NF-κB和MAPK信号通路参与金雀异黄酮诱导乳腺癌MDA-MB-231细胞凋亡的体外研究

目的探讨金雀异黄酮(genistein)诱导人乳腺癌MDA-MB-231细胞凋亡的可能机制。方法采用MTT法观察金雀异黄酮对乳腺癌MDA-MB-231细胞增殖的抑制作用;集落形成法观察金雀异黄酮对MDA-MB-231细胞集落形成能力的影响;金雀异黄酮干预MDA-MB-231细胞36 h后,Western blot检测凋亡相关蛋白Bcl-2、Bax、caspase-3及NF-κB、ERK、p-ERK、JNK、p-JNK蛋白的表达。结果 MTT结果显示,金雀异黄酮呈时间、浓度依赖性抑制乳腺癌MDAMB-231细胞增殖;集落形成实验结果显示,金雀异黄酮能明显抑制乳腺癌MDA-MB-231细胞的集落形成(P<0.05);Western blot结果显示,金雀异黄酮干预乳腺癌MDA-MB-231细胞36 h后,与对照组相比,Bcl-2、NF-κB、p-ERK蛋白表达水平明显下调(P<0.05),Bax、caspase-3、p-JNK蛋白表达水平明显上调(P<0.05)。结论金雀异黄酮能抑制乳腺癌MDA-MB-231细胞生长,且能诱导其凋亡,其机制可能与抑制NF-κB、ERK,激活JNK信号转导通路有关。     

迷迭香酸衍生物RAD-9通过PI3K/Akt和p38 MAPK信号通路诱导胃癌MGC-803细胞凋亡

目的研究迷迭香酸衍生物RAD-9诱导胃癌MGC-803细胞凋亡的作用及其机制。方法 MTT法观察RAD-9对胃癌MGC-803细胞增殖的抑制作用;流式细胞术检测细胞的凋亡;Hoechst 33258染色法观察RAD-9对MGC-803细胞核凋亡形态学的影响;Western blot检测RAD-9干预MGC-803细胞36 h后,对Akt、p-Akt、p38 MAPK、p-p38 MAPK蛋白及凋亡相关蛋白Bcl-2、Bax、caspase-3的影响。结果MTT结果显示,RAD-9呈时间、浓度依赖性抑制胃癌MGC-803细胞增殖;流式细胞术结果显示,RAD-9对胃癌MGC-803细胞有明显的促凋亡作用(P<0.01);Hoechst 33258染色实验结果显示,RAD-9干预胃癌MGC-803细胞36 h后,细胞核呈现典型凋亡形态学改变;Western blot结果显示,RAD-9干预胃癌MGC-803细胞36 h后,Bcl-2蛋白表达水平明显降低,Bax、caspase-3蛋白表达水平明显提高,Akt、p-Akt蛋白表达水平明显下调,p38 MAPK、p-p38 MAPK蛋白表达水平明显上调(P<0.01)。结论 RAD-9能抑制胃癌MGC-803细胞生长,且能诱导其凋亡,其机制可能与抑制PI3K/Akt和激活p38 MAPK信号通路相关。     

亚砷酸钠抑制PI3K/Akt 信号通路促进乳腺癌细胞凋亡的实验研究

目的　探讨亚砷酸钠促进乳腺癌细胞凋亡的作用及机制。方法　用不同浓度的亚砷酸钠分别作用于乳腺癌细胞（MCF-1/MDA-MB-231）48 h，MTT 法检测细胞增殖情况；用4 μg/mL的亚砷酸钠作用于乳腺癌细胞6 h、12 h、24 h、48 h 和72 h 后，MTT 法检测细胞增殖情况；同时采用流式细胞术检测细胞凋亡情况，Western blot 检测PI3K、p-PI3K、Akt 及p-Akt 的表达水平。结果　0.5 μg/mL、1 μg/mL、2 μg/mL、4 μg/mL、8 μg/mL亚砷酸钠作用48 h 后，MCF-1、MDAMB-231 细胞存活率均明显低于对照组（P<0.05）。MCF-1、MDA-MB-231 细胞经亚砷酸钠作用后细胞凋亡率明显高于对照组（P<0.01）。亚砷酸钠作用后的乳腺癌细胞中PI3K、Akt 的表达水平与对照组比较无显著差异（P>0.05），而p-PI3K、p-Akt的表达水平明显低于对照组（P<0.01）。结论　亚砷酸钠可抑制乳腺癌细胞增殖，且其效应随作用时间的增加而增强；亚砷酸钠可促进乳腺癌细胞凋亡，其作用机制可能与抑制PI3K/Akt 信号通路的激活有关。     

Gab2通过PI3K/Akt/ARK5/MMP途径影响乳腺癌的侵袭和转移

目的探讨Gab2在乳腺浸润性导管癌侵袭和转移中的分子机制,为临床预防乳腺癌的侵袭和转移提供理论依据。方法采用免疫组织化学SP法检测80例乳腺浸润性导管癌组织及癌旁相对正常组织(>5 cm)中Gab2蛋白表达情况,并分析其表达与乳腺浸润性导管癌临床病理参数的相关性,分析浸润性导管癌中Gab2、MMP-2及MMP-9蛋白表达的相关性。采用Western blot检测乳腺癌细胞系MCF-7、MDA-MB-231中Gab2蛋白的表达情况。采用RNAi技术将小RNA干扰质粒瞬时转染乳腺癌细胞系MDA-MB-231,应用Western blot检测转染成功后各组细胞中MMP-2和MMP-9蛋白的表达情况,应用Transwell体外侵袭实验检测各组细胞的侵袭性,用EGF刺激细胞后Western blot检测Akt及ARK5的磷酸化情况。结果浸润性导管癌组织中Gab2蛋白的阳性表达率明显高于癌旁正常组织(P<0.01),浸润性导管癌中Gab2蛋白的表达与ER、组织学分期及淋巴结转移密切相关(P<0.05),且其表达与MMP-2及MMP-9蛋白的表达呈正相关;MDA-MB-231细胞系中Gab2蛋白表达量高于MCF-7细胞系中表达量;转染干扰质粒24 h后,与转染空载细胞组相比,SiG ab2/MDA-MB-231细胞组中Gab2蛋白的表达量明显降低,结果显示转染成功。同时,SiG ab2/MDA-MB-231细胞组穿过Transwell小室人工基膜数量明显减少(P<0.05),并伴有MMP-2、MMP-9蛋白表达降低(P<0.05)。用EGF刺激各组细胞,结果显示:在SiG ab2/MDAMB-231细胞组Akt及ARK5的磷酸化明显受到抑制。结论Gab2通过PI3K/Akt/ARK5信号通路影响MMP-2、MMP-9的表达从而促进乳腺癌的侵袭与转移。     

冬凌草甲素通过PI3K/Akt通路诱导MDA-MB-231细胞的凋亡

目的：研究冬凌草甲素对人乳腺癌MDA-MB-231细胞增殖产生的影响,初步探讨其作用机理。方法：体外培养人乳腺癌MDA-MB-231细胞,采用6、12、24μmol/L冬凌草甲素对其进行处理,采用倒置显微镜进行细胞形态学观察,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blotting检测凋亡相关蛋白procaspase-3、PARP及Akt、p-Akt、p-GSK3β表达的变化。结果：冬凌草甲素作用MDA-MB-231细胞24h后,可观察到细胞凋亡的形态学改变,以24μmol/L组最为明显。实验组与对照组相比,细胞存活率显著降低、凋亡率显著升高（P〈0.01）,具有时间和剂量依赖性,凋亡相关蛋白procaspase-3下调,caspase-3底物PARP被逐步剪切,并伴随p-Akt、p-GSK3β蛋白水平下调（P〈0.05）。结论：冬凌草甲素可有效抑制人乳腺癌MDA-MB-231细胞的增殖,诱导其凋亡,机制与PI3K/Akt通路的抑制有关。     

乳腺癌细胞培养上清液促进人脂肪间充质干细胞迁移和增殖的机制探讨

目的 探讨MDA-MB-231乳腺癌细胞培养上清液对人脂肪间充质干细胞（hAdMSC）迁移、增殖和凋亡的影响及分子机制。方法 将MDA-MB-231细胞上清液和不含胎牛血清的RPMI-1640培养基以1∶4的体积比混匀后培养的hAdMSC为MDA-MB-231上清液组。向MDA-MB-231上清液组添加10μmol/L Reparixin(CXCR1/2抑制剂)为CXCR1/2抑制剂组;向MDA-MB-231上清液组添加10 nmol/L GSK690693(Akt抑制剂)为Akt抑制剂组。无任何刺激进行培养的hAdMSC为对照组。细胞划痕实验检测各组hAdMSC的迁移能力,CCK-8实验检测各组hAdMSC增殖情况,Annexin V-FITC/PI双标记流式细胞凋亡实验检测各组hAdMSC凋亡率,Western blot检测各组hAdMSC的mTOR/磷酸化mTOR(p-mTOR)和Akt/磷酸化Akt(p-Akt)蛋白表达。结果 与对照组相比,MDA-MB-231上清液组hAdMSC的24 h和48 h细胞划痕闭合面积、细胞增殖水平以及p-Akt和p-mTOR蛋白相对表达量均增加...     

胡桃醌衍生物MAM抑制人乳腺癌MDA-MB-231细胞增殖的体外研究

目的　探讨虎杖提取物2- 甲氧基-6- 乙酰基-7- 甲基胡桃醌（2-Methoxy-6-acetyl-7-methyljuglone,MAM）对人乳腺癌细胞MAD-MB-231 体外增殖的影响及可能机制。方法　体外培养正常人肝细胞LO2、人乳腺癌MDA-MB-231 和MDA-MB-468 细胞,采用Cell Counting Kit-8（CCK8）法检测MAM 对细胞增殖的影响,采用Hoechst33258 染色检测MAM 对MDA-MB-231 细胞凋亡的影响,采用流式细胞术检测MAM 对MDA-MB-231 细胞周期的影响,western blot 分析相关蛋白表达的变化。结果　MAM 对人乳腺癌MDA-MB-231 细胞生长有较强的抑制作用,呈剂量和时间依赖性。MAM 作用24 h 可诱导MDA-MB-231 细胞发生典型的凋亡形态学改变,抑制MDAMB-231 细胞周期在G2/M 期,同时显著下调细胞内Cdk1 和CyclinB1 蛋白的表达、上调p-Cdk1（Thr14）的蛋白表达水平（P<0.05）。结论　MAM 能够通过抑制Cdk1-CyclinB1 复合物的形成,使细胞周期阻滞在G2/M 期,干扰细胞周期进程,从而抑制MDA-MB-231 细胞的增殖。     

EGCG对三阴乳腺癌细胞MDA-MB-231的增殖抑制作用及机制

目的探讨表没食子儿茶素没食子酸酯[(-)-epigallo-catechin-3-gallate,EGCG]对三阴乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其可能作用机制。方法通过CCK-8实验观察不同浓度EGCG(10、20、40、80、160 mg.L-1)对MDA-MB-231细胞增殖的影响;Hoechst33258染色法观察EGCG对MDA-MB-231细胞凋亡的影响;JC-1法测定细胞线粒体膜电位;caspase-3活性检测试剂盒检测caspase-3活性的变化;Western blot法检测葡萄糖调节蛋白78(glucose reg-ulatd protein 78,GRP78)和caspase-3蛋白表达变化。结果EGCG能明显抑制MDA-MB-231细胞增殖,且随EGCG浓度的增加和作用时间的延长而增强,EGCG作用MDA-MB-231细胞12、24、48 h的IC50分别为69.1、40.4、29.4 mg.L-1。EGCG作用MDA-MB-231细胞24 h后,细胞出现体积变小、染色质聚集、细胞核边缘化等典型的凋亡形态学改变,且随EGCG浓度的增加,MDA-MB-231细胞凋亡率逐渐增加,线粒体膜电位明显降低,caspase-3活性明显增强,West-ern blot结果显示EGCG可抑制GRP78蛋白表达,增强活性caspase-3蛋白表达。结论 EGCG能够促进MDA-MB-231凋亡,其机制可能与内质网应激(Endoplasmic reticulumstress,ERS)引起的caspase-3激活有关。     

迷迭香酸类似物-11通过抑制ERK/MAPK通路诱导人胃癌MGC-803细胞凋亡

目的对迷迭香酸类似物-11(rosmarinic acid analogue-11,RAA-11)诱导人胃癌MGC-803细胞凋亡及其机制进行初步探究。方法 MTT法和克隆形成法观察RAA-11对人胃癌MGC-803细胞增殖的抑制作用;Hoechst 33258染色法观察RAA-11对人胃癌MGC-803细胞核凋亡形态学的影响;流式细胞术检测RAA-11对人胃癌MGC-803细胞凋亡率的影响;Western blot观察RAA-11对人胃癌MGC-803细胞凋亡相关蛋白caspase-3、Bcl-2、Bax及通路蛋白ERK、p-ERK表达的影响。结果 MTT结果提示RAA-11可以抑制人胃癌MGC-803细胞增殖(P<0.01),且呈时间浓度依赖性;克隆形成实验结果提示,RAA-11能抑制人胃癌MGC-803细胞的集落形成能力;Hoechst 33258染色结果提示,RAA-11干预人胃癌MGC-803细胞48 h后,细胞核呈现典型凋亡形态学改变;流式细胞术结果提示,RAA-11对人胃癌MGC-803细胞有明显的促凋亡作用; Western blot结果显示,RAA-11上调人胃癌MGC-803细胞促凋亡相关蛋白caspase-3、Bax(P<0.01),下调抑凋亡相关蛋白Bcl-2的表达水平,并降低了通路蛋白ERK、p-ERK的表达水平(P<0.01)。结论 RAA-11能抑制人胃癌MGC-803细胞的增殖,并能够诱导凋亡,其机制可能与抑制ERK/MAPK通路有关。     

磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶信号通路参与核因子-κB受体活化因子配体诱导的MDA-MB-231乳腺癌细胞迁移

目的探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)信号通路在核因子-κB受体活化因子配体(RANKL)诱导的乳腺癌细胞迁移中的作用。方法 Transwell法测定RANKL刺激后MDA-MB-231细胞迁移能力的改变。蛋白印迹法检测MDA-MB-231细胞表面RANK蛋白的表达及RANKL刺激后pAkt及Akt的表达;检测数据用x珚±s表示,采用SPSS 16.0软件进行t检验。结果 MDA-MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强,RANKL的圈套受体骨保护素(OPG)可阻断RANKL诱导的细胞迁移(P<0.01)。RANKL刺激后MDA-MB-231细胞p-Akt表达升高,PI3K抑制剂LY294002抑制RANKL诱导的细胞迁移(P<0.01)。结论 PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞MDA-MB-231迁移。     

Cerebral metabolism of [3H]-p-chloroamphetamine

The time-dependent metabolism of intraventricularly administered $\text{[}{}^3\text{H}\text{]-p-}\text{chloroamphetamine}$ was followed. The parent compound and its metabolites were recovered by high pressure liquid chromatography and characterized by high pressure liquid chromatography, thin-layer chromatography, and gas chromatography-mass spectrometry. By 4 hr after injection, two major toluene-soluble metabolites were present in brain. Their biological half-lives were different from the parent compound. On the basis of their analyses, one of the metabolites is p-chloronorephedrine, the other (P₃) is as yet unidentified. Pretreatment with Lilly 110140 prevented or markedly reduced the synthesis of both p-chloronorephedrine and P₃. Iprindole prevented the synthesis of p-chloronorephedrine. The P₃ appeared first in the brain then in the liver, suggesting that both of these organs can metabolize p-chloroamphetamine to this compound. The metabolites were recovered primarily from the nuclear and microsomal fractions following subcellular fractionation of the brain, with small quantities present in the synaptosomal fraction. The level of metabolites was higher in the brainstem than in the neocortex. Glutathione, administered simultaneously with p-chloroamphetamine either intraventricularly or intraperitoneally failed to alter the toxicity of p-chloroamphetamine.     

Clevudine University of Georgia/Abbott/Bukwang/Triangle/Yale University

The pyrimidine analog, clevudine (L-FMAU: 2'-fluoro-5-methyl-beta-L-arabinofuranosyluridine) is a potent antihepatitis B virus (HBV) and anti-Epstein-Barr virus (EBV) agent, discovered by researchers at the University of Georgia, in collaboration with Yale University and Bukwang. Bukwang transferred its technology to Triangle Pharmaceuticals in 1998 together with a license to develop clevudine worldwide except Korea [279649], [281942]. In June 1999, Triangle and Abbott Laboratories entered into a strategic alliance to copromote antiviral products including L-FMAU [326798]. In September 2000, Triangle Pharmaceuticals Inc initiated a 30-day phase I/II evaluation of clevudine in HBV-infected patients [381755]. Clevudine is a much less toxic derivative of the toxic agent P-D-FMAU. The mechanism of action of clevudine is not yet clear, but the agent induces a rapid decrease in HBV nucleic acid as doses increase from 0.3 to 10 mg/kg [319145]. It is believed that the target for clevudine lies in the viral replication mechanism. Clevudine is phosphorylated to the triphosphate form intracellularly. This is removed slowly from the cells, thus exerting a sustained inhibitory antiviral activity [178173], [320720], [320721].     

Spotlight from the American Society for Preventive Cardiology on Key Features of the 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guidelines on the Management of Blood Cholesterol

The 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol retains focus on recommendations for statin treatment in the original four statin-eligible groups [those with atherosclerotic cardiovascular disease (ASCVD), diabetes, low-density lipoprotein cholesterol (LDL-C) ≥ 190 mg/dL, and higher risk primary prevention] without the use of treatment initiation or target LDL-C levels from the earlier 2013 American College of Cardiology/American Heart Association (ACC/AHA) guideline, but has several new features. First, patients with primary prevention are divided into those who are at low (< 5%), borderline (5% to < 7.5%), intermediate (7.5% to < 20%), and high (≥ 20%) risk based on the ASCVD risk estimator. Moreover, the new guideline goes further to consider a wider range of factors [now called “risk enhancers”—premature family history of ASCVD, persistently high LDL-C, chronic kidney disease (CKD), metabolic syndrome, conditions specific to women, inflammatory diseases, and high-risk ethnicities] that can be used to better inform the treatment decision. Moreover, more detailed recommendations on how the results of coronary calcium scanning can be used to inform the treatment decision are provided, including how it may be used to “de-risk” certain patients for delaying or avoiding the use of statin therapy. There are also specific sections for cholesterol management in other patient subgroups including women, children, certain ethnic groups, those with CKD, chronic inflammatory disorders and HIV, as well as discussion on the management of hypertriglyceridemia. Importantly, for persons with known ASCVD, a distinction is made for those who are at “very high risk” based on having had two major ASCVD events or one major event and two or more other high risk conditions, such as diabetes or other major risk factors, or bypass surgery or percutaneous intervention. Finally, the concept of a threshold LDL-C for initiating a non-statin therapy (after considering highest tolerated statin dosage) is provided, with ezetimibe recommended as the key non-statin to be added if the LDL-C still remains ≥ 70 mg/dL for all ASCVD patients, and in those who are at “very high risk”, further consideration for using a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. While the new guideline does have greater detail (and arguably, complexity), the refinements provide a strategy for guiding the clinician to target both statin and non-statin therapy to those most likely to derive benefit.     

Pitavastatin Nissan/Kowa Yakuhin/Novartis/Sankyo

Pitavastatin (nisvastatin) is an HMG CoA reductase inhibitor being developed jointly by Nissan, Kowa Kogyo, Novartis and Sankyo for the potential treatment of atherosclerosis and hyperlipidemia.     

Amlodipine/Valsartan/Hydrochlorothiazide

Amlodipine/valsartan/hydrochlorothiazide (HCTZ) is a fixed-dose combination of the well established antihypertensive agents amlodipine (a calcium channel antagonist), valsartan (an angiotensin II receptor antagonist), and HCTZ (a thiazide diuretic). In patients with moderate or severe hypertension, triple combination therapy with amlodipine + valsartan + HCTZ produced significantly greater reductions from baseline in mean sitting systolic and diastolic BP (msSBP and msDBP) than either valsartan + HCTZ, amlodipine + HCTZ, or amlodipine + valsartan in a large, 8-week, randomized, double-blind, multinational, phase III trial. Furthermore, the proportion of patients achieving overall BP control at endpoint was significantly greater with the triple combination regimen than with any of the dual regimens, with significantly more triple combination recipients achieving msSBP and msDBP control at each assessment throughout the trial. Subgroup analyses of this study suggested that amlodipine + valsartan + HCTZ was generally more effective in reducing BP and providing overall BP control than the dual combination therapies, irrespective of age, race, gender, ethnicity, or hypertension severity. Several smaller studies provide data that support the efficacy of amlodipine + valsartan + HCTZ in patients whose BP is inadequately controlled with amlodipine + valsartan, amlodipine + HCTZ, or valsartan + HCTZ dual combination therapy. Treatment with amlodipine + valsartan + HCTZ for up to 8 weeks was generally well tolerated in the large, phase III trial, with most adverse events being transient and of mild to moderate severity.     

N-Nitroso-N-Methyldodecylamine and N-Nitroso-N-Methyltetradecylamine in household dishwashing liquids

Eleven household dishwashing liquids and four household surface cleaners were analysed for N-nitroso-N-methyldodecylamine and N-nitroso-N-methyltetradecylamine by gas chromatography with detection using a Thermal Energy Analyzer. Both nitrosamines were found in three of the dishwashing detergents and one of the surface cleaners. [1-¹⁴C]-N-Nitroso-N-methyldodecylamine was used to determine recoveries, which were between 65 and 88%. Levels of N-nitroso-N-methyldodecylamine ranged from 112 to 661 ppb and those of N-nitroso-N-methyltetradecylamine from 46 to 151 ppb. A simple method was developed to screen the products for N,N-dimethyldodecylamine-N-oxide, a surfactant ingredient suspected of being the source of these nitrosamines. By application of this method it was established that all of the products formulated with this amine oxide contained these two nitrosamines, whereas in products that did not contain this ingredient, these nitrosamines were not detected.     

PROTON AND POTASSIUM TRANSPORT BY H+/K+-ATPases

1. H⁺/K⁺-ATPases are members of the P-type ATPase multigene family. The prototypical H⁺/K⁺-ATPase is the protein that acidifies gastric luminal contents. The physiological and pharmacological significance of this pump has led to a detailed investigation of its biochemistry and molecular and cell biology. 2. Recently, a number of closely related H⁺/K⁺-ATPase isoforms have been discovered. These isoforms are present in organs other than the stomach, including the colon and kidney, where they contribute to acid—base and potassium homeostasis. The structure, expression and physiological roles of the gastric H⁺/K⁺-ATPase and other isoforms are reviewed.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

EFFECTS OF THE OPIOID PEPTIDES [MET5]ENKEPHALIN-ARG6-PHE7 AND [MET5]ENKEPHALIN-ARG6-GLY7-LEU8 ON CHOLINERGIC NEUROTRANSMISSION IN THE RABBIT ISOLATED ATRIA

1. The effect of the opioid peptides [Met5]enkephalin-Arg6-Phe7 (MEAP) and [Met5]enkephalin-Arg6-Gly7-Leu8 (MEAGL) were compared with those of [Leu5]enkephalin and [D-Ala2,Met5]enkephalinamide (DAME) on cholinergic neurotransmission in the rabbit isolated atria. 2. Rabbit isolated atria had a resting rate of 190 beats/min. In the presence of the beta-adrenoceptor antagonist propranolol (0.3 mumol/l), atria responded to electrical field stimulation with a cholinergically mediated negative chronotropic response. The opioid peptides had no effect on the resting rate, but inhibited the negative chronotropic response to field stimulation. The IC50 values for inhibiting the cholinergic responses were 1.4 mumol/l for [Leu5]enkephalin (LE), 1.4 mumol/l for MEAP, 1.3 mumol/l for MEAGL and 0.2 mumol/l for DAME. Responses of a similar magnitude to exogenous acetylcholine were unaffected. 3. Thus, MEAP, MEAGL and LE had similar potencies but DAME was about seven times more potent in inhibiting cholinergic neurotransmission in the rabbit isolated atria. The site of inhibition appears to be prejunctional.