Longitudinal Smooth Muscle Cells Research Articles

Bisphenol A inhibits duodenal movement ex vivo of rat through nitric oxide-mediated soluble guanylyl cyclase and α-adrenergic signaling pathways.

The gastrointestinal tract is directly exposed to bisphenol A (BPA)-tainted foods and beverages stored in polycarbonate plastic containers. The effect of BPA on the movement of small intestine has not been reported until now. We report here the effect of BPA on the movement of the duodenum ex vivo in a rat model. We found significant inhibition of duodenal movement by BPA (10-320 µM). We suggest that BPA-induced inhibition of duodenal movement might be due to the suppression of stimulatory and/or activation of inhibitory motor neurons in enteric plexuses innervating the longitudinal and circular visceral smooth muscle cells in the duodenal wall. We observed a significant reversal of BPA-induced depression of duodenal movement by methylene blue, a soluble guanylyl cyclase blocker and N-ω-nitro-L-arginine methyl ester, a nitric oxide (NO) synthase inhibitor; but significant potentiation of the movement by sodium nitroprusside, a NO donor. From the results, we may suggest that BPA-induced inhibition of the movement might be partially due to activation of inhibitory motor neurons that secrete NO, a relaxant, on to smooth muscle cells. Furthermore, we found significant reversal of BPA-induced depression of the movement in phentolamine, an α-adrenergic receptor blocker, pretreated preparation. This result proves that norepinephrine secreting motor neurons may also be involved in BPA-induced inhibition of the movement. From the results, we conclude that BPA inhibits the movement of the duodenum through NO-mediated soluble guanylyl cyclase and α-adrenergic signaling pathways in visceral smooth muscle cells.

Hydroxy-α sanshool induces colonic motor activity in rat proximal colon: a possible involvement of KCNK9.

Various colonic motor activities are thought to mediate propulsion and mixing/absorption of colonic content. The Japanese traditional medicine daikenchuto (TU-100), which is widely used for postoperative ileus in Japan, accelerates colonic emptying in healthy humans. Hydroxy-α sanshool (HAS), a readily absorbable active ingredient of TU-100 and a KCNK3/KCNK9/KCNK18 blocker as well as TRPV1/TRPA1 agonist, has been investigated for its effects on colonic motility. Motility was evaluated by intraluminal pressure and video imaging of rat proximal colons in an organ bath. Distribution of KCNKs was investigated by RT-PCR, in situ hybridization, and immunohistochemistry. Current and membrane potential were evaluated with use of recombinant KCNK3- or KCNK9-expressing Xenopus oocytes and Chinese hamster ovary cells. Defecation frequency in rats was measured. HAS dose dependently induced strong propulsive “squeezing” motility, presumably as long-distance contraction (LDC). TRPV1/TRPA1 agonists induced different motility patterns. The effect of HAS was unaltered by TRPV1/TRPA1 antagonists and desensitization. Lidocaine (a nonselective KCNK blocker) and hydroxy-β sanshool (a geometrical isomer of HAS and KCNK3 blocker) also induced colonic motility as a rhythmic propagating ripple (RPR) and a LDC-like motion, respectively. HAS-induced “LDC,” but not lidocaine-induced “RPR,” was abrogated by a neuroleptic agent tetrodotoxin. KCNK3 and KCNK9 were located mainly in longitudinal smooth muscle cells and in neural cells in the myenteric plexus, respectively. Administration of HAS or TU-100 increased defecation frequency in normal and laparotomy rats. HAS may evoke strong LDC possibly via blockage of the neural KCNK9 channel in the colonic myenteric plexus.

Spontaneous transient hyperpolarizations in the rabbit small intestine.

Four types of electrical activity were recorded and related to cell structure by intracellular recording and dye injection into impaled cells in muscles of rabbit small intestine. The specific cell types from which recordings were made were longitudinal smooth muscle cells (LSMCs), circular smooth muscle cells (CSMCs), interstitial cells of Cajal distributed in the myenteric region (ICC-MY) and fibroblast-like cells (FLCs). Slow waves (slow wavesSMC) were recorded from LSMCs and CSMCs. Slow waves (slow wavesICC) were of greatest amplitude (>50mV) and highest maximum rate of rise (>10Vs(-1)) in ICC-MY. The dominant activity in FLCs was spontaneous transient hyperpolarizations (STHs), with maximum amplitudes above 30mV. STHs were often superimposed upon small amplitude slow waves (slow wavesFLC). STHs displayed a cyclical pattern of discharge irrespective of background slow wave activity. STHs were inhibited by MRS2500 (3μm), a P2Y1 antagonist, and abolished by apamin (0.3μm), a blocker of small conductance Ca(2+)-activated K(+) channels. Small amplitude STHs (<15mV) were detected in smooth muscle layers, whereas STHs were not resolved in cells identified as ICC-MY. Electrical field stimulation evoked purinergic inhibitory junction potentials (IJPs) in CSMCs. Purinergic IJPs were not recorded from ICC-MY. These results suggest that FLCs may regulate smooth muscle excitability in the rabbit small intestine via generation of rhythmic apamin-sensitive STHs. Stimulation of P2Y1 receptors modulates the amplitudes of STHs. Our results also suggest that purinergic inhibitory motor neurons regulate the motility of the rabbit small intestine by causing IJPs in FLCs that conduct to CSMCs.

Cytokine-induced iNOS and ERK1/2 inhibit adenylyl cyclase type 5/6 activity and stimulate phosphodiesterase 4D5 activity in intestinal longitudinal smooth muscle.

This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1β and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1β and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1β or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation.

Hexamethonium-induced augmentation of the electrical twitch response in the guinea-pig ileum longitudinal muscle–myenteric plexus strip

Longitudinal muscle–myenteric plexus strips of the guinea-pig ileum were used to investigate the nature of the hexamethonium-induced augmentation of the twitch response. All preparations were set up in Tyrode solution and intermittent longitudinal twitch contractions were evoked by single pulse electrical field stimulation. Hexamethonium, a blocker of nicotinic ganglionic transmission, at 300μmol/l and 1mmol/l augmented the twitch contractions by 21% and 35%, respectively. First we tested for a possible nicotinic drive onto an inhibitory neuronal component to the longitudinal smooth muscle cells. However, guanethidine (5μmol/l), naloxone (1μmol/l), or l-NAME (300μmol/l) were without effect on the hexamethonium-induced augmentation. The P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS), 25–100μmol/l, without altering the control twitch responses, dose-dependently reduced the hexamethonium-induced augmentation; at 100μmol/l a statistically significantly inhibition was observed. Based on these functional experiments we found no evidence that blocking nicotinic transmission removed a tonic adrenergic, opioidergic or nitrergic inhibitory input to the longitudinal muscle. However, we provide evidence for a hexamethonium-induced augmentation of the P2 purinergic input to cholinergic motoneurons of the guinea-pig ileum longitudinal muscle. The P2-nicotinic receptor interaction presents a novel modulatory mechanism to cholinergic myenteric motor neurons.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pretreated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1β or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1β or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.

Cholinergic neuromuscular transmission mediated by interstitial cells of Cajal in the myenteric layer in mouse ileal longitudinal smooth muscles

To elucidate the roles played by the interstitial cells of Cajal in the myenteric layer (ICC-MY) in cholinergic neuromuscular transmission, we recorded mechanical and electrical activities in response to electrical field stimulation (EFS) of the ileal longitudinal muscle strips from WBB6F1-W/W(V) (W/W(V)) mutant mice, that lacked ICC-MY and compared with those in WBB6F1-+/+ (+/+) control mice. In +/+ muscle strips, EFS induced phasic contractions, which were abolished or strongly attenuated by atropine or tetrodotoxin. In W/W(V) preparations, EFS induced similar phasic contractions, but the cholinergic component was smaller than that in +/+ strips. This was despite of the fact that the contractions because of exogenous applications of carbachol and high K(+) solution in W/W(V) strips were comparable to or rather greater than those in the +/+ preparations. EFS induced atropine-sensitive excitatory junction potentials (EJPs) in the +/+ longitudinal smooth muscle cells but not in W/W(V) cells. In the presence of eserine, EFS induced atropine-sensitive EJPs in W/W(V) cells. These results suggest that ICC-MY mediate the cholinergic neuromuscular transmission in mouse ileal longitudinal smooth muscles. In addition, the other pathway in which ICC-MY are not involved can operate concomitantly.

Role of PGE2 in the colonic motility: PGE2 generates and enhances spontaneous contractions of longitudinal smooth muscle in the rat colon

The aim of this study was to determine which PGE2 receptors (EP1-4 receptors) influence colonic motility. Mucosa-free longitudinal smooth muscle strips of the rat middle colon spontaneously induced frequent phasic contractions (giant contractions, GCs) in vitro, and the GCs were almost completely abolished by a cyclooxygenase inhibitor, piroxicam, and by an EP3 receptor antagonist, ONO-AE3-240, but enhanced by tetrodotoxin (TTX). In the presence of piroxicam, exogenous PGE2, both ONO-AE-248 (EP3 agonist), and ONO-DI-004 (EP1 agonist) induced GC-like contractions, and increased the frequency and amplitude. These effects of EP receptor agonists were insensitive to TTX and ω-conotoxins. In immunohistochemistry, the EP1 and EP3 receptors were expressed in the longitudinal smooth muscle cells. These results suggest that the endogenous PGE2 spontaneously generates and enhances the frequent phasic contractions directly activating the EP1 and EP3 receptors expressed on longitudinal smooth muscle cells in the rat middle colon.

In vitro effects of lidocaine on contractility of circular and longitudinal equine intestinal smooth muscle

The purpose of the study was to compare the contractility-enhancing effects of lidocaine in equine jejunal circular (CSM) and longitudinal smooth muscle (LSM) in vitro. In previous studies, more pronounced effects of lidocaine were observed in ischaemia–reperfusion (IR) injured smooth muscle. Therefore in this study, effects were examined in both non-injured control tissues and tissues challenged by a defined, artificial IR injury. Isometric contractile performance of CSM and LSM, assessed by frequency (F), amplitude (A) and mean active force (MAF) of contractions, was defined as contractility.LSM featured lower basic contractility compared to CSM. Lidocaine provoked contractility-enhancing effects in both smooth muscle layers, but except for F at high lidocaine concentrations, contractility of LSM remained lower throughout the trial. Additionally, higher lidocaine concentrations were required to cause significant effects in LSM. No differences were observed in contractility of control and IR injured smooth muscle, but higher lidocaine concentrations were needed to provoke effects in IR injured smooth muscle. In contrast to CSM, contractility of LSM did not decrease at comparably high lidocaine concentrations.Differences in basic contractility of CSM and LSM might be explained by physiologically lower activity of LSM per se or by a thinner LSM layer with fewer smooth muscle cells taking part in contractions. The smaller thickness of the LSM layer may also contribute to persisting discrepancies in contractility following lidocaine application. Additionally, variations in lidocaine concentrations necessary for inducing significant effects could result from differences in the molecular structure of CSM and LSM cells.

Brain‐derived neurotrophic factor augments the contraction of intestinal longitudinal muscle strips induced by Carbachol

BackgroundWe have demonstrated that intestinal longitudinal smooth muscle cells synthesize and secrete brain derived neurotrophic factor (BDNF) in response to neuropeptide transmitters. But the function of this BDNF in the gastrointestinal tract is undetermined.Aimto test the effect of BDNF on agonist‐induced contraction in intestinal longitudinal smooth muscle.Material and methodRabbit longitudinal muscle strips were prepared from the intestine. The contraction of the muscle strips was recorded in response to substance P 10–6M (SP 10–6) and Carbachol 10–5M (CCh 10–5). Muscle strips were preincubated with 10 nM exogenous BDNF and the changes in SP/CCh‐induced contraction were examined. A Trk antagonist and two Trk B specific agonists were used to investigate the role of the Trk B receptor in the effect of BDNF on contraction.ResultsBDNF significantly enhanced the contraction of longitudinal smooth muscle induced by CCh 10–5. However, contraction induced by SP 10–6 was not affected by pretreatment with BDNF. The pan Trk receptor inhibitor (K252a) inhibited the effect of BDNF on CCh‐induced contraction. Moreover, two Trk B agonists (LM 22A4 &amp; 7, 8‐dihydroxyflavone) augmented the CCh‐induced contraction of the longitudinal muscle strips.ConclusionBDNF enhances the cholinergic‐induced longitudinal muscle contraction through the activation of its cognate Trk B receptor.

Ultrastructural Changes of Smooth Muscles in Varicocele Veins

Background: Varicocele is a dilatation of the pampiniform venous plexus and internal spermatic veins. It affects about 15–20% of male population and can cause infertility.Objective: To describe the most significant ultrastructural changes of the smooth muscle cells in grade 3 varicocele veins.Methods: The authors analyzed 2- to 3-cm tracts of pampiniform venous plexus from 20 patients who underwent varicocelectomy for left varicocele. Light microscopic examination was performed with Van Gieson’s stain. Ultrastructural examination was done using scanning and transmission electron microscopy.Results: Light microscopic examination revealed irregularity and separation of medial smooth muscle cells by abundant collagen fibers in varicocele veins. On scanning electron microscopy, the medial layer of varicocele veins showed hypertrophy, irregularity, and separation of the outer longitudinal smooth muscle cells and deposition of numerous fatty globules in between muscle fibers. Transmission electron microscopy showed marked indentation and chromatin condensation of the nucleus, presence of clear vacuoles and myelin figures in the cytoplasm and plasmalemmal projections and formation of ghost bodies. Furthermore, smooth muscle cells were found to have pseudopodia-like projections around adjacent elastic and collagen fibers.Conclusions: The degenerative changes observed in smooth muscle cells and presence of abundant collagen fibers in the medial layer may contribute to the development of the varicocele of pampiniform venous plexus. Further molecular studies are required to shed more light for the underlying mechanism.

Myogenic regional responsiveness to cholinergic and vipergic stimulation in human colon

Differences in the actions of enteric neurotransmitters on colonic circular and longitudinal muscle layers have not been clearly determined, nor the possible existence of intrinsic myogenic phenotypes that might contribute to regional differences in human colon motor activity. The aim of this study was to analyze the direct pharmaco-mechanical coupling of carbachol (CCh) and vasoactive intestinal polypeptide (VIP) on human colonic smooth muscle strips and cells. Circular and longitudinal muscle strips and cells were obtained from 15 human specimens of ascending and sigmoid colon. Both isometric tension on muscle strips and contraction and relaxation on cells were measured in response to increasing CCh and VIP concentrations. Circular muscle strips of ascending colon were more sensitive to the effect of CCh than that of sigmoid colon, EC(50) values being, respectively, 4.15μmolL(-1) and 8.47μmolL(-1) (P<0.05), although there were no differences in maximal responses. No regional differences were observed in longitudinal muscle strips or in smooth muscle cells. Maximal responses to CCh were higher on circular than longitudinal muscle strips and cells throughout the colon. A greater sensitivity to VIP was observed in ascending colon compared with sigmoid colon, both in circular (EC(50:) 0.041 and 0.15μmolL(-1) , respectively, P<0.01) and longitudinal (EC(50:) 0.043 and 0.09μmolL(-1) , respectively, P<0.05) strips, and similar differences were observed in longitudinal smooth muscle cells (EC(50:) 44.85 and 75.24nmolL(-1) , respectively, P<0.05). Regional myogenic differences in pharmaco-mechanical coupling between the enteric neurotransmitters and smooth muscle contribute to the complex regional motor patterns of human colon.

Substance P‐induced increase in Brain Derived Neurotrophic Factor expression and secretion by intestinal smooth muscle is mediated by a rise in intracellular Ca2+

BackgroundWe have previously shown that intestinal longitudinal smooth muscle cells express and secrete brain derived neurotrophic factor (BDNF) and addition of substance P (SP) to cultured smooth muscle cells induces further expression and secretion of BDNF above basal levels. It is not known, however, what cellular signaling pathways mediate SP‐induced expression and secretion of BDNF.AimTo identify the signaling pathways involved in BDNF synthesis and secretion by smooth muscle in response to SP.MethodCultured intestinal smooth muscle cells were treated with 10–100 nM SP for 24 hours in the presence and absence of 1,2 bis (o‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid (BAPTA) to chelate intracellular Ca2+. BDNF protein expression and secretion were measured by in‐cell western and ELISA, and BDNF mRNA expression was measured by qRT‐PCR.ResultsThe expression of BDNF protein was increased by 10–121% and BDNF secretion was increased by 50% by SP. Consistent with this, BDNF mRNA expression was increased by 161% by SP. Both BDNF protein and mRNA responses to SP were blocked by BAPTA.ConclusionSP stimulation of BDNF expression and secretion requires increased in intracellular Ca2+. The source of the intracellular Ca2+ increased remains to be determined. Supported by grants DK15564 and DK34153 from the National Institutes of Health.

Spatiotemporal expression of the serine protease inhibitor, SERPINE2, in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation

BackgroundSERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent serpins that modulates the activity of the plasminogen activator (PA) and was implicated in tissue remodeling. In this study, we investigated the expression patterns of SERPINE2 in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation.MethodsSERPINE2 was purified from mouse seminal vesicle secretion using liquid chromatography (LC) and identified by LC/tandem mass spectrometry. The antiserum against the SERPINE2 protein was raised in rabbits. To reveal the uterine and placental expression of SERPINE2, tissues at various stages were collected for real-time PCR quantification, Western blotting, and immunohistochemical staining.ResultsSerpine2 mRNA was the major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation, although Serpine1 mRNA had higher expression levels than Serpine2 mRNA in the placenta. Plat seemed to be the major PA in the mouse uterus and placenta. Antiserum against the SERPINE2 protein specifically recognized two forms of SERPINE2 and an extra 75-kDa protein, which was probably a complex of SERPINE2 with a certain protease, from among thousands of protein components in the tissue extract as demonstrated by Western blotting. In the uterus, SERPINE2 was primarily localized in luminal and glandular epithelial cells but it also was detected in circular and longitudinal smooth muscle cells during the estrous cycle and lactation. It was prominently expressed in decidual stroma cells, the metrial gland, and endometrial epithelium of the pregnant uterus. In the placenta, SERPINE2 was expressed in trophoblasts of the labyrinth and spongiotrophoblasts. However, its expression was remarkably reduced in giant cells which existed in the giant cell-decidual junction zone. In contrast, prominent expression of SERPINE2 seemed to be detected on clusters of glycogen cells near the junction zone. In addition, yolk sac membranes also showed high expression of SERPINE2.ConclusionsThese findings indicate that SERPINE2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. It may participate in the PA-modulated tissue remodeling process in the mouse placenta and uterus.

Bioengineered Three-Dimensional Physiological Model of Colonic Longitudinal Smooth Muscle In Vitro

The objective of this study was to develop a physiological model of longitudinal smooth muscle tissue from isolated longitudinal smooth muscle cells arranged in the longitudinal axis. Longitudinal smooth muscle cells from rabbit sigmoid colon were isolated and expanded in culture. Cells were seeded at high densities onto laminin-coated Sylgard surfaces with defined wavy microtopographies. A highly aligned cell sheet was formed, to which addition of fibrin resulted in delamination. (1) Acetylcholine (ACh) induced a dose-dependent, rapid, and sustained force generation. (2) Absence of extracellular calcium attenuated the magnitude and sustainability of ACh-induced force by 50% and 60%, respectively. (3) Vasoactive intestinal peptide also attenuated the magnitude and sustainability of ACh-induced force by 40% and 60%, respectively. These data were similar to force generated by longitudinal tissue. (4) Bioengineered constructs also maintained smooth muscle phenotype and calcium-dependence characteristics. This is a novel physiologically relevant in vitro three-dimensional model of colonic longitudinal smooth muscle tissue. Bioengineered three-dimensional longitudinal smooth muscle presents the ability to generate force, and respond to contractile agonists and relaxant peptides similar to native longitudinal tissue. This model has potential applications to investigate the underlying pathophysiology of dysfunctional colonic motility. It also presents as a readily implantable band-aid colonic longitudinal muscle tissue.

Proteinase-activated receptor-2 activation evokes oesophageal longitudinal smooth muscle contraction via a capsaicin-sensitive and neurokinin-2 receptor-dependent pathway

Intraluminal acid evokes sustained oesophageal longitudinal smooth muscle (LSM) contraction and oesophageal shortening, which may play a role in oesophageal pain and the aetiology of hiatus hernia. In the opossum model, this reflex has been shown to involve mast cell activation and release of neurokinins from capsaicin-sensitive neurons. The aim of this study was to determine whether proteinase-activated receptor-2 (PAR-2) activation evokes reflex LSM contraction via similar mechanisms. Tension recording studies were performed using opossum oesophageal LSM strips in the presence and absence of pharmacological agents. In addition, the effect of trypsin on single isolated LSM cells was determined using videomicroscopy, and the expression of PAR-2 in oesophageal tissue was examined using immunohistochemistry. The PAR-2 agonist trypsin evoked sustained, concentration-dependent contraction of LSM muscle strips, but had no effect on isolated LSM cells. The trypsin-induced contraction was blocked by capsaicin desensitization, substance P (SP) desensitization or application of the selective neurokinin-2 (NK-2) receptor antagonist MEN 10376. Immunohistochemistry revealed co-localization of SP, calcitonin gene-related peptide and PAR-2 in axons of opossum oesophageal LSM. Longitudinal smooth muscle contraction induced by trypsin involves capsaicin-sensitive neurons and subsequent activation of NK-2, which is identical to the pathway involved in acid-induced LSM contraction and oesophageal shortening. This suggests that acid-induced LSM contraction may involve mast cell-derived mediators that activate capsaicin-sensitive neurons via PAR-2.

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release.

Platelet‐derived growth factor signals play critical roles in differentiation of longitudinal smooth muscle cells in mouse embryonic gut

In the development of mouse gut, longitudinal smooth muscle cells (LMC) and interstitial cells of Cajal (ICC) originate from common precursor cells expressing c-Kit. Recently, some gastrointestinal stromal tumours, which develop from smooth muscle layers of the gut and have gain-of-function mutations of c-kit, have been reported to have gain-of-function mutations of platelet-derived growth factor (PDGF) receptor alpha gene. These data raise the possibility that PDGF signalling might be involved in the development of LMC. Therefore, we examined the expression pattern of the PDGF signal family of embryonic gut by immunohistochemistry and in situ hybridization, and investigated the role of PDGF signals in the development of smooth muscle layers in mouse gut using a new organ culture system. During embryonic development, the circular muscle layer expressed PDGF-A, enteric neurons expressed PDGF-B and common precursor cells of LMC and ICC expressed both PDGF receptor alpha and beta. The selective PDGF receptor inhibitor AG1295 suppressed the differentiation of LMC in gut explants. We conclude that PDGF signals play critical roles in the differentiation of LMC in mouse embryonic gut.

Role of cADPR in sodium nitroprusside-induced opossum esophageal longitudinal smooth muscle contraction

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca(2+) mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC(50) of 1 nM and peak contraction of 57 +/- 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10(-6) M. The EC(50) of cADPR caused contraction (18 +/- 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 muM) induced contraction of LSM strips that averaged 67 +/- 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.

Differential modulation by Ca 2+ ‐sensitive K + channels in longitudinal and transverse bladder strip contractions: implications in age‐related over‐active bladder symptoms

Over-active bladder (OAB) is affecting up to one-third of middle-aged adults worldwide. Physiological changes to the detrusor portion of the bladder are often associated with OAB symptoms. In the detrusor, smooth muscle cells (SMCs) are oriented both longitudinally and transversely. Evidence has pointed to the disparity in contractile responses between longitudinal SMCs (LSMCs) and transverse SMCs (TSMCs) in old adult rat bladders. The differences in contractility between LSMCs and TSMCs may also exist in young bladders, however only to be exacerbated during the aging process. Differences in the modulation of LSMC and TSMC contractility were hereby identified in the young adult rat detrusor. Detrusor strips of identical sizes were mounted on a myograph system where contractions were measured from SMCs in both orientations, i.e. LSMC- and TSMC-oriented. A single application of the muscarinic agonist carbachol (CCh, 0.01-50 micromol/L) yielded no differences in contractions between TSMC- and LSMC-oriented strips. However, blockade of KCa with tetraethylammonium (0.1–30 mmol/L) in TSMC-oriented strips showed more prominent contractions than the LSMC-oriented ones. The present findings indicated that young adult rat bladder strip contractions are modulated by KCa to different extents, depending on the SMC orientation. It is of interests to determine which KCa subtype(s) and if other types of K+ channels contribute to the observed differential LSMC and TSMC contractility. The K+ channel-associated differences in LSMC and TSMC contractions may have implications in the initiation and progression of OAB accompanied with aging. Thus, future studies examining bladders from a wider age group of rats will provide more insights into the pathophysiology of OAB.