Lu Xue

Involvement of Large-Conductance Ca2+-Activated K+ Channels in Chloroquine-Induced Force Alterations in Pre-Contracted Airway Smooth Muscle

The participation of large-conductance Ca2+ activated K+ channels (BKs) in chloroquine (chloro)-induced relaxation of precontracted airway smooth muscle (ASM) is currently undefined. In this study we found that iberiotoxin (IbTx, a selective inhibitor of BKs) and chloro both completely blocked spontaneous transient outward currents (STOCs) in single mouse tracheal smooth muscle cells, which suggests that chloro might block BKs. We further found that chloro inhibited Ca2+ sparks and caffeine-induced global Ca2+ increases. Moreover, chloro can directly block single BK currents completely from the intracellular side and partially from the extracellular side. All these data indicate that the chloro-induced inhibition of STOCs is due to the blockade of chloro on both BKs and ryanodine receptors (RyRs). We also found that low concentrations of chloro resulted in additional contractions in tracheal rings that were precontracted by acetylcholine (ACH). Increases in chloro concentration reversed the contractile actions to relaxations. In the presence of IbTx or paxilline (pax), BK blockers, chloro-induced contractions were inhibited, although the high concentrations of chloro-induced relaxations were not affected. Taken together, our results indicate that chloro blocks BKs and RyRs, resulting in abolishment of STOCs and occurrence of contraction, the latter will counteract the relaxations induced by high concentrations of chloro.

Cortex phellodendri Extract Relaxes Airway Smooth Muscle.

Cortex phellodendri is used to reduce fever and remove dampness and toxin. Berberine is an active ingredient of C. phellodendri. Berberine from Argemone ochroleuca can relax airway smooth muscle (ASM); however, whether the nonberberine component of C. phellodendri has similar relaxant action was unclear. An n-butyl alcohol extract of C. phellodendri (NBAECP, nonberberine component) was prepared, which completely inhibits high K+- and acetylcholine- (ACH-) induced precontraction of airway smooth muscle in tracheal rings and lung slices from control and asthmatic mice, respectively. The contraction induced by high K+ was also blocked by nifedipine, a selective blocker of L-type Ca2+ channels. The ACH-induced contraction was partially inhibited by nifedipine and pyrazole 3, an inhibitor of TRPC3 and STIM/Orai channels. Taken together, our data demonstrate that NBAECP can relax ASM by inhibiting L-type Ca2+ channels and TRPC3 and/or STIM/Orai channels, suggesting that NBAECP could be developed to a new drug for relieving bronchospasm.

Deficiency of myotubularin-related protein 14 influences body weight, metabolism, and inflammation in an age-dependent manner

BackgroundMyotubularin-related protein 14 (MTMR14) is a novel phosphoinositide phosphatase with roles in the maintenance of normal muscle performance, autophagy, and aging in mice. Our initial pilot study demonstrated that MTMR14 knock out (KO) mice gain weight earlier than their wild-type (WT) littermates, which suggests that this gene may also be involved in metabolism regulation.ResultsThe present study evaluated the role of MTMR14 in the development of aging-associated obesity. We found that aged MTMR14 KO mice fed a normal chow diet exhibited increased serum triglyceride, total cholesterol, and glucose levels compared to age-matched WT controls. Lipid accumulation was also increased in aged KO mice. Several inflammatory cytokines and adipokines were dramatically dysregulated in the metabolic tissues of aged MTMR14 KO mice compared to control mice. Circulating inflammatory cytokines were significantly elevated and plasma adipokine levels were abnormally regulated in aged MTMR14 KO mice. These data suggest that MTMR14 deficiency caused a late-onset inflammation and metabolic dysfunction. Further study demonstrated that this exacerbated metabolic dysfunction and inflammation may be regulated by the phosphoinositide 3 kinase/protein kinase B and extracellular signal-regulated protein kinase signaling pathways.ConclusionsOur current research suggests that MTMR14 deletion induces overweight and adult obesity accompanied by chronic inflammation in an age-dependent manner.

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.

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⁺ (V_Na) 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 V_Na 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, V_Na channels, Ca²⁺-activated Cl^- channels, NCX, Na⁺/K⁺ ATPase, IP₃Rs-mediated Ca²⁺ release, and extracellular Ca²⁺.

Distinct Effects of Ca2+ Sparks on Cerebral Artery and Airway Smooth Muscle Cell Tone in Mice and Humans

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the β1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.

Hypertonic saline inhibits airway smooth muscle contraction by inhibiting Ca2+ sensitization

The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)‐induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca2+ that was further enhanced by 5% NaCl, indicating that the HS‐induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca2+. The Rho‐associated kinase (ROCK) inhibitor Y‐27632 relaxed ACh‐induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh‐induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca2+ sensitization, not by decreasing intracellular Ca2+.

Relaxing Effect of TSU-68, an Antiangiogenic Agent, on Mouse Airway Smooth Muscle

Background/Aims: Recently, some small-molecule compounds that were designed for cancer therapy have acquired new roles in the treatment of pulmonary diseases. However, drug screening aimed at abnormal muscle contraction is still limited. TSU-68 is a potent, orally administered, small-molecule agent that can reduce the vascular endothelial growth factor (VEGF)-induced Ca2+ increase in endothelial cells. We questioned whether TSU-68 could also affect calcium influx and relax airway smooth muscle (ASM) cells. The current study aimed to investigate these effects and to explore the underlying mechanisms. Methods: The effects of TSU-68 on ASM cells were studied in mice using a series of biophysiological techniques, including force measurement and patch-clamp experiments. Results: TSU-68 inhibited high K+ or acetylcholine chloride (ACh)-induced pre-contracted mouse tracheal rings in a concentration-dependent manner. Further research demonstrated that the TSU-68-induced ASM relaxation was mediated by calcium, which was decreased by blocking voltage-dependent Ca2+ channels (VDCCs) and non-selective cation channels (NSCCs). Conclusion: Our data indicated that TSU-68 relaxes tense ASM by reducing the intracellular Ca2+ concentration through blocking VDCCs and NSCCs, which suggested that this small molecule might be useful in the treatment of abnormal smooth muscle.