浙江蝮蛇(Agkistrodon halys Pallas)蛇毒中舒缓激肽增强肽的研究 Ⅱ.舒缓激肽增强肽组分Ⅰ结构与功能的关系

1.浙江蝮蛇毒冻干粉的70%甲醇提取液,经Sephadex G-15层析柱,可分离出三个舒缓激肽增强肽的组分。对其中BPP_1的结构进行了测定,其氨基酸顺序为: -Glu·Gly·Arg·Pro·Pro·Gly·Pro·Pro·Ile·Pro·Pro 2.研究了BPP_1结构与功能的关系。当用焦谷氨肽酶移去BPP_1N末端的焦谷氨酸残基时,对舒缓激肽的增强效应反而升高一倍左右;此后随着逐步进行Edman降解,活力也随之下降,但降解至C末端三肽时,生物活力仍保留约90%,至C末端二肽时,活力就骤然丧失。对BPP_1C末端用羧肽酶Y水解时,一小时后活力降低二分之一。这些都说明,BPP_1C末端结构的完整,是其生物活力所必需的。     

浙江蝮蛇(Agkistrodon halys Pallas)蛇毒中舒缓激肽增强肽的研究——Ⅱ.舒缓激肽增强肽组分Ⅰ结构与功能的关系

1.浙江蝮蛇毒冻干粉的70%甲醇提取液,经Sephadex G-15层析柱,可分离出三个舒缓激肽增强肽的组分。对其中BPP_1的结构进行了测定,其氨基酸顺序为: (?)GIu·Gly·Arg·Pro·Pro·Gly·Pro·Pro·Ile·Pro·Pro 2.研究了BPP_1结构与功能的关系。当用焦谷氨肽酶移去BPP_1 N末端的焦谷氨酸残基时,对舒缓激肽的增强效应反而升高一倍左右;此后随着逐步进行Edman降解,活力也随之下降,但降解至C末端三肽时,生物活力仍保留约90%,至C末端二肽时,活力就骤然丧失。对BPP_1C末端用羧肽酶Y水解时,一小时后活力降低二分之一。这些部说明,BPP_1 C末端结构的完整,是其生物活力所必需的。     

人组织激肽释放酶成熟蛋白在大肠杆菌中的高效表达

将编码人组织激肽释放酶成熟蛋白的基因片段扩增并分别克隆到原核表达载体pET2 8(b)及分泌型表达载体pET2 0 (b)中 ,使其C端融合 6×HisTag序列 .转化不同受体菌 ,IPTG诱导表达后利用SDS PAGE、免疫印记等方法对重组蛋白进行分析 .在 6株基因工程菌株中 ,均表达出分子量约30kD的激肽释放酶融合蛋白 ,其中激肽释放酶在pET2 8载体中的表达水平高于pET2 0载体 .pET2 8和pET2 0载体表达的重组激肽释放酶蛋白分别占菌体总蛋白约 2 6 %和 10 % .Western印迹分析表明 ,目的蛋白可与抗人血清KK单克隆抗体发生特异性反应 .未经纯化的激肽释放酶融合蛋白具有一定的水解苯甲酰精胺酸乙酯 (BAEE)的能力 .在大肠杆菌中获得了人组织激肽释放酶的高效表达 ,表达产物具有免疫原性和生物活力 ,这为研究其生物功能和开发基因工程药物奠定基础     

菜花烙铁头蛇毒中一种具有激肽释放酶与纤维蛋白原溶酶活性的Jerdonase

通过DEAESephadexA 5 0阴离子交换、超细SephadexG 10 0分子筛和反相高效液相C4 色谱层析 ,从菜花烙铁头蛇毒冻干粉中纯化出一种具有激肽释放酶活性和α纤维蛋白原溶酶活性的丝氨酸蛋白酶 ,命名为Jerdonase。在 12 .5 %胶浓度的SDS还原电泳条件下 ,该酶分子量大约为 5 5kD ,在非还原电泳条件下 ,分子量大约为 5 3kD。此酶是一种糖蛋白 ,含有约 35 .8%的中性糖。它的N末端氨基酸序列为IIGGDEENINEHPFLVALYDA ,其序列和蛇毒中其他丝氨酸蛋白酶具有非常高的序列相似性。Jerdonase能够催化BAEE、S 2 2 38和S 2 30 2的水解 ,其水解活性可被PMSF抑制 ,但是EDTA对此没有影响。Jerdonase能优先水解人纤维蛋白原的Aα链 ,同时伴随有微弱的Bβ链水解活性。另外 ,此酶能够水解牛低分子量的激肽原 ,释放舒缓激肽。总之 ,所有的结果表明Jerdonase是一个具有多功能活性的蛇毒丝氨酸蛋白酶     

重组水蛭素相关肽Hi-lys的表达与纯化(英文)

为开发一种新的有临床应用价值的抗血栓药物,根据水蛭素保持抗凝活性的2 0肽片段,设计并构建了水蛭素相关肽(Hi lys)与天冬酰胺酶C端的融合表达系统.为方便目的肽与融合伙伴的分离,增加了富含带电序列的8肽(KRKRKKSR)及酸敏感的天冬氨酰 脯氨酸(Asp Pro)位点,获得了表达质粒pED P8 Hi lys.将其转化E .coliBL 2 1,玉米浆培养基(kanr)培养,乳糖诱导获得融合蛋白(AnsB C P8 Hi lys)的高效表达.通过细菌裂解、包涵体洗涤、尿素溶解、乙醇沉淀、酸水解和DEAE 纤维素5 2柱层析纯化获得目的肽Hi lys ,用凝血酶测定法测得其抗凝活性为5 0ATU mg .     

固相法合成促黄体生成素释放激素及其类似物

哺乳动物下丘脑分泌的促黄体生成素释放激素能促使垂体前叶分泌促黄体生成素和促滤泡成熟素,因此在临床和畜牧业生产中有着重要的应用价值。本文报导了采用固相肽合成的改进方法合成了促黄体生成素释放激素,即pGlu·His·Trp·Ser·Tyr·Gly·Leu·Arg·Pro·Gly·NH_2,及其类似物[D-Ala~6][去Gly·NH_2~10]促黄体生成素释放激素的乙基酰胺,即pGlu·His·Trp·Ser·Tyr·D-Ala·Leu·Arg·Pro·NHC_2H_5,都获得了较满意的结果。对大白鼠和蟾蜍等动物诱导排卵试验的结果说明,该促黄体生成素释放激素类似物的活性较促黄体生成素释放激素提高了上百倍。     

抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2 6在延长小鼠出血时间上具有较明显的作用     

硫激肽及其受体调控褐飞虱取食行为

【目的】明确硫激肽(sulfakinin, SK)及硫激肽受体(sulfakinin receptor, SKR)在褐飞虱Nilaparvata lugens取食行为中的作用。【方法】PCR克隆褐飞虱硫激肽基因Nlsk及其受体基因Nlskr cDNA全长序列并进行生物信息学分析；利用qRT-PCR检测Nlsk及Nlskr在褐飞虱不同发育阶段(卵、1-5龄若虫、雄成虫和雌成虫)和雌成虫不同组织(头、触角、翅、口针、足、肠道和马氏管)中的表达量；褐飞虱3龄若虫注射dsNlskr进行基因沉默，qRT-PCR检测4龄若虫中Nlskr的表达量，测定4龄若虫的取食量；基于已构建的Nlskr RNAi后的4龄若虫转录组数据库进行差异表达基因(differentially expressed genes, DEGs)的GO和KEGG分析以及取食相关基因的qRT-PCR验证。【结果】PCR克隆得到了褐飞虱Nlsk(GenBank登录号：AB817281)及Nlskr(GenBank登录号：BAO01059.1)的cDNA全长序列。序列比对结果显示，褐飞虱NlSK成熟肽具有与其他物种保守的C端FMRFam...     

中国蝮蛇(Agkistrodon halys Pallas)蛇毒中舒缓激肽增强肽的研究

1.中国浙江产蝮蛇(Agkistrodon halys Pallas)蛇毒中至少含有两种以上的舒缓激肽增强肽,对其中之一的组分进行了分离提纯,经鉴定为11肽,N 端为环谷,其氨基酸组成与日本蝮蛇(Agkistrodon halys bromhoffii)蛇毒中舒缓激肽增强肽的组分A 极相似,仅多一个脯氨酸。2.豚鼠回肠纵行肌中含有能使舒缓激肽降解的各种激肽酶,经鉴定其中包括有氨肽酶,激肽酶Ⅰ及其他肽酶,但不具有激肽酶Ⅱ的活力,它们都属于含金属的外切酶,经邻二氮菲处理后活力不可逆地丧失。3.豚鼠回肠纵行肌经邻二氮菲处理后,其所含的激肽酶活力虽丧失,但对舒缓激肽增强肽的效应却仍保留,这说明在体外舒缓激肽增强肽的效应与激肽酶的活力无关。     

激肽系统活性产物

随着研究的不断深入,发现激肽释放酶-激肽系统参与了机体众多的生物学过程,除了对机体心血管系统、凝血系统、纤溶系统和肾功能等正常生理过程的调节作用外,在高血压、炎症、疼痛、血管生成、细胞增殖、凋亡和肿瘤发生等诸多的病理生理过程中也陆续发现有着不可替代的作用。有关激肽释放酶等上游活性物质的生理功能报道较多,本文从人激肽释放酶-激肽系统开始,重点对下游活性产物缓激肽、活性高分子量激肽原等在生理及病理过程中的作用和调节作一综述。     

Conformational and biological properties of Bauhinia bauhinioides kallikrein inhibitor fragments with bradykinin‐like activities

Proteinase inhibitors extracted form medicinal plants are an interesting source of new drugs. Modifications in the structure of some of this kind of macromolecules could also lead to compounds of interesting biological properties. In this work, we synthesized and tested one fragment containing the reactive site of the Bauhinia bauhinioides kallikrein inhibitor (BbKI), denoted BbKI_51–62, and a related analog (P₂) in which a proline residue was inserted in order to mimic the N‐terminal region of the bradykinin molecule. The related retro‐inverso counterparts Ri‐BbKI_51–62 and Ri‐P₂ were also included. The ability of these peptides to induce contraction of stomach fundus isolated from mouse was evaluated as well as their capability to induce calcium release from a cell culture of smooth muscle from guinea pig ileum. The conformational properties of the peptides were evaluated by circular dichroism and their resistance to enzymatic degradation by exposure to human blood plasma. Our results show that neither the parent BbKI_51–62 nor its Ri‐BbKI_51–62 analog exhibit bradykinin‐like activity, although the retro‐inverso isomer was resistant to blood plasma degradation. On the other hand, the peptides P₂ and Ri‐P₂ presented contractile activities on gastric smooth muscle from stomach fundus possibly by acting via B‐2 receptor. Both compounds also induce calcium release from guinea pig ileum muscle cells in a manner similar to bradykinin. Moreover, both compounds also inhibited porcine pancreatic kallikrein. However, conformational analysis did not reveal any direct correlation between structure and biological effects. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Solution conformations of bradykinin antagonists modified with Calpha-Calpha cyclized nonaromatic residues

The conformations of four BK antagonists, [D-Arg 0, Hyp3, Thi5, D-Phe7, Acc8]BK (1), Aaa[D-Arg 0, Hyp3, Thi5, D-Phe7, Acc8]BK (2), [D-Arg 0, Hyp3, Thi5, 8, Apc7]BK (3), and Aaa[D-Arg(0), Hyp(3), Thi(5, 8), Apc7]BK (4) were studied by using 2D NMR spectroscopy and MD simulations with time-averaged (TAV) restraints. According to the results of the NMR measurements, the BK antagonists contain 7-30% of minor conformation resulting from cis/trans isomerization of the peptide bonds preceding either Pro or Hyp residues. The major conformation of each peptide possesses all peptide bonds in trans configuration. Peptides modified with the Apc residue at position 7 (peptides 3 and 4) possess a higher percentage of minor isomer.Peptide 1 exhibits the strongest vasodepressor potency among the analogs studied and as a single one forms the betaII-turn in the 2-5 fragment, which is believed to be crucial for antagonistic activity. This peptide is also the most compact. The radius of gyration (Rg) amounts to 6.9 A and is by ca 1.5 A lower than that of the remaining analogs. With peptide 4, the ST-turn of type I within the Ser6-Thi8 fragment was found.     

Bradykinin-bradykinin receptor (B1R) signalling is involved in the blood–brain barrier disruption in moyamoya disease

Moyamoya disease (MMD) is a rare disorder of the cerebrovascular system. It is a steno-occlusive disease that involves angiogenesis and blood–brain barrier (BBB) disruption. Bradykinin (BK), its metabolite des-Arg9-BK, and receptor (B1R) affect angiogenesis and BBB integrity. In this study, we aimed to investigate the changes in BK, B1R and des-Arg9-BK levels in the serum and brain tissues of patients with MMD and explore the underlying mechanism of these markers in MMD. We obtained the serum samples and superficial temporal artery (STA) tissue of patients with MMD from the Department of Neurosurgery of the Jining First People's Hospital. First, we measured BK, des-Arg9-BK and B1R levels in the serum of patients by means of ELISA. Next, we performed immunofluorescence to determine B1R expression in STA tissues. Finally, we determined the underlying mechanism through Western blot, angiogenesis assay, immunofluorescence, transendothelial electrical resistance and transcytosis assays. Our results demonstrated a significant increase in the BK, des-Arg9-BK and B1R levels in the serum of patients with MMD compared to healthy controls. Furthermore, an increase in the B1R expression level was observed in the STA tissues of patients with MMD. BK and des-Arg9-BK could promote the migratory and proliferative abilities of bEnd.3 cells and inhibited the formation of bEnd.3 cell tubes. In vitro BBB model showed that BK and des-Arg9-BK could reduce claudin-5, ZO-1 and occluding expression and BBB disruption. To the best of our knowledge, our results show an increase in BK and B1R levels in the serum and STA tissues of patients with MMD. BK and Des-Arg9-BK could inhibit angiogenesis, promote migratory and proliferative capacities of cells, and disrupt BBB integrity. Therefore, regulating BK, des-Arg9-BK and B1R levels in the serum and the brain could be potential strategies for treating patients with MMD.     

The human bronchus model in vitro. Pharmacological approach of various components involved in the functional response

Studying the human bronchi in vitro, and therefore sheltered from the toxicity problems inherent in human experiments, makes it possible to conduct a monofactorial analysis, disregarding the perturbations engendered by reflex phenomena, hemodynamic changes, etc. Analysing the effects of mediators on tissues may be less simple that it looks, due to the multiplicity of the cell types that are present. For example, in studying the effects of bradykinin we have shown that bradykinin is a potent contractile agent of small-diameter isolated bronchi, whereas it has no significant contractile effect on larger bronchi. The bradykinin-induced contraction results from a contractile component due to stimulation of the TP receptor, and of a relaxant component due to rrelaxant prostanoids. The two components of the bradykinin effects are produced by stimulation of B₂ receptors. In vitro stimulation of bronchi by LPS or interleukin-1 permits us to obtain hyperractivity to bradykinin due to induction of thromboxane synthetase or isomerase rather than to induction of B₂ receptors or cyclooxygenase. Involvement of the nervous system may persist in the in vitro bronchial model, and indeed we have shown, for example, that pentamidine, well known for its tussigenic effect, is an indirect parasympathomimetic compound. Thus, study of the isolated bronchus permits an approach to the mechanisms of action of medicinal drugs. Despite the simplification provided compared to the in vivo study, analysis of bronchoreactivity on the isolated bronchus must take into account numerous parameters which interfere with the proper effects of the substances.     

Investigation of bradykinin metabolism in human and rat plasma in the presence of the dual ACE/NEP inhibitors GW660511X and omapatrilat.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.     

Molecular features characterizing non-peptide selectivity to the human B1 and B2 bradykinin receptors

Bradykinin is produced in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases. Actions of this peptide are mediated through two different G-protein coupled receptors, named B1 and B2 that have different pharmacological characteristics. The former is up-regulated during inflammation episodes or tissue trauma whereas, the latter is constitutively expressed in a variety of cell types. In a previous work we have characterized the molecular features that explain the observed structure-activity results for both receptors by means of molecular modeling studies, using diverse ligands for both receptors. These results were summarized in the form of two different pharmacophores that provided new insights to be used for the design of novel molecules with antagonistic profile. In the present work, we compare these pharmacophores to understand the features that characterize ligand selectivity to the two bradykinin receptors. The study shows that most of the residues involved in the binding pocket are similar in both receptors and consequently are the pharmacophores obtained. The main difference between the two pharmacophores remains on point #5 that involves a polar moiety for the B1 receptor and an aromatic ring for the B2 receptor. Accordingly, analysis of the prospective bound conformation of several non-selective small molecule ligands of the bradykinin receptors permits to conclude that fulfilment of point#5 is a requirement to produce selective ligands. However, the study also shows that this is a necessary condition only, since ligands need also to be bulky enough to avoid binding to these receptors in diverse poses. These results provide new insights for a better understanding of the molecular features that ligands are required to exhibit to be selective bradykinin ligands.     

Bradykinin Generation Triggered by Pseudomonas Proteases Facilitates Invasion of the Systemic Circulation by Pseudomonas aeruginosa

To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo.     

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. II. Effects of thapsigargin on the cellular Ca2+ homeostasis

Summary Evidence was provided, in the preceding paper (Thuringer & Sauvé, 1992), that the external Ca²⁺-dependent phase of the Ca²⁺ signals evoked by bradykinin (BK) or caffeine in bovine aortic endothelial cells (BAE), differ in their respective sensitivity to procaine. To examine whether the emptying of the InsP₃-sensitive Ca²⁺ store is the signal for activating the agonist-evoked Ca²⁺ entry, we have investigated the effects of thapsigargin (TSG), a known inhibitor of the microsomal Ca²⁺-ATPase activity in a variety of cell types, via the activity of calcium-activated potassium channels [K(Ca²⁺) channels]. In cell-attached experiments, the external application of TSG caused a sustained or oscillatory activation of K(Ca²⁺) channels depending on both the cells and doses tested. The TSG-evoked channel activity could be reversibly blocked by removing extracellular Ca²⁺, and strongly decreased by adding 10 mm procaine to the bath medium. In Ca²⁺-free external conditions, TSG did not promote an apparent Ca²⁺ discharge from internal stores but prevented in a dose- and timedependent manner the subsequent agonist-evoked channel activity related to the release of internally sequestered Ca²⁺. These results confirm that TSG and BK release Ca²⁺ from the same internal stores but with different kinetics. Because the channel response to caffeine was found to be poorly sensitive to procaine, in contrast to that evoked by BK and TSG, it may be concluded that both BK and TSG activate the same Ca²⁺ entry pathway. Therefore, the emptying of the InsP₃-sensitive Ca²⁺ store is likely to be the main signal for activating the agonist-evoked Ca²⁺ entry in BAE cells.The authors wish to thank Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

Bradykinin enhances cell migration in human chondrosarcoma cells through BK receptor signaling pathways

Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. BK has recently been shown to be involved in carcinogenesis and cancer progression. In this study, we found that BK increased the migration and the expression of α2β1 integrin in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significantly higher expression of the B1 and B2 receptors comparing to normal cartilage. BK‐mediated migration and integrin up‐regulation was attenuated by B1 and B2 BK receptor siRNA or antagonist. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after BK treatment was demonstrated, and BK‐induced integrin expression and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. Taken together, our results indicated that BK enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the BK receptors/PLC/PKCδ/NF‐κB signal transduction pathway. J. Cell. Biochem. 109: 82–92, 2010. © 2009 Wiley‐Liss, Inc.     

Modulation of bradykinin signaling by EP24.15 and EP24.16 in cultured trigeminal ganglia

Metalloendopeptidases expressed in neural tissue are characterized in terms of their neuropeptide substrates. One such neuropeptide, bradykinin (BK), is an important inflammatory mediator that activates the type-2 BK receptor (B2R) on the terminal endings of specialized pain-sensing neurons known as nociceptors. Among several metalloendopeptidases that metabolize and inactivate BK, EP24.15 and EP24.16 are known to associate with the plasma membrane in several immortalized cell lines. Potentially, the colocalization of EP24.15/16 and B2R at plasma membrane microdomains known as lipid rafts in a physiologically relevant nociceptive system would allow for discrete, peptidase regulation of BK signaling. Western blot analysis of crude subcellular fractions and lipid raft preparations of cultured rat trigeminal ganglia demonstrate similar expression profiles between EP24.15/16 and B2R on a subcellular level. Furthermore, the treatment of primary cultures of trigeminal ganglia with inhibitors of EP24.15/16 led to the potentiation of several bradykinin-induced events that occur downstream of B2R activation. EP24.15/16 inhibition by N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-AlalTyr-p-aminobenzoate (cFP) resulted in a 1000-fold increase in B2R sensitivity to BK as measured by inositol phosphate accumulation. In addition, cFP treatment resulted in a 31.1+/-5.0% potentiation of the ability of BK to inhibit protein kinase B (Akt) activity. Taken together, these data demonstrate that EP24.15/16 modulate intracellular, peptidergic signaling cascades through B2R in a physiologically relevant nociceptive system.