Citrate Improves Biomimetic Mineralization Induced by Polyelectrolyte-Cation Complexes Using PAsp-Ca&Mg Complexes.

Magnesium ions are highly enriched in early stage of biological mineralization of hard tissues. Paradoxically, hydroxyapatite (HAp) crystallization is inhibited significantly by high concentration of magnesium ions. The mechanism to regulate magnesium-doped biomimetic mineralization of collagen fibrils has never been fully elucidated. Herein, it is revealed that citrate can bioinspire the magnesium-stabilized mineral precursors to generate magnesium-doped biomimetic mineralization as follows: Citrate can enhance the electronegativity of collagen fibrils by its absorption to fibrils via hydrogen bonds. Afterward, electronegative collagen fibrils can attract highly concentrated electropositive polyaspartic acid-Ca&Mg (PAsp-Ca&Mg) complexes followed by phosphate solution via strong electrostatic attraction. Meanwhile, citrate adsorbed in/on fibrils can eliminate mineralization inhibitory effects of magnesium ions by breaking hydration layer surrounding magnesium ions and thus reduce dehydration energy barrier for rapid fulfillment of biomimetic mineralization. The remineralized demineralized dentin with magnesium-doped HAp possesses antibacterial ability, and the mineralization mediums possess excellent biocompatibility via cytotoxicity and oral mucosa irritation tests. This strategy shall shed light on cationic ions-doped biomimetic mineralization with antibacterial ability via modifying collagen fibrils and eliminating mineralization inhibitory effects of some cationic ions, as well as can excite attention to the neglected multiple regulations of small biomolecules, such as citrate, during biomineralization process.

Density Alters Impacts of Genotypic Evenness on Productivity in an Experimental Plant Population.

Genetic diversity plays important roles in maintaining population productivity. While the impact of genotypic richness on productivity has been extensively tested, the role of genotypic evenness has not been considered. Plant density can also affect population productivity, but its interaction with genotypic diversity has not been tested. We constructed experimental populations of the clonal plant Hydrocotyle vulgaris with either low or high richness (consisting of four vs. eight genotypes), either low or high evenness (each genotype had a different number vs. the same number of ramets), and either low or high density (consisting of 16 vs. 32 ramets) in a full factorial design. Total biomass of plant populations did not differ between four- and eight-genotype mixtures. When the initial plant density was low, total biomass of populations with high genotypic evenness was significantly greater than total biomass of those with low genotypic evenness. However, this difference disappeared when the initial plant density was high. Moreover, total biomass increased linearly with increasing plant density at harvest, but was negatively correlated to variation in leaf area. We conclude that genotypic evenness but not genotypic richness can benefit population productivity, and that plant density can alter the impact of genotypic evenness on population productivity.

Co-selective Pressure of Cadmium and Doxycycline on the Antibiotic and Heavy Metal Resistance Genes in Ditch Wetlands.

Abuse of heavy metals and antibiotics results in the dissemination of metal resistance genes (MRGs) and antibiotic resistance genes (ARGs). Ditch wetlands are important sinks for heavy metals and antibiotics. The relationships between bacterial communities and MRG/ARG dissemination under dual stresses of heavy metals and antibiotics remain unclear. The responses of MRGs and ARGs to the co-selective pressure of cadmium (Cd) and doxycycline (DC) in ditch wetlands were investigated after 7-day and 84-day exposures. In ecological ditches, residual rates of Cd and DC varied from 0.4 to -5.73% and 0 to -0.61%, respectively. The greatest total relative abundance of ARGs was observed in the Cd 5 mg L-1 + DC 50 mg L-1 group. A significant level of DC (50 mg L-1) significantly reduced the total relative abundances of MRGs at a concentration of 5 mg L-1 Cd stress. Redundancy analysis indicated that Cd and DC had strong positive effects on most ARGs and MRGs after a 7-day exposure. Meanwhile, the class 1 integron gene (intI1) exhibited strong positive correlations with most ARGs and cadmium resistance genes (czcA) after an 84-day exposure. Network analysis showed that Acinetobacter and Pseudomonas were the potential dominant host genera for ARGs and MRGs, and tetracycline resistance genes (tetA), czcA, and intI1 shared the same potential host bacteria Trichococcus after an 84-day exposure.

[Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting].

S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F0 mice were obtained and identified by PCR identification and gene sequencing. F0 mice were further mated with wild-type C57BL/6 mice to obtain F1 heterozygous mice, and then homozygous offspring were obtained through F1 mice self-crossing. Real-time PCR, Western blot and immunohistochemistry (IHC) were used to verify the expression and distribution of S100A9. In order to observe the pathological changes of mouse lung tissue using HE staining, an allergic asthma model was induced by ovalbumin from chicken egg white (OVA). The results showed that the 2 492 bp of exons 2, 3 of the S100A9 gene was successfully knocked out, and S100A9-/- mice with stable inheritance were obtained. Furthermore, it was found that S100A9 gene was highly expressed in the lung and spleen of wild-type mice. The expression of S100A9 mRNA and protein was not detected in the lung and spleen of S100A9-/- mice. However, compared with wild-type mice, the lungs of S100A9-/- mice showed a significantly worse inflammatory phenotype, and the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly increased in response to the treatment of OVA. These results suggest we have successfully constructed a new strain of S100A9-/- mice, and preliminarily confirmed that the lack of S100A9 function can aggravate airway inflammation in asthmatic mice, providing a new mouse model for further study of S100A9 gene function.

Functional analysis of the ocnE gene involved in nicotine-degradation pathways in Ochrobactrum intermedium SCUEC4 and its enzymatic properties.

The SCUEC4 strain of Ochrobactrum intermedium is a newly isolated bacterium that degrades nicotine can use nicotine as the sole carbon source via a series of enzymatic catalytic processes. The mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in the nicotine-degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4 and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of the OcnE protein was detected by SDS-PAGE analysis, and study of the function of this protein was spectrophotometrically carried out by monitoring the changes of 2,5-dihydroxypyridine. Moreover, the effects of temperature, pH, and metal ions on the biological activities of the OcnE protein were analyzed. The optimal conditions for the biological activities of OcnE, a protein of approximately 37.6 kDa, were determined to be 25 °C, pH 7.0, and 25 µmol/L Fe2+, and the suitable storage conditions for the OcnE protein were 0 °C and pH 7.0. In conclusion, the ocnE gene is responsible for the ability of 2,5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in O. intermedium SCUEC4.

Dihydropyridine Enhances the Antioxidant Capacities of Lactating Dairy Cows under Heat Stress Condition.

Heat stress (HS), a nonspecific response to environmental heat, can seriously affect dairy cow health. Feed additives may alleviate HS in dairy cows by improving rumen fermentation efficacy, stimulating feed consumption, enhancing vasodilation, and/or improving antioxidant capacity. The temperature-humidity index (THI) indicates that spring is a non-HS season, and summer is an HS season. HS results in the decrease in dairy cow antioxidant capacities. Our results indicated the decrease in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidation (T-AOC) levels and the increase in malondialdehyde (MDA) level during HS season. Meanwhile, antioxidant indexes (SOD, GSH-Px, and T-AOC) were positively correlated with milk yield (p < 0.01), whereas MDA exhibited a significant negative correlation with milk yield (p < 0.05). In addition, the effects of dihydropyridine (DHP) on antioxidant capacity and ruminal microbial communities in dairy cows under HS were investigated. During summer, dairy cows were randomly assigned into two groups under HS, including a standard diet (S-ND) group and standard diet with 3 g/day/cow DHP (S-D) group. DHP treatment significantly restored SOD and GSH-Px levels under HS. Denaturing gradient gel electrophoresis results indicated that the DHP altered ruminal bacterial community mainly composed Proteobacteria and Firmicutes in dairy cows under HS. Our results suggest that DHP can enhance the antioxidant abilities of dairy cows with favorable effects on ruminal microbial communities under HS, further alleviating HS on dairy cows.

Folium Sennae and emodin reverse airway smooth muscle contraction.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

C, N, and P stoichiometry and their interaction with different plant communities and soils in subtropical riparian wetlands.

Ecological stoichiometry represents the balance of nutrient elements under ecological interactions, which are crucial for biogeochemical cycles in ecosystems. Little is known about carbon (C), nitrogen (N), and phosphorus (P) ecological stoichiometry in aboveground biomass, roots, and soil, especially in the subtropical riparian wetlands. Here, eight dominate plant communities in riparian wetlands were chosen, and C, N, and P contents, and C:N:P ratios of aboveground biomass, roots, and soil were investigated. The results demonstrated that plant community had remarkable effects on the C:N:P stoichiometry in aboveground biomass, roots, and soil, which varied widely. C, N, and P concentrations in aboveground biomass were mostly higher than that in roots, while no significant difference was detected in C:N:P ratios. Moreover, there were higher soil C, N, and P contents in Cannabis indica plant communities; while lower soil N:P ratios suggested that riparian wetlands were more susceptible to N limitation, rather than P. Pearson correlation analysis and redundancy analysis (RDA) showed that there were strong associations among C, N, and P contents, and C:N:P ratios in aboveground biomass, roots, and soil, indicating that C, N, and P ecological stoichiometry of aboveground biomass were regulated by soil C, N, and P contents through the roots. In addition, the contents of C and N, and N and P exhibited a strong relationship according to linear regression. These findings suggested that the interactions among the C, N, and P stoichiometry were existed in the plant-soil system.

Paracetamol inhibits Ca2+ permeant ion channels and Ca2+ sensitization resulting in relaxation of precontracted airway smooth muscle.

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.

Integrated 3D bioprinting-based geometry-control strategy for fabricating corneal substitutes.

BACKGROUND: The shortage of donor corneas is a severe global issue, and hence the development of corneal alternatives is imperative and urgent. Although attempts to produce artificial cornea substitutes by tissue engineering have made some positive progress, many problems remain that hamper their clinical application worldwide. For example, the curvature of tissue-engineered cornea substitutes cannot be designed to fit the bulbus oculi of patients. OBJECTIVE: To overcome these limitations, in this paper, we present a novel integrated three-dimensional (3D) bioprinting-based cornea substitute fabrication strategy to realize design, customized fabrication, and evaluation of multi-layer hollow structures with complicated surfaces. METHODS: The key rationale for this method is to combine digital light processing (DLP) and extrusion bioprinting into an integrated 3D cornea bioprinting system. A designable and personalized corneal substitute was designed based on mathematical modelling and a computer tomography scan of a natural cornea. The printed corneal substitute was evaluated based on biomechanical analysis, weight, structural integrity, and fit. RESULTS: The results revealed that the fabrication of high water content and highly transparent curved films with geometric features designed according to the natural human cornea can be achieved using a rapid, simple, and low-cost manufacturing process with a high repetition rate and quality. CONCLUSIONS: This study demonstrated the feasibility of customized design, analysis, and fabrication of a corneal substitute. The programmability of this method opens up the possibility of producing substitutes for other cornea-like shell structures with different scale and geometry features, such as the glomerulus, atrium, and oophoron.

NS8593 inhibits Ca2+ permeant channels reversing mouse airway smooth muscle contraction.

AIMS: This study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism. MAIN METHODS: ASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system. KEY FINDINGS: High K+ (80 mM K+) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs). SIGNIFICANCE: Our results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator.

Correction: Sodium butyrate interrupts the maturation of oocytes and enhances the development of preimplantation embryos.

[This corrects the article DOI: 10.1371/journal.pone.0220479.].

Relaxant Action of Diclofenac Sodium on Mouse Airway Smooth Muscle.

Diclofenac sodium (DCF) is a nonsteroidal anti-inflammatory drug (NSAID) and is widely used as an analgesic and anti-inflammatory agent. Herein, we found that DCF could relax high K+ (80 mM K+)-/ACh-precontracted tracheal rings (TRs) in mice. This study aimed to elucidate the underlying mechanisms of DCF-induced relaxations. The effects of DCF on airway smooth muscle (ASM) cells were explored using multiple biophysiological techniques, such as isometric tension measurement and patch-clamping experiments. Both high K+- and ACh-evoked contraction of TRs in mice were relaxed by DCF in a dose-dependent manner. The results of isometric tension and patch-clamping experiments demonstrated that DCF-induced relaxation in ASM cells was mediated by cytosolic free Ca2+, which was decreased via inhibition of voltage-dependent L-type Ca2+ channels (VDLCCs), nonselective cation channels (NSCCs), and Na+/Ca2+ exchange. Meanwhile, DCF also enhanced large conductance Ca2+ activated K+ (BK) channels, which led to the relaxation of ASMs. Our data demonstrated that DCF relaxed ASMs by decreasing the intracellular Ca2+ concentration via inhibition of Ca2+ influx and Na+/Ca2+ exchange. Meanwhile, the enhanced BK channels also played a role in DCF-induced relaxation in ASMs. These results suggest that DCF is a potential candidate for antibronchospasmic drugs used in treating respiratory diseases such as asthma and chronic obstructive pulmonary disease.

Sodium butyrate interrupts the maturation of oocytes and enhances the development of preimplantation embryos.

Histone acetylation is one of the most important posttranslational modifications that contribute to transcriptional initiation and chromatin remodeling. In the present study, we aimed to investigate the effect of sodium butyrate (NaBu), a natural histone deacetylase inhibitor (HDACi), on the maturation of oocytes, preimplantation embryonic development, and expression of important developmental genes. The results indicated that NaBu decreased the rates of GVBD and the first polar body extrusion (PBE) in vitro in a dose-dependent manner. Meanwhile, NaBu treatment led to an abnormality in the spindle apparatus in oocytes in MI. However, the ratio of phosphor-extracellular signal-regulated kinases (p-ERK)/ERK significantly decreased in oocytes treated with 2.0 mM NaBu for 8 h. Furthermore, NaBu treatment at 2.0 mM improved the quality of embryos and the mRNA expression levels of important developmental genes such as HDAC1, Sox2, and Pou5f1. These data suggest that although a high concentration NaBu will impede the meiosis of oocytes, 2.0 mM NaBu will promote the development of embryos in vitro. Further investigation is needed to clarify the direct/indirect effects of NaBu on the regulation of important developmental genes and their subsequent impacts on full-term development in mammals.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

Semen cassiae Extract Inhibits Contraction of Airway Smooth Muscle.

ß2-adrenoceptor agonists are commonly used as bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, they induce severe side effects. Therefore, developing new bronchodilators is essential. Herbal plants were extracted and the extracts' effect on airway smooth muscle (ASM) precontraction was assessed. The ethyl alcohol extract of semen cassiae (EESC) was extracted from Semen cassia. The effects of EESC on the ACh- and 80 mM K+-induced sustained precontraction in mouse and human ASM were evaluated. Ca2+ permeant ion channel currents and intracellular Ca2+ concentration were measured. HPLC analysis was employed to determine which compound was responsible for the EESC-induced relaxation. The EESC reversibly inhibited the ACh- and 80 mM K+-induced precontraction. The sustained precontraction depends on Ca2+ influx, and it was mediated by voltage-dependent L-type Ca2+ channels (LVDCCs), store-operated channels (SOCs), TRPC3/STIM/Orai channels. These channels were inhibited by aurantio-obtusin, one component of EESC. When aurantio-obtusin removed, EESC's action disappeared. In addition, aurantio-obtusin inhibited the precontraction of mouse and human ASM and intracellular Ca2+ increases. These results indicate that Semen cassia-contained aurantio-obtusin inhibits sustained precontraction of ASM via inhibiting Ca2+-permeant ion channels, thereby, which could be used to develop new bronchodilators.

Deficiency of MTMR14 impairs male fertility in Mus musculus.

Calcium signalling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and acrosome reactions are all mediated by increases in intracellular Ca2+. Our previous reports have shown that deficiency of MTMR14, a novel phosphoinositide phosphatase, induces a muscle disorder by disrupting Ca2+ homeostasis. Recently, we found that MTMR14 is also expressed in the testes; however, whether deficiency of MTMR14 in the testes also alters the Ca2+ concentration and impairs male fertility remains entirely unknown. In the present study, we found that MTMR14 is also expressed in the testes and mature sperm cells, suggesting that deficiency of MTMR14 might have some effect on male fertility. Both in vivo fertility and in vitro fertilization tests were then performed, and we found that MTMR14-/- male mice showed decreased fertility. A series of experiments were then arranged to test the testis and sperm parameters; we found that MTMR14 deficiency caused small size of the testes, small numbers of both total and immotile sperm, expanded membrane of sperm tail, a decreased proportion of acrosome reaction, and in contrast, an increased proportion of abnormal sperm and augmented apoptosis, etc. Further study also found that the muscle force of the vas deferens decreased significantly in KO mice. Intracellular calcium homeostasis in the testes and epididymis was impaired by MTMR14 deletion; moreover, the relative mRNA expression levels of Itpr1, Itpr2, and Ryr3 were dramatically decreased in MTMR14 KO mice. Thus, MTMR14 deletion impairs male fertility by causing decreased muscle force of the vas deferens and intracellular calcium imbalance.

Generation and Role of Oscillatory Contractions in Mouse Airway Smooth Muscle.

BACKGROUND/AIMS: Tetraethylammonium chloride (TEA) induces oscillatory contractions in mouse airway smooth muscle (ASM); however, the generation and maintenance of oscillatory contractions and their role in ASM are unclear. METHODS: In this study, oscillations of ASM contraction and intracellular Ca2+ were measured using force measuring and Ca2+ imaging technique, respectively. TEA, nifedipine, niflumic acid, acetylcholine chloride, lithium chloride, KB-R7943, ouabain, 2-Aminoethoxydiphenyl borate, thapsigargin, tetrodotoxin, and ryanodine were used to assess the mechanism of oscillatory contractions. RESULTS: TEA induced depolarization, resulting in activation of L-type voltage-dependent Ca2+ channels (LVDCCs) and voltage-dependent Na+ (VNa) channels. The former mediated Ca2+ influx to trigger a contraction and the latter mediated Na+ entry to enhance the contraction via activating LVDCCs. Meanwhile, increased Ca2+-activated Cl- channels, inducing depolarization that resulted in contraction through LVDCCs. In addition, the contraction was enhanced by intracellular Ca2+ release from Ca2+ stores mediated by inositol (1,4,5)-trisphosphate receptors (IP3Rs). These pathways together produce the contractile phase of the oscillatory contractions. Furthermore, the increased Ca2+ activated the Na+-Ca2+ exchanger (NCX), which transferred Ca2+ out of and Na+ into the cells. The former induced relaxation and the latter activated Na+/K+-ATPase that induced hypopolarization to inactivate LVDCCs causing further relaxation. This can also explain the relaxant phase of the oscillatory contractions. Moreover, the depolarization induced by VNa channels and NCX might be greater than the hypopolarization caused by Na+/K+-ATPase alone, inducing LVDCC activation and resulting in further contraction. CONCLUSIONS: These data indicate that the TEA-induced oscillatory contractions were cooperatively produced by LVDCCs, VNa channels, Ca2+-activated Cl- channels, NCX, Na+/K+ ATPase, IP3Rs-mediated Ca2+ release, and extracellular Ca2+.

Polygonum aviculare L. extract and quercetin attenuate contraction in airway smooth muscle.

Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease.

Nuciferine Relaxes Tracheal Rings via the Blockade of VDLCC and NSCC Channels.

This study aimed to elucidate the mechanisms of nuciferine (a main aporphine alkaloid of lotus leaf extract), which can induce relaxation in contracted tracheal rings. Under Ca2+-free and 2 mM Ca2+ conditions, we found that nuciferine had no effect on the resting muscle tone of tracheal rings. In contrast, nuciferine relaxed high K+-contracted mouse tracheal rings in a dose-dependent manner and inhibited both Ca2+ influx and voltage-dependent L-type Ca2+ channel currents induced by high K+. Similarly, nuciferine also inhibited acetylcholine-induced contractions in mouse tracheal rings in a dose-dependent manner. Meanwhile, both acetylcholine-induced intracellular Ca2+ influx and whole-cell currents of nonselective cation channels were blocked by nuciferine. Together, the results indicate that nuciferine-induced relaxation in tracheal rings mainly occurred due to the inhibition of extracellular Ca2+ influx through the blockade of voltage-dependent L-type Ca2+ channels and/or nonselective cation channels. These results suggest that nuciferine has a therapeutic effect on respiratory diseases associated with the aberrant contraction of airway smooth muscles and/or bronchospasm.