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British Heart Journal Oct 1953
Topics: Hypertension; Protoveratrines; Veratrum Alkaloids
PubMed: 13093878
DOI: 10.1136/hrt.15.4.439 -
American Journal of Physiology. Cell... Sep 2022Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked...
Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na was substituted with -Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The Vs of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC = 1.5 nM) and the Na1.7 subtype-selective blocker, PF-05089771 (IC = 8.6 nM), consistent with Na1.7 as the underlying pore-forming α subunit. Two Na1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-Na antibody. RT-PCR, performed on groups of ∼15 isolated SMCs, revealed transcripts for Na1.7 in 7/8 samples. Veratridine (30 µM), a nonselective Na channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE (prostaglandin E), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express Na1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.
Topics: Animals; Atropine Derivatives; Bronchi; Mice; Myocytes, Smooth Muscle; Sodium; Tetrodotoxin; Veratridine
PubMed: 35876287
DOI: 10.1152/ajpcell.00011.2022 -
The Journal of General Physiology Jan 1986Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na... (Review)
Review
Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.
Topics: Animals; Bungarotoxins; Chloramines; In Vitro Techniques; Ion Channels; Kinetics; Muscles; Neurotoxins; Rana pipiens; Rats; Sodium; Tosyl Compounds; Veratridine; Veratrine
PubMed: 2419478
DOI: 10.1085/jgp.87.1.1 -
PloS One 2011GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA...
BACKGROUND AND AIMS
GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs.
METHODS
Expressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered.
RESULTS
IHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC.
CONCLUSION
DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.
Topics: Adenomatous Polyposis Coli; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Gene Expression Regulation, Neoplastic; Humans; Oncogene Proteins; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Small Interfering; Trans-Activators; Up-Regulation; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 22110720
DOI: 10.1371/journal.pone.0027684 -
Acta Pharmacologica Sinica May 2012To investigate the role of Hedgehog (Hh) signaling pathway in the invasion and metastasis of human hepatocellular carcinoma (HCC).
AIM
To investigate the role of Hedgehog (Hh) signaling pathway in the invasion and metastasis of human hepatocellular carcinoma (HCC).
METHODS
Eighty six HCC tissues samples and HCC cell line Bel-7402 were examined. The protein expression of sonic hedgehog (Shh), nuclear glioma-associated oncogene-1 (Gli1), MMP-9 and p-ERK1/2 in HCC was analyzed using immunohistochemistry and Western blot analysis. Boyden chamber assay and wound-healing assay were used to quantify the invasion and metastasis of Bel-7402 cells.
RESULTS
In 86 HCC tissue samples, the positive ratio of Shh and nucleus Gli1 was 67.44% (58/86) and 60.47% (52/86), respectively; the expression of nucleus Gli1 was correlated with the tumor pathological grade (P=0.034), and with the ability of the tumor to invade and metastasize (P=0.001); the expression of nucleus Gli1 was also correlated with p-ERK1/2 (P=0.031) and with MMP-9 (P=0.034). Neither Shh, nor nucleus Gli1 was observed in normal liver tissue. KAAD-cyclopamine (KAAD-cyc), a specific inhibitor of the Hh pathway, at the concentrations of 1 and 4 μmol/L inhibited the invasion and migration of Bel-7402 cells and decreased the expression of Gli1 in nucleus and MMP-9, p-ERK1/2 proteins in Bel-7402 cells. On the other hand, Shh, a ligand of the Hh pathway, at the concentration of 0.5 μg/mL produced opposite effects. The MAPK pathway inhibitors U0126 and PD98059 at the concentrations of 5 and 10 μmol/L inhibited invasion and metastasis of Bel-7402 cells induced by Shh, and decreased the expression of p-ERK1/2 and MMP-9. However, U0126 and PD98059 had no effect on the expression of Gli1.
CONCLUSION
Hh signaling pathway mediates invasion and metastasis of human HCC by up-regulating the protein expression of MMP-9 via ERK pathway.
Topics: Adult; Aged; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cinnamates; Dose-Response Relationship, Drug; Female; Flavonoids; Hedgehog Proteins; Humans; Immunohistochemistry; Liver Neoplasms; MAP Kinase Signaling System; Male; Matrix Metalloproteinase 9; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Grading; Neoplasm Invasiveness; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 22543708
DOI: 10.1038/aps.2012.24 -
Nucleic Acids Research Jan 2018Because the transcription factor activator protein-1 (AP-1) regulates a variety of protein-encoding genes, it is a participant in many cellular functions, including...
Because the transcription factor activator protein-1 (AP-1) regulates a variety of protein-encoding genes, it is a participant in many cellular functions, including proliferation, transformation, epithelial mesenchymal transition (EMT), and apoptosis. Inhibitors targeting AP-1 have potential use in the treatment of cancer and other inflammatory diseases. Here, we identify veratramine as a potent natural modulator of AP-1, which selectively binds to a specific site (TRE 5'-TGACTCA-3') of the AP-1 target DNA sequence and regulates AP-1-dependent gene transcription without interfering with cystosolic signaling cascades that might lead to AP-1 activation. Moreover, RNA-seq experiments demonstrate that veratramine does not act on the Hedgehog signaling pathway in contrast to its analogue, cyclopamine, and likely does not harbor the same teratogenicity and toxicity. Additionally, veratramine effectively suppresses EGF-induced AP-1 transactivation and transformation of JB6 P+ cells. Finally, we demonstrate that veratramine inhibits solar-ultraviolet-induced AP-1 activation in mice. The identification of veratramine and new findings in its specific regulation of AP-1 down stream genes pave ways to discovering and designing regulators to regulate transcription factor.
Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; DNA; Gene Expression Profiling; HeLa Cells; Humans; Mice; Protein Binding; Signal Transduction; Transcription Factor AP-1; Transcriptional Activation; Ultraviolet Rays; Veratrum Alkaloids
PubMed: 29237043
DOI: 10.1093/nar/gkx1241 -
International Journal of Molecular... Aug 2023The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This...
The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This transformation occurs in the cortical marginal zone (MZ), a layer that contains the Cajal-Retzius neurons and their axonal projections. Cajal-Retzius neurons (CRNs) are well known for their critical role in secreting Reelin, a glycoprotein that controls dendritogenesis and cell positioning in many regions of the developing brain. In this study, we examine the possibility that CRNs in the MZ may provide additional signals to arriving CPNs, that may promote the maturation of CPNs and thus shape the development of the cortex. We use whole embryonic hemisphere explants and multiphoton microscopy to confirm that CRNs display intracellular calcium transients of <1-min duration and high amplitude during early corticogenesis. In contrast, developing CPNs do not show high-amplitude calcium transients, but instead show a steady increase in intracellular calcium that begins at the time of dendritic initiation, when the leading process of the migrating CPN is encountering the MZ. The possible existence of CRN to CPN communication was revealed by the application of veratridine, a sodium channel activator, which has been shown to preferentially stimulate more mature cells in the MZ at an early developmental time. Surprisingly, veratridine application also triggers large calcium transients in CPNs, which can be partially blocked by a cocktail of antagonists that block glutamate and glycine receptor activation. These findings outline a model in which CRN spontaneous activity triggers the release of glutamate and glycine, neurotransmitters that can trigger intracellular calcium elevations in CPNs. These elevations begin as CPNs initiate dendritogenesis and continue as waves in the post-migratory cells. Moreover, we show that the pharmacological blockade of glutamatergic signaling disrupts migration, while forced expression of a bacterial voltage-gated calcium channel (CavMr) in the migrating neurons promotes dendritic growth and migration arrest. The identification of CRN to CPN signaling during early development provides insight into the observation that many autism-linked genes encode synaptic proteins that, paradoxically, are expressed in the developing cortex well before the appearance of synapses and the establishment of functional circuits.
Topics: Calcium Signaling; Calcium; Veratridine; Neurons; Dendrites; Calcium, Dietary; Glutamic Acid
PubMed: 37629145
DOI: 10.3390/ijms241612965 -
International Journal of Oncology Dec 2018Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic...
Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinicopathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; RNA, Small Interfering; Signal Transduction; Survival Analysis; Up-Regulation; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 30272371
DOI: 10.3892/ijo.2018.4573 -
PloS One 2012Many prostate cancers relapse due to the generation of chemoresistance rendering first-line treatment drugs like paclitaxel (PTX) ineffective. The present study aims to...
Many prostate cancers relapse due to the generation of chemoresistance rendering first-line treatment drugs like paclitaxel (PTX) ineffective. The present study aims to determine the role of miRNAs and Hedgehog (Hh) pathway in chemoresistant prostate cancer and to evaluate the combination therapy using Hh inhibitor cyclopamine (CYA). Studies were conducted on PTX resistant DU145-TXR and PC3-TXR cell lines and clinical prostate tissues. Drug sensitivity and apoptosis assays showed significantly improved cytotoxicity with combination of PTX and CYA. To distinguish the presence of cancer stem cell like side populations (SP), Hoechst 33342 flow cytometry method was used. PTX resistant DU145 and PC3 cells, as well as human prostate cancer tissue possess a distinct SP fraction. Nearly 75% of the SP cells are in the G0/G1 phase compared to 62% for non-SP cells and have higher expression of stem cell markers as well. SP cell fraction was increased following PTX monotherapy and treatment with CYA or CYA plus PTX effectively reduced their numbers suggesting the effectiveness of combination therapy. SP fraction cells were allowed to differentiate and reanalyzed by Hoechst staining and gene expression analysis. Post differentiation, SP cells constitute 15.8% of total viable cells which decreases to 0.6% on treatment with CYA. The expression levels of P-gp efflux protein were also significantly decreased on treatment with PTX and CYA combination. MicroRNA profiling of DU145-TXR and PC3-TXR cells and prostate cancer tissue from the patients showed decreased expression of tumor suppressor miRNAs such as miR34a and miR200c. Treatment with PTX and CYA combination restored the expression of miR200c and 34a, confirming their role in modulating chemoresistance. We have shown that supplementing mitotic stabilizer drugs such as PTX with Hh-inhibitor CYA can reverse PTX chemoresistance and eliminate SP fraction in androgen independent, metastatic prostate cancer cell lines.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Hedgehog Proteins; Humans; Male; MicroRNAs; Models, Biological; Paclitaxel; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Side-Population Cells; Signal Transduction; Veratrum Alkaloids
PubMed: 22768203
DOI: 10.1371/journal.pone.0040021 -
The American Journal of Pathology Apr 2012The Hedgehog (Hh) signaling pathway regulates tissue patterning during development, including patterning and growth of limbs and face, but whether Hh signaling plays a...
The Hedgehog (Hh) signaling pathway regulates tissue patterning during development, including patterning and growth of limbs and face, but whether Hh signaling plays a role in adult kidney remains undefined. In this study, using a panel of hedgehog-reporter mice, we show that the two Hh ligands (Indian hedgehog and sonic hedgehog ligands) are expressed in tubular epithelial cells. We report that the Hh effectors (Gli1 and Gli2) are expressed exclusively in adjacent platelet-derived growth factor receptor-β-positive interstitial pericytes and perivascular fibroblasts, suggesting a paracrine signaling loop. In two models of renal fibrosis, Indian Hh ligand was upregulated with a dramatic activation of downstream Gli effector expression. Hh-responsive Gli1-positive interstitial cells underwent 11-fold proliferative expansion during fibrosis, and both Gli1- and Gli2-positive cells differentiated into α-smooth muscle actin-positive myofibroblasts. In the pericyte-like cell line 10T1/2, hedgehog ligand triggered cell proliferation, suggesting a possible role for this pathway in the regulation of cell cycle progression of myofibroblast progenitors during the development of renal fibrosis. The hedgehog antagonist IPI-926 abolished Gli1 induction in vivo but did not decrease kidney fibrosis. However, the transcriptional induction of Gli2 was unaffected by IPI-926, suggesting the existence of smoothened-independent Gli activation in this model. This study is the first detailed description of paracrine hedgehog signaling in adult kidney, which indicates a possible role for hedgehog-Gli signaling in fibrotic chronic kidney disease.
Topics: Animals; Cell Line; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Fibroblasts; Fibrosis; Hedgehog Proteins; Kidney; Kidney Tubules; Kruppel-Like Transcription Factors; Ligands; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Paracrine Communication; Patched Receptors; Pericytes; Receptors, Cell Surface; Signal Transduction; Up-Regulation; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2
PubMed: 22342522
DOI: 10.1016/j.ajpath.2011.12.039