Colonic Smooth Muscle Cells Research Articles

Src kinase‐dependent gating properties of single Ca2+ channels are altered by tyrosine nitration in colitis

Reduced L‐type Ca2+ ‐channel (LTCC) function contributes to colonic dysmotility during inflammation. We have previously shown that nitration of tyrosine residues within the carboxy terminus of LTCC (Y1837 and Y2134) impairs c‐Src kinase regulation during colitis. We examined the gating properties of single LTCC, using cell‐attached patch clamp mode, in mouse colon smooth muscle cells (control vs colitis‐TNBS) and CHO cells transfected with hCaV1.2b (wild type) or mutated hCaV1.2b (Y1837F:Y2134F). Inflammation significantly reduced LTCC ensemble average current (−0.33 vs −0.22 pA, P=0.05), without affecting the single‐channel properties: mean amplitude (−2.4pA), slope conductance (23 vs 27pS) or the open‐time constants (τ1: 1.6 vs 1.2; τ2: 6.1 vs 3.9 ms). However, the availability (slow gating) of LTCC was significantly reduced by inflammation (0.19 vs 0.06; P=0.02) without affecting the open probability of modal gating. Similar effects were seen with inhibition of c‐Src kinase by PP2 or by tyrosine nitration induced by SIN‐1+ONOO− combination in smooth muscle cells. Mutation of tyrosine residues (Y1837/2134F) in hCaV1.2b, which prevents c‐Src kinase regulation, resulted in diminished channel availability. This study demonstrates that src kinase‐ phosphorylation enhances the availability of LTCC, that is impaired by tyrosine nitration during colonic inflammation. Supported by NIH DK046367.

Exposure of Toll‐like receptors 4 to bacterial lipopolysaccharide (LPS) impairs human colonic smooth muscle cell function

Endotoxemia by bacterial lipopolysaccharide (LPS) has been reported to affect gut motility specifically depending on Toll-like receptor 4 activation (TLR4). However, the direct impact of LPS ligation to TLR4 on human smooth muscle cells (HSMC) activity still remains to be elucidated. The present study shows that TLR4, its associated molecule MD2, and TLR2 are constitutively expressed on cultured HSMC and that, once activated, they impair HSMC function. The stimulation of TLR4 by LPS induced a time- and dose-dependent contractile dysfunction, which was associated with a decrease of TLR2 messenger, a rearrangement of microfilament cytoskeleton and an oxidative imbalance, i.e., the formation of reactive oxygen species (ROS) together with the depletion of GSH content. An alteration of mitochondria, namely a hyperpolarization of their membrane potential, was also detected. Most of these effects were partially prevented by the NADPH oxidase inhibitor apocynin or the NFkappaB inhibitor MG132. Finally, a 24 h washout in LPS-free medium almost completely restored morphofunctional and biochemical HSMC resting parameters, even if GSH levels remained significantly lower and no recovery was observed in TLR2 expression. Thus, the exposure to bacterial endotoxin directly and persistently impaired gastrointestinal smooth muscle activity indicating that HSMC actively participate to dysmotility during infective burst. The knowledge of these interactions might provide novel information on the pathogenesis of infection-associated gut dysmotility and further clues for the development of new therapeutic strategies.

Abnormal distribution of sarcoglycan subcomplex in colonic smooth muscle cells of aganglionic bowel

Hirschsprung's disease (HD) is a development disorder of the enteric nervous system in which the altered innervation explains the inability of the aganglionic segment to relax. Impairment of cytoskeleton in SMC of aganglionic bowel has been shown. Sarcoglycan subcomplex (SG) may support the development and maintenance of muscle cells. We examined the SG subunit expression in colonic aganglionic and ganglionic specimens obtained from patients with HD. Full-thickness bowel specimens were obtained from six patients with HD. Six normal colon specimens were used as controls. Immunofluorescent analysis and reverse transcriptase polymerase chain reaction evaluation were performed for alpha-, beta-, gamma-, delta- and epsilon-SG. In control colon, the indirect immunofluorescence showed a strong staining pattern of beta- gamma- delta- and epsilon-SG while a weak positivity of alpha-SG was recorded. In aganglionic bowel, immunofluorescence intensity values documented a significant lack of epsilon-SG while an enhanced alpha-SG, coupled to a loss of epsilon-SG, was recorded in ganglionic bowel in HD-affected patients. Our observations underscore the assumption that non-neuronal elements of the colon might play a key role in the pathogenesis of HD and loss of epsilon-SG might critically alter the cytoskeleton in the aganglionic bowel segment. Up-regulation of alpha-SG is probably an acquired phenomenon to reinforce the sarcolemma and to perform a forceful contraction in dilated ganglionic HD-affected colon, related to chronic pseudo-obstruction, contributing to the intestinal dysmotility that persists in 20% of patients after resection of the aganglionic bowel.

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca2+ concentration ([Ca2+](c)). Local Ca2+ release from an InsP(3) receptor (InsP(3)R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP(3)R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP(3)-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP(3)R. Mitochondria may regulate InsP(3)-mediated Ca2+ signals by operating between or within InsP(3)R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP(3)-mediated Ca2+ release by operating within (local) or between (global) InsP(3)R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP(3) in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5-4.0 F/F(0), durations of approximately 112 ms at half-maximum amplitude, and were abolished by the InsP(3)R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized DeltaPsi(M) to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by approximately 66 or approximately 60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by approximately 62%. The "fast" Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acted like mitochondria to prolong InsP(3)-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP(3)R communication at the intra-cluster level and that mitochondria regulate both local and global InsP(3)-mediated Ca2+ signals.

Increased colonic motility in a rat model of irritable bowel syndrome is associated with up-regulation of L-type calcium channels in colonic smooth muscle cells

This paper aimed to investigate the relationship between up-regulation of L-type calcium channels and altered motility disorder in a rat model of irritable bowel syndrome (IBS). Male Sprague-Dawley rats were subjected to neonatal maternal separation (NMS) from postnatal day 2-14 or normal handling (NH), and used when weighted 250-300 g. Colonic smooth muscle contractions was studied in an organ bath system. L-type Ca(2+) channel alpha(1c) subunit expression in smooth muscles from rat colon were studied by immunofluorescence and Western blotting analysis. The intracellular calcium concentration ([Ca(2+)](i)) of enzymatically isolated single colonic smooth muscle cell was studied with laser confocal fluorescent microscopy. The fecal pellets during 1 h water avoidance stress (WAS) were significantly increased; the amplitude of spontaneous contractions and contractions induced by Bay K 8644 (10 nM-1 microM), KCl (10-60 mM) and ACh (100 nM-10 microM) were significantly increased in NMS rats, when comparing with that of NH rats. [Ca(2+)]i induced by Bay K 8644 (1 microM), KCl (40 mM), and ACh (10 microM) significantly increased in muscle cells of NMS rats than NH rats. Further, alpha(1c) protein expression was significantly up-regulated in colonic smooth muscle of NMS rats than NH rats. These results suggest that NMS lead to up-regulation of L-type Ca(2+) channels expression in the colon, which contributes to the colonic motility disorder. Our findings provide direct evidence to help understanding the underlying mechanism of chronic stress-induced colonic motility disorder in IBS.

Cloning and characterization of rabbit Rgs4 promoter in gut smooth muscle

Regulator of G-protein signaling 4 (Rgs4) regulates the strength and duration of G-protein signaling, and plays an important role in cardiac development, smooth muscle contraction and psychiatric disorders. Rgs4 expression is regulated at both mRNA and protein levels. In order to examine the transcriptional mechanism of Rgs4 expression, we have cloned and characterized rabbit Rgs4 promoter. The coding sequence of rabbit Rgs4 was obtained by degenerative RT-PCR and used for Northern blot and 5′-RACE analysis. A single transcript was identified in rabbit colonic smooth muscle cells. The 5′-untranslated region (UTR) extended 120 bp nucleotides upstream of the Rgs4 start codon. A putative promoter sequence (1389 bp) showed a consensus TATA box and cis-acting binding sites for several potential transcriptional factors. Reporter gene assay identified strong promoter activity in various cell types. Further analysis by deletion mutagenesis suggested that the proximal region had a highest core promoter activity while the distal region is suppressive. IL-1β significantly increased the promoter activity. The in vitro and in vivo binding activities for NF-κB transcription factor were validated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay respectively. Mutation of NF-κB site reduced the promoter activity. These data suggest that the cloned rabbit Rgs4 promoter is functionally active and NF-κB binding site possesses enhancer activity in regulating Rgs4 transcription. Our studies provide an important basis for further understanding of Rgs4 regulation and function in different diseases.

Phospholipase C-independent effects of 3M3FBS in murine colon

The muscarinic receptor subtype M 3 is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5) P 3 . This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide ( m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation. We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide ( o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments. Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K + channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K + currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca 2+ currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca 2+. Pretreatment with U73122 did not prevent the decrease in Ca 2+ currents upon m-3M3FBS application. In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca 2+] i in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level.

Role of thin-filament regulatory proteins in relaxation of colonic smooth muscle contraction

Coordinated regulation of smooth muscle contraction and relaxation is required for colonic motility. Contraction is associated with phosphorylation of myosin light chain (MLC(20)) and interaction of actin with myosin. Thin-filament regulation of actomyosin interaction is modulated by two actin-binding regulatory proteins: tropomyosin (TM) and caldesmon (CaD). TM and CaD are known to play crucial role in actomyosin interaction promoting contraction. Contraction is associated with phosphorylation of the small heat shock protein HSP27, concomitant with the phosphorylation of TM and CaD. Phosphorylation of HSP27 is attributed as being the prime modulator of thin-filament regulation of contraction. Preincubation of colonic smooth muscle cells (CSMC) with the relaxant neurotransmitter vasoactive intestinal peptide (VIP) showed inhibition in phosphorylation of HSP27 (ser78). Attenuation of HSP27 phosphorylation can result in modulation of thin-filament-mediated regulation of contraction leading to relaxation; thus the role of thin-filament regulatory proteins in a relaxation milieu was investigated. Preincubation of CSMC with VIP exhibited a decrease in phosphorylation of TM and CaD. Furthermore, CSMC preincubated with VIP showed a reduced association of TM with HSP27 and with phospho-HSP27 (ser78) whereas there was reduced dissociation of TM from CaD and from phospho-CaD. We thus propose that, in addition to alteration in phosphorylation of MLC(20), relaxation is associated with alterations in thin-filament-mediated regulation that results in termination of contraction.

Ezrin overexpression in gastrointestinal stromal tumors: an independent adverse prognosticator associated with the non-gastric location

Ezrin, a member of the ezrin–radixin–moesin family, acts as a link between the cell membrane and actin cytoskeleton to integrate cell adhesion-mediated signaling. It implicates tumor progression, metastatic dissemination, and adverse outcomes in several cancer types, including pediatric and adult sarcomas. Although ezrin upregulation was shown by cDNA expression profiling, no study has systematically evaluated the significance of ezrin expression in a large cohort of gastrointestinal stromal tumors (GISTs). Ezrin immunostaining was carried out on tissue microarrays of primary GISTs and assessable in 347 cases, 188 of which were successfully evaluated for mutation variants of KIT and PDGFRA receptor tyrosine kinase (RTK) genes by sequencing with or without screening by denatured high-performance liquid chromatography. These GISTs with known RTK genotypes were dichotomized into two prognostically different groups. The endogenous expression and phosphorylation of ezrin in GIST cell lines were analyzed by western blotting. By immunohistochemistry, ezrin overexpression was present in 66% of GISTs and significantly associated with the non-gastric location (P=0.002) and decreased disease-free survival (P=0.032, univariately). However, it was not related to the National Institute of Health (NIH) risk category, Ki-67 labeling index, RTK genotypes, and other variables. In multivariate analyses, ezrin overexpression remained independently predictive of adverse outcome (P=0.008, risk ratio=2.363), together with Ki-67 labeling index >5% (P<0.001, risk ratio=3.581), high-risk category (P<0.001, risk ratio=2.156), and the non-gastric location (P=0.029, risk ratio=1.899). Despite the variation in the ezrin expression level, phosphorylated ezrin at threonine567 was only detectable in GIST882 and GIST48 cells, but not in colonic smooth muscle cells. In conclusion, ezrin is frequently overexpressed in GISTs, especially those arising from the non-gastric sites. Given that its impact is independent of the NIH risk category, cell proliferation, and tumor location, ezrin immunoreactivity represents a valuable prognostic adjunct of GISTs, suggesting a causative role in conferring an aggressive phenotype.

Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors

Mitochondria modulate cellular Ca 2+ signals by accumulating the ion via a uniporter and releasing it via Na +- or H +-exchange. In smooth muscle, inhibition of mitochondrial Ca 2+ uptake inhibits Ca 2+ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP 3R). At least two mechanisms may explain this effect. First, localised uptake of Ca 2+ by mitochondria may prevent negative feedback by cytosolic Ca 2+ on IP 3R activity, or secondly localised provision of Ca 2+ by mitochondrial efflux may maintain IP 3R function or SR Ca 2+ content. To distinguish between these possibilities the role of mitochondrial Ca 2+ efflux on IP 3R function was examined. IP 3 was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na +–Ca 2+ exchanger inhibited with CGP-37157 (10 μM). Mitochondria accumulated Ca 2+ during IP 3-evoked [Ca 2+] c rises and released the ion back to the cytosol (within ∼15 s) when mitochondrial Ca 2+ efflux was active. When mitochondrial Ca 2+ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca 2+ occurred after IP 3-evoked Ca 2+ release. IP 3-evoked [Ca 2+] c rises were initially unaffected, then only slowly inhibited by CGP-37157. IP 3R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP 3 release did not inhibit IP 3R. CGP-37157 directly inhibited voltage-gated Ca 2+ channel activity, however SR Ca 2+ content was unaltered by the drug. Thus, the gradual decline of IP 3R function that followed mitochondrial Na +–Ca 2+ exchanger inhibition resulted from a gradual overload of mitochondria with Ca 2+, leading to a reduced capacity for Ca 2+ uptake. Localised uptake of Ca 2+ by mitochondria, rather than mitochondrial Ca 2+ efflux, appears critical for maintaining IP 3R activity.

Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein

Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca 2+. In many smooth muscles, increasing intracellular Ca 2+ activates Ca 2+-activated Cl − channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4′, 4′-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway.

W1702 Reversible and Persistent Cellular Alterations Induced By Bacterial Lipopolysaccharide (LPS) On Human Colonic Smooth Muscle Cells

W1702 Reversible and Persistent Cellular Alterations Induced By Bacterial Lipopolysaccharide (LPS) On Human Colonic Smooth Muscle Cells

W1596 GATA-6 Mediates IL-1β-Induced Up-Regulation of RGS4 in Rabbit Colonic Smooth Muscle Cells

W1596 GATA-6 Mediates IL-1β-Induced Up-Regulation of RGS4 in Rabbit Colonic Smooth Muscle Cells

Norepinephrine mediates the transcriptional effects of heterotypic chronic stress on colonic motor function.

Chronic stress precipitates or exacerbates the symptoms of functional bowel disorders, including motility dysfunction. The cellular mechanisms of these effects are not understood. We tested the hypothesis that heterotypic chronic stress (HeCS) elevates the release of norepinephrine from the adrenal medulla, which enhances transcription of the gene-regulating expression of Ca(v)1.2 (L-type) channels in colonic circular smooth muscle cells, resulting in enhanced colonic motor function. The experiments were performed in rats using a 9-day heterotypic chronic stress (HeCS) protocol. We found that HeCS, but not acute stress, time dependently enhances the contractile response to ACh in colonic circular smooth muscle strips and in single dissociated smooth muscle cells, the plasma levels of norepinephrine and the mRNA and protein expressions of the alpha(1C) subunit of Ca(v)1.2 channels. These effects result in faster colonic transit and increase in defecation rate. The effects of HeCS are blocked by adrenalectomy but not by depletion of norepinephrine in sympathetic neurons. The inhibition of receptors for glucocortocoids, corticotropin-releasing hormone or nicotine also does not block the effects of heterotypic chronic stress. Norepinephrine acts on alpha- and beta(3)-adrenergic receptors to induce the transcription of alpha(1C) subunit. We conclude that HeCS alters colonic motor function by elevating the plasma levels of norepinephrine. Colonic motor dysfunction is associated with enhanced gene transcription of Ca(v)1.2 channels in circular smooth muscle cells. These findings suggest the potential cellular mechanisms by which heterotypic chronic stress may exacerbate motility dysfunction in patients with irritable bowel syndrome.

Nuclear Myosin II Regulates the Assembly of Preinitiation Complex for ICAM-1 Gene Transcription

Actin-myosin II motor converts chemical energy into force/motion in muscle and nonmuscle cells. The phosphorylation of 20-kilodalton regulatory myosin light chain (MLC(20)) is critical to the cytoplasmic functions of these motors. We do not know whether myosin II and actins in the nucleus function as motors to generate relative motion, such as that between RNA polymerase II holoenzyme and DNA, for assembly of the preinitiation complex. The experiments were performed on primary cultures of human colonic circular smooth muscle cells and rat colonic circular muscle strips. We show that myosin II and alpha- and beta-actins are present in the nuclei of colonic smooth muscle cells. The nuclear myosin II is tethered to recognition sequence AGCTCC (-39/-34) in the intercellular adhesion molecule 1 (ICAM-1) core promoter region. The actins are known to complex with RNA polymerase II, and they are tethered to the nucleoskeleton. The dephosphorylation of MLC(20) increases the transcription of ICAM-1, whereas its phosphorylation decreases it. Colonic inflammation suppresses nuclear myosin light chain kinase, which increases the unphosphorylated form of nuclear MLC(20), resulting in enhanced transcription of ICAM-1. Myosin II is a core transcription factor. The phosphorylation/dephosphorylation of nuclear MLC(20) results in the sliding of myosin and actin molecules past each other, producing relative motion between DNA bound to the myosin II and RNA polymerase II holoenzyme bound to actins and nucleoskeleton.

Mitochondria Act Within InsP3R Clusters To Maintain Ca2+ Release In Smooth Muscle

Many smooth muscle activities including contraction, transcription, growth and apoptosis are regulated by transient inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). InsP3 binds to receptors (InsP3R) present on the sarcoplasmic reticulum to evoke Ca2+ release. InsP3R exist in clusters and Ca2+ released from one receptor may activate nearby InsP3R within this cluster in a CICR-like process to evoke a ‘puff’. Ca2+ released may also diffuse to adjacent clusters to trigger further Ca2+ release and generate a Ca2+ rise throughout the cell. Mitochondrial Ca2+ uptake limits a negative feedback process operative on InsP3R to maintain Ca2+ release. Inhibition of mitochondrial Ca2+ uptake decreases InsP3-mediated Ca2+ waves by ≥50%. We addressed whether mitochondria act to maintain release by operating within or between InsP3R clusters. Ca2+ puffs were evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells in which [Ca2+]c and ΔΨM were measured simultaneously. EGTA, a slow Ca2+ buffer, was used to functionally uncouple puff sites to prevent the formation of Ca2+ waves. EGTA was used at a concentration ([300 μM]) which does not affect the magnitude or kinetics of Ca2+ puffs. InsP3-evoked Ca2+ puffs had amplitudes of 0.5-5.0 F/F0 and durations of ∼200 ms at half-maximum amplitude. Puffs were abolished by the InsP3R inhibitor 2-APB. The protonophore CCCP and the mitochondrial inhibitor rotenone, each used with oligomycin, depolarized the mitochondrial membrane potential (ΔΨM) and prevented mitochondrial Ca2+ uptake. Depolarizing ΔΨM with CCCP attenuated Ca2+ puffs by ∼65% while rotenone inhibited them by ∼60%. These results indicate mitochondrial Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level. Supported by the Wellcome Trust and British Heart Foundation.

Airway Smooth Muscle Cells Synthesize Hyaluronan Cable Structures Independent of Inter-α-inhibitor Heavy Chain Attachment

The covalent association of inter-alpha-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan "cable" structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan "cables" in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass.

Gene plasticity in colonic circular smooth muscle cells underlies motility dysfunction in a model of postinfective IBS

The cellular mechanisms of motility dysfunction in postinfectious irritable bowel syndrome (PI-IBS) are not known. We used a rat model of neonatal inflammation to test the hypothesis that gene plasticity in colonic circular smooth muscle cells underlies motility dysfunction in PI-IBS. Mild/moderate or severe inflammation was induced in neonatal and adult rats. Experiments were performed in tissues obtained at 7 days (short term) and 6-8 wk (long term) after the induction of inflammation. Severe inflammation in neonatal rats induced persistent long-term smooth muscle hyperreactivity to acetylcholine (ACh), whereas that in adult rat caused smooth muscle hyporeactivity that showed partial recovery in the long term. Mild/moderate inflammation had no effect in neonatal rats, but it induced smooth muscle hyporeactivity to ACh in adult rats, which recovered fully in the long term. Smooth muscle hyperreactivity to ACh resulted in accelerated colonic transit and increase in defecation rate, whereas hyporeactivity had opposite effects. Smooth muscle hyperreactivity to ACh was associated with increase in transcription rate of key cell-signaling proteins of the excitation-contraction coupling alpha1C subunit of Cav1.2 (L-type) calcium channels, Galphaq, and 20-kDa myosin light chain (MLC20), whereas hyporeactivity was associated with their suppression. Inflammation in adult rats induced classical inflammatory response, which was absent in neonatal rats. Severe neonatal inflammation enhanced plasma norepinephrine and muscularis propria vasoactive intestinal polypeptide in the long term. We conclude that severe, but not mild/moderate, inflammation in a state of immature or impaired stress and immune response systems alters the transcription rate of key cell-signaling proteins of excitation-contraction coupling in colonic circular smooth muscle cells to enhance their contractility and accelerate colonic transit and defecation rate.

Insulin-like growth factor-1 upregulates the expression of stem cell factor in rat colonic smooth muscle cells

Insulin-like growth factor-1 upregulates the expression of stem cell factor in rat colonic smooth muscle cells

Direct association of calponin with specific domains of PKC-α

Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-alpha in the membrane. In the present study, we have determined the domain of PKC-alpha involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Calponin was found to bind directly to the full-length PKC-alpha. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-alpha. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-alpha showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-alpha and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-alpha and that the functional association of PKC-alpha with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.