This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

The present study was aimed to investigate the effect of Janus kinase 3 (JAK3) on the migration of breast cancer cells and the underlying mechanism. The expression of JAK3 in breast cancer MCF-7 cells was silenced by siRNA (siJAK3). The migration ability of MCF-7 cells was detected by scratch test. The activity of store-operated calcium channel (SOCC) was detected by fluorescence calcium imaging. The expression levels of Orai1 and STIM1, key molecules in the process of store-operated calcium entry (SOCE) were detected by Western blot and RT-PCR. The results showed that 2-APB, an inhibitor of SOCC, could inhibit the migration ability of MCF-7 cells. siJAK3 transfection significantly inhibited the migration ability of MCF-7 cells, decreased the activity of SOCC, and down-regulated mRNA and protein expression levels of Orai1 and Stim1. Over-expression of Orai1 or STIM1 in JAK3-silenced cells restored their migration ability. These results suggest that JAK3 facilitates the migration of breast cancer cells by SOCC.
Breast Neoplasms ; enzymology ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cell Movement ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Janus Kinase 3 ; genetics ; metabolism ; MCF-7 Cells ; ORAI1 Protein ; genetics

This study aims to validate our hypothesis that acid-sensing ion channels (ASICs) may contribute to the symptom of pain in patients with chronic prostatitis (CP). We first established a CP rat model, then isolated the L5-S2 spinal dorsal horn neurons for further studies. ASIC1a was knocked down and its effects on the expression of neurogenic inflammation-related factors in the dorsal horn neurons of rat spinal cord were evaluated. The effect of ASIC1a on the Ca²⁺ ion concentration in the dorsal horn neurons of rat spinal cord was measured by the intracellular calcium ([Ca²⁺]i) intensity. The effect of ASIC1a on the p38/mitogen-activated protein kinase (MAPK) signaling pathway was also determined. ASIC1a was significantly upregulated in the CP rat model as compared with control rats. Acid-induced ASIC1a expression increased [Ca²⁺]i intensity in the dorsal horn neurons of rat spinal cord. ASIC1a also increased the levels of neurogenic inflammation-related factors and p-p38 expression in the acid-treated dorsal horn neurons. Notably, ASIC1a knockdown significantly decreased the expression of pro-inflammatory cytokines. Furthermore, the levels of p-p38 and pro-inflammatory cytokines in acid-treated dorsal horn neurons were significantly decreased in the presence of PcTx-1, BAPTA-AM, or SB203580. Our results showed that ASIC1a may contribute to the symptom of pain in patients with CP, at least partially, by regulating the p38/MAPK signaling pathway.
Acid Sensing Ion Channel Blockers/pharmacology* ; Acid Sensing Ion Channels/genetics* ; Animals ; Calcium/metabolism* ; Chelating Agents/pharmacology* ; Chronic Disease ; Cytokines/metabolism* ; Disease Models, Animal ; Egtazic Acid/pharmacology* ; Gene Knockdown Techniques ; Imidazoles/pharmacology* ; Inflammation/metabolism* ; MAP Kinase Signaling System/genetics* ; Male ; Pain/genetics* ; Peptides/pharmacology* ; Phosphorylation/drug effects* ; Posterior Horn Cells/metabolism* ; Prostatitis/complications* ; Protein Kinase Inhibitors/pharmacology* ; Pyridines/pharmacology* ; Rats ; Spider Venoms/pharmacology* ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism*

Calcium Channels, L-Type ; genetics ; metabolism ; Esophageal Achalasia ; genetics ; metabolism ; Esophagus ; metabolism ; Humans ; Nitric Oxide Synthase ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Calcium-Sensing ; genetics ; metabolism

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.
Animals ; Calcium ; metabolism ; Cryoelectron Microscopy ; Endocytosis ; Endosomes ; metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; metabolism ; Mice ; Models, Biological ; Mucolipidoses ; genetics ; metabolism ; pathology ; Nanostructures ; chemistry ; ultrastructure ; Phosphatidylinositols ; metabolism ; Transgenes ; Transient Receptor Potential Channels ; chemistry ; genetics ; metabolism

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.
Animals ; Calcium Channels, L-Type ; chemistry ; genetics ; metabolism ; Channelopathies ; genetics ; metabolism ; Humans ; Mutation ; NAV1.1 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; Protein Domains ; Rabbits ; Structure-Activity Relationship

TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products, including gingerol, a main active constituent of ginger. Ginger could improve the digestive function; however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated. In the present study, gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium. TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPA1-specific siRNA were also used for mechanistic studies. The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA. Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red, EGTA, and TRPA1-specific siRNA. In summary, our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.
Calcium ; metabolism ; Calcium Channels ; genetics ; metabolism ; Catechols ; pharmacology ; Cell Line ; Fatty Alcohols ; pharmacology ; Gastrointestinal Tract ; drug effects ; metabolism ; Ginger ; chemistry ; Humans ; Nerve Tissue Proteins ; genetics ; metabolism ; Plant Extracts ; pharmacology ; TRPA1 Cation Channel ; Transient Receptor Potential Channels ; genetics ; metabolism

This study was amid to construct the pharmacophore model of L-type calcium channel antagonist in the application of screening Drugbank and TCMD. This paper repositions the approved drugs resulting from virtual screening and discusses the relocation-based drug discovery methods, screening antihypertensive drugs with L-type calcium channel function from TCMD. Qualitative hypotheses wre generated by HipHop separately on the basis of 12 compounds with antagonistic action on L-type calcium channel expressed in rabbit cardiac muscle. Datebase searching method was used to evaluate the generated hypotheses. The optimum hypothesis was used to search Drugbank and TCMD. This paper repositions the approved drugs and evaluates the antihypertensive effect of the chemical constituent of traditional Chinese medicine resulting from virtual screening by the matching score and literature. The results showed that optimum qualitative hypothesis is with six features, which were two hydrogen-bond acceptors, four hydrophobic groups, and the CAI value of 2.78. Screening Drugbank achieves 93 approved drugs. Screening TCMD achieves 285 chemical constituents of traditional Chinese medicine. It was concluded that the hypothesis is reliable and can be used to screen datebase. The approved drugs resulting from virtual screening, such as pravastatin, are potentially L-type calcium channels inhibitors. The chemical constituents of traditional Chinese medicine, such as Arctigenin III and Arctigenin are potentially antihypertensive drugs. It indicates that Drug Repositioning based on hypothesis is possible.
Animals ; Antihypertensive Agents ; chemistry ; pharmacology ; Calcium Channel Blockers ; chemistry ; pharmacology ; Calcium Channels, L-Type ; genetics ; metabolism ; Drug Repositioning ; methods ; Molecular Structure ; Myocardium ; metabolism ; Rabbits

To build the Dendrobium nobile -T2DM network, and elucidate the molecular mechanism of D. nobile to type 2 diabetes (T2DM). Collect the chemical composition of D. nobile and the targets on T2DM by retrieving database and documents, build the network of D. nobile to T2DM using the entity grammar systems inference rules. The molecular mechanism of D. nobile to T2DM includes: (1) regulating lipid metabolism by lowering triglyceride; (2) reducing insulin resistance; (3) protecting islet cells; (4) promoting the glucose-dependent insulin tropic peptide (GIP) secretion; (5) inhibiting calcium channel. Under the guidance of network pharmacology, through entity grammar systems inference rules we elucidate the molecular mechanism of D. nobile to T2DM, and provide the basis for the further development of health care products based on D. nobile.
Animals ; Calcium Channels ; genetics ; metabolism ; Databases, Factual ; Dendrobium ; chemistry ; Diabetes Mellitus, Type 2 ; drug therapy ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Gene Regulatory Networks ; drug effects ; Humans ; Hypoglycemic Agents ; administration & dosage ; chemistry ; Insulin Resistance ; Islets of Langerhans ; metabolism ; Triglycerides ; metabolism

Orai1 is the key subunit of the Ca2+-release-activated Ca2+ channel. Our previous report has demonstrated that Orai1 expression in the airway was upregulated in the ovalbumin (OVA)-induced allergic rhinitis (AR) mouse models. To observe whether inhibition of Orai1 expression in the airway could suppress symptoms in a murine model of AR and to assess the impacts of this inhibition on the responses of local and systemic immunocytes, we administered recombinant lentivirus vectors that encoded shRNA against ORAI1 (lenti-ORAI1) into the nostrils of OVA-sensitized mice before the challenges, and analyzed its effect on allergic responses, as compared with the unsensitized mice and untreated AR mice. Administration of lenti-ORAI1 into the nasal cavity successfully infected cells in the epithelial layer of the nasal mucosa, and significantly decreased the frequencies of sneezing and nasal rubbing of the mice. Protein levels of leukotriene C4, OVA-specific IgE, and IL-4 in the nasal lavage fluid and serum and eosinophil cation protein in the serum were also significantly reduced by lenti-ORAI1, as were the mRNA levels of these factors in the nasal mucosa and spleen. These data suggested that administration of lenti-ORAI1 into the nasal cavity effectively decreased Orai1 expression in the nasal mucosa, alleviated AR symptoms, and partially inhibited the hyperresponsiveness of the local and systemic immune cells including T cells, B cells, mast cells and eosinophils that are involved in the pathogenesis of AR.
Animals ; Calcium Channels/analysis/*genetics/immunology ; *Down-Regulation ; Eosinophil Cationic Protein/blood/genetics ; Glutathione Transferase/blood/genetics/immunology ; Immunoglobulin E/blood/genetics/immunology ; Interleukin-4/blood/genetics/immunology ; Lentivirus/genetics ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa/immunology/metabolism ; Ovalbumin/immunology ; RNA, Messenger/genetics ; RNA, Small Interfering/*administration & dosage/genetics ; Rhinitis, Allergic, Perennial/*genetics/immunology ; Spleen/immunology/metabolism ; *Transfection