Deletion of Dicer in smooth muscle affects voiding pattern and reduces detrusor contractility and neuroeffector transmission

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+) channels in the detrusor.     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

The effect of ovariectomy on the contractile response of the rat isolated detrusor muscle and urethra

Contractile responses induced by carbachol on the detrusor muscle and by noradrenaline on the isolated urethra were compared between ovariectomized rats pretreated with estradiol (50 μg/animal s.c. twice daily for five days), untreated ovariectomized rats and intact animals. In the detrusor muscle, contractions induced by 30μM carbachol, when normalized with respect to KCl 100 mM-induced contraction, were similar for the three groups. Furthermore, contractions induced by 100 μM noradrenaline in the isolated urethra were not significatively different between groups. However, the pD₂ value for noradrenaline was greater in urethral tissue from ovariectomized rats compared with ovariectomized -estrogen treated and control rats. A similar result was found for pD₂ values for carbachol-induced contractions on the detrusor muscle. These results suggest that ovariectomy increases the sensitivity of the urinary bladder and urethra to the contractile effects of carbachol and noradrenaline, respectively and that this effect is reversed by in vivo estrogen pretreatment.     

Bladder dysfunction after acute urinary retention in the rats: a novel over active bladder model

As there is increasing evidence that benign prostatic hyperplasia and its related acute urinary retention (AUR) induce over active bladder (OAB) syndrome, we investigated the effects of AUR on bladder function over a 4-week period in a rat model. Ten-week-old female Sprague-Dawley rats were used in this study. AUR was induced by clamping the distal urethra of each rat with a small clip, and then infusing 3 ml (0.6 ml/min) of saline with an infusion pump through a transurethral catheter (22G). The obstruction was sustained for 60 min and the clip was removed and then the bladder was allowed to drain through the catheter. The bladder function was estimated by voiding behavior studies (at 3 days, 1, 2, 3, and 4 weeks), cystometric studies (at 2 and 4 weeks) and organ bath studies using KCl and carbachol (at 2 and 4 weeks). Furthermore, we evaluated histological changes in the rat bladder 2 and 4 weeks after the induction of AUR. The same parameters were also measured in non-AUR rats (control group). The rat bladder weight in the AUR group at 2 weeks was significantly larger than that of the controls, and returned to the control level 4 weeks after the AUR episode. The voiding behavior studies showed significant increase in micturition frequency per day and decrease in single voiding volume 3 days after the induction of AUR, and this voiding behavior was continued for more than 2 weeks. The cystometric studies showed a significant decrease in single-voided volume at 2 weeks rat. However, no significant changes of the other parameters were observed in the rats. The histological studies showed significant infiltration of neutrophils and lymphocytes, as well as increase in turnover of epithelium in AUR rats at 2 weeks, while significant increases in fibrosis in submucosal layer were observed in AUR rats at 4 weeks. This study demonstrated that bladder dysfunction in the rat model caused by AUR needs more than 2 weeks of recovery period. The AUR-associated alterations in the bladder may represent a key clue to understand the underlying pathophysiological mechanisms, which take place in OAB syndrome.     

Relationship between connexin43-derived gap junction proteins in the bladder and age-related detrusor underactivity in rats

Aims

To confirm the mechanisms of age-associated detrusor underactivity (DU), we examined the differences in bladder activity and connexin-43 (Cx43)-derived gap junctions in the bladders of young and old rats.

Main methods

Female Sprague–Dawley rats aged 3 months (young) and 12 months (old) were used. Continuous cystometry was performed under urethane anesthesia in both ages of rats. In addition, isovolumetric cystometry was performed in young rats during the intravesical application of carbenoxolone, a gap junction blocker, to confirm the role of gap junction proteins in the bladder. Western blotting analyses were performed to assess Cx43 protein expression in the bladders of both groups of rats. Bladders were also analyzed using Masson's trichrome staining and immunostaining for Cx43.

Key findings

Cystometric evaluations revealed that compared with young rats, bladder contractility was reduced by 27% and residual urine volume was significantly increased in old rats. However, the intercontraction intervals did not differ between the two groups. Under isovolumetric conditions, bladder contraction was suppressed after the intravesical application of carbenoxolone. In the bladders of old rats, increase of smooth muscle cell hypertrophy and fibrous tissue was observed compared with young rats. In association with these findings, immunostaining for smooth muscle Cx43 and its protein level were decreased by 28% compared with young rats.

Significance

These results suggest that age-related DU might be caused by the downregulation of gap junctional intercellular communication in the bladder. Consequently, the normal signals that contribute to voiding function might not be transported between detrusor muscles.     

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death

Partial bladder outlet obstruction of the rabbit bladder results in a rapid increase in mass characterized by remodeling of the bladder wall.In this study we investigated the effect of partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium, and on blood flow using a fluorescent microsphere technique. Transverse sections of bladder wall were examined after 0 (unobstructed), 1, 3, 5, 7, and 14 days of obstruction. The microvasculature of obstructed rabbit bladder mucosa and detrusor smooth muscle apparently increased relative to augmentation of these compartments, while new vessels appeared in the thickening serosa. These vascular changes correlated with results showing that, at 1 week after obstruction, blood flow (ml/min/g tissue) to the mucosa and detrusor was unchanged.Thickening of the serosa, apparent after 1 day of obstruction, began before its vascularization. Then, 1 week post-obstruction, there was significant microvessel formation in the transition region between the detrusor smooth muscle and the increasing serosa; after 2 weeks, the entire serosa was vascularized. The vascularization of the muscle-serosal transition region and then the remaining serosa apparently precedes fibroblast differentiation, providing blood supply and thus metabolic support for this process.All obstructed rabbit bladders in this study were in a state of compensated function based on their weights. Our working hypothesis is that blood flow per unit tissue mass is normal in compensated obstructed bladders, thus allowing for normal contractile function and cellular metabolism. The results of this study indicate the presence of an augmented microvasculature in compensated obstructed rabbit bladders that provides adequate blood perfusion for normal function.     

Aging and neural control of the GI tract: V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins

The object of this theme is to offer new perspectives on the effect of aging on signal-transduction pathways associated with agonist-induced contraction of smooth muscle cells from the colon. Smooth muscle cells from old rats (32 mo old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal-transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKC pathway, and a reduced association of contractile proteins [heat shock protein 27 (HSP27)-tropomyosin, HSP27-actin, actin-myosin]. Levels of HSP27 phosphorylation are also reduced compared with adult rats.     

<Emphasis Type="Italic">Octodon degus</Emphasis>, a new model to study the agonist and plexus-induced response in the urinary bladder

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm²) at 60 mM. In the case of cumulative dose–response of bethanecol, the maximum response (37.42 mN/mm²) was reached at 3.2 × 10^?4 M. The response to ATP was clearly smaller (3.8 mN/mm²). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans.     

Phasic Contractions in Urinary Bladder from Juvenile versus Adult Pigs

Aims

Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs).

Methods

Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib.

Results

Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition.

Conclusions

We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals.     

The effects of isoproterenol on intracellular calcium concentration

beta-Adrenergic agonist, isoproterenol (ISO), is a potent relaxant of tracheal smooth muscle and inhibits carbachol-induced contraction. The effect of ISO on intracellular free Ca2+ concentration ([Ca2+]i) was examined in bovine tracheal smooth muscle strips, employing aequorin as Ca2+ indicator. Surprisingly, 10 microM ISO induces a 5-fold increase in [Ca2+]i which then gradually declines but still remains higher than basal after 1 h of stimulation. The ISO-induced increase in [Ca2+]i is dose-dependent, and the ED50 is approximately 50 nM. The ISO-induced increase in [Ca2+]i is inhibited by a beta-receptor blocker, propranolol, not by an alpha-blocker, phentolamine. The ISO-induced rise in [Ca2+]i is dependent on extracellular Ca2+. Forskolin, an adenylate cyclase activator, and vasoactive intestinal peptide, which is known to stimulate adenylate cyclase via a specific receptor in this tissue, have similar effects on [Ca2+]i, suggesting that a rise in cyclic AMP concentration mediates this effect of ISO on [Ca2+]i. Pretreatment of muscle with 10 microM ISO inhibits both the initial Ca2+ transient and the contractile response induced by 0.3 microM carbachol. Conversely, in carbachol-pretreated muscle strips, addition of ISO causes a fall rather than a rise in [Ca2+]i, and an inhibition of contraction. These results indicate that ISO has effects on cellular Ca2+ metabolism at more than a single site in bovine tracheal smooth muscle, that these effects are different in control and carbachol-pretreated muscle, and that the relaxing effect of ISO is not due solely to its effect on Ca2+ metabolism.     

Effects of nicorandil on isolated smooth muscle cells from guinea-pig taenia caeci

1. The effects of nicorandil on guinea-pig taenia caeci were investigated with the use of isolated smooth muscle cells and glycerin-treated muscle fiber bundles. 2. Nicorandil inhibited high K-, Ca2+- and carbachol-induced contractions in a dose-dependent manner without affecting 45Ca fluxes in isolated cells. 3. Nicorandil had no effect on ATP-induced contraction of glycerin-treated muscle fiber bundles. 4. The present results suggest that nicorandil may inhibit the contraction by action on the contractile proteins in an indirect manner in guinea-pig taenia caeci.     

Accelerated communication the direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca²⁺ release, and verapamil, a Ca²⁺ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED₅₀ was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca²⁺-release from intracellular calcium stores in the contractile response to GRP.     

Altered distribution of interstitial cells and innervation in the rat urinary bladder following spinal cord injury

Changes in the distribution of interstitial cells (IC) are reportedly associated with dysfunctional bladder. This study investigated whether spinal cord injury (SCI) resulted in changes to IC subpopulations (vimentin-positive with the ultrastructural profile of IC), smooth muscle and nerves within the bladder wall and correlated cellular remodelling with functional properties. Bladders from SCI (T8/9 transection) and sham-operated rats 5 weeks post-injury were used for ex vivo pressure-volume experiments or processed for morphological analysis with transmission electron microscopy (TEM) and light/confocal microscopy. Pressure-volume relationships revealed low-pressure, hypercompliance in SCI bladders indicative of decompensation. Extensive networks of vimentin-positive IC were typical in sham lamina propria and detrusor but were markedly reduced post-SCI; semi-quantitative analysis showed significant reduction. Nerves labelled with anti-neurofilament and anti-vAChT were notably decreased post-SCI. TEM revealed lamina propria IC and detrusor IC which formed close synaptic-like contacts with vesicle-containing nerve varicosities in shams. Lamina propria and detrusor IC were ultrastructurally damaged post-SCI with retracted/lost cell processes and were adjacent to areas of cellular debris and neuronal degradation. Smooth muscle hypertrophy was common to SCI tissues. In conclusion, IC populations in bladder wall were decreased 5 weeks post-SCI, accompanied with reduced innervation, smooth muscle hypertrophy and increased compliance. These novel findings indicate that bladder wall remodelling post-SCI affects the integrity of interactions between smooth muscle, nerves and IC, with compromised IC populations. Correlation between IC reduction and a hypercompliant phenotype suggests that disruption to bladder IC contribute to pathophysiological processes underpinning the dysfunctional SCI bladder.     

TPC2 Proteins Mediate Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)- and Agonist-evoked Contractions of Smooth Muscle

Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca²⁺ from sarcoplasmic reticulum. However, the additional Ca²⁺-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca²⁺-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2^−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.     

HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction

We have investigated the role of heat shock protein 27 (HSP27) phosphorylation and the association of HSP27 with contractile proteins actin, myosin, and tropomyosin. Smooth muscle cells were labeled with [(32)P]orthophosphate. C2-ceramide (0.1 microM), an activator of protein kinase C (PKC), induced a sustained increase in HSP27 phosphorylation that was inhibited by calphostin C. C2-ceramide-induced (0.1 microM) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of HSP27 with tropomyosin and in the association of HSP27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of HSP27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin, and HSP27 followed by confocal microscopy. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that phosphorylation of HSP27 could affect myosin association with actin. In conclusion 1) HSP27 phosphorylation appears to be necessary for reorganization of HSP27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway, and 2) agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin.     

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

Effects of Synbiotic2000™ Forte on the Intestinal Motility and Interstitial Cells of Cajal in TBI Mouse Model

The main objective of this study was to investigate the effects of Synbiotic2000? Forte on the intestinal motility and interstitial cells of Cajal (ICC) in traumatic brain injury (TBI) mouse model. Kunming mice were randomly divided into sham operation group (S group), enteral nutrition group with TBI (E group), and Synbiotic2000? Forte group with TBI (P group). The contractile activity of the intestinal smooth muscle, densities and ultrastructure of the ICC, kit protein concentration, weight, and defecation of mice were monitored and analyzed. TBI markedly suppressed contractile activity of the intestinal smooth muscle (P < 0.01), which led to a reduction of defecation (P < 0.01) and weight (P < 0.01). However, application of Synbiotic2000? Forte significantly improved contractile activity of the small intestine (P < 0.01), which may be related to protective effects to the interstitial cells of Cajal, smooth muscle cells, and enteric neurons. TBI impaired ICC networks and densities (P < 0.01), events that were protected by the application of Synbiotic2000? Forte. Synbiotic2000? Forte may attenuate TBI-mediated inhibition of the kit protein pathway. Synbiotic2000? Forte may improve intestinal motility and protect the ICC in the TBI mouse. These findings provide a novel support for the application of Synbiotic2000? Forte in intestinal motility disturbance after TBI.     

Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle

Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin. acetylcholine; fusion proteins; serine     

Ultrastructural basis of airway smooth muscle contraction

The sliding filament theory of contraction that was developed for striated muscle is generally believed to be also applicable to smooth muscle. However, the well-organized myofilament lattice (i.e., the sarcomeric structure) found in striated muscle has never been clearly delineated in smooth muscle. There is evidence that the myofilament lattice in some smooth muscles, such as airway smooth muscle, is malleable; it can be reshaped to fit a large range of cell dimensions while the maximal overlap between the contractile filaments is maintained. In this review, some early models of the structurally static contractile apparatus of smooth muscle are described. The focus of the review, however, is on the recent findings supporting a model of structurally dynamic contractile apparatus and cytoskeleton for airway smooth muscle. A list of unanswered questions regarding smooth muscle ultrastructure is also proposed in this review, in the hope that it will provide some guidance for future research.