呼吸道迷走神经感受器概述

肺以及气道与外界环境之间存在着巨大的界面,因此需要有效的防御反射机制。呼吸道感受器是肺部神经反射的起始点,其重要性不言而喻,采用组织,解剖与电生理学方法,经过一个世纪的研究,我们对于呼吸道感受器的认识,特别对其结构的认识,仍然有限,据电生理实验结果,肺部感受器至少可被分为三大类;慢适应感受器,快适应感受器以及C纤维感受器,按血供来源,后者又可分为气道（体循环）与肺（肺循环）两类,近来发现呼吸道中存在着第四类感受器,它们由迷走神经的Aδ传入纤维传递冲动,其放电活动不同于上述各类,对肺充气反应阈值高,故称之为高阈值Aδ感受器,功能上前两类基本属于机械性感受器,而后两类可归为化学敏感性感受器,另外,用组织学方法,观察到气道内有一些神经内分泌细胞,它们可以散在分布,亦可集聚成小体。这些神经上皮小体受多种神经支配,其结构复杂,形态酪似感受器,虽然我们对其形态了解颇深,但对其放电形式一无所知,本文对以上各类感受器进行了评述与探讨。     

采用共聚焦显微术对家兔呼吸道感受器进行观察

呼吸道感受器在机体对肺部物理和化学环境变化作出反应时起到重要作用,它们引起的反射具有调节或保护作用,或产生病理效应。基于电生理研究,可将呼吸道感受器分为四类,但它们的组织结构不详。由于对感受器形态的认识不足,阻碍了对其生理功能的理解。近来,我们采用共聚焦显微术与免疫组织化学方法（用钠钾ATP酶作为标记),首次高清晰度地展示了呼吸感受器结构。本文采用这种新方法在家兔中进行了系统观察。结果显示,各级气道通均有多种不同的感受器结构。大气道中的结构往往比小气道中的复杂,虽然它们的大小、所在部位与走向不尽相同,但都有多个终端末梢。有些感受器埋置在气道平滑肌中,其结构便于感受对气道的机械性刺激：有些覆盖在气道上皮的表面,其形态适合于感受通过气道的刺激性物质;另一些则位于气道粘膜下,在上皮与平滑肌之间。有些传入神经轴突可支配多个感受器结构。据此,传入单纤维中记录到的电活动是来自一个感觉单位。后者可以由多个感受器组成。除了感受器之外,我们还观察到气道内神经节,它们与感受神经的轴突密切相关。本文证实气道内存在不同形态结构的感受器,并探讨了其生理分类。     

一类新的第二信使——花生四烯酸脂氧合酶代谢物

海免感觉神经元对神经活性肽四肽FMRF酰胺的抑制性应答,是以花生四烯酸脂氧合酶代谢物为第二信使介导的。这类新的第二信使既可在胞内也可在胞问传递信号。同种离子通道(S通道)的开或关,受两种不同的第二信使系统调节控制。     

花生四烯酸对钾离子通道的调节作用

花生四烯酸是一个重要的炎症介质,它可以对广泛存在于各种细胞膜表面的外离子通道进行直接的或间接的调节。花生四烯酸与通道的直接作用,改变了通道蛋白的构象;直接学可与通道周围的细胞膜作用,影响钾离子通道的功能。花生四烯酸还可以通过其环氧酶和脂氧酶的代谢产物、蛋白激酶Ｃ（ＰＫＣ）及「Ｃａ＾２＋」间接影响了子通道。这些环节为药理学研究提供了新的可能的靶点。     

花生四烯酸高产菌株的选育

本研究以一株能产生花生四烯酸的被孢霉菌为出发菌株,通过紫外线诱变筛选出一株高产花生四烯酸的突变株Ｍ１０,发酵试验结果表明：突变株Ｍ１０的每升培养液中干菌体得率为３１ｇ,油脂含量为８．３ｇ,而原菌株仅为２０．３ｇ和５．４ｇ,气相色谱分析结果Ｍ１０所产花生四烯酸的量占总脂的１０．０６％（０．８３ｇ／Ｌ培养液）。同时对Ｍ１０菌株的生长和发酵特性及发酵过程中菌丝体形态变化作了一定的探讨。     

花生四烯酸的生物活性及其钙信号转导作用

花生四烯酸（arachidonic acid,AA）以酯化形式在膜磷脂中,细胞兴奋时多种信号转导途径可引起游离AA释放,并迅速代谢为具有生物活性的炎症物质,参与细胞免疫和炎症反应。目前大量研究表明,AA本身还直接参与细胞内生物功能的调节,包括影响酶功能,调控各种离子通道,尤其是直接导致细胞内信号转导,成为细胞膜受体兴奋－细胞内生物反应偶联的第二信使。但AA的作用机制及其生理和病理生理意义有待进一步研究。     

离子注入花生四烯酸产生菌诱变选育

利用离子束注入生物技术对花生四烯酸产生菌(Mortierella alpina)进行诱变高产菌筛选。筛选到高产菌I₄₉N₁₈,该菌每升培养液可得生物量30.80g(约4%的含水量),干菌体油脂含量为25.8%,其中花生四烯酸的含量占总.脂的45.37%。30L和250L发酵罐发酵试验,该高产菌的花生.四烯酸得率为4.0g/L。     

KLa对高山被孢霉产花生四烯酸的影响

研究了体积溶氧系数(K1a)对被孢霉产生四烯酸的影响。结果表明在摇瓶水平放大培养中,K1a、被孢霉生长、花生四烯酸（AA）产量均降低。利用0.3％Tween20作为氧载体提高各级的K1a,可以较大幅度提高AA产量。     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

呼吸道神经与炎症及癌变的关系(英文)

肺癌是主要医学难题之一,尽管分子生物学和药理学技术在进步,肺癌的治疗结果却不尽人意。临床上,炎症与肿瘤密切相关,炎症能够促进肿瘤的形成。从遗传角度讲,这两个过程受到同一个基因座的调控。越来越多的证据表明,神经和免疫两个系统存在交互作用,其中迷走神经起着重要作用。在临床及动物实验中分别观察到:切除迷走神经后肺部的肿瘤发生率增高,转移增加。表明迷走神经具有保护作用,能抑制肿瘤生长。气道感受器是生物感应器,能感受肺部炎症及肿瘤生长过程中的多种介质和细胞因子。这些信号通过迷走神经传递给脑,提供肿瘤生长的信息,随后产生一系列的反应调节炎症的广度和强度以及肿瘤生长速度。肿瘤细胞表达神经递质的受体,能提供底物与神经元直接相互作用。因此,免疫反应的神经调节既可以靶向炎症又可以靶向肿瘤。认识肺部神经如何监控肿瘤的生长并且产生神经免疫相互作用以调节肿瘤的进展及转移,将提高肺癌的治疗水平。     

Proteolytic enzymes and arachidonic acid metabolites produced by MRC-5 cells on various microcarrier substrates

Summary Human diploid fibroblasts were cultured on microcarriers made from DEAE-dextran, denatured collagen, DEAE-dextran linked to denatured collagen, and glass. Cells grown on these four substrates were examined for the production of proteolytic enzymes and arachidonic acid metabolites. Culture fluids from cells grown on the DEAE-dextran microcarriers contained the highest amounts of proteolytic enzyme activity. Both plasminogen-independent and plasminogen-dependent fibrinolytic activities were present and the plasminogen-dependent activity seemed to result from the presence of both urokinase and tissue plasminogen activator. Culture fluid from the cells grown on the glass microcarriers contained the least amount of protease activity, and nearly all of the plasminogen-activator activity seemed to be of the urokinase type. Protease activity in the culture fluids of cells grown on the other two substrates were intermediate. With regard to arachidonic acid metabolites, cells grown on the DEAE-dextran microcarriers produced the highest amounts of cyclooxygenase products but very low levels of lipoxygenase metabolites. Cells grown on the other three substrates produced comparable amounts of various cyclooxygenase products (lower than that produced by cells on the DEAE-dextrans substrate). Cells grown on the glass microcarriers also produced detectable amounts of two lipoxygenase metabolites—leukotriene B₄ and leukotriene C₄. Inasmuch as both proteolytic enzymes and arachidonic acid metabolites regulate basic cell properties, the differential amount of these metabolites observed in the culture fluids on the various substrates may contribute to the biological differences that exist on these substrates. This study was supported in part by grants R44 CA 36656 and IK08HL01332-01 from the Public Health Service, U. S. Department of Health and Human Services and by grant BC-512 from the American Cancer Society. JDH is a research fellow of the American Lung Association.     

Effect of arachidonic acid,fatty acids,prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells

Summary Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE₂ inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE₂-induced Na uptake. PGE₂ has no effect on the volume-activated K and Cl permeabilities. LTB₄, LTC₄ and LTE₄ inhibit RVD insignificantly in hypotonically swollen cells. LTD₄, more-over, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD₄ even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.     

Uptake of arachidonic acid into membrane phospholipids: Effect on chloride transport across cornea

Summary We demonstrate that arachidonic acid (AA) stimulation of chloride transport across frog cornea is mediated via two independent pathways: (1) stimulation of prostaglandins and cAMP synthesis, and (2) a direct physical change in the membrane produced by substitution of different phospholipid acyl chains. AA is well known as a precursor in the synthesis of prostaglandins, which have been shown to stimulate cAMP synthesis and chloride transport in frog cornea. We show that frog cornea can convert exogenous AA to PGE₂, but that in the presence of 10^–5 m indomethacin both the conversion to PGE₂ and stimulation of cAMP are completely blocked. However, with indomethacin the action of AA to stimulate chloride transport (as measured by SCC) remains, but peak height of the response is reduced to 57% of that found when AA alone is given. Similarly, we show that propranolol completely blocks cAMP stimulation, but stimulation of SCC is reduced to 45% of the original response. Therefore, cAMP appears to be responsible for roughly half of the observed stimulation in SCC. By gas chromatographic analysis we show that significant quantities of AA can rapidly substitute into membrane phospholipids of corneal epithelium and L929 cells following the addition of AA to the medium. Modification of membrane phospholipid structure can affect membrane viscosity, membrane-bound enzyme activity, and the distribution and lateral mobility of integral proteins. It seems likely that such alterations in the properties of the membrane may modulate the rate of chloride transport, and this may constitute the second mechanism. Upon addition of AA, both mechanisms appear to stimulate chloride transport simultaneously, and are apparently additive. We show that prolonged exposure to AA results in a large incorporation of AA into phospholipid and consequently, a perturbation in the ratio of unsaturated to saturated fatty acids. We also find evidence of a compensatory cellular mechanism that alters the ratio of endogenously synthesized fatty acids and tends to reduce the membrane-perturbing effect of AA.     

Substrate specificity of human ceramide kinase

Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. To date, CERK has not been well characterized in vitro. In this study, we investigated the substrate specificity of CERK using baculovirus-expressed human CERK (6 x His) and a newly designed assay based on mixed micelles of Triton X-100. The results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific. A minimum of a 12 carbon acyl chain was required for normal CERK activity, and the 4-5 trans double bond was important for substrate recognition. A significant discrimination by CERK was not observed between ceramides with long saturated and long unsaturated fatty acyl chains. Methylation of the primary hydroxyl group resulted in a loss of activity, confirming that CERK produces ceramide-1-phosphate versus ceramide-3-phosphate. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation by CERK. These results also indicated that the free hydrogen of the secondary amide group is critical for substrate recognition. Lastly, the sphingoid chain was also required for substrate recognition by CERK. Together, these results indicate a very high specificity for substrate recognition by CERK, explaining the use of ceramide and not sphingosine or diacylglycerol as substrates.     

微生物油脂中花生四烯酸含量的准确快速测定

建立了快速、准确测定微生物油脂中花生四烯酸(ARA)含量的气相色谱检测方法。选用DB-23毛细管色谱柱,设置合适的载气压力,采用FID检测器,对ARA含量进行了定量分析。测定结果表明:油脂中各脂肪酸组分可有效分离,分析时间仅需20 min,ARA的回收率为90.146%~100.634%,相对标准偏差为4.175%。     

Arachidonic and other long-chain polyunsaturated fatty acids in spermatophores and spermathecae of Teleogryllus commodus: Significance in prostaglandin-mediated reproductive behaviour

Estimates of weights of fatty acids, especially arachidonic acid, in spermathecae from virgin and mated T. commodus indicate substantial elevation in all fatty acids and particularly arachidonic acid following mating. Analysis of spermatophore lipids suggests that this can be in part accounted for by the contents of one or several spermatophores. Fractionation of total lipids from spermatophores showed that arachidonic acid comprised 24% of phosphotidylcholine and 4% of phosphotidylethanolamine, but was not detected in neutral lipids whereas it was approximately equally distributed over phosphotidylcholine and phosphotidylethanolamine in lipids from spermathecae. These data indicate that in addition to prostaglandin synthetase, the spermatophore contains a physiologically significant quantity of prostaglandin precursor, arachidonic acid, esterified to phospholipid and presumably unavailable for enzymatic action during mating transfer. We also note that proportions of arachidonic acid in the phosphotidylcholine of spermatophores are the highest recorded for this fatty acid in the insect literature, which in conjunction with recent work emphasizes the likely physiological significance of long-chain polyunsaturated fatty acids in insects generally.     

Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability

Chronic administration of mood stabilizers to rats down‐regulates the brain arachidonic acid (AA) cascade. This down‐regulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E₂ concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1‐¹⁴C]AA was infused intravenously for 5 min, arterial plasma was collected and high‐energy microwaved brain was analyzed. CLZ increased incorporation coefficients and rates J_in,i of plasma unesterified AA into brain phospholipids i, while decreasing plasma unesterified but not esterified AA. These effects disappeared after washout. Thus, CLZ and OLZ similarly down‐regulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by down‐regulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.