Qing-Hua Liu

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca2+ channels (VDCCs) inducing Ca2+ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca2+ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca2+ release, leading to a significant increase in the activity of Ca2+ sparks and contraction in airway SMCs. The increased Ca2+ sparks are independent of VDCCs and the associated extracellular Ca2+ influx. This format of local Ca2+ release results from a direct activation of G protein-coupled, M3 muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP3), inducing initial Ca2+ release through IP3 receptors and then further Ca2+ release via RyR2 due to a local Ca2+-induced Ca2+ release process. These findings demonstrate an important mechanism for Ca2+ signaling and attendant physiological function in SMCs.

Protein Kinase C-ε Regulates Local Calcium Signaling in Airway Smooth Muscle Cells

Protein kinase C (PKC) is known to regulate ryanodine receptor (RyR)-mediated local Ca(2+) signaling (Ca(2+) spark) in airway and vascular smooth muscle cells (SMCs), but its specific molecular mechanisms and functions still remain elusive. In this study, we reveal that, in airway SMCs, specific PKCepsilon peptide inhibitor and gene deletion significantly increased the frequency of Ca(2+) sparks, and decreased the amplitude of Ca(2+) sparks in the presence of xestospogin-C to eliminate functional inositol 1,4,5-triphosphate receptors. PKCepsilon activation with phorbol-12-myristate-13-acetate significantly decreased Ca(2+) spark frequency and increased Ca(2+) spark amplitude. The effect of PKCepsilon inhibition or activation on Ca(2+) sparks was completely lost in PKCepsilon(-/-) cells. PKCepsilon inhibition or PKCepsilon activation was unable to affect Ca(2+) sparks in RyR1(-/-) and RyR1(+/-) cells. Modification of RyR2 activity by FK506-binding protein 12.6 homozygous or RyR2 heterozygous gene deletion did not prevent the effect of PKCepsilon inhibition or activation. RyR3 homogenous gene deletion did not block the effect of PKCepsilon inhibition and activation, either. PKCepsilon inhibition promotes agonist-induced airway muscle contraction, whereas PKCepsilon activation produces an opposite effect. Taken together, these results indicate that PKCepsilon regulates Ca(2+) sparks by specifically interacting with RyR1, which plays an important role in the control of contractile responses in airway SMCs.

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca2+ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca2+ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca2+-free conditions, and, following a restoration of Ca2+, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca2+ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K

It has been reported that bitter tastants decrease blood pressure and relax precontracted vascular smooth muscle. However, the underlying mechanisms remain unclear. The aim of the present study was to determine the mechanism underlying the vasorelaxant effect of the bitter tastants. Thoracic aortic rings were isolated from Wistar rats and contractions were measured using an isometric myograph. Intracellular Ca2+ ([Ca2+]i) in single rat thoracic aortic smooth muscle cells was recorded by calcium imaging. Calcium currents in single cells were recorded using patch‐clamp techniques. High K+ (140 mmol/L) induced contractions in rat thoracic aortic rings that were inhibited by 3 mmol/L chloroquine, 3 mmol/L denatonium and 10 μmol/L nifedipine. In single rat thoracic aortic smooth muscle cells, high K+ increased [Ca2+]i and this effect was also blocked by 3 mmol/L chloroquine and 10 μmol/L nifedipine. Under Ca2+‐free conditions, high K+ failed to induce contractions in rat thoracic aortic rings. On its own, chloroquine had no effect on the muscle tension of rat aortic rings and [Ca2+]i. The vasorelaxant effects of chloroquine on precontracted rat thoracic aortic rings were not altered by either 1 μg/mL pertussis toxin (PTX), an inhibitor of Gαo/i ‐protein, or 1 mmol/L gallein, an inhibitor of Gβγ‐protein. The results of patch‐clamp analysis in single cells indicate that 1 mmol/L chloroquine blocks voltage‐dependent L‐type Ca2+ channel (VDLCC) currents from both extracellular and intracellular sides. Together, the results indicate that chloroquine can block VDLCC, independent of PTX‐ and gallein‐sensitive G‐proteins, resulting in relaxation of high K+‐precontracted thoracic aortic smooth muscle.

Reactive oxygen species induce a Ca2+-spark increase in sensitized murine airway smooth muscle cells

The level of reactive oxygen species (ROS) and the activity of spontaneous, transient, localized Ca(2+) increases (known as Ca(2+) sparks) in tracheal smooth muscle cells (TSMCs) in an experimental allergic asthma mouse model has not yet been investigated. We used laser confocal microscopy and fluorescent dyes to measure ROS levels and Ca(2+) sparks, and we found that both events were significantly increased in TSMCs obtained from ovalbumin (OVA)-sensitized/-challenged mice compared with control mice. ROS levels began to increase in TSMCs after the first OVA challenge, and this increase was sustained. However, this elevation and Ca(2+)-spark increase was abolished after the administration of the ROS scavenger N-acetylcysteine amide (NACA) for 5days. Furthermore, a similar inhibition was also observed following the direct perfusion of NACA into cells isolated from the (OVA)-sensitized mice that were not treated with NACA. Moreover, we used 0.1-mM caffeine treatment to increase the Ca(2+) sparks in single TSMCs and observed cell shortening. In addition, we did not find increases in the mRNA levels of ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors in the tracheal smooth muscle cells of (OVA)-sensitized mice compared with controls. We concluded that ROS and Ca(2+) sparks increased in (OVA)-sensitized TSMCs. We found that ROS induces Ca(2+) sparks, and increased Ca(2+) sparks resulted in the contraction of (OVA)-sensitized TSMCs, resulting in the generation of airway hyperresponsiveness (AHR). This effect may represent a novel mechanism for AHR pathogenesis and might provide insight into new methods for the clinical prevention and treatment of asthma and asthmatic AHR.

Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM) was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM), GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor) and U73122 (PLC inhibitor). However, 2-APB (IP3R blocker) only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

Chloroquine Inhibits Ca 2+ Signaling in Murine CD4 + Thymocytes

Background/Aims: Bitter-tasting chloroquine can suppress T cell activation by inhibiting Ca²⁺ signaling. However, the mechanism of inhibition remains largely unclear. Methods: In this study, CD4⁺ T cells were isolated from the thymus, and the calcium content of CD4⁺ thymocytes was measured using fura-2 AM and a TILL imaging system. Pyrazole-3 (Pyr3), thapsigargin (TG), and caffeine were used to assess the effects of chloroquine on the intracellular Ca²⁺ content of CD4⁺ T cells. Results: In murine CD4⁺ thymocytes, chloroquine decreased the TG-triggered intracellular Ca²⁺ increase in a dose-dependent manner. In the absence of chloroquine under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA), TG induced a transient Ca²⁺ increase. After restoration of the extracellular Ca²⁺ concentration to 2 mM, a dramatic Ca²⁺ increase occurred. This elevation was completely blocked by chloroquine and was markedly inhibited by Pyr3, a selective antagonist of transient receptor potential C3 (TRPC3) channel and stromal interaction molecule (STIM)/Orai channel. Furthermore, the TG-induced transient Ca²⁺ increase under Ca²⁺-free conditions was eliminated in the presence of chloroquine. Chloroquine also blocked the dialyzed inositol-1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ increase. However, chloroquine was not able to decrease the caffeine-induced Ca²⁺ increase. Conclusion: These data indicate that chloroquine inhibits the elevation of intracellular Ca²⁺ in thymic CD4⁺ T cells by inhibiting IP₃ receptor-mediated Ca²⁺ release from intracellular stores and TRPC3 channel-mediated and/or STIM/Orai channel-mediated Ca²⁺ influx.

Ventricular hypertrophy amplifies transmural dispersion of repolarization by preferentially increasing the late sodium current in endocardium

BACKGROUNDnThe late sodium current (INa-L) contributes importantly to rate-dependent change in action potential duration (APD) and transmural dispersion of repolarization (TDR). However, little is known about the mechanisms of increased APD rate-dependence and amplified TDR in left ventricular hypertrophy (LVH) and failure. The purpose of this study was to investigate the role of INa-L in rate-adaptation of transmural APD heterogeneity.nnnMETHODSnAPD, its rate-dependence and INa-L current were examined in myocytes isolated from the endocardium and epicardium of the control and LVH rabbits. AP was recorded using the standard microelectrode technique, and INa-L was recorded using the whole-cell patch clamp technique.nnnRESULTSnEarly afterdepolarizations (EADs) were frequently recorded in the isolated myocytes of the LVH rabbits but not in those of controls. LVH prolonged APD more significantly in the endocardial myocytes than in the epicardium (31.7±3.4 vs. 21.6±1.5% n=6, p<0.05), leading to a marked increase in TDR. LVH endocardial myocytes exhibited a greater rate-dependent change in APD compared to the epicardial myocytes. INa-L densities were significantly increased in both LVH endocardium and epicardium. However, LVH increased the INa-L density preferentially in the endocardial myocytes compared to the epicardial myocytes (54.5±4.8% vs. 39.2±3.3%, n=6, p<0.05).nnnCONCLUSIONSnOur results demonstrate that LVH increased the INa-L preferentially in the endocardium over the epicardium, which contributes importantly to the stronger rate-dependent change in repolarization and longer APD in the endocardium. This results in an amplified TDR capable of initiating EAD and ventricular arrhythmias.

Methods to measure and analyze ciliary beat activity: Ca2+ influx-mediated cilia mechanosensitivity.

Airway ciliary beat activity (CBA) plays a pivotal role in protecting the body by removing mucus and pathogens from the respiratory tract. Since CBA is complicated and cannot be characterized by merely frequency, we recorded CBA using laser confocal line scanning and defined six parameters for describing CBA. The values of these parameters were all above 0 when measured in beating ciliated cells from mouse tracheae. We subsequently used 10xa0μM adenosine-5′-triphosphate (ATP) to stimulate ciliated cells and simultaneously recorded intracellular Ca2+ levels and CBA. We found that intracellular Ca2+ levels first increased, followed by an increase in CBA. Among the six parameters, frequency, amplitude, and integrated area significantly increased, whereas rise time, decay time, and full duration at half maximum markedly decreased. The results suggest that these six parameters are appropriate for assessing CBA and that increased intracellular Ca2+ levels might enhance CBA. We next used our established methods to observe changes in mechanically stimulated cilia tips. We found that mechanical stimulation-induced changes in both intracellular Ca2+ levels and CBA were not only similar to those induced by ATP, but were also blocked by treatment with a Ca2+ chelator, BAPTA-AM, (10 μM) for 10xa0min. Moreover, while the same blockage was observed under Ca2+-free conditions, addition of 2xa0mM Ca2+ into the chamber restored increases in both intracellular Ca2+ levels and CBA. Taken together, we have provided a novel method for real-time measurement and complete analysis of CBA as well as demonstrated that mechanical stimulation of cilia tips resulted in Ca2+ influx that led to increased intracellular Ca2+ levels, which in turn triggered CBA enhancement.

Relaxant Action of Plumula Nelumbinis Extract on Mouse Airway Smooth Muscle

The traditional herb Plumula Nelumbinis is widely used in the world because it has many biological activities, such as anti-inflammation, antioxidant, antihypertension, and butyrylcholinesterase inhibition. However, the action of Plumula Nelumbinis on airway smooth muscle (ASM) relaxation has not been investigated. A chloroform extract of Plumula Nelumbinis (CEPN) was prepared, which completely inhibited precontraction induced by high K+ in a concentration-dependent manner in mouse tracheal rings, but it had no effect on resting tension. CEPN also blocked voltage-dependent L-type Ca2+ channel- (VDCC-) mediated currents. In addition, ACh-induced precontraction was also completely blocked by CEPN and partially inhibited by nifedipine or pyrazole 3. Besides, CEPN partially reduced ACh-activated nonselective cation channel (NSCC) currents. Taken together, our data demonstrate that CEPN blocked VDCC and NSCC to inhibit Ca2+ influx, resulting in relaxation of precontracted ASM. This finding indicates that CEPN would be a candidate of new potent bronchodilators.