ABSTRACT
Background: Forest musk deer (FMD, Moschus Berezovskii) is a critically endangered species world-widely, the death of which can be caused by pulmonary disease in the farm. Pulmonary fibrosis (PF) was a huge threat to the health and survival of captive FMD. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been involved in the regulation of immune genes and disease development. However, the regulatory profiles of mRNAs and miRNAs involved in immune regulation of FMD are unclear. Methods: In this study, mRNA-seq and miRNA-seq in blood were performed to constructed coexpression regulatory networks between PF and healthy groups of FMD. The hub immune- and apoptosis-related genes in the PF blood of FMD were explored through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Further, protein-protein interaction (PPI) network of immune-associated and apoptosis-associated key signaling pathways were constructed based on mRNA-miRNA in the PF blood of the FMD. Immune hub DEGs and immune hub DEmiRNAs were selected for experimental verification using RT-qPCR. Results: A total of 2744 differentially expressed genes (DEGs) and 356 differentially expressed miRNAs (DEmiRNAs) were identified in the PF blood group compared to the healthy blood group. Among them, 42 DEmiRNAs were negatively correlated with 20 immune DEGs from a total of 57 correlations. The DEGs were significantly associated with pathways related to CD molecules, immune disease, immune system, cytokine receptors, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, and NOD-like receptor signaling pathway. There were 240 immune-related DEGs, in which 186 immune-related DEGs were up-regulated and 54 immune-related DEGs were down-regulated. In the protein-protein interaction (PPI) analysis of immune-related signaling pathway, TYK2, TLR2, TLR4, IL18, CSF1, CXCL13, LCK, ITGB2, PIK3CB, HCK, CD40, CD86, CCL3, CCR7, IL2RA, TLR3, and IL4R were identified as the hub immune genes. The mRNA-miRNA coregulation analysis showed that let-7d, miR-324-3p, miR-760, miR-185, miR-149, miR-149-5p, and miR-1842-5p are key miRNAs that target DEGs involved in immune disease, immune system and immunoregulation. Conclusion: The development and occurrence of PF were significantly influenced by the immune-related and apoptosis-related genes present in PF blood. mRNAs and miRNAs associated with the development and occurrence of PF in the FMD.
Subject(s)
Deer , Gene Expression Profiling , Gene Regulatory Networks , MicroRNAs , Pulmonary Fibrosis , RNA, Messenger , Transcriptome , Animals , MicroRNAs/genetics , Deer/genetics , Deer/immunology , RNA, Messenger/genetics , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/immunology , Protein Interaction Maps , Gene Expression Regulation , Computational Biology/methodsABSTRACT
To explore the active substances exerting anti-tumour effect in lemon essential oil and the molecular mechanism inhibiting the proliferation of head and neck cancer cells SCC15 and CAL33, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT) was utilized to identify the active component inhibiting the proliferation of head and neck cancer cells, namely citral. The IC_(50) of citral inhibiting the proliferation of head and neck cancer cells and normal cells were also determined. In addition, a 5-ethynyl-2'-deoxyuridine(EdU) staining assay was used to detect the effect of citral on the proliferation rate of head and neck cancer cells, and a colony formation assay was used to detect the effect of citral on tumor sphere formation of head and neck cancer cells in vitro. The cell cycle arrest and apoptosis induction of head and neck cancer cells by citral were evaluated by flow cytometry, and Western blot was used to detect the effect of citral on the expression levels of cell cycle-and apoptosis-related proteins in head and neck cancer cells. The findings indicated that citral could effectively inhibit the proliferation and growth of head and neck cancer cells, with anti-tumor activity, and its half inhibitory concentrations for CAL33 and SCC15 were 54.78 and 25.23 µg·mL~(-1), respectively. Furthermore, citral arrested cell cycle at G_2/M phase by down-regulating cell cycle-related proteins such as S-phase kinase associated protein 2(SKP2), C-MYC, cyclin dependent kinase 1(CDK1), and cyclin B. Moreover, citral increased the cysteinyl aspartate-specific proteinase-3(caspase-3), cysteinyl aspartate-specific proteinase-9(caspase-9), and cleaved poly ADP-ribose polymerase(PARP). It up-regulated the level of autophagy-related proteins including microtubule associated protein 1 light chain 3B(LC3B), sequestosome 1(P62/SQSTM1), autophagy effector protein Beclin1(Beclin1), and lysosome-associate membrane protein 1(LAMP1), suggesting that citral could effectively trigger cell apoptosis and cell autophagy in head and neck cancer cells. Furthermore, the dual-tagged plasmid system mCherry-GFP-LC3 was used, and it was found that citral impeded the fusion of autophagosomes and lysosomes, leading to autophagic flux blockage. Collectively, our findings reveal that the main active anti-proliferation component of lemon essential oil is citral, and this component has a significant inhibitory effect on head and neck cancer cells. Its underlying molecular mechanism is that citral induces apoptosis and autophagy by cell cycle arrest and ultimately inhibits cell proliferation.
Subject(s)
Acyclic Monoterpenes , Apoptosis , Cell Proliferation , Head and Neck Neoplasms , Monoterpenes , Oils, Volatile , Humans , Cell Proliferation/drug effects , Acyclic Monoterpenes/pharmacology , Acyclic Monoterpenes/chemistry , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/genetics , Apoptosis/drug effects , Cell Line, Tumor , Oils, Volatile/pharmacology , Oils, Volatile/chemistry , Monoterpenes/pharmacology , Monoterpenes/chemistry , Cell Cycle Checkpoints/drug effects , Citrus/chemistry , Plant Oils/pharmacology , Plant Oils/chemistryABSTRACT
N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, voltage-dependent channels of the ionotropic glutamate receptor family. The present study explored whether NMDAR activation induced ferroptosis in vascular endothelial cells and its complicated mechanisms in vivo and in vitro. Various detection approaches were used to determine the ferroptosis-related cellular iron content, lipid reactive oxygen species (LOS), siRNA molecules, RNA-sequence, MDA, GSH, and western blotting. The AMPK activator Acadesine (AICAR), HMGB1 inhibitor glycyrrhizin (GLY), PP2A inhibitor LB-100, and NMDAR inhibitor MK801 were used to investigate the involved in vivo and in vitro pathways. The activation of NMDAR with L-glutamic acid (GLU) or NMDA significantly promoted cellular ferroptosis, iron content, MDA, and the PTGS2 expression, while decreasing GPX4 expression and GSH concentration in human umbilical vein endothelial cells (HUVECs), which was reversed by ferroptosis inhibitors Ferrostatin-1(Fer-1), Liproxstatin-1 (Lip-1), or Deferoxamine (DFO). RNA-seq revealed that ferroptosis and SLC7A11 participate in NMDA or GLU-mediated NMDAR activation. The PP2A-AMPK-HMGB1 pathway was majorly associated with NMDAR activation-induced ferroptosis, validated using the PP2A inhibitor LB-100, AMPK activator AICAR, or HMGB1 siRNA. The role of NMDAR in ferroptosis was validated in HUVECs induced with the ferroptosis activator errasin or RSL3 and counteracted by the NMDAR inhibitor MK-801. The in vivo results showed that NMDA- or GLU-induced ferroptosis and LOS production was reversed by MK-801, LB-100, AICAR, MK-801, and GLY, confirming that the PP2A-AMPK-HMGB1 pathway is involved in NMDAR activation-induced vascular endothelium ferroptosis. In conclusion, the present study demonstrated a novel role of NMDAR in endothelial cell injury by regulating ferroptosis via the PP2A-AMPK-HMGB1 pathway.
ABSTRACT
CONTEXT: Concanavalin A (Con A) exhibited multiple roles in cancer cells. However, the role of Con A in endothelial cells was not reported. OBJECTIVE: Our present study investigated the potential angiogenic role of Con A in endothelial cells and ischaemic hind-limb mice. MATERIALS AND METHODS: Human umbilical vein endothelial cells and Ea.hy926 cells were employed to determine the effect of Con A (0.3, 1, and 3 µg/mL) or vehicle on angiogenesis and cell proliferation with tube formation, ELISA, flow cytometry, EdU, and western blot. Hind-limb ischaemic mice were conducted to determine the pro-angiogenic effect of Con A (10 mg/kg) for 7 days. RESULTS: Con A promoted tube formation to about three-fold higher than the control group and increased the secretion of VEGFa, PDGFaa, and bFGF in the medium. The cell viability was promoted to 1.3-fold by Con A 3 µg/mL, and cell cycle progression of G0G1 phase was decreased from 77% in the vehicle group to 70% in Con A 3 µg/mL, G2M was promoted from 15 to 19%, and S-phase was from 7 to 10%. Con A significantly stimulated phosphorylation of Akt and ERK1/2 and expression of cyclin D1 and decreased the expression of p27. These effects of Con A were antagonised by the PI3K inhibitor LY294002 (10 µM) and MEK pathway antagonist PD98059 (10 µM). Moreover, Con A (10 mg/kg) exhibited a repair effect in ischaemic hind-limb mice. DISCUSSION AND CONCLUSIONS: This study will provide a new option for treating ischaemic disease by local injection with Con A.
Subject(s)
Angiogenesis Inducing Agents/pharmacology , Cell Proliferation/drug effects , Concanavalin A/pharmacology , Neovascularization, Physiologic/drug effects , Angiogenesis Inducing Agents/administration & dosage , Animals , Cell Survival/drug effects , Chromones/pharmacology , Concanavalin A/administration & dosage , Cyclin D1/metabolism , Dose-Response Relationship, Drug , Flavonoids/pharmacology , Hindlimb , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Ischemia/drug therapy , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Inbred C57BL , Morpholines/pharmacology , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
BACKGROUND: Our recent study demonstrated that the nonselective cation current mediated by the transient receptor potential canonical 1 (TRPC1) channel is activated by endothelin-1 (ET-1) in human atrial myocytes; however, the related signal molecules involved are unknown. OBJECTIVE: The purpose of this study was to investigate how the TRPC1 channel is regulated by ET-1 and whether it is upregulated in human atria from patients with atrial fibrillation (AF). METHODS: Whole-cell patch technique and molecular biology techniques were used in the study. RESULTS: The ET-1-evoked TRPC1 current was inhibited by the ET-1 type A (ETA) receptor antagonist BQ123 and the ET-1 type B (ETB) receptor antagonist BQ788 as well as the protein kinase C inhibitor chelerythrine. ETA receptor-mediated TRPC1 channel activity was selectively inhibited by the phosphoinositide-3-kinase inhibitor wortmannin, while ETB receptor-mediated TRPC1 activity was inhibited by the phospholipase C inhibitor U73122. The messenger RNAs and proteins of the TRPC1 channel and ETA receptor, but not the ETB receptor, were significantly upregulated in atria from patients with AF. The basal TRPC1 current increased in AF myocytes, and the response to ET-1 was greater in AF myocytes than in sinus rhythm myocytes. ET-1 induced a delayed repolarization in 20% of AF myocytes. CONCLUSION: These results demonstrate for the first time that TRPC1 activation by ET-1 is mediated by protein kinase C through the distinct phospholipids pathways phosphoinositide-3-kinase and phospholipase C and that the TRPC1 channel and ETA receptor are upregulated in AF atria, which are likely involved in atrial electrical remodeling in patients with AF.
Subject(s)
Atrial Fibrillation/genetics , Endothelin-1/pharmacology , Protein Kinase C/metabolism , Signal Transduction/genetics , TRPC Cation Channels/genetics , Atrial Fibrillation/physiopathology , Blotting, Western , Cells, Cultured , Heart Atria/metabolism , Humans , Myocytes, Cardiac/metabolism , Real-Time Polymerase Chain Reaction/methods , Reference Values , Sensitivity and Specificity , Up-Regulation/geneticsABSTRACT
The inhibition of transient outward potassium current (Ito) is the major ionic mechanism for quinidine to treat Brugada syndrome; however, quinidine is inaccessible in many countries. The present study compared the inhibitory effect of the nonselective ß-adrenergic blocker carvedilol with quinidine on human Kv4.3 (hKv4.3, encoding for Ito) channel and action potential notch using a whole-cell patch technique in HEK 293 cell line expressing KCND3 as well as in ventricular epicardial myocytes of rabbit hearts. It was found that carvedilol and quinidine inhibited hKv4.3 current in a concentration-dependent manner. The IC50 of carvedilol was 1.2⯵M for inhibiting hKv4.3 charge area, while the IC50 of quinidine was 2.9⯵M (0.2â¯Hz). Both carvedilol and quinidine showed typical open channel blocking properties (i.e. decreasing the time to peak of activation and increasing the inactivation of hKv4.3), negatively shifted the V1/2 of activation and inactivation, and slowed the recovery from inactivation of the channel. Although carvedilol had weaker in use- and rate-dependent inhibition of hKv4.3 peak current than quinidine, its reduction of the charge area was more than quinidine at all frequencies (0.2-3.3â¯Hz). Moreover, the inhibitory effect of carvedilol on action potential notch was greater than quinidine. These results provide the novel information that carvedilol, like quinidine, significantly inhibits hKv4.3 and action potential notch, suggesting that carvedilol is likely an alternative drug for preventing malignant ventricular arrhythmias in patients with Brugada syndrome in countries where quinidine is unavailable.
Subject(s)
Carvedilol/pharmacology , Potassium Channel Blockers/pharmacology , Quinidine/pharmacology , Shal Potassium Channels/antagonists & inhibitors , Shal Potassium Channels/genetics , Action Potentials/drug effects , Animals , Gene Expression , HEK293 Cells , Heart Ventricles/cytology , Humans , Inhibitory Concentration 50 , Ion Channel Gating/drug effects , Kinetics , Male , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Protein Stability/drug effects , Rabbits , Shal Potassium Channels/metabolismABSTRACT
As the first examination of distribution, guanine-cytosine (GC) pattern, and variation analysis of microsatellites (SSRs) in different genomic regions of six bovid species, SSRs displayed nonrandomly distribution in different regions. SSR abundances are much higher in the introns, transposable elements (TEs), and intergenic regions compared to the 3'-untranslated regions (3'UTRs), 5'UTRs and coding regions. Trinucleotide perfect SSRs (P-SSRs) were the most frequent in the coding regions, whereas, mononucleotide P-SSRs were the most in the introns, 3'UTRs, TEs, and intergenic regions. Trifold P-SSRs had more GC-contents in the 5'UTRs and coding regions than that in the introns, 3'UTRs, TEs, and intergenic regions, whereas mononucleotide P-SSRs had the least GC-contents in all genomic regions. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs showed significantly different distributions in different regions (P < 0.01). Except for the coding regions, mononucleotide P-SSRs had the most RCNs, followed by the pattern: di- > tri- > tetra- > penta- > hexanucleotide P-SSRs in the same regions. The analysis of coefficient of variability (CV) of SSRs showed that the CV variations of RCN of the same mono- to hexanucleotide SSRs were relative higher in the intronic and intergenic regions, followed by the CV variation of RCN in the TEs, and the relative lower was in the 5'UTRs, 3'UTRs, and coding regions. Wide SSR analysis of different genomic regions has helped to reveal biological significances of their distributions.
Subject(s)
3' Untranslated Regions , 5' Untranslated Regions , Genome , Introns , Microsatellite Repeats , Ruminants/genetics , AnimalsABSTRACT
Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ ( Cai2+ ) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store-operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Cai2+ via ER-IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit+ progenitor cells.
Subject(s)
Bradykinin/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Proto-Oncogene Proteins c-kit/genetics , Stem Cells/drug effects , Cations, Divalent , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , Female , Gene Expression Regulation , Humans , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Ion Transport/drug effects , Male , Middle Aged , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Myocardium/cytology , Myocardium/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , ORAI1 Protein/antagonists & inhibitors , ORAI1 Protein/genetics , ORAI1 Protein/metabolism , Primary Cell Culture , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/metabolism , Quinolizines/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Stromal Interaction Molecule 1/antagonists & inhibitors , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolism , TRPC Cation Channels/antagonists & inhibitors , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolismABSTRACT
BACKGROUND AND PURPOSE: Adrenergic regulation of cell volume-regulated chloride current (ICl.vol ) is species-dependent. The present study investigates the mechanism underlying adrenergic regulation of ICl.vol in human atrial myocytes. EXPERIMENTAL APPROACH: Conventional whole-cell patch voltage-clamp techniques were used to record membrane current in human atrial myocytes. ICl.vol was evoked by hyposmotic bath solution (0.6 times isosmotic, 0.6 T). KEY RESULTS: ICl.vol was augmented by noradrenaline (1 µM) during cell swelling in 0.6 T but not under isosmotic (1 T) conditions. Up-regulation of ICl.vol in 0.6 T was blocked by the ß-adrenoceptor antagonist propranolol (2 µM), but not by the α1 -adrenoceptor antagonist prazosin (2 µM). This ß-adrenergic response involved cAMP but was independent of PKA; the protein kinase inhibitor H-89 (2 µM) or PKI (10 µM in pipette solution) failed to prevent ICl.vol up-regulation by noradrenaline. Moreover, the PI3K/PKB inhibitor LY294002 (50 µM) and the PKG inhibitor KT5823 (10 µM) did not affect noradrenaline-induced increases in ICl.vol . Interestingly, the exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2'-O-Me-cAMP (50 µM) also up-regulated ICl.vol , and the noradrenaline-induced increase of ICl.vol in 0.6 T was reversed or prevented by the Epac inhibitor ESI-09 (10 µM). CONCLUSION AND IMPLICATIONS: These data show that ICl.vol evoked by cell swelling of human atrial myocytes is up-regulated by noradrenaline via a PKA-independent cAMP/Epac pathway in human atrial myocytes. cAMP/Epac-induced ICl.vol is expected to shorten action potential duration during human atrial myocytes swelling and may be involved in abnormal cardiac electrical activity during cardiac pathologies that evoke ß-adrenoceptor signalling.
Subject(s)
Cyclic AMP/physiology , Guanine Nucleotide Exchange Factors/physiology , Myocytes, Cardiac/drug effects , Norepinephrine/pharmacology , Cells, Cultured , Chlorides/metabolism , Cyclic AMP-Dependent Protein Kinases , Heart Atria/cytology , Humans , Myocytes, Cardiac/physiology , Up-Regulation/drug effectsABSTRACT
The natural flavone acacetin inhibits several voltage-gated potassium currents in atrial myocytes, and has anti-atrial fibrillation (AF) effect in experimental AF models. The present study investigates whether acacetin inhibits the Ca2+-activated potassium (KCa) currents, including small conductance (SKCa1, SKCa2, and SKCa3), intermediate conductance (IKCa), and large-conductance (BKCa) channels stably expressed in HEK 293 cells. The effects of acacetin on these KCa channels were determined with a whole-cell patch voltage-clamp technique. The results showed that acacetin inhibited the three subtype SKCa channel currents in concentration-dependent manner with IC50 of 12.4 µM for SKCa1, 10.8 µM for SKCa2, and 11.6 µM for SKCa3. Site-directed mutagenesis of SKCa3 channels generated the mutants H490N, S512T, H521N, and A537V. Acacetin inhibited the mutants with IC50 of 118.5 µM for H490N, 275.2 µM for S512T, 15.3 µM for H521N, and 10.6 µM for A537V, suggesting that acacetin interacts with the P-loop helix of SKCa3 channel. However, acacetin at 3-10 µM did not decrease, but induced a slight increase of BKCa (+70 mV) by 8% at 30 µM. These results demonstrate the novel information that acacetin remarkably inhibits SKCa channels, but not IKCa or BKCa channels, which suggests that blockade of SKCa by acacetin likely contributes to its anti-AF property previously observed in experimental AF.
ABSTRACT
The present study investigated the effects of equol on cardiovascular K+ channel currents. The cardiovascular K+ channel currents were determined in HEK 293 cells stably expressing cloned differential cardiovascular K+ channels with conventional whole-cell patch voltage-clamp technique. We found that equol inhibited hKv1.5 (IC50: 15.3 µM), hKv4.3 (IC50: 29.2 µM and 11.9 µM for hKv4.3 peak current and charge area, respectively), IKs (IC50: 24.7 µM) and IhERG (IC50: 31.6 and 56.5 µM for IhERG.tail and IhERG.step, respectively), but not hKir2.1 current, in a concentration-dependent manner. Interestingly, equol increased BKCa current with an EC50 of 0.1 µM. It had no significant effect on guinea pig ventricular action potentials at concentrations of ≤3 µM. These results demonstrate that equol inhibits several cardiac K+ currents at relatively high concentrations, whereas it increases BKCa current at very low concentrations, suggesting that equol is a safe drug candidate for treating patients with cerebral vascular disorders.
Subject(s)
Equol/pharmacology , Potassium Channels/drug effects , Action Potentials/drug effects , Animals , Guinea Pigs , HEK293 Cells , Heart Ventricles/drug effects , Humans , Potassium Channels/physiologyABSTRACT
The present study was designed to investigate whether large conductance Ca2+ -activated K+ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK-HEK 293 cells expressing both functional α-subunits and the auxiliary ß1-subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK-HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α- and ß1-subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre-contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Tyrphostins/pharmacology , Animals , Basilar Artery/cytology , Cell Separation , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Genistein/pharmacology , HEK293 Cells , Humans , Indoles/pharmacology , Ion Channel Gating/drug effects , Male , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Protein Subunits/metabolism , Rats, Sprague-Dawley , Vanadates/pharmacology , Vasodilation/drug effectsABSTRACT
BACKGROUND AND PURPOSE: The ultra-rapidly activating delayed rectifier K+ current IKur (encoded by Kv 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACH: Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial IKur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTS: Human atrial IKur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of IKur induced by 30 µM genistein or 10 µM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hKv 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 µM) slightly enhanced IKur and hKv 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hKv 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONS: The PTK inhibitors genistein and AG556 decrease human atrial IKur and cloned hKv 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases IKur and hKv 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial IKur .
Subject(s)
Delayed Rectifier Potassium Channels/antagonists & inhibitors , ErbB Receptors/antagonists & inhibitors , Genistein/pharmacology , Heart Atria/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Tyrphostins/pharmacology , Cells, Cultured , Delayed Rectifier Potassium Channels/metabolism , ErbB Receptors/metabolism , Genistein/chemistry , HEK293 Cells , Heart Atria/metabolism , Humans , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Tyrphostins/chemistryABSTRACT
BACKGROUND AND PURPOSE: Clemizole, a histamine H1 receptor antagonist has a potential therapeutic effect on hepatitis C infection and also potently inhibits TRPC5 ion channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and thus affects cardiac repolarization. EXPERIMENTAL APPROACH: Whole-cell patch techniques was used to examine the effects of clemizole on hERG channel current, IKs and Kv 1.5 channel current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. Isolated hearts from guinea pigs were used to determine the effect on the ECG. KEY RESULTS: Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50 : 0.07 µM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50 : 0.82, 0.89, 1.49 and 2.98 µM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and human Kv 1.5 channel current. Moreover, clemizole increased the duration of the ventricular action potential in guinea pig hearts and the QTc interval in isolated perfused hearts from guinea pigs, in a concentration-dependent manner (0.1-1.0 µM). CONCLUSION AND IMPLICATIONS: Our results provide the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs , delays cardiac repolarization and thereby prolongs QT interval. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.
Subject(s)
Benzimidazoles/pharmacology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Histamine H1 Antagonists/pharmacology , Potassium Channel Blockers/pharmacology , Action Potentials/drug effects , Animals , Benzimidazoles/administration & dosage , Dose-Response Relationship, Drug , Electrocardiography , Female , Guinea Pigs , HEK293 Cells , Heart Ventricles/metabolism , Histamine H1 Antagonists/administration & dosage , Humans , Inhibitory Concentration 50 , Long QT Syndrome/chemically induced , Male , Patch-Clamp Techniques , Potassium Channel Blockers/administration & dosageABSTRACT
The morbidity and mortality of patients with ischemic cardiomyopathy resulted from ischemia/reperfusion injury are very high. The present study investigates whether our previously synthesized water-soluble phosphate prodrug of acacetin was cardioprotective against ischemia/reperfusion injury in an in vivo rat model. We found that intravenous administration of acacetin prodrug (10 mg/kg) decreased the ventricular arrhythmia score and duration, reduced ventricular fibrillation and infarct size, and improved the impaired heart function induced by myocardial ischemia/reperfusion injury in anesthetized rats. The cardioprotective effects were further confirmed with the parent compound acacetin in an ex vivo rat regional ischemia/reperfusion heart model. Molecular mechanism analysis revealed that acacetin prevented the ischemia/reperfusion-induced reduction of the anti-oxidative proteins SOD-2 and thioredoxin, suppressed the release of inflammation cytokines TLR4, IL-6 and TNFα, and decreased myocyte apoptosis induced by ischemia/reperfusion. Our results demonstrate the novel evidence that acacetin prodrug confer significant in vivo cardioprotective effect against ischemia/reperfusion injury by preventing the reduction of endogenous anti-oxidants and the release of inflammatory cytokines, thereby inhibiting cardiomyocytes apoptosis, which suggests that the water-soluble acacetin prodrug is likely useful in the future as a new drug candidate for treating patients with acute coronary syndrome.
Subject(s)
Flavones/therapeutic use , Myocardial Reperfusion Injury/prevention & control , Prodrugs/therapeutic use , Protective Agents/therapeutic use , Animals , Apoptosis/drug effects , Disease Models, Animal , Flavones/chemistry , Flavones/metabolism , Flavones/pharmacology , Heart/drug effects , Heart Rate/drug effects , Interleukin-6/metabolism , Male , Models, Biological , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocardium/cytology , Myocardium/metabolism , Myocardium/pathology , Oxidative Stress/drug effects , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacology , Protective Agents/chemistry , Protective Agents/metabolism , Protective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolism , Thioredoxins/metabolism , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolism , Ventricular Pressure/drug effectsABSTRACT
We previously reported that duodenal administration of the natural flavone acacetin can effectively prevent the induction of experimental atrial fibrillation (AF) in canines; however, it may not be used intravenously to terminate AF due to its poor water-solubility. The present study was to design a water-soluble prodrug of acacetin and investigate its anti-AF effect in beagle dogs. Acacetin prodrug was synthesized by a three-step procedure. Aqueous solubility, bioconversion and anti-AF efficacy of acacetin prodrug were determined with different methodologies. Our results demonstrated that the synthesized phosphate sodium salt of acacetin prodrug had a remarkable increase of aqueous solubility in H2O and clinically acceptable solution (5% glucose or 0.9% NaCl). The acacetin prodrug was effectively converted into acacetin in ex vivo rat plasma and liver microsome, and in vivo beagle dogs. Intravenous infusion of acacetin prodrug (3, 6 and 12 mg/kg) terminated experimental AF without increasing ECG QTc interval in beagle dogs. The intravenous LD50 of acacetin prodrug was 721 mg/kg in mice. Our preclinical study indicates that the synthesized acacetin prodrug is highly water-soluble and safe; it effectively terminates experimental AF in beagle dogs and therefore may be a promising drug candidate for clinical trial to treat patients with acute AF.
Subject(s)
Atrial Fibrillation/drug therapy , Flavones/chemical synthesis , Flavones/therapeutic use , Prodrugs/chemical synthesis , Prodrugs/therapeutic use , Water/chemistry , Animals , Atrial Fibrillation/blood , Dogs , Flavones/blood , Flavones/pharmacokinetics , Humans , Mice, Inbred ICR , Potassium Channels/metabolism , Prodrugs/pharmacokinetics , Rats , Solubility , Toxicity Tests, Acute , Vagus Nerve/drug effectsABSTRACT
The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells.
Subject(s)
Myocardium/cytology , Proto-Oncogene Proteins c-kit/metabolism , Stem Cells/metabolism , TRPV Cation Channels/metabolism , Calcium/metabolism , Calcium Signaling , Cell Movement , Cell Proliferation , Gene Expression Regulation , Gene Silencing , Humans , Intracellular Space/metabolism , RNA, Small Interfering/metabolism , Stem Cells/cytology , TRPV Cation Channels/geneticsABSTRACT
SKF-96365 is a TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in different systems, which was recently reported to induce QTc prolongation on ECG by inhibiting TRPC channels. The present study investigates whether the blockade of cardiac repolarization currents would be involved in the increase of QTc interval. Cardiac repolarization currents were recorded in HEK 293 cells stably expressing human ether-à-go-go-related gene potassium (hERG or hKv11.1) channels, hKCNQ1/hKCNE1 channels (IKs) or hKir2.1 channels and cardiac action potentials were recorded in guinea pig ventricular myocytes using a whole-cell patch technique. The potential effect of SKF-96365 on QT interval was evaluated in ex vivo guinea pig hearts. It was found that SKF-96365 inhibited hERG current in a concentration-dependent manner (IC50, 3.4µM). The hERG mutants S631A in the pore helix and F656V of the S6 region reduced the inhibitory sensitivity with IC50s of 27.4µM and 11.0µM, suggesting a channel pore blocker. In addition, this compound inhibited IKs and hKir2.1currents with IC50s of 10.8 and 8.7µM. SKF-96365 (10µM) significantly prolonged ventricular APD90 in guinea pig ventricular myocytes and QTc interval in ex vivo guinea pig hearts. These results indicate that the TRPC channel antagonist SKF-96365 exerts blocking effects on hERG, IKs, and hKir2.1 channels. Prolongation of ventricular APD and QT interval is related to the inhibition of multiple repolarization potassium currents, especially hERG channels.
Subject(s)
Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Imidazoles/pharmacology , Potassium Channel Blockers/pharmacology , Animals , Electrocardiography/drug effects , Guinea Pigs , HEK293 Cells , Heart/physiology , Heart Rate/drug effects , Humans , Male , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiologyABSTRACT
BACKGROUND: Several mammalian species display distinct biophysical properties between atrial and ventricular voltage-gated sodium current (INa); however, the potential mechanism behind this phenomenon is unknown. OBJECTIVE: The purpose of this study was to investigate the potential molecular identities of the different INa in atrial and ventricular myocytes of rat hearts. METHODS: Whole-cell patch voltage-clamp and molecular biology techniques were used in the study. RESULTS: Ventricular INa exhibited a slower inactivation, more positive potential of inactivation, and quicker recovery from inactivation compared to atrial INa. Real-time polymerase chain reaction and western blot analysis revealed that mRNA and protein levels of NaVß2 and NaVß4 subunits, but not NaV1.5, were greater in ventricular myocytes than in atrial myocytes. INa in heterologous HEK 293 cell expression system with coexpressing hNaV1.5 and hNaVß2/hNaVß4 showed similar biophysical properties to ventricular INa. Greater protein expression of NaVß2 and NaVß4 subunits was also observed in human ventricles. Interestingly, pharmacologic study revealed that the antiarrhythmic drug dronedarone (10 µM) inhibited atrial INa more (by 73%) than ventricular INa (by 42%), and shifted its inactivation to more negative voltages (-4.6 mV) compared to ventricular INa. CONCLUSION: The results of this study demonstrate the novel information that the distinctive biophysical properties of INa in atrial and ventricular myocytes can be attributed to inhomogeneous expression of NaVß2 and NaVß4 subunits, and that atrial INa is more sensitive to inhibition by dronedarone.
Subject(s)
Amiodarone/analogs & derivatives , Heart Atria/metabolism , Heart Ventricles/metabolism , Myocytes, Cardiac/metabolism , Voltage-Gated Sodium Channels/metabolism , Amiodarone/pharmacology , Animals , Cells, Cultured , Disease Models, Animal , Dronedarone , Heart Atria/drug effects , Heart Atria/pathology , Heart Ventricles/drug effects , Heart Ventricles/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Patch-Clamp Techniques , Rats , Sodium Channel Blockers/pharmacology , Voltage-Gated Sodium Channels/drug effectsABSTRACT
Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.