results Whilst maximum anal resting pressure (MARP) increased slowly in control pigs during the experimental period, reflecting weight gain, a fall was observed in treated pigs. In vitro, IAS strips from control pigs generated 400 mg of spontaneous tone per gram of tissue (± 45; standard error), compared to 250 (± 25) mg/g tissue from treated pigs (P < 0.01). Electric Field Stimulation at 50Hz produced 150 (± 22) mg contraction/gram tissue in IAS strips from control pigs compared to 53 (± 13) mg/g tissue in treated pigs (P < 0.0005). This contractile response was blocked by guanethidine. Conclusion Botulinum toxin has a significant action on the IAS. It reduces myogenic tone and contractile responses of this tissue to sympathetic nerve stimulation. Further studies are required to clarify its mechanism of action more precisely.     

Role of nitric oxide in the internal anal sphincter of Hirschsprung's disease

It is not clear what contribution the internal anal sphincter (IAS) makes to the impaired motility observed in patients with Hirschsprung's disease (HD). Nitric oxide (NO) has recently been shown to be a neurotransmitter in the nonadrenergic noncholinergic (NANC) inhibitory nerves in the human gut. To clarify the physiologic significance of NO in the IAS of HD (aganglionosis), we investigated the enteric nerve responses on lesional (aganglionic) and normal IAS muscle strips above the dentate line. Lesional and normal IAS muscle strips above the dentate line were derived from patients with HD (10 cases) and patients who underwent rectal amputation for low rectal cancer (12 cases). A mechanographic technique was used to evaluate in vitro muscle responses to electrical field stimulation (EFS) before and after treatment with various autonomic nerve blockers, N(G)-L-nitroarginine, and L-arginine. The following results were obtained: (1) Cholinergic nerves are mainly involved in the regulation of enteric nerve responses to EFS in the normal IAS. (2) The aganglionic IAS of patients with HD was more strongly innervated by cholinergic nerves than the normal IAS (p < 0.05). (3) NANC inhibitory nerves were found to act on the normal IAS but had no effect on the enteric nerves in patients with aganglionosis. (4) NO was found to act on normal IAS, but no effect was observed in the aganglionic IAS. These findings suggest that innervation of the cholinergic nerves and a loss of NO mediation of NANC inhibitory nerves play an important role in the impaired motility observed in the IAS with HD.     

L-arginine-induced relaxation of the internal anal sphincter is not mediated by nitric oxide

BACKGROUND: Topical application of L-arginine, the precursor of nitric oxide, reduces resting anal pressure without significant side-effects and may therefore be of benefit in the treatment of anal fissure. This in vitro study investigated the effect of L-arginine on sheep and human isolated internal anal sphincter (IAS) to ascertain the role played by nitric oxide and guanosine 3',5'-cyclic monophosphate. METHODS: Strips of sheep and human IAS were mounted in isolated organ baths. The effects on myogenic tone of increasing concentrations of L-arginine, D-arginine and other amino acids were evaluated. RESULTS: L-Arginine, D-arginine and other basic amino acids (L-lysine and L-ornithine) all caused a concentration-dependent reduction in myogenic tone. L-Arginine was the most effective and produced a mean(s.e.m.) maximal reduction in myogenic tone of 78.2(7.1) and 40.2(9.3) per cent in sheep and human tissue respectively. These responses were not affected by N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase. Changes in pH per se were unable to explain the relaxation fully, but an equiosmolar sodium chloride solution produced a concentration-response relationship similar to that of L-arginine. CONCLUSION: The ability of L-arginine to reduce myogenic tone is independent of nitric oxide. This effect may be partially pH dependent but the osmolality of the solution appears to be a major factor. Hyperosmolar solutions might be worthy of further investigation as agents that affect anal tone.     

gamma-Aminobutyric acid enhances the tone of human internal anal sphincter.

The effect of gamma-aminobutyric acid (GABA) on the human internal anal sphincter was investigated. Cumulative applications of GABA produced concentration-dependent contractions (10(-8)-10(-5) M) of the isolated human sphincter. Pretreatment with bicuculline (GABAA antagonist) turned them to relaxation. Muscimol, a GABAA agonist, induced concentration-dependent contractions (10(-8)-10(-5) M); however, baclofen (GABAB agonist, 10(-8)-10(-5) M) promoted concentration-dependent relaxation of the strips. These results suggested that both excitatory GABAA receptors and inhibitory GABAB receptors exist in the internal anal sphincter. Oral administration of sodium valproate (1600 mg/day), a GABA transaminase inhibitor, enhanced the anal canal resting pressure in 10 normal volunteers. Anal manometry showed a significant elevation in tonus without affecting amplitudes or frequencies. These results indicated that endogenous GABA, which was increased by sodium valproate, produced elevations in the anal canal resting pressure through its specific receptors in the human internal anal sphincter.     

Effects of some calcium channel blockers on isolated human penile erectile tissues

The effects of the calcium channel blockers (CCBs) verapamil, nifedipine and diltiazem on contractile activation of isolated human penile erectile tissues were investigated. Specimens of the corpus spongiosum (CS) and corpora cavernosa (CC) were obtained from men with a history of normal penile erection undergoing cystourethrectomy because of bladder malignancy. Preparations were mounted in organ baths and isometric tension was recorded. Deprivation of extracellular calcium abolished electrically induced contractions in both CS and CC preparations within 15 min.; norepinephrine (NE)-induced contractions were reduced by 90% (CS) and 83% (CC) after 30 min. All the CCBs reduced electrically induced contractions concentration-dependently, nifedipine being the most potent agent. Contractions induced by exogenous NE were depressed by about 50%, whereas high K+ (124 mM) induced responses were abolished. It is concluded that contraction in penile erectile tissues is mediated mainly by neuronally released NE stimulating postjunctional alpha-adrenoceptors. The contraction is highly dependent on extracellular calcium and can partly be inhibited by CCBs. It cannot be excluded that some CCBs injected intracavernosally may be useful for diagnosis and even treatment of erectile dysfunction. However, calcium channel blockade may not be as effective as a therapeutic principle as blockade of alpha-adrenoceptors.     

Modulation of intrinsic cavernous tone and nitric oxide production by arginase in rabbit corpus cavernosum

PURPOSE: Arginase shares the common substrate L-arginine with nitric oxide synthase (NOS). We investigated the roles of NOS and arginase for modulating intrinsic and vasoconstrictor tone in rabbit corpus cavernosum (RCC). MATERIALS AND METHODS: Isolated RCC tissues were used for isometric tension experiments, and NOS and arginase activities. The endothelium lining RCC lacunar spaces was disrupted and/or removed by saponin treatment. RESULTS: Following stretch of approximately 1gm NG-nitro-L-arginine methyl ester (L-NAME) as a NOS inhibitor caused endothelium dependent contraction, while the potent and specific arginase inhibitor N omega-hydroxy-nor-L-arginine (nor-NOHA) caused endothelium dependent relaxation. Relaxation with nor-NOHA was reversed by L-NAME. In the presence of 10 mM L-arginine 0.1 mM nor-NOHA was ineffective. Pretreatment with 0.1 mM L-NAME and 0.1 mM nor-NOHA did not significantly affect the vasoconstrictor response to phenylephrine. The magnitude of contraction with 0.1 mM L-NAME and relaxation with 0.1 mM nor-NOHA during contraction induced by phenylephrine were not significantly different from changes with L-NAME and nor-NOHA under intrinsic basal tone. In the enzymatic study NOS and arginase were detectable in cavernous homogenates. Nor-NOHA inhibited arginase but not NOS activity. CONCLUSIONS: Results indicate that basal nitric oxide production from the endothelium regulates intrinsic cavernous tone and endogenous arginase activity in the endothelium modulates tone by inhibiting nitric oxide production, presumably through competition with constitutive NOS for the common substrate L-arginine.     

Mechanism of action of botulinum toxin on the internal anal sphincter

BACKGROUND: Botulinum toxin is an effective treatment for anal fissure. Manometric studies support an apparent action of botulinum toxin on the internal anal sphincter (IAS). This aim of this study was to establish the underlying mechanism. METHODS: Porcine IAS strips were suspended in a superfusion organ bath and allowed to equilibrate. Electrical field stimulation (EFS) was applied with parameters that induced nitrergic relaxation followed by noradrenaline-mediated contraction. These responses were compared before and after addition of botulinum toxin. RESULTS: All strips developed myogenic tone, which was slightly increased following the addition of botulinum toxin. EFS-induced nitrergic relaxation was unaffected by toxin treatment. However, EFS-induced contraction was significantly reduced by toxin treatment. 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic agonist, caused muscle strip contraction, which was blocked by guanethidine, implying the presence of sympathetic ganglia within the IAS. Botulinum toxin significantly attenuated DMPP-induced contraction. CONCLUSION: In the treatment of anal fissure the major effect of botulinum toxin on the IAS is blockade of sympathetic (noradrenaline mediated) neural output. This is probably a postganglionic action, involving a reduction in noradrenaline release at the neuromuscular junction. Botulinum toxin has no significant effect on nitrergic transmission, which is probably not vesicular in nature.     

Immunomodulating effects of second-generation calcium channel blockers on experimental heart transplantation.

The administration of second-generation calcium channel blockers (CCBs) to counteract the adverse effects of conventional immunosuppression gains more and more acceptance. Since these newly developed molecules differ in their chemical structure and possess specific pharmacokinetic profiles, we hypothesized that exposure to clinically relevant concentrations may have a significant immunomodulatory potential. The effects of various second-generation CCBs, felodipine, amlodipine, mibefradil and clentiazem, on cardiac allograft survival were therefore evaluated. Inbred male Lewis rats were used as recipients and Brown-Norway rats as donors. After abdominal implantation of the donor heart, allograft recipients were exposed to felodipine (31 microg/kg/day), amlodipine (25 microg/kg/day), mibefradil (3 mg/kg/day) or clentiazem (2.5 mg/kg/day). Other allograft recipients were treated with low-dose cyclosporine (CsA) alone (2 mg/kg/day) or with low-dose CsA combined with amlodipine (25 microg/kg/day), mibefradil (3 mg/kg/day) or clentiazem (2.5 mg/kg/day). All drugs were given daily by gavage. Median survival time of untreated cardiac allografts was 6.5 days. When given alone, not all the second-generation CCBs elicited a positive effect: the dihydropyridines felodipine and amlodipine were ineffective (median survival time was 6.5 and 7.0 days, respectively), the T- and L-type CCB mibefradil had a significant but minor impact (median survival time = 9.0 days, p <0.0015) while the benzothiazepine clentiazem produced the most significant result (median survival time = 16.0 days, p <0.0033). Neither amlodipine nor mibefradil modified the extent of survival provided by low-dose CsA (median survival time = 9.0 days), while clentiazem had a significant positive effect. These data indicate that second-generation CCBs differ in their immunomodulatory potential. These observations of pharmacodynamic specificity appear to be related to differences in their chemical structure as well as their interaction with other sites than the calcium channel.     

钙通道阻滞剂对低温保存大鼠肝脏的保护作用

目的：研究钙通道阻滞剂是否减轻低温保存下肝细胞的钙超载并起到保护肝脏的作用。方法：低温保存大鼠肝细胞于不同时限,按保存液的不同成分分组：（1）对照组：DMEM液;（2）实验Ⅰ组：DMEM液＋Verapamil(维拉帕米）;（3）实验Ⅱ组：DMEM液＋Nifedipine(硝苯地平）;（4）实验Ⅲ组：DMEM液＋Diltiazem(硫氮卓酮）。用Fura-2法测定低温保存下的大鼠肝细胞内钙和肝功能,并在光镜和电镜下观察肝脏的结构。结果：细胞内钙及肝功能测定,各实验组与对照组差异显著;组织学观察,保存0－48h各实验组损伤明显低于对照组。结论钙通道阻滞剂对低温保存肝脏有保护作用,且三类药物中帕米作用最强,硝苯地平和硫氮卓酮次之。     

Recovery of the internal anal sphincter and continence after restorative proctocolectomy

The internal anal sphincter (IAS) was assessed prospectively using electromyography and manometry in 66 patients (48 men) undergoing restorative proctocolectomy to determine its role in the gradual return of continence. Twenty-nine patients received a J pouch and 37 a W reservoir. Some 38 pouches (J, ten; W, 28) were hand-sewn (mucosal proctectomy with endoanal anastomosis) and 28 (J, 19; W, nine) stapled (end-to-end pouch-anal anastomosis 1 cm above the dentate line). Twelve patients underwent a one-stage procedure (all J pouches), while the remainder had a covering loop ileostomy. Each patient was reassessed immediately after restorative proctocolectomy and again at 7 days, 1 month, 4 months, 9 months and 18 months after pouch formation. Internal sphincter electromyographic activity was greatly reduced after pouch-anal anastomosis (median preoperative frequency 0·51 Hz versus immediate postoperative frequency 0·21 Hz, P < 0·003) and gradually recovered from 4 months after surgery. At 18 months, measurements of IAS function had not fully recovered to preoperative values (median frequency 0·31 Hz; P < 0·03). Resting anal pressures (median preoperative value 99 cmH₂O) decreased by over 50 per cent after surgery (median immediate postoperative resting pressure 44 cmH₂O) and recovered gradually but incompletely (median pressure at 18 months 63 cmH₂O). Eleven patients reported leakage in the follow-up period. The median (range) resting pressure in these patients (54 (40–71) cmH₂O) was not significantly different at 9 months from that of those who were continent either before or after operation (59 (46–68) cmH₂O). Prolonged recordings in patients with faecal leakage revealed evidence of high-pressure pouch waves that overwhelmed anal sphincter pressures and coincided with leakage. These episodes were most common during sleep, when anal sphincter activity was reduced.     

The effects of calcium channel blockers on random skin flap survival in the pig model

Summary This study was designed to evaluate the influence of two calcium channel blockers, verapamil and nifedipine, on skin flap survival. These agents were selected because they inhibit the passage of calcium through calcium selective channels in the plasma membrane, thereby blocking calcium mediated electromechanical coupling in contractile tissue and resulting in peripheral arterial vasodilation. Three groups of pigs were used in this study. All skin flaps in this study were 3 cm wide and 12 cm long. The first group (10 flaps) served as controls with no pharmacologic manipulations. Pigs in group II (15 flaps) received verapamil (80 mg orally, three times a day) for 7 days postoperatively. Pigs in group III (15 flaps) received nifedipine (10 mg orally, three times a day) for 7 days postoperatively. Statistical analysis of the results demonstrated that both verapamil and nifedipine resulted in significant enhancement of skin flap survival. The increased survival of the skin flaps produced by nifedipine as compared to verapamil was statistically significant.This study is supported in part by PHS Research Grant DE00853 from the National Institute of Dental Research     

Effects of potassium channel inhibitors on the relaxation induced by the nitric oxide donor diethylamine nitric oxide in isolated human cerebral arteries

OBJECT: The goal of this study was to investigate whether K+ channels are involved in nitric oxide (NO)-induced relaxation of isolated human cerebral arteries. METHODS: Successive concentration-response curves relating to the use of the NO donor diethylamine NO (DEA/NO) were established in the absence and presence of different K+ channel inhibitors after mounting human cerebral arteries onto a wire myograph. The arteries were obtained from macroscopically intact tissue that had been removed during brain tumor operations. A high K+ concentration partially inhibited the relaxant effects of DEA/NO. Different K+ channel inhibitors (tetraethylammonium [TEA], 10(-3) M; charybdotoxin, 10(-7) M; glibenclamide, 10(-6) M; 4-aminopyridine [4-AP], 10(-3) M; BaCl2, 5 x 10(-5) M; and apamin, 10(-6) M) alone failed to affect the responses to DEA/NO. However, a combination of TEA, glibenclamide, 4-AP, and BaCl2 partially blocked the relaxant effects of DEA/NO. In addition, the effects of DEA/NO were inhibited by the thromboxane A2 analog U46619 (3 x 10(-7) M). CONCLUSIONS: Inhibitors of the large-conductance or small-conductance Ca++-activated K+ channels, the adenosine triphosphate-sensitive K+ channels, and the delayed-rectifier or inward-rectifier K+ channels failed to alter the effects of DEA/NO when only one K+ channel blocker was used. However, a regimen of a combination of K+ channel blockers that possess selectivity for different channels demonstrated that different K+ channel types are involved; these channels may function in a redundant manner and compensate for each other. Selective thromboxane A2 agonists are capable of inhibiting the relaxant response to the NO donor.     

Fissurectomy and anal advancement flap for anterior chronic anal fissure without hypertonia of the internal anal sphincter in females

Aim Lateral internal sphincterotomy is considered the surgical treatment of choice for chronic anal fissure after failure of medical therapy but it risks continence. The aim of the study was to evaluate fissurectomy with advancement flap for anterior chronic anal fissure (CAAF) resistant to medical therapy. Method Sixteen women with CAAF without hypertonia of the internal anal sphincter, unresponsive to previous medical treatment, were included in the study. Absence of hypertonia was defined as a maximum anal resting pressure (MRP) of less than 85 mmHg. All patients underwent fissurectomy with an advancement skin flap. Results Complete healing occurred in all patients within 30 days. The intensity and the duration of pain after defecation reduced from the first postoperative defecation. MRP before surgery and at 6 months showed no significant difference. At 1 month, four patients experienced a continence disturbance, two of whom had it preoperatively. At 12 months, two (12.5%) patients continued to experience a continence disturbance. Conclusion Fissurectomy with skin advancement flap resulted in complete healing and full relief of symptoms in all patients. There was a low incidence of continence disturbance.