Carbachol and Bradykinin Increase the Production of Diacylglycerol from Sources Other than Inositol-Containing Phospholipids in PC12 Cells

Both carbachol and bradykinin increased diacylglycerol formation in PC12 pheochromocytoma cells. The effect of carbachol was apparent only in cells that had been treated with nerve growth factor. Incubation of the cells in Ca2(+)-free medium attenuated carbachol-stimulated diacylglycerol formation but did not reduce the response to bradykinin. Pretreatment of the cells with pertussis toxin did not affect either carbachol- or bradykinin-stimulated diacylglycerol formation; therefore, the inhibitory guanine nucleotide Gi probably does not mediate this response. The time course of carbachol-stimulated diacylglycerol accumulation did not coincide with the time course of inositol 1,4,5-trisphosphate (IP3) production. IP3 was elevated at the earliest time measured, 15 s, and then slowly declined so that by 5 min IP3 levels were only 50% of maximal. Diacylglycerol levels, in contrast, were not elevated for the first 2 min and then peaked at 5 min. These data indicate that hydrolysis of phosphatidylinositol 4,5-bisphosphate was not the major source of the diacylglycerol peak at 5 min. To investigate the source of diacylglycerol, I examined the fatty acid composition of the diacylglycerol by prelabeling the cells with [3H]palmitic acid and [14C]stearic acid. The 14C/3H ratio in diacylglycerol should reflect the phospholipid(s) from which it is derived. The 14C/3H ratio of the increment in diacylglycerol produced by carbachol and bradykinin was intermediate between the 14C/3H ratios of phosphatidylcholine and phosphatidylinositol. The 14C/3H ratio in triacylglycerol was similar to that of phosphatidylcholine. These data indicate that carbachol and bradykinin stimulate the formation of diacylglycerol from sources other than inositol-containing phospholipids; phosphatidylcholine and triacylglycerol are two possible sources of this diacylglycerol.     

瘦素上调动脉内皮细胞环氧合酶-2 及其产物的实验研究

目的:研究瘦素对大鼠主动脉内皮细胞(RAECs)环氧化酶-2(COX-2)及其下游产物前列环素和血栓素A2表达的影响。方法:酶消化法分离RAECs;免疫荧光检测细胞Ⅷ因子的表达;实时荧光定量PCR检测不同浓度瘦素(10~(-10)M、10~(-9)M、10~(-8)M)和时间(36h、48 h)处理后对RAECs COX-2 mRNA表达的影响;Western blot检测各组COX-2蛋白的改变;酶联免疫吸附测定(ELISA)检测各组细胞上清液中6-酮前列腺素F1α(6-Keto PGF1α)和血栓素B2(TXB2)的水平。结果:酶消化法6天后见细胞呈长梭形或多边形,培养10天后细胞呈典型的"铺路石"样改变,免疫荧光检测发现Ⅷ因子在胞浆中特异性表达,内皮细胞纯度达90%。与对照组(PBS处理)相比较,瘦素处理后,各组大鼠主动脉内皮细胞COX-2 m RNA及蛋白表达量均有升高趋势,两者表达量均随浓度增加和处理时间延长而升高(P0.05)。而6-Keto PGF1α水平升高及TXB2/6-Keto PGF1α比例降低仅见于相对高浓度瘦素(10-9、10-8M)处理后(P0.05),但瘦素对RAECs TXB2表达无影响(P0.05)。结论:瘦素增加RAECs炎性标志物COX-2及其下游舒张血管产物6-Keto PGF1α的表达,降低TXB2(收缩血管物质)/6-Keto PGF1α比例。COX-2可能在瘦素介导的高血压发病中起重要作用,但因其下游产物则有利于血管舒张,故选择性COX-2抑制剂可能会增加高血压风险。     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction Between TRPC Channel Subunits in Endothelial Cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

两种不同内皮祖细胞的分离培养与鉴定

目的:探讨从小鼠骨髓中分离、培养、诱导分化及鉴定两种内皮祖细胞的方法,为进一步研究和临床应用奠定基础。方法:密度梯度离心法分离小鼠骨髓单个核细胞,接种于内皮祖细胞条件培养基,通过贴壁培养法培养出早期内皮祖细胞和晚期内皮祖细胞,并在0 d、6 d、10 d流式鉴定早期内皮祖细胞,在第8周流式鉴定晚期内皮祖细胞。结果:通过体外贴壁扩增培养,从小鼠骨髓细胞中成功培养出EEPC(早期内皮祖细胞)和EOC(晚期内皮祖细胞),表达CD34+/CD133+/VEGFR2+的EEPC比例从最初的0.08%能够增长至70%;EOC大约出现于3-4周,5-8周时呈现指数增长,具有典型的内皮细胞鹅卵石样形态,表达CD31、VEGFR2等内皮细胞表面标志而不表达CD34、CD133等干细胞表面标志。结论:确立了内皮祖细胞体外分离培养和诱导分化的实验方法,为进一步研究奠定基础。     

内皮祖细胞的分离培养与鉴定

内皮祖细胞的分离方法有免疫磁珠分离法、淋巴细胞分离液分离法(1.077)和差速贴壁法,这3种方法已被人们广泛使用,均可分离到一定的目的细胞。分离到的目的细胞在培养过程中逐渐分化、成熟、发育为内皮细胞。在内皮细胞和内皮祖细胞的鉴别区分,使用CD34+/CD133+/KDR+鉴定为内皮祖细胞,同时使用内皮祖细胞吞噬D il-ac-LDLFITC-UEA双阳性的方法也可鉴定为内皮祖细胞。     

Biosynthesis of Prostacyclin in Rat Cerebral Microvessels and the Choroid Plexus

Abstract: Microvessels, predominantly capillaries, were isolated from rat cerebrum by a modification of published procedures. The morphology and purity of the preparations were monitored by light and electron microscopy and by enrichment in alkaline phosphatase, γ-glutamyl transpeptidase, and prostacyclin synthetase. A reversed-phase high-pressure liquid chromatographic method was used in the purification of prostaglandins after extraction from aqueous incubation solutions. Prostacyclin synthesis in brain is localized in cerebral blood vessels and capillaries. The endogenous biosynthetic capacity of the isolated cerebral capillary fractions for prostacyclin, measured as its chemically stable breakdown product, 6-keto-prostaglandin F_1α, was 11 ng/mg protein/10 min. Choroid plexus and intact surface vessels synthesized 6-keto-prostaglandin F_1α at 37 and 35 ng/mg protein/10 min, respectively. The prostacyclin-synthesizing enzyme of the cerebral capillaries also converted the exogenously added prostaglandin endoperoxides to 6-keto-prostaglandin F_1α. Comparison of the synthesis of prostaglandins 6-keto-F_1α, E₂, and F_2α showed that 6-keto-prostaglandin F_1α was the major prostaglandin formed in the microvessels, in the larger surface vessels, and in the choroid plexus. Prostaglandin D₂ was not detected. Prostacyclin synthesis by the cerebral vasculature is similar to that in other blood vessels and cultured human endothelial cells. Possible physiological roles of prostacyclin in the cerebral microvasculature are discussed with special regard to the autoregulation of cerebral blood flow.     

Calcium/Calmodulin-Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that α-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.     

血管内皮祖细胞与糖尿病微血管病变研究进展

糖尿病微血管病变严重影响了患者生活质量,是患者致死致残主要原因。微血管病变主要表现在视网膜、肾、神经、心肌组织。微血管病变的机制尚未完全清楚,近年越来越多研究发现血管内皮祖细胞(endothelial progenitor cells,EPCs)是该病发病重要原因。EPCs有分化为成熟的内皮细胞并且参与新血管形成和新生的能力。正常情况下内皮损失和EPCs对内皮的修复作用处于动态平衡状态,一旦EPCs受损,内皮损害和修复之间的平衡被打破,内皮层的完整性遭到破坏,必然参与糖尿病血管病变的发生发展。国内外大量研究证明糖尿病合并大血管病变EPCs数目功能改变,而糖尿病合并微血管病变EPCs的怎样变化?本文就EPCs与糖尿病微血管病变的关系进行系统综述。     

内皮祖细胞的生物学特性及其临床应用前景

内皮祖细胞（Endothelial Progenitor Cells,EPCs）是内皮细胞（endothelial cells,ECs）的前体细胞,即能分化为成熟ECs的祖细胞,它在血管内皮再生中发挥着重要作用。随着EPCs研究的深入,其在临床诊断、预后判断和各种缺血性疾病的治疗方面将会有广阔的应用前景。然而,关于EPCs的定义、来源、表面标记以及培养鉴定方法目前仍存在争议。     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Generation of Hematopoietic Stem Cells from Purified Embryonic Endothelial Cells by a Simple and Efficient Strategy

Recent progress by versatile approaches supports the new hypothesis that multi-potent hematopoietic stein cells （HSCs） are directly formed from a rare population of endothelial cells in mid-gestation mouse embryos. This process is therefore known as the endothelial-to- hematopoietic transition （EHT）. Nevertheless, there is no functional evidence that documents the HSC transition from purified endothelial cells. In this study, we developed an OP9-DLl-based co-culture system that was able to facilitate the HSC specification and/or expansion in vitro of mouse embryonic day 10.5 （El0.5） Tie2~ cells remarkably. Then, the immunophenotypically defined endothelial ceils were harvested by a combination of surface markers （Flkl＋CD31 ~CD41 CD45 Ter119 ） from the caudal half of EI0.0-EI 1.0 mouse embryos. The transplantation of the endothelia/OP9-DL1 co-cultures led to long-term, high-level, multi-lineage, and multi-organ he- matopoietic reconstitution in the irradiated adult recipients. The induced HSC activity was initially observed at El0.5, and a significant increase was detected at El 1.0, which suggests a temporally specific regulation. Taken together, tbr the first time, we provide functional evidence showing the HSC potential of purified embryonic endothelial cells, which is indispensable for the emerging EHT concept. Moreover, the newly defined co-culture system will aid the exploration of the key molecules governing the HSC transition from embryonic and even postnatal endothelial cells, which has enormous significance in basic and translational research.     

内皮祖细胞在支架术后再内皮化中应用的研究进展

经皮冠状动脉介入治疗的应用改善了冠心病患者的临床症状及预后,但现在困扰人们的问题是作为其术后并发症之一的支架内再狭窄发病率仍然很高。大量的研究证实,内膜增生在支架内再狭窄的形成中起主导作用,所以提高受损内膜再内皮化的速度是防止支架内再狭窄的一个重要措施。新近的研究表明,内皮祖细胞能参与损伤后血管内皮修复,促进受损血管内膜的再内皮化,因此,在防止支架内再狭窄中将得到进一步的研究与应用。因此,本文就内皮祖细胞在支架术后再内皮化中应用的研究进展做一综述。     

氧化应激诱导内皮细胞凋亡microRNA表达改变的研究

目的:研究氧化应激诱导的内皮细胞micro RNA的表达变化。方法:ECM(Endothelial Cell Medium)培养人脐静脉内皮细胞,利用不同浓度双氧水(0μmol/L,200μmol/L,500μmol/L,800μmol/L)刺激24小时后应用流式细胞术检测其凋亡水平。提取细胞总RNA,利用实时定量PCR(Quantitive real-time PCR;q RT-PCR)检测micro RNA表达量变化,并利用生物信息学软件预测可能的靶基因。结果:加入不同浓度双氧水处理24 h后的内皮细胞总凋亡率均显著高于对照组,200μmol/L、500μmol/L和800μmol/L组的凋亡率分别为(13.31%vs 4.75%,35.9%vs 4.75%,89.75%vs 4.75%,P0.01)。200μmol/L的双氧水处理内皮细胞后,micro RNA的表达出现了明显的改变。其中mi R-92a、mi R-126的表达明显下调(P0.05),mi R-181a、mi R-217、mi R-34a和mi R-320的表达明显上调(P0.05)。靶基因预测显示mi R-320、mi R-92a可能调控多个和内皮细胞凋亡相关的基因表达。结论:在氧化应激诱导的内皮细胞凋亡中,mi RNA表达发生改变并可能参与调控内皮细胞功能。     

Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons

Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo-[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 microM bradykinin for time intervals of 5-300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion-exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10-fold increased accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl-monoacylglycerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyceride suggested the involvement of di- and monoglyceride lipases in arachidonic acid mobilization. A role for phospholipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin-stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 microM), or EDTA (2.5 mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 microM).     

Stathmin 在微血管内皮细胞的表达与胶质瘤恶性程度关系

目的:检测Stathmin在正常脑组织及不同级别胶质瘤微血管内皮细胞中的表达情况。方法:利用结合CD105单克隆抗体的免疫磁珠内皮细胞分选系统特异性分选出68例胶质瘤微血管内皮细胞(其中低级别胶质瘤(WHO分级Ⅰ-Ⅱ)24例,高级别胶质瘤(WHO分级Ⅲ-Ⅳ)44例)和20例正常脑组织微血管内皮细胞。应用免疫组化、RT-PCR和Western blot检测Stathmin在胶质瘤微血管内皮细胞和正常脑组织微血管内皮细胞中的表达。结果:免疫组化证实Stathmin在正常脑组织微血管内皮细胞、低级别胶质瘤微血管内皮细胞和高级别胶质瘤微血管内皮细胞的表达百分率分别是20%,66%和95%(P<0.05)。RT-PCR和Western blot法检测显示,Stathmin在胶质瘤微血管内皮细胞中的表达明显增高。低级别胶质瘤组、高级别胶质瘤组分别与正常组比较,均有显著性差异(P<0.01);且低级别胶质瘤组与高级别胶质瘤组比较,有显著性差异(P<0.01),随着胶质瘤恶性程度的增加,Stathmin表达上调,具有统计学意义。结论:Stathmin在脑胶质瘤微血管内皮细胞中表达随肿瘤恶性程度增高而增加,可能为脑胶质瘤的生物治疗提供一个新靶点。     

Direct Cytotoxicity of Ethylcholine Mustard Aziridinium in Cerebral Microvascular Endothelial Cells

Abstract: The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibitor of choline uptake in brain synaptosomes and is used as a neurotoxin to produce animal models of cholinergic hypofunction. However, previous studies have shown that intraocular administration of AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of lactate dehydrogenase activity in the culture medium, AF64A produced similar concentration-dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human cholinergic neuroblastoma cell line SK-N-MC, but not in bovine cerebral smooth muscle cells. The toxic effect of AF64A correlated well with the affinity of the choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell choline carrier, as demonstrated by the following observations: (a) AF64A inhibited [³H]choline uptake in a concentration-dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of choline or hemicholinium-3 to the culture medium prevented the AF64A-induced toxicity in endothelial cell cultures.     

内皮祖细胞分离培养及应用技术新进展

内皮祖细胞(Endothelial progenitor cells,EPCs)是能够增殖、迁移、粘附并分化为血管内皮细胞潜能的一种原始细胞,在修复血管内皮和促进血管生成中具有重要的作用。EPC来源于骨髓,存在于骨髓、外周血、脐带血,新研究表明在脂肪组织、心肌中也能发现EPC的存在。EPC与干细胞的细胞表面标志物相似,功能上亦接近干细胞,但不具有自我更新的功能。近年来EPC已成为热点问题,对疾病诊断,预后判断和靶向治疗方面发挥重要作用,在冠状动脉粥样硬化性疾病、糖尿病血管病变、恶性肿瘤等治疗中全身或局部注射EPC具有更广泛的前景和应用价值。但关于分离培养EPC的方法及细胞表面标志物不完全相同,报道较少,至今尚没有形成统一的标准,本文就对于内皮祖细胞基本现状、分离培养技术、分选鉴定及临床应用方面做一综述。     

过氧化氢对内皮祖细胞凋亡相关蛋白表达的影响

目的:比较不同浓度过氧化氢(hydrogen peroxide,H2O2)对人外周血来源的内皮祖细胞(endothelial progenitor cells,EPCs)生存能力的改变及其对凋亡相关蛋白表达的影响。方法:采用密度梯度离心法和差速贴壁法从人外周静脉血中分离培养内皮祖细胞。选取传代后第3代EPCs作为研究对象,以终浓度分别为50μmol/L、100μmol/L、150μmol/L、200μmol/L、300μmol/L和400μmol/L的过氧化氢处理内皮祖细胞12 h,同时设立正常处理对照组。CCK-8法检测各组内皮祖细胞生存能力的差异;Western blot分析各组内皮祖细胞中凋亡相关蛋白Bax、Bcl-2和p53的蛋白表达情况。结果:与对照组相比,在浓度为50μmol/L、100μmol/L和150μmol/L过氧化氢处理组中细胞存活能力逐渐增强,促凋亡蛋白Bax、p53随之下调,抗凋亡蛋白Bcl-2则显著上调;在浓度为200μmol/L、300μmol/L和400μmol/L过氧化氢处理组中细胞生存能力相对于正常对照组逐渐减弱,促凋亡蛋白Bax、p53表达水平逐渐增加,抗凋亡蛋白Bcl-2表达水平下降。结论:过氧化氢对内皮祖细胞存活能力和凋亡相关蛋白Bax、Bcl-2、p53表达的影响均呈双相性变化。