Background & Aims
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The mechanisms underlying abdominal pain perception in irritable bowel syndrome (IBS) are poorly understood. Intestinal mast cell infiltration may perturb nerve function leading ...to symptom perception. We assessed colonic mast cell infiltration, mediator release, and spatial interactions with mucosal innervation and their correlation with abdominal pain in IBS patients.
Methods
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IBS patients were diagnosed according to Rome II criteria and abdominal pain quantified according to a validated questionnaire. Colonic mucosal mast cells were identified immunohistochemically and quantified with a computer-assisted counting method. Mast cell tryptase and histamine release were analyzed immunoenzymatically. Intestinal nerve to mast cell distance was assessed with electron microscopy.
Results
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Thirty-four out of 44 IBS patients (77%) showed an increased area of mucosa occupied by mast cells as compared with controls (9.2% ± 2.5% vs. 3.3 ± 0.8%, respectively;
P < 0.001). There was a 150% increase in the number of degranulating mast cells (4.76 ± 3.18/field vs. 2.42 ± 2.26/field, respectively;
P = 0.026). Mucosal content of tryptase was increased in IBS and mast cells spontaneously released more tryptase (3.22 ± 3.48 pmol/min/mg vs. 0.87 ± 0.65 pmol/min/mg, respectively;
P = 0.015) and histamine (339.7 ± 59.0 ng/g vs. 169.3 ± 130.6 ng/g, respectively;
P = 0.015). Mast cells located within 5 μm of nerve fibers were 7.14 ± 3.87/field vs. 2.27 ± 1.63/field in IBS vs. controls (
P < 0.001). Only mast cells in close proximity to nerves were significantly correlated with severity and frequency of abdominal pain/discomfort (
P < 0.001 and
P = 0.003, respectively).
Conclusions
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Colonic mast cell infiltration and mediator release in proximity to mucosal innervation may contribute to abdominal pain perception in IBS patients.
The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR2), which is required for postendocytic sorting of activated receptors to lysosomes, where ...degradation terminates signaling. The mechanisms of PAR2 deubiquitination and its importance in trafficking and signaling of endocytosed PAR2 are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR2 ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR2. Trapping PAR2 in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR2 with β-arrestin2 or the duration of PAR2-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR2 but not for regulating PAR2 dissociation from β-arrestin2 or PAR2-mediated ERK2 activation.
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•SUMO-1 increased CaV2.2 Ca2+ current and caused a shift in activation gating.•SUMO-1 effects are abolished in a mutant lacking 5 highest probability SUMO sites.•K394 within the ...CaV2.2 I-II loop is one molecular determinant for SUMO-1 effects.•SUMO-1 had effects in SCG neurons consistent with raised presynaptic Ca2+ current.•We provide evidence that CaV2.2 is a functional target for SUMOylation pathways.
SUMOylation is an important post-translational modification process involving covalent attachment of SUMO (Small Ubiquitin-like MOdifier) protein to target proteins. Here, we investigated the potential for SUMO-1 protein to modulate the function of the CaV2.2 (N-type) voltage-gated calcium channel (VGCC), a protein vital for presynaptic neurotransmitter release. Co-expression of SUMO-1, but not the conjugation-deficient mutant SUMO-1ΔGG, increased heterologously-expressed CaV2.2 Ca2+ current density, an effect potentiated by the conjugating enzyme Ubc9. Expression of sentrin-specific protease (SENP)-1 or Ubc9 alone, had no effect on recombinant CaV2.2 channels. Co-expression of SUMO-1 and Ubc9 caused an increase in whole-cell maximal conductance (Gmax) and a hyperpolarizing shift in the midpoint of activation (V1/2). Mutation of all five CaV2.2 lysine residues to arginine within the five highest probability (>65 %) SUMOylation consensus motifs (SCMs) (construct CaV2.2-Δ5KR), produced a loss-of-function mutant. Mutagenesis of selected individual lysine residues identified K394, but not K951, as a key residue for SUMO-1-mediated increase in CaV2.2 Ca2+ current density. In synaptically-coupled superior cervical ganglion (SCG) neurons, SUMO-1 protein was distributed throughout the cell body, axons and dendrites and presumptive presynaptic terminals, whilst SUMO-1ΔGG protein was largely confined to the cell body, in particular, the nucleus. SUMO-1 expression caused increases in paired excitatory postsynaptic potential (EPSP) ratio at short (20−120 ms) inter-stimuli intervals in comparison to SUMO-1ΔGG, consistent with an increase in residual presynaptic Ca2+ current and an increase in release probability of synaptic vesicles. Together, these data provide evidence for CaV2.2 VGCCs as novel targets for SUMOylation pathways.
Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR sub(2)) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR ...sub(2)-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR sub(2) sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR sub(2) was colocalized with protein kinase (PK) C epsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR sub(2) agonists promoted translocation of PKC epsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR sub(2) couples to phospholipase C beta , leading to stimulation of PKC, we also observed that PAR sub(2) agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR sub(2) agonists enhanced capsaicin-stimulated increases in Ca super(2+) sub(i) and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR sub(2) agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKC epsilon and PKA prevented sensitization of TRPV1 Ca super(2+) signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR sub(2) activates PKC epsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.
Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders ...like Alzheimer’s disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-β peptide 1-42 as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.
Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, ...multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca
oscillations and the manipulation of Ca
signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca
oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca
imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval-IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca
dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca
oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca
dynamics, such as the fraction of cells showing intracellular Ca
oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca
dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca
associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca
dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca
dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca
dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.
A distinct feature of the Toll-like receptor 4 (TLR4) is its ability to trigger both MyD88-dependent and MyD88-independent signalling, culminating in activation of pro-inflammatory NF-κB and/or the ...antiviral IRF3. Although TLR4 agonists (lipopolysaccharides; LPSs) derived from different bacterial species have different endotoxic activity, the impact of LPS chemotype on the downstream signalling is not fully understood. Notably, different TLR4 agonists exhibit anti-tumoural activity in animal models of glioma, but the underlying molecular mechanisms are largely unknown.
Thus, we investigated the impact of LPS chemotype on the signalling events in the human glioma cell line U251. We found that LPS of Escherichia coli origin (LPSEC) leads to NF-κB-biased downstream signalling compared to Salmonella minnesota-derived LPS (LPSSM). Exposure of U251 cells to LPSEC resulted in faster nuclear translocation of the NF-κB subunit p65, higher NF-κB-activity and expression of its targets genes, and higher amount of secreted IL-6 compared to LPSSM. Using super-resolution microscopy we showed that the biased agonism of TLR4 in glioma cells is neither a result of differential regulation of receptor density nor of formation of higher order oligomers. Consistent with previous reports, LPSEC-mediated NF-κB activation led to significantly increased U251 proliferation, whereas LPSSM-induced IRF3 activity negatively influenced their invasiveness. Finally, treatment with methyl-β-cyclodextrin (MCD) selectively increased LPSSM-induced nuclear translocation of p65 and NF-κB activity without affecting IRF3.
Our data may explain how TLR4 agonists differently affect glioma cell proliferation and migration.
•Activation of Toll-like receptor 4 in glioma cells results in biased signalling.•Diverse LPS chemotypes differentially affect the activity of NF-κB - and IRF3 and their respective target gene expression.•Biased signalling via TLR4 is not a result of different receptor oligomerisation•Depletion of cholesterol-rich membrane microdomains is sufficient to invert the signalling bias.
Background In rat middle cerebral and mesenteric arteries the K.sub.Ca 2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an ...effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K.sub.Ca 2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K.sub.Ca 2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). Methods MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. Results Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K.sub.Ca 2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K.sub.Ca 2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. Conclusions Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K.sub.Ca 2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K.sub.Ca 2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.
The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR2), which is required for post-endocytic sorting of PAR2 to lysosomes, where degradation arrests signaling. The mechanisms of ...post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR2. In HEK-PAR2 cells, PAR2 activating peptide (PAR2-AP) induced PAR2 trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR2 and c-Cbl accumulated, and PAR2 failed to traffic to lysosomes. Overexpression of HRS prevented PAR2-AP-induced degradation of PAR2, as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR2 in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR2Δ14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR2Δ14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR2 and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR2 and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.
Volatile anesthetics such as isoflurane and halothane have been in clinical use for many years and represent the group of drugs most commonly used to maintain general anesthesia. However, despite ...their widespread use, the molecular mechanisms by which these drugs exert their effects are not completely understood. Recently, a seemingly paradoxical effect of general anesthetics has been identified: the activation of peripheral nociceptors by irritant anesthetics. This mechanism may explain the hyperalgesic actions of inhaled anesthetics and their adverse effects in the airways.
To test the hypothesis that irritant inhaled anesthetics activate the excitatory ion-channel transient receptor potential (TRP)-A1 and thereby contribute to hyperalgesia and irritant airway effects, we used the measurement of intracellular calcium concentration in isolated cells in culture. For our functional experiments, we used models of isolated guinea pig bronchi to measure bronchoconstriction and withdrawal threshold to mechanical stimulation with von Frey filaments in mice.
Irritant inhaled anesthetics activate TRPA1 expressed in human embryonic kidney cells and in nociceptive neurons. Isoflurane induces mechanical hyperalgesia in mice by a TRPA1-dependent mechanism. Isoflurane also induces TRPA1-dependent constriction of isolated bronchi. Nonirritant anesthetics do not activate TRPA1 and fail to produce hyperalgesia and bronchial constriction.
General anesthetics induce a reversible loss of consciousness and render the patient unresponsive to painful stimuli. However, they also produce excitatory effects such as airway irritation and they contribute to postoperative pain. Activation of TRPA1 may contribute to these adverse effects, a hypothesis that remains to be tested in the clinical setting.
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