Human RecQL4 helicase plays multifaceted roles in the genomic stability of normal and cancer cells.

Human RecQ helicases that share homology with E. coli RecQ helicase play critical roles in diverse biological activities such as DNA replication, transcription, recombination and repair. Mutations in three of the five human RecQ helicases (RecQ1, WRN, BLM, RecQL4 and RecQ5) result in autosomal recessive syndromes characterized by accelerated aging symptoms and cancer incidence. Mutational inactivation of Werner (WRN) and Bloom (BLM) genes results in Werner syndrome (WS) and Bloom syndrome (BS) respectively. However, mutations in RecQL4 result in three human disorders: (I) Rothmund-Thomson syndrome (RTS), (II) RAPADILINO and (III) Baller-Gerold syndrome (BGS). Cells from WS, BS and RTS are characterized by a unique chromosomal anomaly indicating that each of the RecQ helicases performs specialized function(s) in a non-redundant manner. Elucidating the biological functions of RecQ helicases will enable us to understand not only the aging process but also to determine the cause for age-associated human diseases. Recent biochemical and molecular studies have given new insights into the multifaceted roles of RecQL4 that range from genomic stability to carcinogenesis and beyond. This review summarizes some of the existing and emerging knowledge on diverse biological functions of RecQL4 and its significance as a potential molecular target for cancer therapy.

STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis.

CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.

PCSK5 mutation in a patient with the VACTERL association.

BACKGROUND: The VACTERL association is a typically sporadic, non-random collection of congenital anomalies that includes vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula with esophageal atresia, renal anomalies, and limb abnormalities. Although several chromosomal aberrations and gene mutations have been reported as disease-causative, these findings have been sparsely replicated to date. CASE PRESENTATION: In the present study, whole exome sequencing of a case with the VACTERL association uncovered a novel frameshift mutation in the PCSK5 gene, which has been reported as one of the causative genes for the VACTERL association. Although this mutation appears potentially pathogenic in its functional aspects, it was also carried by the healthy father. Furthermore, a database survey revealed several other deleterious variants in the PCSK5 gene in the general population. CONCLUSIONS: Further studies are necessary to clarify the etiological role of the PCSK5 mutation in the VACTERL association.

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.

Cytochemistry of sialoglycoconjugates and lysozyme in canine anal glands as studied by electron microscopic methods.

The distribution of sialoglycoconjugates and lysozyme in the secretory cells of canine anal glands was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry and immunocytochemistry. Sialic acids were predominantly present in the secretory granules, Golgi bodies, surface coat of the plasma membrane and luminal secretions. In addition, within these structures, the secretory granules, Golgi bodies and luminal secretions exhibited high levels of sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc or Siaα2-3Galß1-4GlcNAc. In the secretory cells, reactive gold particles representing lysozyme were mainly detectable in the secretory granules and Golgi bodies. Sialic acids possess diverging functional properties, whereas lysozyme contributes to the non-specific defense against microorganisms. Therefore, their presence and secretion are suggestive of protective effects of both secretory products at the anal mucosa.

Nitric oxide synthase expression in rat anorectal tissue after sacral neuromodulation.

BACKGROUND: Sacral neuromodulation is becoming established as a valid treatment option for patients with anorectal disorders. Nevertheless, despite its efficacy, little is known regarding its mechanism of action. The purpose of this study was to evaluate whether chronic sacral neuromodulation is able to influence the expression of nitric oxide synthetase (NOS) in the anorectum of rats. MATERIALS AND METHODS: Twenty-six female Sprague-Dawley rats were divided into three groups; normal control rats (n = 6); sham treatment (n =10) and group in whom, electrical sacral neuromodulation was performed (n = 10). Bilateral electrode wires were placed in the S1 and electrical stimulation was performed for 14 d. At the end of the procedures the rats were sacrificed, proctectomy was performed, and anorectal specimens were sent to the laboratory for immunostaining with n-NOS and i-NOS. RESULTS: In the anal and rectal specimens, n-NOS and i-NOS expression was significantly increased in epithelial and muscle cells after neuromodulation of the anus and rectum of the animals. CONCLUSION: Our results showed that this model can be applied in further experimental studies to better understand the mechanism of action of sacral neuromodulation in anorectal disorders.

Bioengineered internal anal sphincter derived from isolated human internal anal sphincter smooth muscle cells.

BACKGROUND & AIMS: The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional. METHODS: 3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured. RESULTS: The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not. CONCLUSIONS: These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions.

COX-1 vs. COX-2 as a determinant of basal tone in the internal anal sphincter.

Prostanoids, produced endogenously via cyclooxygenases (COXs), have been implicated in the sustained contraction of different smooth muscles. The two major types of COXs are COX-1 and COX-2. The COX subtype involved in the basal state of the internal anal sphincter (IAS) smooth muscle tone is not known. To identify the COX subtype, we examined the effect of COX-1- and COX-2-selective inhibitors, SC-560 and rofecoxib, respectively, on basal tone in the rat IAS. We also determined the effect of selective deletion of COX-1 and COX-2 genes (COX-1(-/-) and COX-2(-/-) mice) on basal tone in murine IAS. Our data show that SC-560 causes significantly more efficacious and potent concentration-dependent decreases in IAS tone than rofecoxib. In support of these data, significantly higher levels of COX-1 than COX-2 mRNA were found in the IAS. In addition, higher levels of COX-1 mRNA and protein were expressed in rat IAS than rectal smooth muscle. In wild-type mice, IAS tone was decreased 41.4 +/- 3.4% (mean +/- SE) by SC-560 (1 x 10(-5) M) and 5.4 +/- 2.2% by rofecoxib (P < 0.05, n = 5). Basal tone was 0.172 +/- 0.021 mN//mg in the IAS from wild-type mice and significantly less (0.080 +/- 0.015 mN/mg) in the IAS from COX-1(-/-) mice (P < 0.05, n = 5). However, basal tone in COX-2(-/-) mice was not significantly different from that in wild-type mice. We conclude that COX-1-related products contribute significantly to IAS tone.

Cellular regulation of basal tone in internal anal sphincter smooth muscle by RhoA/ROCK.

Sustained contractions of smooth muscle cells (SMC) maintain basal tone in the internal anal sphincter (IAS). To examine the molecular bases for the myogenic tone in the IAS, the present studies focused on the role of RhoA/ROCK in the SMC isolated from the IAS vs. the adjoining phasic tissues of the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM) of rat. We also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP, RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex formation, levels of p(Thr696)-MYPT1, and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ROCK were higher at the cell membrane in the IAS SMC compared with those from the RSM and ASM. C3 exoenzyme (RhoA inhibitor) and Y 27632 (ROCK inhibitor) caused a concentration-dependent relaxation of the IAS SMC. In addition, active ROCK-II (primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3 exoenzyme increased RhoA-RhoGDI binding and reduced the levels of RhoA-GTP and p(Thr696)-MYPT1. ROCK inhibitor attenuated PKC-induced contractions in IAS SMC. Conversely, a PKC inhibitor (Gö 6850, which causes partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, active form of RhoA (RhoA-GTP), ROCK-II, 20-kDa myosin regulatory light chain (MLC(20)), phospho-MYPT1, and phospho-MLC(20) in the IAS vs. RSM and ASM SMC. However, the trend was the reverse with the levels of inactive RhoA (GDP-RhoA-RhoGDI complex) and MYPT1. We conclude that RhoA/ROCK play a critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light chain phosphatase.

Rho kinase as a novel molecular therapeutic target for hypertensive internal anal sphincter.

BACKGROUND & AIMS: An increase in Rho kinase (ROK) activity has been associated with agonist-induced sustained contraction of the smooth muscle, but its role in the pathophysiology of spontaneously tonic smooth muscle is not known. METHODS: Present studies examined the effects of ROK inhibitor Y-27632 in the tonic smooth muscle of the rat internal anal sphincter (IAS) versus in the flanking phasic smooth muscle of the rectum. In addition, studies were performed to determine the relationship between the decreases in the basal IAS tone and the ROK activity. Confocal microscopic studies determined the cellular distribution of the smooth muscle-predominant isoform of ROK (ROCK-II) in the smooth muscle cells (SMCs). RESULTS: In in vitro studies using neurohumoral inhibitors and tetrodotoxin and the use of SMCs demonstrate direct relaxation of the IAS SMCs by Y-27632. The ROK inhibitor was more potent in the IAS than in the rectal smooth muscle. The IAS relaxation by Y-27632 correlated specifically with the decrease in ROK activity. Confocal microscopy revealed high levels of ROCK-II toward the periphery of the IAS SMCs. In in vivo studies, the lower doses of Y-27632 caused a potent and selective decrease in the IAS pressures without any adverse cardiovascular systemic effects. The ROK inhibitor also caused potent relaxation of the hypertensive IAS. CONCLUSIONS: RhoA/ROK play a crucial role in the maintenance of the basal tone in the IAS, and ROK inhibitors have a therapeutic potential in the IAS dysfunction characterized by the hypertensive IAS.

Nitric oxide deficiency in the internal anal sphincter of patients with chronic anal fissure.

Anal fissure is a common condition affecting young to middle-aged adults. It causes severe pain on defecation and rectal bleeding. The aetiology remains uncertain. Spasm of the internal anal sphincter is a constant feature. Nitric oxide (NO) is the major inhibitory neurotransmitter of the internal anal sphincter (IAS). In other spasmodic conditions of the GI tract a lack of normal nitric oxide synthase (NOS) activity has been reported. The aim of this preliminary study was to compare the presence of NOS in the internal sphincters of patients with and without chronic anal fissure. Internal anal sphincter biopsies were taken under general anaesthesia from patients having lateral internal sphincterotomy for chronic anal fissure and from sphincter of patients having abdominoperineal resections as controls. Sections of IAS were stained to show the presence of NADPH diaphorase (and hence presence of NOS). Internal anal sphincter was taken from 6 patients with chronic anal fissure and 6 controls. IAS taken from patients with chronic anal fissure showed little NOS presence compared with controls. It may be that there is an abnormal failure of relaxation of internal sphincter in those patients who develop chronic anal fissure caused by an intrinsic lack of neural NOS in the internal anal sphincter.

Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle.

1. The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms. 2. The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC(50) values against CCh and Tg of 8.4+/-3.3 (n=6) and 6.1+/-2.1 (n=7) micro M, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69+/-7% (n=4) inhibition using 10 micro M Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A. 3. In beta-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 micro M) and GTP (100 micro M). These responses to CCh and Tg were inhibited by Y27632 (10 micro M). 4. Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane. 5. These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus.

Cyclooxygenase cloning in dogfish shark, Squalus acanthias, and its role in rectal gland Cl secretion.

The present studies were carried out with the aims to determine the cDNA sequence for cyclooxygenase (COX) in an elasmobranch species and to study its role in regulation of chloride secretion in the perfused shark rectal gland (SRG). With the use of long primers (43 bp) derived from regions of homology between zebrafish and rainbow trout COX-2 genes, a 600-bp product was amplified from SRG and was found to be almost equally homologous to mammalian COX-1 and COX-2 (65%). The full-length cDNA sequence was obtained by 5'-RACE and by analyzing an EST clone generated by the EST Project of the Mt. Desert Island Biological Laboratory Marine DNA Sequencing Center. The longest open reading frame encodes a 593-amino acid protein that has 68 and 64% homology to mammalian COX-1 and COX-2, respectively. The gene and its protein product is designated as shark COX (sCOX). The key residues in the active site (Try(385), His(388), and Ser(530)) are conserved between the shark and mammalian COX. sCOX contains Val(523) that has been shown to be a key residue determining the sensitivity to COX-2-specific inhibitors including NS-398. The mRNA of sCOX, detected by RT-PCR, was found in all tissues tested, including rectal gland, kidney, spleen, gill, liver, brain, and heart, but not in fin. In the perfused SRG, vasoactive intestinal peptide (VIP) at 5 nM induced rapid and marked Cl(-) secretion (basal: <250 microeq x h(-1) x g(-1); peak response: 3,108 +/- 479 microeq x h(-1) x g(-1)). In the presence of 50 microM NS-398, both the peak response (2,131 +/- 307 microeq x h(-1) x g(-1)) and the sustained response to VIP were significantly reduced. When NS-398 was removed, there was a prompt recovery of chloride secretion to control values. In conclusion, we have cloned the first COX in an elasmobranch species (sCOX) and shown that sCOX inhibition suppresses VIP-stimulated chloride secretion in the perfused SRG.

Inducible and neuronal nitric oxide synthase involvement in lipopolysaccharide-induced sphincteric dysfunction.

We examined the effect of endotoxin lipopolysaccharide (LPS) on the basal tone and on the effects of different stimuli and agonists and transcriptional and translational expression of nitric oxide (NO) synthase (NOS) isozymes in the lower esophageal sphincter (LES), pyloric sphincter (PS), and internal anal sphincter (IAS). NO release was also examined before and after LPS. LPS caused a dose-dependent fall in the basal tone and augmentation of the relaxation caused by nonadrenergic, noncholinergic (NANC) nerve stimulation in the LES and IAS. In the PS, LPS had no significant effect on the basal tone and caused an attenuation of the NANC relaxation and an augmentation of the contractile response of muscarinic agonist. Interestingly, the smooth muscle relaxation by atrial natriuretic factor was suppressed in the LES and IAS but not in the PS. These changes in the sphincteric function following LPS may be associated with increase in the inducible NOS (iNOS) expression since they were blocked by iNOS inhibitor L-canavanine. Augmentation of NANC relaxation in the LES and IAS smooth muscle by LPS may be due to the increased activity of neuronal NOS and NO production.

Influence of heme oxygenase inhibitors on the basal tissue enzymatic activity and smooth muscle relaxation of internal anal sphincter.

We examined the actions of different heme oxygenase (HO) inhibitors on the basal HO activity in the opossum internal anal sphincter (IAS), rectum, and liver tissues and the IAS smooth muscle relaxation in response to nonadrenergic noncholinergic (NANC) nerve stimulation and different agonists. All the tissues examined were found to have significant levels of basal HO activity. Among different HO inhibitors, tin protoporphyrin IX (SnPP IX) was found to be most selective in inhibiting the HO activity in the IAS smooth muscle. Conversely, in the liver, all the HO inhibitors except SnPP IX caused significant inhibition of HO activity. Consistent with HO activity inhibition, the IAS smooth muscle relaxations caused by NANC nerve stimulation and vasoactive intestinal polypeptide also were inhibited by zinc protoporphyrin IX and SnPP IX. Zinc protoporphyrin IX also caused a significant attenuation of the IAS smooth muscle relaxation caused by isoproterenol. The IAS smooth muscle relaxation caused by nitric oxide was not significantly modified by any of the HO inhibitors. The data show the presence of HO activity in the IAS and other gastrointestinal tissues. The differential attenuation of HO activity by different HO inhibitors in the IAS smooth muscle and liver confirms the presence of different isozymes of HO in different tissues. Suppression of basal HO activity and the IAS smooth muscle relaxation induced by NANC nerve stimulation or VIP but not NO suggest that some of the stimuli that cause IAS smooth muscle relaxation may involve HO activity.

Heme oxygenase activity in the internal anal sphincter: effects of nonadrenergic, noncholinergic nerve stimulation.

BACKGROUND & AIMS: To date, the exact role of carbon monoxide (CO) in the nonadrenergic, noncholinergic (NANC) relaxation is not known. This is partly related to the lack of an appropriate method to measure heme oxygenase (HO) activity in the gastrointestinal tissues. METHODS: HO activity of the opossum internal anal sphincter (IAS) smooth muscle was determined using a newly developed assay system that used radiolabeled hemin as a substrate. Enzyme activity of the IAS tissues was measured in the basal state, after electric field stimulation (EFS), ganglionic stimulant dimethyl diphenyl piperazinium iodide (DMPP), and neuropeptide vasoactive intestinal polypeptide (VIP). The presence and localization of HO was examined by Western blot analysis and immunocytochemistry. RESULTS: NANC nerve stimulation of the IAS smooth muscle by EFS (0.25-5 Hz), DMPP, and VIP caused a significant increase in the HO activity of the IAS. The increase in HO activity by EFS was inhibited by the HO inhibitor Tin protoporphyrin (1 x 10(-4) mol/L). Both HO-1 and HO-2 were present in the IAS tissue extracts, and both enzymes were localized in the neurons of the myenteric plexus. The method for HO activity determination used in the present study was found to be reliable and reproducible. CONCLUSIONS: The data suggest that the HO pathway may have a role in neurally mediated relaxation of the IAS. The exact site of involvement and the source of HO activity, however, remains to be determined.

End-to-side pudendal nerve anastomosis for the creation of a new reinforcing anal sphincter in dogs.

BACKGROUND: An ideal reinforcing neo-sphincter should be innervated by the pudendal nerve to work in coordination with the external anal sphincter. The aim of this study was to create a skeletal muscle innervated by the pudendal nerve without inducing external anal sphincter morbidity. METHODS: Seven dogs were used. On the right side of each dog, the distal end of the transected nerve innervating the biceps femoris muscle was anastomosed to the perineural window of the pudendal nerve, where the epineurium was excised over a small area without injuring the funiculus. Reinnervation was studied 5 months after the operations. The left side of the dogs was used as a control. RESULTS: For all 7 dogs, there was no macroscopic difference detected in the external anal sphincter. For 6 dogs, the biceps femoris muscle was preserved after end-to-side pudendal nerve anastomosis. After the pudendal nerve central to the anastomosis site was electrically stimulated, the external anal sphincter contracted in all dogs. After end-to-side pudendal nerve anastomosis, the biceps femoris muscle contracted with the evoked potential in 5 dogs (71%) and demonstrated electric activity at rest in 3 dogs (43%), but there was no reflex activity after anal stimulation. The ratio of type 1 to type 2 muscle fiber in the biceps femoris muscle after end-to-side pudendal nerve anastomosis significantly changed and became the same as that in the external anal sphincter. The diameter of type 2 muscle fibers in the biceps femoris muscle significantly decreased after surgery. In addition, regenerated myelinated axons were observed in a cross section of the anastomosed nerve in 6 dogs. CONCLUSIONS: End-to-side pudendal nerve anastomosis is a promising technique for the creation of an anal sphincter in patients who have fecal incontinence. The technique preserved the original external anal sphincter and created a skeletal muscle that was innervated by the pudendal nerve in 71% of the dogs studied. This newly innervated skeletal muscle was capable of contracting in coordination with the original external anal sphincter on electric stimulation and also demonstrated characteristics closely similar to those of the external anal sphincter.

Heme oxygenase-2 distribution in anorectum: colocalization with neuronal nitric oxide synthase.

Recent investigations have suggested carbon monoxide (CO) as a putative messenger molecule. Although several studies have implicated the heme oxygenase (HO) pathway, responsible for the endogenous production of CO, in the neuromodulatory control of the internal anal sphincter (IAS), its exact role is not known. Nitric oxide, produced by neuronal nitric oxide synthase (nNOS) of myenteric neurons, is an important inhibitory neural messenger molecule mediating nonadrenergic noncholinergic (NANC) relaxation of the IAS. The present studies were undertaken to investigate in detail the presence and coexistence of heme oxygenase-2 (HO-2) with nNOS in the opossum anorectum. In perfusion-fixed, frozen-sectioned tissue, HO-2 immunoreactive (IR) and nNOS IR nerves were identified using immunocytochemistry. Ganglia containing HO-2 IR neuronal cell bodies were present in the myenteric and submucosal plexuses throughout the entire anorectum. Colocalization of HO-2 IR and nNOS IR was nearly 100% in the IAS and decreased proximally from the anal verge. In the rectum, colocalization of HO-2 IR and nNOS IR was approximately 70%. Additional confocal microscopy studies using c-Kit staining demonstrated the localization of HO-2 IR and nNOS IR in interstitial cells of Cajal (ICC) of the anorectum. From the high rate of colocalization of HO-2 IR and nNOS IR in the IAS as well as the localization of HO-2 IR and nNOS IR in ICC in conjunction with earlier studies of the HO pathway, we speculate an interaction between HO and NOS pathways in the NANC inhibitory neurotransmission of the IAS and rectum.

Biochemical and functional profile of a newly developed potent and isozyme-selective arginase inhibitor.

An increase in arginase activity has been associated with the pathophysiology of a number of conditions, including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle. An arginase inhibitor may rectify this condition. We compared the effects of a newly designed arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH), with the currently available N(omega)-hydroxy-L-arginine (L-HO-Arg), on the NANC nerve-mediated internal anal sphincter (IAS) smooth-muscle relaxation and the arginase activity in the IAS and other tissues. Arginase caused an attenuation of the IAS smooth-muscle relaxations by NANC nerve stimulation that was restored by the arginase inhibitors. L-HO-Arg but not ABH caused dose-dependent and complete reversal of N(omega)-nitro-L-arginine-suppressed IAS relaxation that was similar to that seen with L-arginine. Both ABH and L-HO-Arg caused an augmentation of NANC nerve-mediated relaxation of the IAS. In the IAS, ABH was found to be approximately 250 times more potent than L-HO-Arg in inhibiting the arginase activity. L-HO-Arg was found to be 10 to 18 times more potent in inhibiting the arginase activity in the liver than in nonhepatic tissues. We conclude that arginase plays a significant role in the regulation of nitric oxide synthase-mediated NANC relaxation in the IAS. The advent of new and selective arginase inhibitors may play a significant role in the discrimination of arginase isozymes and have important pathophysiological and therapeutic implications in gastrointestinal motility disorders.

A unique myosin isoform transition in cat striated external anal sphincter muscle induced by denervation.

Denervation of limb and trunk muscles leads to characteristic changes in their biochemical properties. However, the effects of denervation of the striated external anal sphincter (EAS) muscle have not been studied, even though denervation is a common etiology for fecal incontinence. The present study reports effects ofdenervation of the cat EAS and compares them with changes in a denervated limb muscle [cat extensor digitorum longus (EDL)]. Nerves supplying the EAS and the EDL were sectioned in anesthetized cats, which were allowed to recover and live for 10, 30, or 60 days. Their muscles were then excised, and myofibrillar proteins were isolated. Myosin heavy chain (MHC) isoform composition was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EDL and EAS showed progressive increases in MHC type IIA and progressive decreases in MHC type IIB at 10, 30, and 60 days. MHC type I progressively increased in the EDL but showed no change in the EAS. We hypothesize that this distinctive transition is related to the unique embryological origin and function of the EAS.