Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of μ-opioid receptors in mature brain neurons

Background and purpose:

The ability of an agonist to induce desensitization of the µ-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist).

Experimental approach:

MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)α knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs).

Key results:

Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCα, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCα knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM.

Conclusions and implications:

In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCα-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.     

Differences in gut microbial metabolism are responsible for reduced hippurate synthesis in Crohn's disease

Background  

Certain urinary metabolites are the product of gut microbial or mammalian metabolism; others, such as hippurate, are mammalian-microbial 'co-metabolites'. It has previously been observed that Crohn's disease (CD) patients excrete significantly less hippurate than controls. There are two stages in the biosynthesis of this metabolite: 1) gut microbial metabolism of dietary aromatic compounds to benzoate, and 2) subsequent hepatorenal conjugation of benzoate with glycine, forming hippurate. Differences in such urinary co-metabolites may therefore reflect systemic consequences of altered gut microbial metabolism, though altered host metabolic pathways may also be involved.     

The use of the Flexi-Seal? Faecal Management System in laparostomy wounds involving enterocutaneous fistula

We present an innovative application of the Flexi-Seal^® Faecal Management System (FMS) for the diversion of upper and lower gastrointestinal secretions from enterocutaneous fistulae associated with complex wounds. Fistula is a common complication after the formation of a laparostomy, secondary to cases of severe intra-abdominal sepsis, acute mesenteric ischaemia, necrotising infection of the abdominal wall, or intra-abdominal hypertension. A significant mortality rate is associated with such fistula. With the successful continent diversion of gastrointestinal secretions by the Flexi-Seal^® FMS, abdominal wounds can be successfully skin-grafted, and wound healing expedited.     

恶性黑色素瘤和非皮肤型恶性肿瘤的相关性

背景：黑色素瘤患者继发恶性肿瘤的发病率增减的报道不一：为了解恶性黑色素瘤与非皮肤型恶性肿瘤是否有关联，作者调查了印第安纳大学肿瘤中心的数据库。目的：作者搜寻黑色素瘤患者发生非皮肤型恶性肿瘤的证据。方法：分析1987年1月至2001年3月间病检确诊的恶性黑色素瘤患者。在这些患者中调查被诊断为黑色素瘤前后发生非皮肤型恶性肿瘤的数据。用实际继发恶性肿瘤人数和预期继发恶性肿瘤的比率作标准化发病率（SIR）的计算，从累积泊松分布估计SIR的95％CI。结果：14年间共955例黑色素瘤患者（其中男498例，女457例）记录在案。59例（6．2％）黑色素瘤患者（男39例，女20例）证实有69个非皮肤型恶性肿瘤。在男性，非霍奇金淋巴瘤（SIR=1．91，95％C10．88～3．62）和肾细胞癌（SIR=2．41，95％C10．97～4．97）有较高的危险度。但在女性患者中，未见非皮肤型恶性肿瘤的较高危险度。结论：本研究未显示黑色素瘤患者有发生前列腺癌、胃肠癌、白血病、子宫内膜癌或神经和神经内分泌系统肿瘤的较高危险度。女性患者无发生非皮肤型肿瘤的较高危险度。     

Effect of K+, and other ligands on the thiol reactivity and tryptic cleavage pattern of scallop sarcoplasmic reticulum

Summary The kinetics of reaction of the thiol groups of both membranous and non-ionic detergent-solubilized Ca-ATPase of scallop sarcoplasmic reticulum towards 5,5-dithiobis(2-nitrobenzoate) were greatly modified in different ways by the presence of the following combinations of ligands: Ca²⁺, EGTA (no Ca²⁺), (ATPMg^2–+EGTA) and (ATP+Ca²⁺). K⁺ was found to influence greatly the pattern of reactivity of the thiol groups of the scallop Ca-ATPase, modifying the kinetics of reaction differently according to the types of other ligand present. While all the thiol groups on the non-ionic detergent-solubilized Ca-ATPase were available for reaction in the absence of K⁺, whatever the combination of ligands, in the presence of K⁺, several groups became completely unreactive towards the reagent. In some cases the rate of inactivation of Ca-ATPase activity could be related to the rates of reaction of different kinetic classes of thiol group, according to the ligands present. Large differences were also seen in the tryptic cleavage pattern in the presence of the different ligands, and K⁺ led to major modifications in the products of digestion in the absence of nucleotide. An 80 kDa tryptic fragment was observed, as with lobster Ca-ATPase but unlike rabbit skeletal muscle Ca-ATPase. A complete amino-acid analysis of the scallop Ca-ATPase was carried out. Differences in the non-polar amino-acid content from rabbit skeletal muscle Ca-ATPase may relate to the different lipid composition of the two membranes.Abbreviations FSR fragmented sarcoplasmic reticulum - C₁₂E₈ dodecyl octaethylene glycol monoether - C₁₂E₉ dodecyl nonaethylene glycol monoether - TES N-tris(hydroxymethyl)methyl-2-aminomethanesulphonic acid) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - TRIS tris(hydroxymethyl)aminomethane - PIPES piperazine-N,N-bis(2-ethanesulphonic acid) - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - AMP-PNP 5-adenylyl imidodiphosphate - SDS sodium dodecyl sulphate - DTT dithiothreitol - DTNB 5,5-dithiobis(2-nitrobenzoate)     

Myosin filament ATPase is enhanced by intramolecularly cross-linked actin

Summary Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallp myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.Abbreviations ANB-NOS N-5-azido-2-nitrobenzoyloxy-succinimide - G-actin monomeric actin - F-actin filamentous actin - S-1 subfragment I - HMM heavy meromyosin - EGTA ethyleneglycol(bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid) - DTT dithiothreitol - DMSO dimethyl sulfoxide - NADodSO₄ sodium dodecyl sulphate - TEMED N,N,N,N-tetramethylethylenediamine - TB tris(hydroxmethy)aminomethane boric acid - Tris tris(hydroxymethy)aminomethane - PTH phenylthiohydantoin - TPCK N-tosyl-phenylalanine chloromethyl ketone - HPLC highperformance liquid chromatography - TFA trifluoroacetic acid     

阿司匹林和塞莱昔布对幽门螺杆菌的体外影响

目的:探讨阿斯匹林和选择性COX-2抑制剂塞莱昔布对体外培养的幽门螺杆菌(H pylori)生长、毒力因子及外膜蛋白的影响．方法:不同浓度的阿司匹林及塞莱昔布与H pylori共同培养,以活菌计数、分光光度计法检测H pylori的生长状态,分光光度计检测A 560nm值判断尿素酶活性,Hela细胞空泡变性实验和中性红吸收试验检测空泡毒素的活性,以SDS-PAGE电泳检测H pylori外膜蛋白的变化．结果:阿司匹林及塞莱昔布可以抑制H pylori的生长,此过程为剂量依赖性效应．阿司匹林0．5 mmol／L及塞莱昔布0．01 mmol／L时与DMSO对照相比H pylori24 h和48 h的菌落计数开始降低,随着两药剂量的加大菌落计数降低得更加明显,阿司匹林2．0 mmol/L和塞莱昔布0．04 mmol／L时H pylori被完全杀灭．阿司匹林及塞莱昔布可剂量依赖性的抑制H pylori的尿素酶活性及空泡毒素的活性．在对H pylori外膜蛋白的研究中发现,NSAIDs可能对H pylori的某种外膜蛋白表达有影响．结论:阿司匹林和塞莱昔布可抑制H pylori的生长、毒力因子的活性,并可能改变H pylori外膜蛋白的表达．     

体表高频超声技术评价家兔血管成形术后再狭窄模型与病理学结果的相关性

目的:分析体表高频超声检测家兔血管内球囊成形术后再狭窄程度与组织病理学分析的相关性。评估体表超声检测的可行性、可靠性及应用价值。方法:实验于2002-03/2003-12在北京中医药大学中医内科学重点学科实验室完成。①日本大耳白兔26只,随机分为正常组10只、假手术组6只、模型组10只。②模型组电刺激兔颈总动脉,电流1.2mA,刺激12～15min,术后第2天喂饲高脂饲料共8周,假手术组仅剥离颈总动脉,不做电刺激,喂高脂饲料,正常组不施加任何干预因素。③模型组和假手术组根据B超选择颈总动脉有斑块或血流明显改变者行球囊血管内成形术。分别于电刺激后8周、血管成形术后4周行超声检查动脉内径和动脉内膜厚度。④所有动物于血管成形术后4周处死取材,进行病理学半定量分析,并与超声测量结果进行相关分析。结果:进入结果分析数量24只,正常组中途死亡1只,原因为牙齿畸型影响进食。模型组1只因电刺激8周时超声评价颈动脉未形成斑块及血流无明显改变而剔出实验。①超声检测电刺激8周时正常组内膜厚为(0.028±0.004)cm,模型组管壁明显增厚(0.043±0.014)cm,差异有显著性(P<0.05),至血管成形术后膜厚增加更明显(0.064±0.002)cm,内径稍有扩大,但差异不显著。②超声检测模型组颈动脉内径与膜厚的测量结果与病理学测量结果呈正相关关系(OR=0.361,P<0.05;OR=0.526,P<0.01),病理狭窄率与超声是否检测到斑块呈正相关关系(OR=0.796,P<0.01)。结论:体表高频超声在评价家兔颈动脉狭窄诊断中有一定应用价值,与病理学半定量分析结果相关性良好。