Epigenetic combined with transcriptomic analysis of the m6A methylome after spared nerve injury-induced neuropathic pain in mice.

Epigenetic changes in the spinal cord play a key role in the initiation and maintenance of nerve injury-induced neuropathic pain. N6-methyladenosine (m6A) is one of the most abundant internal RNA modifications and plays an essential function in gene regulation in many diseases. However, the global m6A modification status of mRNA in the spinal cord at different stages after neuropathic pain is unknown. In this study, we established a neuropathic pain model in mice by preserving the complete sural nerve and only damaging the common peroneal nerve. High-throughput methylated RNA immunoprecipitation sequencing results showed that after spared nerve injury, there were 55 m6A methylated and differentially expressed genes in the spinal cord. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway results showed that m6A modification triggered inflammatory responses and apoptotic processes in the early stages after spared nerve injury. Over time, the differential gene function at postoperative day 7 was enriched in "positive regulation of neurogenesis" and "positive regulation of neural precursor cell proliferation." These functions suggested that altered synaptic morphological plasticity was a turning point in neuropathic pain formation and maintenance. Results at postoperative day 14 suggested that the persistence of neuropathic pain might be from lipid metabolic processes, such as "very-low-density lipoprotein particle clearance," "negative regulation of cholesterol transport" and "membrane lipid catabolic process." We detected the expression of m6A enzymes and found elevated mRNA expression of Ythdf2 and Ythdf3 after spared nerve injury modeling. We speculate that m6A reader enzymes also have an important role in neuropathic pain. These results provide a global landscape of mRNA m6A modifications in the spinal cord in the spared nerve injury model at different stages after injury.

Single-cell analyses reveal the dynamic functions of Itgb2+ microglia subclusters at different stages of cerebral ischemia-reperfusion injury in transient middle cerebral occlusion mice model.

Introduction: The underlying pathophysiological mechanisms of cerebral ischemia reperfusion injury (CIRI) is intricate, and current studies suggest that neuron, astrocyte, microglia, endothelial cell, and pericyte all have different phenotypic changes of specific cell types after ischemic stroke. And microglia account for the largest proportion after CIRI. Previous transcriptomic studies of ischemic stroke have typically focused on the 24 hours after CIRI, obscuring the dynamics of cellular subclusters throughout the disease process. Therefore, traditional methods for identifying cell types and their subclusters may not be sufficient to fully unveil the complexity of single-cell transcriptional profile dynamics caused by an ischemic stroke. Methods: In this study, to explore the dynamic transcriptional profile of single cells after CIRI, we used single-cell State Transition Across-samples of RNA-seq data (scSTAR), a new bioinformatics method, to analyze the single-cell transcriptional profile of day 1, 3, and 7 of transient middle cerebral artery occlusion (tMCAO) mice. Combining our bulk RNA sequences and proteomics data, we found the importance of the integrin beta 2 (Itgb2) gene in post-modeling. And microglia of Itgb2+ and Itgb2- were clustered by the scSTAR method. Finally, the functions of the subpopulations were defined by Matescape, and three different time points after tMCAO were found to exhibit specific functions. Results: Our analysis revealed a dynamic transcriptional profile of single cells in microglia after tMCAO and explored the important role of Itgb2 contributed to microglia by combined transcriptomics and proteomics analysis after modeling. Our further analysis revealed that the Itgb2+ microglia subcluster was mainly involved in energy metabolism, cell cycle, angiogenesis, neuronal myelin formation, and repair at 1, 3, and 7 days after tMCAO, respectively. Discussion: Our results suggested that Itgb2+ microglia act as a time-specific multifunctional immunomodulatory subcluster during CIRI, and the underlying mechanisms remain to be further investigated.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke.

Stroke is the second leading cause of death worldwide, and oxidative stress plays a crucial role. Celastrol exhibits strong antioxidant properties in several diseases; however, whether it can affect oxidation in cerebral ischemic-reperfusion injury (CIRI) remains unclear. This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms. Here, we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2 (Nrf2). Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry, we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4 (Nedd4) and then released Nrf2 from Nedd4 in astrocytes. Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI, which was significantly blocked by celastrol. Furthermore, by inhibiting oxidative stress and astrocyte activation, celastrol effectively rescued neurons from axon damage and apoptosis. Our study uncovered Nedd4 as a direct target of celastrol, and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

Celastrol-encapsulated microspheres prepared by microfluidic electrospray for alleviating inflammatory pain.

Inflammatory pain is induced by trauma, infection, chemical stimulation, etc. It causes severe physical and psychological agony to patients. Celastrol has powerful anti-inflammatory property and has achieved good results in various inflammation-related diseases. However, the low water solubility and multi-system toxicity limit its clinical application. Herein, we proposed alginate microspheres with core-shell structure which encapsulated celastrol by microfluidic electrospray to effectively overcome the shortcomings and improve the therapeutic effect. The microspheres had uniform size and good biocompatibility, and could release the loaded drugs in the gut. The behavioral tests showed that the celastrol-loaded microspheres effectively alleviated inflammatory pain, and the hematoxylin and eosin staining (HE staining), immunofluorescence and detection of inflammatory cytokines showed the anti-inflammatory effect. These results indicated that the microspheres could reduce dose and toxicity without affecting efficacy, and facilitate the application of celastrol in different clinical situations.

miR-26a-5p alleviates CFA-induced chronic inflammatory hyperalgesia through Wnt5a/CaMKII/NFAT signaling in mice.

BACKGROUND: Inflammation often leads to the occurrence of chronic pain, and many miRNAs have been shown to play a key role in the development of inflammatory pain. However, whether miR-26a-5p relieves pain induced by inflammation and its possible mechanism are still unclear. METHODS: The complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model was employed. Intrathecal or subcutaneous injection of miR-26a-5p agomir was performed after modeling to study its antinociceptive effect and the comparison of different administration methods. Bioinformatics analysis of miRNAs was performed to study the downstream mechanisms of miR-26a-5p. HE staining, RT-qPCR, Western blotting, and immunofluorescence were used for further validation. RESULTS: A single intrathecal and subcutaneous injection of miR-26a-5p both reversed mechanical hypersensitivity and thermal latency in the left hind paw of mice with CFA-induced inflammatory pain. HE staining and immunofluorescence studies found that both administrations of miR-26a-5p alleviated inflammation in the periphery and spinal cord. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. Wnt5a was mainly expressed in neurons and microglia in the spinal cord of mice with inflammatory pain. Intrathecal injection of miR-26a-5p could significantly reduce the expression level of Wnt5a and inhibit the downstream molecules of noncanonical Wnt signaling Camk2/NFAT, inhibiting the release of spinal cord inflammatory factors and alleviating the activation of microglia. In addition, miR-26a-5p could also inhibit lipopolysaccharide (LPS)-stimulated BV2 cell inflammation in vitro through a noncanonical Wnt signaling pathway. CONCLUSIONS: miR-26a-5p is a promising therapy for CFA-induced inflammatory pain. Both intrathecal and subcutaneous injections provide relief for inflammatory pain. miR-26a-5p regulated noncanonical Wnt signaling to be involved in analgesia partly through antineuroinflammation, suggesting a pain-alleviating effect via noncanonical Wnt signaling pathway in the CFA-induced inflammatory pain model in vivo.

Human PMSCs-derived small extracellular vesicles alleviate neuropathic pain through miR-26a-5p/Wnt5a in SNI mice model.

BACKGROUND: Mesenchymal stem cell (MSCs)-derived small Extracellular Vesicles (sEVs) are considered as a new cell-free therapy for pain caused by nerve injury, but whether human placental mesenchymal stem cell-derived sEVs relieve pain in sciatic nerve injury and its possible mechanism are still unclear. In this study, we investigated the roles of hPMSCs-derived sEVs and related mechanisms in neuropathic pain. METHODS: The spared nerve injury (SNI) mouse model was employed. Intrathecal injection of sEVs or miR-26a-5p agomir was performed on the seventh day of modeling, to study its anti-nociceptive effect. sEVs' miRNA sequencing (miRNA-Seq) and bioinformatics analysis were performed to study the downstream mechanisms of miRNAs. RT-qPCR, protein assay and immunofluorescence were used for further validation. RESULTS: A single intrathecal injection of sEVs durably reversed mechanical hypersensitivity in the left hind paw of mice with partial sciatic nerve ligation. Immunofluorescence studies found that PKH26-labeled sEVs were visible in neurons and microglia in the dorsal horn of the ipsilateral L4/5 spinal cord and more enriched in the ipsilateral. According to miRNA-seq results, we found that intrathecal injection of miR-26a-5p agomir, the second high counts microRNA in hPMSCs derived sEVs, significantly suppressed neuropathic pain and neuroinflammation in SNI mice. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. The results showed that overexpression of miR-26a-5p in vivo could significantly reduce the expression level of Wnt5a. In addition, Foxy5, a mimetic peptide of Wnt5a, can significantly reverse the inhibitory effect of miR-26a-5p on neuroinflammation and neuropathic pain, and at the same time, miR-26a-5p can rescue the effect of Foxy5 by overexpression. CONCLUSIONS: We reported that hPMSCs derived sEVs as a promising therapy for nerve injury induced neuropathic pain. In addition, we showed that the miR-26a-5p in the sEVs regulated Wnt5a/Ryk/CaMKII/NFAT partly take part in the analgesia through anti-neuroinflammation, which suggests an alleviating pain effect through non-canonical Wnt signaling pathway in neuropathic pain model in vivo.

Effect of Celastrol on LncRNAs and mRNAs Profiles of Cerebral Ischemia-Reperfusion Injury in Transient Middle Cerebral Artery Occlusion Mice Model.

Celastrol plays a significant role in cerebral ischemia-reperfusion injury. Although previous studies have confirmed that celastrol post-treatment has a protective effect on ischemic stroke, the therapeutic effect of celastrol on ischemic stroke and the underlying molecular mechanism remain unclear. In the present study, focal transient cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in mice and celastrol was administered immediately after reperfusion. We performed lncRNA and mRNA analysis in the ischemic hemisphere of adult mice with celastrol post-treatment through RNA-Sequencing (RNA-Seq). A total of 50 differentially expressed lncRNAs (DE lncRNAs) and 696 differentially expressed mRNAs (DE mRNAs) were identified between the sham and tMCAO group, and a total of 544 DE lncRNAs and 324 DE mRNAs were identified between the tMCAO and tMCAO + celastrol group. Bioinformatic analysis was done on the identified deregulated genes through gene ontology (GO) analysis, KEGG pathway analysis and network analysis. Pathway analysis indicated that inflammation-related signaling pathways played vital roles in the treatment of ischemic stroke by celastrol. Four DE lncRNAs and 5 DE mRNAs were selected for further validation by qRT-PCR in brain tissue, primary neurons, primary astrocytes, and BV2 cells. The results of qRT-PCR suggested that most of selected differentially expressed genes showed the same fold change patterns as those in RNA-Seq results. Our study suggests celastrol treatment can effectively reduce cerebral ischemia-reperfusion injury. The bioinformatics analysis of lnRNAs and mRNAs profiles in the ischemic hemisphere of adult mice provides a new perspective in the neuroprotective effects of celastrol, particularly with regards to ischemic stroke.

Celastrol Protects against Cerebral Ischemia/Reperfusion Injury in Mice by Inhibiting Glycolysis through Targeting HIF-1α/PDK1 Axis.

Cerebral ischemia/reperfusion (I/R) injury is closely related to dysfunctional glucose metabolism. Celastrol is a bioactive compound that has been found to exhibit neuroprotective effects in cerebral ischemia, while whether it can protect against cerebral I/R injury by regulating glycolysis remains unclear. The goal of this study is to investigate the role of celastrol on cerebral I/R injury and its underlying mechanisms in transient middle cerebral artery occlusion (tMCAO) mice. Methods. To observe the protective effect of celastrol and select its optimal dosage for further study, neurological score, TTC staining, and HE staining were used to evaluate neurological function, cerebral infarct volume, and cortical cell damage, respectively. QRT-PCR and Western blot were used to detect the mRNA and protein expression of hypoxia inducible factor-1α (HIF-1α), pyruvate dehydrogenasekinase1 (PDK1), lactate dehydrogenase A (LDHA), glucose transporter1 (GLUT1), and hexokinase2 (HK2), respectively. The lactate production, ATP level, and glucose content were assessed by assay kits. Results. Our results indicated that celastrol dose-dependently improved neurological function and reduced cerebral infarct volume and cortical cell death of tMCAO mice, and its optimal dosage was 4.5 mg/kg. In addition, celastrol significantly blocked I/R-induced increase of LDHA, GLUT1, HK2, and lactate production as well as decrease of ATP level and glucose content. Moreover, celastrol inhibited the I/R-induced upregulation of HIF-1α and PDK1. Overexpression of HIF-1α by DMOG reversed the protective effect of celastrol on cerebral I/R injury and blocked celastrol-induced suppression of glycolysis. Conclusions. Taken together, these results suggested that celastrol protected against cerebral I/R injury through inhibiting glycolysis via the HIF-1α/PDK1 axis.

Supplemental N-3 Polyunsaturated Fatty Acids Limit A1-Specific Astrocyte Polarization via Attenuating Mitochondrial Dysfunction in Ischemic Stroke in Mice.

Ischemic stroke is one of the leading causes of death and disability for adults, which lacks effective treatments. Dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) exerts beneficial effects on ischemic stroke by attenuating neuron death and inflammation induced by microglial activation. However, the impact and mechanism of n-3 PUFAs on astrocyte function during stroke have not yet been well investigated. Our current study found that dietary n-3 PUFAs decreased the infarction volume and improved the neurofunction in the mice model of transient middle cerebral artery occlusion (tMCAO). Notably, n-3 PUFAs reduced the stroke-induced A1 astrocyte polarization both in vivo and in vitro. We have demonstrated that exogenous n-3 PUFAs attenuated mitochondrial oxidative stress and increased the mitophagy of astrocytes in the condition of hypoxia. Furthermore, we provided evidence that treatment with the mitochondrial-derived antioxidant, mito-TEMPO, abrogated the n-3 PUFA-mediated regulation of A1 astrocyte polarization upon hypoxia treatment. Together, this study highlighted that n-3 PUFAs prevent mitochondrial dysfunction, thereby limiting A1-specific astrocyte polarization and subsequently improving the neurological outcomes of mice with ischemic stroke.

A Simple and Efficient CRISPR Technique for Protein Tagging.

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.

Propofol Exposure Disturbs the Differentiation of Rodent Neural Stem Cells via an miR-124-3p/Sp1/Cdkn1b Axis.

Accumulating studies have indicated that propofol may lead to neurotoxicity and its effect on neural stem cells (NSCs) may play pivotal role in propofol-related neurotoxicity. Previously, we found that propofol could promote NSCs proliferation and could regulate several microRNA expressions. However, the underlying mechanism between microRNAs and NSCs development after propofol exposure is still unclear. Our data first observed that rat primary neural stem cells exposed to propofol exhibited a cell cycle arrest status and an inclination to differentiate into GFAP+ or S100ß+ cells. This phenomenon was accompanying with a lower miR-124-3p expression and could be reversed via overexpression miR-124-3p in NSCs. Using bioinformatic predictions and luciferase assay we confirmed that Sp1 (Specificity Protein 1) is the target gene of miR-124-3p, indicating that miR-124-3p may regulate NSCs development through Sp1. Further, knockdown of Sp1 rescue the effect of propofol on NSCs differentiation. Finally, we demonstrated that Sp1 could bind cdkn1b promoter region through chromatin immunoprecipitation assay, indicating that Sp1 affect NSC's cell cycle through cdkn1b directly. Overall, our study highlights the miR-124-3p/Sp1/cdkn1b axis to be important in propofol interfering the differentiation of NSCs.

GPR39 is coupled to TMEM16A in intestinal fibroblast-like cells.

GPR39 is a GPCR implicated as a regulator of gastrointestinal motility, although the mechanism remains elusive. Here, we report that GPR39 is expressed by a specific cell population cultured from mouse small intestine muscle layers, which was subsequently identified as fibroblast-like cells (FLCs) that have recently been shown to modulate gut motility. Application of the GPR39 agonist, Zn(2+), induced large currents and membrane depolarization in FLCs cultured from wild-type mice, but not Gpr39(-/-) mice. This Zn(2+)-induced current could be suppressed by application of a TMEM16A antagonist, CaCC(inh)-A01, or by silencing Tmem16a expression. These data suggest that GPR39 might modulate gut motility via regulating TMEM16A function in FLCs.

TRPA1 agonist activity of probenecid desensitizes channel responses: consequences for screening.

The transient receptor potential channel subtype A member 1 (TRPA1) is a nonselective cation channel widely viewed as having therapeutic potential, particularly for pain-related indications. Realization of this potential will require potent, selective modulators; however, currently the pharmacology of TRPA1 is poorly defined. As TRPA1 is calcium permeable, calcium indicators offer a simple assay format for high-throughput screening. In this report, we show that probenecid, a uricosuric agent used experimentally in screening to increase loading of calcium-sensitive dyes, activates TRPA1. Prolonged probenecid incubation during the dye-loading process reduces agonist potency upon subsequent challenge. When Chinese Hamster Ovary (CHO)-hTRPA1 or STC-1 cells, which endogenously express TRPA1, were dye loaded in the presence of 2 mM probenecid TRPA1, agonists appeared less potent; EC(50) for allyl isothiocyante agonists in CHO-hTRPA1 was increased from 1.5±0.19 to 7.32±1.20 µM (P<0.01). No significant effect on antagonist potency was observed when using the agonist EC(80) concentration determined under the appropriate dye-loading conditions. We suggest an alternative protocol for calcium imaging using another blocker of anion transport, sulfinpyrazone. This blocker significantly augments indicator dye loading and the screening window, but is not a TRPA1 agonist and has no effect on agonist potency.

Increased prokineticin 2 expression in gut inflammation: role in visceral pain and intestinal ion transport.

BACKGROUND: Prokineticin 2 (PROK2) is an inflammatory cytokine-like molecule expressed predominantly by macrophages and neutrophils infiltrating sites of tissue damage. Given the established role of prokineticin signaling on gastrointestinal function, we have explored Prok2 gene expression in inflammatory conditions of the gastrointestinal tract and assessed the possible consequences on gut physiology. METHODS: Prokineticin expression was examined in normal and colitic tissues using qPCR and immunohistochemistry. Functional responses to PROK2 were studied using calcium imaging and a novel antagonist, Compound 3, used to determine the role of PROK2 and prokineticin receptors in inflammatory visceral pain and ion transport. KEY RESULTS: Prok2 gene expression was up-regulated in biopsy samples from ulcerative colitis patients, and similar elevations were observed in rodent models of inflammatory colitis. Prokineticin receptor 1 (PKR1) was localized to the enteric neurons and extrinsic sensory neurons, whereas Pkr2 expression was restricted to sensory ganglia. In rats, PROK2-increased intracellular calcium levels in cultured enteric and dorsal root ganglia neurons, which was blocked by Compound 3. Moreover, PROK2 acting at prokineticin receptors stimulated intrinsic neuronally mediated ion transport in rat ileal mucosa. In vivo, Compound 3 reversed intracolonic mustard oil-induced referred allodynia and TNBS-induced visceral hypersensitivity, but not non-inflammatory, stress-induced visceral pain. CONCLUSIONS & INFERENCES: Elevated Prok2 levels, as a consequence of gastrointestinal tract inflammation, induce visceral pain via prokineticin receptors. This observation, together with the finding that PROK2 can modulate intestinal ion transport, raises the possibility that inhibitors of PROK2 signaling may have clinical utility in gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.

TRPC1 transcript variants, inefficient nonsense-mediated decay and low up-frameshift-1 in vascular smooth muscle cells.

BACKGROUND: Transient Receptor Potential Canonical 1 (TRPC1) is a widely-expressed mammalian cationic channel with functional effects that include stimulation of cardiovascular remodelling. The initial aim of this study was to investigate variation in TRPC1-encoding gene transcripts. RESULTS: Extensive TRPC1 transcript alternative splicing was observed, with exons 2, 3 and 5-9 frequently omitted, leading to variants containing premature termination codons. Consistent with the predicted sensitivity of such variants to nonsense-mediated decay (NMD) the variants were increased by cycloheximide. However it was notable that control of the variants by NMD was prominent in human embryonic kidney 293 cells but not human vascular smooth muscle cells. The cellular difference was attributed in part to a critical protein in NMD, up-frameshift-1 (UPF1), which was found to have low abundance in the vascular cells. Rescue of UPF1 by expression of exogenous UPF1 was found to suppress vascular smooth muscle cell proliferation. CONCLUSIONS: The data suggest: (i) extensive NMD-sensitive transcripts of TRPC1; (ii) inefficient clearance of aberrant transcripts and enhanced proliferation of vascular smooth muscle cells in part because of low UPF1 expression.

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers.

AIM: The aim of the study was to determine the potential for K(V)1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. METHODS AND RESULTS: Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptase-polymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function. K(V)1.3 was unique among the K(V)1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of K(V)1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC(50) of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. CONCLUSION: K(V)1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events.

Pregnenolone sulphate- and cholesterol-regulated TRPM3 channels coupled to vascular smooth muscle secretion and contraction.

RATIONALE: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in beta cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. OBJECTIVE: To determine the relevance and regulation of TRPM3 in vascular biology. METHODS AND RESULTS: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. beta-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. CONCLUSIONS: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

Glial cell-derived neurotrophic factor enhances synaptic communication and 5-hydroxytryptamine 3a receptor expression in enteric neurons.

BACKGROUND & AIMS: Glial cell-derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system during embryogenesis. We have observed the presence of Gdnf transcripts in the gastrointestinal tract of adult mice, and its early up-regulation after inflammation. We therefore investigated the effects of GDNF on enteric neuronal function in vitro. METHODS: Primary neuronal cultures were established from isolated myenteric plexi, and characterized by immunostaining and Ca(2+) imaging. Gene expression of several ion channels was analyzed by quantitative polymerase chain reaction (PCR) and the electrophysiologic properties of the neurons were studied by patch clamp. RESULTS: GDNF enhanced synaptogenesis and intercellular communication in primary myenteric neuronal cultures. Expression profiling revealed that GDNF exposure results in an up-regulation of Htr3a expression in the cultures and a similar increase was observed in inflamed colonic tissue where Gdnf expression was also increased. The increased Htr3a expression was accompanied by a functional increase in the response of neurons to acute challenge with 5-hydroxytryptamine (5-HT). GDNF treatment also caused inhibition of delayed rectifying voltage-gated potassium (Kv) currents, which correlated with the up-regulation of Htr3a and 5-HT-induced responses. Furthermore, pharmacologic blockade of Kv channels mimicked the effect of GDNF by increasing Htr3a expression as well as enhancing 5-HT-induced responses in the cultured myenteric neurons. CONCLUSIONS: GDNF promotes synaptic communication in cultured myenteric neurons. It also up-regulates 5-HT(3a)-receptor expression via modulation of Kv channel activity. Up-regulation of Gdnf after gastrointestinal inflammation might play an important role in the pathophysiology of gastrointestinal diseases.

Robotic multiwell planar patch-clamp for native and primary mammalian cells.

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

TRPC channel activation by extracellular thioredoxin.

Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5-TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.