Corrosion casts as a method for investigation of the insect tracheal system

Summary The tracheal systems of five insect species (two species of ants, worker bee, housefly and the cabbage butterfly) have been studied by scanning electron microscopy of corrosion casts. This technique, which is commonly used for the investigation of vertebrate vasculature, is adapted to demonstrate the ultrastructure of the insect respiratory organ. The problem of filling a blind ending system was solved by injecting the resin Mercox into the evacuated tracheae through a thoracal spiracle. After polymerization of the resin, the tissue was digested enzymatically and chemically. The three-dimensional structure of the tracheal system was investigated by scanning electron microscopy. The technique used here displays for the first time the complex morphology of the entire tracheal system in fine detail, especially the structure of spiracles, airsacs, tracheae and tracheoles. Smooth-walled terminal tracheoles show up in flight muscles. The finest tracheoles that could be identified have diameters of approximately 70 nm. This approaches the finest tracheoles portrayed by transmission electron micrographs.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

可视软性喉镜与光棒在颈椎损伤手术患者全身麻醉气管插管中的应用效果比较

目的:比较可视软性喉镜与光棒用于颈椎损伤手术患者全身麻醉气管插管的有效性与安全性。方法:选择2017年1月至2019年2月本院60例高位颈椎骨折需行气管插管全身麻醉的患者,随机分为可视软性喉镜组(U组)和光棒组(G组)各30例。术前所有患者颈托固定,U组使用UE可视软性喉镜行气管插管,G组使用光棒行气管插管,确认气管插管成功后接呼吸机机械通气。比较两组气管插管时间、一次性插管成功率、拔管后口咽部并发症、插管前后的皮层体感诱发电位(CSEP)及运动诱发电位(MMEP)的变化。记录两组患者麻醉前、麻醉诱导后、气管插管后即刻、气管插管后1 min、气管插管后3 min的平均动脉压(MAP)和心率(HR)。结果:U组气管插管时间较G组插管时间长(P0.05);U组和G组气管插管一次性成功率分别为95%和100%;插管后即刻G组患者MAP升高较U明显(P0.05);与U组比较,G组插管后即刻及插管后1 min、3 min的HR升高较明显(P0.05);U组患者口咽部并发症较G组少;两组患者插管后SSEP及MMEP与插管前相比无阳性改变。结论:可视软性喉镜较光棒需要更长的气管插管时间,两者的气管插管一次性成功率均较高,但可视软性喉镜插管期间循环波动较小、术后口咽部并发症较轻,值得临床推广应用。     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

全凭静脉麻醉期间右美托咪定对靶控输注丙泊酚用量及血流动力学的影响

目的：探讨右关托咪定（dexmedetomidine,DEX）对行全凭静脉麻醉患者靶控输注（target controlled infusion,TCI）丙泊酚用量及拔管期间血流动力学的影响。方法：选择拟于全麻下行经鼻蝶窦垂体瘤切除术的患者30例（ASA I～II级）,随机分为两组,每组15例。试验组（D组）给予DEX负荷剂量0．5μg·kg-1,注药时间15rain,继以0．3μg·kg-1·h-1持续输注至修补硬膜时;对照纽（N组）在相同时间给予等容量生理盐水。两组麻醉诱导方法相同,术中以脑电双频指数（bispectral index,BIS）为麻醉深度监测指标,根据BIS值调节丙泊酚血浆靶浓度维持麻醉。记录拔管期间收缩压（SBP）、舒张压（DBP）和心率（HR）,并计算心肌氧耗指数（RPP）;记录丙泊酚平均用量、用药前后BIS值、呼吸恢复时间、睁眼时间、拔管时间及术中不良反应。结果：①D组给予负荷剂量后,BIS值由（95±3）降至（77±11）,差异有统计学意义（P〈0．01）。②与N组相比,D组丙泊酚用量减少28％,差异有统计学意义（P〈0．01）。⑧拔管期间SBP、DBP和HR与入室时比较,D组无明显变化,N组HR显著升高（P〈0．05）;D组拔管期间SBP、DBP、HR和RPP明显低于N组（P〈0．05）;④两组患者呼吸恢复时间、睁眼时间及拔管时间差异均无统计学意义;⑤两组不良反应（心动过缓、高血压、低血压）的发生率无显著性差异。结论：术中持续静脉输注DEX可减少TCI丙泊酚用量,能使BIS值进一步降低,产生良好镇静效应;同时可有效减轻拔管期间循环变化,降低RPP,减少心肌耗氧量,提高拔管质量。     

Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers

We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.     

Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.     

Dissociation of Intracellular Ca Release and Ca Entry Response to 5-Hydroxytryptamine in Cultured Canine Tracheal Smooth Muscle Cells

The relationship between the agonist-sensitive Ca²⁺ pool and those discharged by the Ca²⁺-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i), followed by a sustained plateau phase that was dependent on extracellular Ca²⁺. In such cells, TG produced a concentration-dependent increase in [Ca²⁺]_i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca²⁺. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca²⁺]_i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca²⁺-ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca²⁺-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca²⁺ stores is sufficient for activation of Ca²⁺ influx. Some characteristics of the Ca²⁺-influx activated by depletion of internal Ca²⁺ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca²⁺ influx was inhibited by La³⁺, membrane depolarisation, and the novel Ca²⁺-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca²⁺ influx by TG also was blocked by La³⁺, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca²⁺ stores in TSMCs is sufficient for activation of Ca²⁺ influx, and (2) the agonist-activated Ca²⁺ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool are indistinguishable.     

The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.     

rhBMP-2 诱导异位软骨修复重建兔气管缺损的实验研究

目的:探讨重组人骨形成蛋白-2(rhBMP-2)作为激活物诱导异位软骨修复并重建兔气管缺损的可行性。方法:取24只新西兰大白兔,制备气管前壁软骨1/3缺损模型。随机分为A、B组,每组12只,A组为实验组,在气管缺损处前壁颈前肌肉修补,多点注射rhBMP-2;B组于气管软骨缺损部位直接颈前肌群修补。术后观察动物一般情况,于4、8、12周取材进行大体观察、HE染色观察重建区域情况。结果:术后A组动物均存活至实验完成,B组因气道感染及气道分泌物堵管潴留致使实验兔死亡,其余动物出现皮下气肿,呼吸不畅等情况。组织学观察A组有明显的新生软骨细胞及少量软骨样组织,可见结缔组织包绕,周围肌肉组织完整,排列整齐,未见明显坏死组织,有少量淋巴细胞浸润。B组未见软骨组织生成,可见大量肉芽组织增生,结缔组织排列紊乱,伴少量坏死组织,大量淋巴浸润。结论:rhBMP-2可通过注射到颈前肌肉修补肌群中诱导软骨细胞和软骨样组织生成,减轻炎症反应,联合颈前肌瓣修复重建气管缺损能充分维持修复重建后的气道形态,具有减少术后皮下气肿、气管狭窄的作用,有望用于临床修复重建气管组织缺损。