The response of colonic muscle to drugs: an in vitro study of Hirschsprung's disease

An in vitro pharmacologic study was conducted with muscle from 14 cases of Hirschsprung's disease and eight normal controls. The circular muscle from an aganglionic segment was less sensitive to ACh and physostigmine than from segments of proximal ganglionic bowel or normal controls. The degree of insensitivity to ACh had a significant correlation with the clinical severity of obstruction. It appeared that variably increased cholinesterase activity in the aganglionic colon might be responsible for the insensitivity to ACh. These findings suggest the hypothesis that, in addition to the absence of intramural ganglia, there is an imbalance between release of ACh and hydrolysis by ChE which produces the variable clinical symptoms.     

Effect of frusemide on airway smooth muscle contractility in vitro.

Frusemide, an inhibitor of sodium-potassium-chloride (Na-K-Cl) cotransport, has been shown to inhibit the airway response to several constrictor stimuli in asthmatic subjects. The protection seen with frusemide in these studies could be due to an effect on epithelium, inflammatory cells, neural pathways, or airway smooth muscle. To determine whether frusemide inhibits airway smooth muscle contraction, experiments were performed in bovine and human airways in vitro. Fresh bovine tissue was obtained from the abattoir and human tissue from thoracotomy. The effect of 10(-5)M frusemide on histamine, potassium chloride, and hyperosmolar saline induced contractions was studied in bovine tracheal strips without epithelium. Frusemide, at a concentration that specifically inhibits Na-K-Cl cotransport, did not inhibit contraction caused by any of these agents. Frusemide was also without effect on hypertonic saline induced contractions of bovine and human bronchial rings with epithelium intact. These results suggest that modification of Na-K-Cl cotransport does not alter airway smooth muscle contractility and that the protective effect of frusemide on induced bronchoconstriction in vivo is unlikely to be due to a direct effect on airway smooth muscle.     

先天性巨结肠不同节段肠壁神经、平滑肌的分布及临床意义

目的 观察先天性巨结肠(HD)不同节段肠壁神经和平滑肌的病变范围,探讨先天性巨结肠根治术后肠动力功能紊乱原因及手术切除结肠范围.方法 用免疫组织化学和苏木素-伊红(HE)染色法,检测20例先天性巨结肠肠壁神经节细胞、神经纤维和平滑肌细胞病理组织学改变及分布范围.结果 巨结肠不同节段肠壁神经节细胞、神经纤维数量及突触素(Syn)、神经节细胞黏附分子(NCAM)的阳性表达,在距扩张远端8 cm虽未达到正常,但与对照组差异减小(P＞0.05).环肌层和纵肌层出现不同程度增厚,在距扩张远端8 cm仍未正常(P＜0.01).肌层出现空泡样变,与对照组比较差异无统计学意义(P＞0.05).结论 先天性巨结肠切除段肠壁神经、平滑肌层均存在病变,在距扩张段的远端8 cm处,两者病变总体缓解.在允许情况下,手术切除结肠的范围应达到或超过此范围.     

Aberrant colonic expression of MHC class II antigens in Hirschsprung's disease.

The major histocompatibility complex (MHC) Class I and II cell surface antigens responsible for the recognition of self vs non-self were studied in patients with documented Hirschsprung's disease. Monoclonal antibodies reactive with monomorphic determinants of human lymphocyte antigen (HLA)-A,B,C (Class I) and HLA-DR (Class II) were used to demonstrate immunohistochemically the expression of MHC antigens in 27 biopsy specimens from a variety of colorectal disorders. The rectal specimens examined from patients with Hirschsprung's disease showed an unexpected, marked elevation of Class II antigens with abnormal localization in the mucosa and lamina propria. This ectopic expression was not seen in any portion of the small or large bowel of patients who did not have Hirschsprung's disease. Furthermore, proximal normal colon of children with Hirschsprung's disease failed to show increased expression of Class II antigen. In an attempt to better define the effector arm at a cellular level, the distribution of helper T cells (CD4+), cytotoxic/suppressor T cells (CD8+) and natural killer cells (NK; CD16+) was examined in 5 cases. In Hirschsprung's disease, rectal infiltration of CD8+ and CD16+ cells was found, but not CD4+ cells. Ectopic expression of Class II antigen with increased numbers of rectal T cells and NK cells suggested that an early immunologic event may be causal in Hirschsprung's disease.     

Effects of changes in pH on the contractility of vascular smooth muscle

Hydrogen ion concentration (H+) is an important regulator of many cellular functions including contractility of the vascular smooth muscle cells. In many physiological and pathological states, changes in pH may have profound effects on the contractility of blood vessels and thus on the peripheral vascular resistance and blood pressure. Therefore, it is necessary to understand the likely outcome of the situations in the conditions, where pH changes are frequent. In this review, the mechanisms involved in the contractile responses of vascular tissues to pH changes are discussed.     

Hirschsprung's disease: a comparison of the nervous control of ganglionic and aganglionic smooth muscle in vitro

Specimens from aganglionic (constricted) and ganglionic (dilated) gut were obtained from nine patients with Hirschsprung's disease. Transmural nerve stimulation of ganglionic smooth muscle in vitro evoked an initial relaxation followed by a contraction. This contraction was reduced but not abolished by atropine and it was further reduced by substance P antagonists. Guanethidine did not affect the electrically evoked responses. In aganglionic smooth muscle, an atropine-sensitive contraction but no initial relaxation was registered. Tetrodotoxin abolished all responses to electrical stimulation in both ganglionic and aganglionic specimens. Application of carbachol or substance P produced contraction and the adrenergic agonist isoprenaline or vasoactive intestinal peptide produced relaxation in ganglionic as well as aganglionic specimens. Two other gut neuropeptides, neuropeptide Y and galanin, were without effect. The results do not indicate a different receptor set up in ganglionic v aganglionic gut. The results are compatible with a lack of noncholinergic nonadrenergic inhibitory neurons in the aganglionic gut.     

Effect of hypothyroidism on the nitrergic relaxant responses of corpus cavernosal smooth muscle in rabbits

BACKGROUND: The incidence of hormonal dysfunction as a cause of impotence remains controversial. However, several recent studies have reported evidence of hormonal abnormalities in 25-35% of impotent men. Hypothyroidism has been reported to occur in 6% of impotent men. METHODS: In the present study, we examined nitrergic responses in hypothyroidism in rabbit corpus cavernosum and compared them with controls. RESULTS: Carbachol-induced relaxation responses and electrical field stimulation (EFS)-induced frequency-dependent relaxations decreased significantly in hypothyroid rabbits. Papaverine and sodium nitroprusside (SNP)-induced relaxation responses did not change significantly in hypothyroid rabbits. The contraction responses of phenylephrine and EFS-induced frequency-dependent contractions were significantly decreased in the hypothyroid group. CONCLUSIONS: We can speculate that the reduction of relaxant responses to EFS and carbachol in hypothyroid rabbits can depend on a decreased release of nitric oxide (NO) from nitrergic nerves and endothelium or a reduction of muscarinic receptor density. Also, decreases in contraction responses may depend on diminished adrenoceptor density.     

Effect of peritonitis on gallbladder smooth muscle contractility in guinea pigs

BACKGROUND: The mechanisms involved in the impaired gallbladder contractile response in peritonitis are unknown. The aim of this study was to determine the effect of peritonitis on the contraction and relaxation responses to different agonists in gallbladder smooth muscle in guinea pig. MATERIALS AND METHODS: Peritonitis was induced by cecal ligation and puncture (CLP) in 10 guinea pigs. Another group of 10 guinea pigs underwent a sham operation and acted as controls. Twenty-four hours after the operation, the guinea pigs were killed, and gallbladder strips were placed in organ bath. The contraction responses to KCl, carbachol, and histamine, and relaxation responses to cyclooxygenase inhibitors (indomethacin, nimesulide, and DFU) on KCl-induced contractions were recorded. RESULTS: There was no significant difference between the contractile responsiveness to KCl, but maximum contractile responses (E(max)) to carbachol and histamine were significantly reduced. Indomethacin, nimesulide, and DFU concentration dependently inhibited on KCl-induced contractions of gallbladder smooth muscle. E(max) values of indomethacin, nimesulide, and DFU were significantly reduced in the peritonitis group compared with controls (P < 0.05). The inhibitor effects of nimesulide and DFU were considerably similar, but inhibitor effect of indomethacin was significantly less than that measured for nimesulide and DFU in both control and peritonitis groups (P < 0.05). CONCLUSIONS: The contraction responses to carbachol and histamine and relaxation responses to COX inhibitors on gallbladder smooth muscle are significantly decreased by peritonitis. Although the mechanism of the decrease in contraction and relaxation responses in CLP-induced peritonitis is completely unknown, we speculate that impaired smooth muscle responses may be related to an alteration in the regulation of receptor/postreceptor excitation-response coupling and/or through changes on Ca(2+) influx.     

Effects of acid-base changes on human ureteric smooth muscle contractility

Wide fluctuations of both urinary pH and the partial pressure of CO2 (PCO2) occur in normal physiological circumstances and in a variety of pathological conditions. However, the effect of extracellular pH on the contractility of human ureteric muscle has not been clearly defined. This study has established, using a microsuperfusion technique, that an increased superfusate PCO2 increases the magnitude of the phasic contraction to electrical field stimulation. A similar extracellular acidosis induced by alteration of the [HCO3-], at constant [Na+] and free [Ca2+], was without significant effect. Furthermore, when both superfusate PCO2 and [HCO3-] were simultaneously increased at constant pH the contractile response was similar to that when PCO2 alone was raised. These observations suggest that the changes of tension were mediated by intracellular pH changes, providing it is assumed that the ureteric smooth muscle cell membrane is permeable to CO2 but impermeable to H+ and HCO3-. The occurrence of an increase of force in the presence of an acidosis is a highly significant and unusual finding, since it has been assumed that the classical association between acidosis and negative inotropy, seen in cardiac muscle, was also applicable to smooth muscle.     

Enkephalin inhibits presynaptically the contractility of urinary tract smooth muscle

The influence of methionine-enkephalin and leucine-enkephalin on human detrusor and on pig detrusor, trigone, bladder neck and urethral smooth muscle was investigated in vitro. The enkephalins inhibited the smooth muscle contractility presynaptically. Methionine-enkephalin was about 40% more potent than leucine-enkephalin. Phentolamine-resistant contractions were less inhibited by the enkephalins than atropine-resistant contractions and contractions that were not blocked. The inhibitory nerve responses of the trigone, bladder neck and urethra were unaffected.     

Molecular mechanisms for the regulation of penile smooth muscle contractility

Relaxation of penile smooth muscle (arterial and trabecular) initiates and maintains penile erection. Relaxation of smooth muscle is viewed as a 'resetting' of contractile machinery by resumption of a precontractile state accomplished by lowering cytosolic Ca(+2) and/or by a decrease in sensitivity of the contractile machinery to Ca(+2). There are various mechanisms whereby cytosolic Ca(+2) can be reduced and relaxation achieved, but in general, all pathways depend on the accumulation of the nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) or activation of K channels with hyperpolarization. Another mechanism, activation of Na(+)/K(+) adenosine triphosphatase (ATPase) by nitric oxide, has been shown to be involved in relaxation of trabecular smooth muscle. Since Na(+)/K(+) ATPase is electrogenic, its stimulation would cause hyperpolarization. Hyperpolarization will prevent the opening of voltage-dependent calcium channels. Guanylate cyclase, which catalyzes the conversion of guanosine triphosphate to cGMP, is activated by nitric oxide. cGMP activates protein kinase G, which through multiple phosphorylations facilitates calcium sequestration and reduces the entry of calcium into the cell. Other muscle relaxants act by way of a cAMP-dependent mechanism such as prostaglandin E, vasoactive intestinal polypeptide, and catecholamines (via beta-receptors). These substances react with membrane receptors coupled to a GS-type protein that stimulates adenylate cyclase, which catalyzes the accumulation of cAMP. DOI: 10.1038/sj/ijir/3900790     

Histochemical and genetic analysis of colonic mucin glycoproteins in Hirschsprung's disease

BACKGROUND/PURPOSE: Mucin glycoproteins (mucins) recently have been shown to be deficient in the colonic mucosa of patients with Hirschsprung's disease (HD). The authors performed a detailed histo- and immunohistochemical analysis of mucins in the colonic mucosa and studied the expression of mucin genes to characterize histologically mucin quality and gene expression in HD compared with controls. METHODS: Paraffin-embedded 4-microm thick sections from patients with HD (n = 11 ganglionic, 10 aganglionic) and controls (n = 19) were taken. Slides were stained with mild periodic acid Schiff with and without saponification with KOH (reacts with O-actylated mucins), high iron diamine/alcian blue (differentiates sulphated v nonsulphated mucins), the monoclonal antimucin antibodies, PR3A5 (against di- and tri-O-acetylated sialic acids) and 91.9H (against sulphated mucins). O-acetylation and sulphation both confer an increased resistance of mucins to bacterial degradation and are thought to be important in the defensive function of the colonic mucus gel layer. In situ hybridization was used to study expression of the mucin genes MUC 1, 2, 3, 4, 5AC, 5B, 6, 7, and 8. [35S]-sulphate-labelled antisense oligonucleotide 48mer probes designed to the known tandem repeat domains of MUC genes were used. After hybridization and washing the slides were opposed to Hyperfilm MP for 7 days. The autoradiographs were scored by three independent observers for differences in expression and by image analysis. Those with positive findings were dipped in photographic emulsion, developed, and counterstained for photomicrographs. RESULTS: There were different patterns of staining dependent on the region of the colon and especially the age of the patient with three reagents. No significant differences in the histological staining pattern was detected between HD patients and controls. The colonic mucins in HD were found to be primarily O-acetylated and sulphated. The MUC gene expression was similar in patients and controls. MUC2 and 4 were strongly expressed, MUC1, 3, and 5B had moderate to weak expression, and MUC 5AB, 6, 7, and 8 had baseline expression. CONCLUSIONS: The mucin glycoproteins in children with HD, although quantitatively deficient, show no qualitative differences on histo- and immunohistochemical staining from normal controls. The expression of all the known mucin genes, the genetic control of mucin secretion, and the quality of mucins, is similar to normal controls.     

Clinical and genetic differences in total colonic aganglionosis in Hirschsprung's disease

Although apparently the same condition as Hirschsprung's disease (HSCR), total colonic aganglionosis (TCA) patients (2%-14% congenital aganglionosis) display clinical, histopathologic, and genetic differences that may account for altered clinical presentations.

Patients and methods

Clinical, radiologic, and histologic features of 22 TCA patients of 114 HSCR cases (including 16 kindreds) were retrospectively evaluated by chart review. With ethical permission, DNA mutation analysis of the RET and EDNRB genes was carried out. Polymerase Chain Reaction (PCR) products were screened for genetic variation of by Hetroduplex Single Strand conformation polymorphism (HEX/SSCP) analysis and compared with 60 normal population control samples (20/ethnic groups). The SSCP variants were validated with automated sequencing techniques showing conformational variants in acrylamide gel.

Results

Of the 22 patients, 12 (55%) presented within the first 28 days of extrauterine life, but 10 presented later with 3 (14%) presenting more than 6 months of age. The TCA patients evaluated differed clinically, radiologically, and histologically, and misdiagnosis occurred in 23% (5/22). Seven patients (32%) were familial—the remainder being nonrelated. Histologic features varied, and difficulties in diagnosis occurred in 5 (24%), with unclear histologic condition delaying diagnosis in one and a mistaken aganglionic level, requiring repeat surgery in two.RET variations were detected in 82% (18/22)of TCA as opposed to 33% short segment (S-HSCR) with multiple genetic RET variations in 5 (28%). Genetic variations included exon 2 SNPs but less than in S-HSCR. One had an isolated RET A4 variation with no other abnormalities. Intronic RET variations occurred in intron 6 (2 patients) (IVS6+56delG) and intron 16 (2 patients) (IVS16-38delG). A cysteine radical mutation (C620R) (2 patients) was related to Multiple Endocrine Neoplasia Type 2 (MEN2) in the family. In contrast to S-HSCR, genetic variations in TCA aggregated to the important tyrosine kinase (intracellular) region in 5 patients suggesting a possible pathogenetic link. EDNRB variations occurred in 7 patients (32%) all within exon 4 of the gene.

Conclusions

Total colonic aganglionosis differs clinically from other HSCR phenotypes and may lead to misdiagnosis. Potential disease-related RET gene mutations include exon 17-21 genetic variations that suggest the possibility of disrupted downstream signaling pathways from vital gene recruitment sites as possible TCA contributing factors.     

Definitive treatment of extended Hirschsprung's disease or total colonic form

Background: Between December 1990 and March 1999, five laparoscopic Duhamel pull-through procedures for extended or total aganglionosis were performed in our department, one of which had a rectosigmoid form with a short bowel and a colonic resection due to a volvulus. Methods: The aim of this study was to show that even when the extended form of Hirschprung's disease or anatomic difficulties such as a short bowel and anterior colonic resection are encountered, the laparoscopic approach is possible and total colectomy is feasible. The procedure has been described previously. We used one camera port and three working ports. The sigmoid, transverse, and right colon up to the last ileal cove were mobilized laparoscopically. A standard posterior ileo-anal anastomosis was performed, and an endo-GIA stapler was used for the anterior anastomosis. Results: A total of five patients underwent laparoscopic surgery for Hirschprung's disease. There were three total colonic forms, one rectosigmoid form with a short bowel and colon resection attributed to a volvulus, and one transverse variant that required a Deloyers' maneuver for the pull-through. Three of the infants required total parenteral nutrition (TPN) for an average of 49 days (range, 28-60) from diversion until the time of the definitive procedure. Only one patient did not receive TPN. Postoperatively, there were two complications-one wound infection and one hectic fever. The clinical results were good, with no soiling or stool incontinence and no constipation. Conclusion: The laparoscopic procedure for total aganglionosis or the extended form of Hirschprung's disease is safe, feasible, and reproducible.     

Effects of ovariectomy and steroid hormones on vaginal smooth muscle contractility

The role of steroid hormones in regulating vaginal smooth muscle contractility was investigated. Rabbits were kept intact or ovariectomized. After 2 weeks, animals were continuously infused with vehicle or supraphysiological levels of testosterone (100 microg/day), or estradiol (200 microg/day), for an additional 2 weeks. The distal vaginal tissue was used to assess contractility in organ baths and changes in tissue structure were assessed by histology. Ovariectomized animals infused with vehicle exhibited significant atrophy of the muscularis and decreased epithelial height, resulting in thinning of the vaginal wall. Estradiol infusion increased epithelial height, comparable to that of intact animals, but only partially restored the muscularis layer. In contrast, testosterone infusion completely restored the muscularis layer, but only partially restored the epithelial height. In vaginal tissue strips contracted with norepinephrine and treated with bretylium, electrical field stimulation (EFS) caused frequency-dependent relaxation that was slightly attenuated with vehicle, significantly inhibited with estradiol and significantly enhanced with testosterone. VIP-induced relaxation was slightly attenuated in tissues from vehicle and estradiol-infused groups, but was enhanced in tissues from testosterone-infused animals. Contraction elicited by EFS or exogenous norepinephrine was not significantly altered with ovariectomy or steroid hormone infusion when data were normalized to potassium contraction. However, the tissue from testosterone-infused animals developed significantly greater contractile force to norepinephrine. These observations suggest that steroid hormones may be important regulators of vaginal tissue structure and contractility.     

Length dependence of the contractility of pig detrusor smooth muscle fibres

Evidence on the length dependence of the contraction velocity of smooth muscle fibres is contradictory. Nevertheless, a thorough understanding of this dependence is essential for a correct urodynamic diagnosis of voiding problems. We studied muscle fibres of pig urinary bladders (n=23). Force-velocity relations were measured at different muscle lengths with a stop test technique. This method involves measuring force generation of electrically stimulated muscle fibres during controlled shortening from a pre-shortening length at a pre-set velocity to a fixed stop-length. We normalized the length dependence of the measured properties to slack length, optimum length (the length at maximum isometric force generation), and passive force. Isometric force was found to be length dependent with an optimum length of 290 ± 68% of the slack length (n=11, P < 0.05). The maximum shortening velocity was 0.37 ± 0.14 s − 1 related to the slack length and 0.13 ± 0.05 s − 1 related to the optimum length and was not length dependent (n=16, P < 0.05). Slack length is preferable to normalize the length dependence of smooth muscle. Received: 8 August 2000 / Accepted: 6 December 2000     

Long-term outcome and colonic motility after the Duhamel procedure for Hirschsprung's disease

BACKGROUND/PURPOSE: The aim of this study was to investigate long-term functional outcome and colonic motility in children who had undergone the Duhamel-type operation for Hirschsprung's disease (HSCR). METHODS: All patients (n = 91) who underwent the Duhamel or Lester Martin Modified Duhamel operation for HSCR from 1980 to 1991 were included in the study. Twenty-two healthy age-matched children were used as controls. Functional outcome was assessed by questionnaire (response rate 100%). Total and segmental colonic transit time (CTT) was determined using the saturation method (80% participation rate). RESULTS: Outcome scores were significantly worse in the study group for patients with rectosigmoid (RS, P < .001), long segment (LS, P < .001), and total colonic (TC) aganglionosis (P < .05), when compared with controls. The CTT was significantly prolonged in the RS group (P = .01) compared with LS, TC, and control groups; this was caused by prolonged "rectosigmoid" transit in the RS group compared with controls (P = .012). There was a positive linear correlation (P = .0002) between age and outcome score in patients with RS disease unrelated to CTT. Nine patients required a late long-term enterostomy. A satisfactory outcome (defined as outcome score > or = 10th percentile of the control group, and absence of stoma or requirement for major revisional surgery) was seen in only 42% of patients overall and in 79% of patients over 14 years of age. CONCLUSIONS: The Duhamel procedure, in common with other pull-through procedures, is associated with significant long-term morbidity, the aetiology of which is poorly understood.