内皮源性超极化因子对内皮一氧化氮合酶基因表达的调节

目的　以内皮细胞产生NO的关键酶———eNOS(内皮一氧化氮合酶 )为研究目标 ,探讨外源性内皮源性超极化因子EDHF(EETs)对内皮细胞合成NO的影响。方法　在原代培养 3～ 4代以内的牛主动脉内皮细胞中 ,分别加入不同浓度 (5 0～ 2 0 0nmol·L-1)的 8,9 EET、11,12 EET、14 ,15 EET ,作用 1h后用不同的方法收获细胞。用WesternBlot以及NorthernBlot方法检测EETs对eNOS基因表达的影响 ;同时通过检测L [3 H] 精氨酸转化为L [3 H] 瓜氨酸的量研究EETs对NOS活性的影响。结果　显示 8,9 EET、11,12 EET、14 ,15 EET均呈浓度依赖性地增加eNOS蛋白质的表达 ,并提高eNOSmRNA表达水平以及NOS酶活性。结论　外源性EDHF对eNOS基因表达是一种正反馈调节作用 ,从而能够促进内皮细胞NO的产生 ,通过药物调节内皮表氧化酶进而促进eNOS基因表达可作为防治心血管疾病的新策略     

CYP4A/20-HETE和eNOS活化有关的调节蛋白间的相互作用

目的研究CYP4A/20-HETE和eNOS活化有关的调节蛋白间的相互作用。方法培养新生牛肺动脉内皮细胞(PAECs),用免疫沉淀(IP)和免疫印迹(WB)对下列4组的CYP4A,eNOS及eNOS调节蛋白进行测定:对照组:PAECs培养皿中加入与20-HETE处理组体积相同的乙醇溶剂,作用10min;20-HETE处理5min组:PAECs培养皿中20-HETE的浓度为1×10-6mol·L-1,作用5min;20-HET处理10min组:PAECs培养皿中20-HETE的浓度为1×10-6mol·L-1,作用10min;VEGF处理10min组:PAECs培养皿中VEGF的浓度为1×10-8mol·L-1,作用10min。结果经20-HETE处理后:肺动脉内皮细胞eNOS蛋白表达增加、phos-pho-eNOS(Ser1177)合成增加;用VEGF进行的上述实验结果与20-HETE相似;20-HETE促使Hsp、蛋白激酶B(Akt)与eNOS结合。结论免疫沉淀和免疫印迹实验结果表明:CYP4A与eNOS在肺动脉内皮细胞相互结合;20-HETE通过促使Hsp、蛋白激酶B(Akt)与eNOS结合,使eNOS(Ser1177)磷酸化,增加NO释放,舒张血管;VEGF和20-HETE的作用相似。     

纳他卡林激活内皮细胞ATP敏感性钾通道SUR2B/Kir6.1亚型对eNOS磷酸化的调节作用

目的纳他卡林可选择性激活ATP敏感性钾通道(K ATP)SUR2B/Kir6.1亚型,引起胞内Ca2+浓度升高,一氧化氮合酶(eNOS)活性增强,NO释放增加。eNOS活性与eNOS磷酸化密切相关,研究表明eNOS-Ser1177、eNOSSer635、eNOS-Ser615磷酸化增强eNOS活性,eNOS-Thr495、eNOS-Ser116磷酸化抑制eNOS活性。本文研究纳他卡林激活K ATP对eNOS各磷酸化位点的影响,以此认识纳他卡林增强eNOS活性的作用特点。方法在培养的内皮细胞上给予(1)10-9¹0-5mol·L-1不同浓度的纳他卡林孵育5 min,(2)预孵育0.0110-5mol·L-1不同浓度的纳他卡林孵育5 min,(2)预孵育0.01¹μmol·L-1格列苯脲30 min,给予1μmol·L-1纳他卡林孵育5 min,提取细胞总蛋白,用Western blot法检测eNOS-Ser1177、eNOS-Ser635、eNOS-Ser615、eNOSThr495、eNOS-Ser116磷酸化的变化。结果纳他卡林可浓度依赖性的升高eNOS-Ser1177、eNOS-Ser635的磷酸化及eNOS-Thr495的去磷酸化,而对eNOS-Ser615和eNOS-Ser116磷酸化无影响。此作用可被K ATP特异性阻断剂格列苯脲拮抗。结论纳他卡林通过激活内皮细胞K ATP,调节eNOSSer1177、eNOS-Ser635的磷酸化及eNOS-Thr495的去磷酸化,但不影响eNOS-Ser615和eNOS-Ser116磷酸化,从而增强eNOS活性。     

氯沙坦对两肾一夹型高血压大鼠血管内皮功能的影响

目的　探讨特异性血管紧张素Ⅱ受体 1阻断剂氯沙坦调节高血压血管内皮功能与一氧化氮合酶 (nitricoxidesynthase,NOS)的关系。 方法　采用两肾一夹型高血压大鼠(two kedney ,one cliphypertensiverat)为动物模型 ,术后第 2、 4、 6wk测血压 ,6wk后处死大鼠测血浆中NO2 -/NO3 -的含量 ,取胸主动脉作离体血管环张力实验 ,免疫印迹方法检测主动脉内皮型一氧化氮合酶 (endothelialNOS ,eNOS)及诱生型一氧化氮合酶 (inducibleNOS ,iNOS)的表达。结果　模型组大鼠相对于假手术对照组血压由 (15 83± 1 4 6 )kPa升高到 (2 3 6 7± 2 2 6 )kPa ,血浆中NO2 -/NO3 -的含量增加 (10 0 8μmol·L-1vs 37 7μmol·L-1,P <0 0 1) ,乙酰胆碱引起的最大舒张百分比下降 (73 5 %vs2 5 % ,P <0 0 1) ,主动脉中eNOS的表达降低 ,iNOS的表达增强 ;氯沙坦治疗后 ,血压、血浆中NO2 -/NO3 -的含量分别下降到 (17 5 6± 1 19)kPa、4 9 1μmol·L-1(P均 <0 0 1) ,乙酰胆碱引起的最大舒张百分比增加到 6 4 2 % ,改善主动脉中eNOS及iNOS的异常表达。结论　氯沙坦可改善两肾一夹型高血压大鼠的血管内皮功能 ,这种作用可能是通过NOS/NO途径调节的     

注射用丹参总酚酸(冻干)对人CYP450酶和P-糖蛋白体外抑制作用及对大鼠CYP1A2和CYP3A体内诱导作用(英文)

目的探讨注射用丹参总酚酸(冻干)(SLI)对人CYP450酶和P-糖蛋白体外抑制作用以及对大鼠CYP1A2和CYP3A体内诱导作用。方法①应用P450-GloTMCYP450检测试剂盒,通过化学发光法测定SLI和经典抑制剂对细胞色素P4501A2(CYP1A2),CYP2D6,CYP3A4,CYP2C19和CYP2C9的IC50值,通过比较SLI和经典抑制剂对相应细胞色素P450亚型的IC50值来判断SLI对人CYP450酶的体外抑制作用。②Wistar大鼠分别iv给予SLI 3,10和30 mg·kg-1和诱导剂苯巴比妥钠20 mg·kg-1,采用探针底物法,通过比较代谢产物的生成速率来评价SLI对大鼠CYP1A2和CYP3A的诱导作用。③应用ATP酶检测试剂盒,通过化学发光法测定ATP酶活性来评价SLI是否为P-gp的底物或抑制剂。结果①CYP1A2,CYP2C9,CYP2C19,CYP2D6和CYP3A4抑制剂的IC50与SLI对其的IC50进行比较(CYP1A2:0.12μmol·L-1vs 840μmol·L-1;CYP2C9:3.362μmol·L-1vs 704μmol·L-1;CYP2C19:3.236μmol·L-1vs 306μmol·L-1;CYP2D6:0.117μmol·L-1vs 2660μmol·L-1;CYP3A4:0.078μmol·L-1vs 1780μmol·L-1)。②与空白对照组(86.4±6.3)nmol·g-1.min-1相比,SLI 3,10和30 mg·kg-1组CYP1A2活性分别为83.4±6.6,82.5±4.0和(83.4±6.6)nmol·g-1.min-1。与空白对照组(16.1±0.9)nmol·g-1.min-1比较,SLI 3,10和30 mg·kg-1组CYP3A活性分别为15.7±0.6,15.9±0.7和(15.9±1.0)nmol·g-1.min-1,无显著性差异。③以临床血药浓度为依据设计的一系列浓度的SLI 0.0002,0.0006,0.002,0.006,0.017,0.052,0.156和0.468 g.L-1的ATP酶活性分别与空白对照组进行比较(5.8,5.3,5.8,5.5,5.8,5.2,,5.8,5.3,vs 5.75μmol·g-1.min-1),无显著性差异。结论SLI临床给药剂量既不能体外抑制人CYP1A2,CYP2D6,CYP3A4,CYP2C19和CYP2C9酶活性,也不能诱导大鼠CYP1A2和CYP3A,同时也不是P-gp的体外抑制剂或底物。     

奥扎格雷钠对大鼠CYP2D6亚型酶的影响

目的通过奥扎格雷钠的大鼠体内、外实验,观察奥扎格雷钠对大鼠CYP2D6亚型酶的影响。方法对照组和奥扎格雷钠诱导组大鼠分别经口给予生理盐水和奥扎格雷钠1wk,HPLC法测定大鼠尿样及肝微粒体中CYP2D6的探针药物右美沙芬的代谢率,观察奥扎格雷钠对CYP2D6活性的影响。结果①大鼠给予奥扎格雷钠(37mg·kg-1),其尿样中右美沙芬的代谢率明显高于对照组(P<0·01);②奥扎格雷钠诱导组大鼠肝微粒体中加入右美沙芬(0·324mmol·L-1),其右美沙芬的代谢率明显高于对照组(P<0·01);③奥扎格雷钠与CYP2D6特异性抑制剂西米替丁可明显降低右美沙芬的代谢率(P<0·01),并且奥扎格雷钠组其右美沙芬的代谢率低于西米替丁组(P<0·05);④奥扎格雷钠IC50=26·5μmol·L-1,西咪替丁IC50=86·3μmol·L-1,奥扎格雷钠诱导组肝微粒体Km=0·67mmol·L-1,Vmax=2·13nmol·min-1·g-1protein;对照组肝微粒体Km=0·29mmol·L-1,Vmax=0·91nmol·min-1·g-1protein;⑤体内和体外相应实验数据具有很好的相关性(r=0·9811)。结论奥扎格雷钠可诱导CYP2D6酶的活性。     

β肾上腺素受体激动对人脐静脉内皮细胞一氧化氮合酶磷酸化的影响

目的：观察血管内皮β肾上腺素受体激动对内皮一氧化氮合酶（eNOS）蛋白表达及磷酸化水平的影响,阐明β受体激动后eNOS调节的分子机制。方法：异丙肾上腺素（ISO）1μmol/L与培养的人脐静脉内皮细胞（HUVEC）孵育30min后,裂解细胞并用免疫沉淀法分离eNOS蛋白,运用同位素两步色谱法（L-[^3H]精氨酸转化法）检测eNOS活性;蛋白免疫印迹增强化学发光法检测eNOS表达水平和eNOS蛋白丝氨酸磷酸化水平,并观察高选择性β1或β2肾上腺素受体阻断剂对上述作用的影响。结果：ISO与内皮细胞孵育30min引起eNOS活性增高;不影响eNOS蛋白表达,但丝氨酸磷酸化水平明显增加;选择性β2受体阻断剂ICI 118551可完全阻断ISO的作用,而选择性β1受体阻断剂CGP 20712A无影响。结论：ISO通过提高eNOS丝氨酸磷酸化水平增加eNOS活性,这种作用是通过β2肾上腺素受体介导的。     

合用C-Raf和PKC-αmRNA为靶点的反义寡核苷酸增强对肺腺癌A549细胞体外生长的抑制作用

观察了靶向 C- raf m RNA ( ISIS51 32 )和PKC- α m RNA( ISIS352 1 )的反义硫代寡核苷酸合用对肺腺癌 A549细胞体外增殖的抑制作用 ( MTT法 ) .单用药组药物浓度为 50 0 ,2 0 0和 80 nmol·L-1;合用组 A两药浓度分别为 2 50 ,1 0 0及 40 nmol· L-1,即两药药量减半 ,药物摩尔浓度之和与单用药相同 .合用组 B两药浓度分别为 50 0 ,2 0 0及 80nmol·L-1,但作用时间减半 (各 3h) ,两药作用时间之和与单用药相同 .结果表明 ,在合用组 A,两药浓度水平为 1 0 0 nmol· L-1时 ,对 A549细胞生长的抑制作用大于 ISIS352 1单用药组的 2 0 0 nmol· L-1水平 ( P<0 .0 1 ) ;在合用组 B,两药浓度水平为 2 0 0和 80 nmol· L-1时 ,对 A549细胞生长的抑制作用分别大于 ISIS352 1单用药组的 2 0 0 nmol· L-1和ISIS51 32单用药组的 80 nmol· L-1水平 ( P<0 .0 1 ) ;其余合用组各个药物浓度水平的效应 ,与相应的单用药组的效应相近 .提示靶向不同癌基因的反义药物合用 ,可能增强对肿瘤细胞的抑制作用 .     

18α-甘草酸下调“胶原蛋白凝胶三明治”培养的大鼠肝细胞P450酶活性及mRNA表达

研究 18α 甘草酸 (18α GA)对肝细胞主要细胞色素P4 50 (CYP)药物代谢酶的影响 ,并初步探讨其分子机理 .采用“胶原蛋白凝胶三明治”培养的原代大鼠肝细胞 ,加 18α GA孵育 ,酶学测定CYP1A1(7 乙氧基异口恶唑O 脱乙基酶 ,EROD) ,CYP2E1(苯胺羟化酶 ,ANH)和CYP3A(红霉素N 脱甲基酶 ,ERD)活性 ,逆转录聚合酶链反应测定CYP1A1,CYP2E1和CYP3A1mRNA表达水平 .结果可见 ,18α GA浓度依赖性 (50～ 4 0 0mg·L- 1)抑制大鼠肝细胞EROD ,ANH和ERD活性 ,2 0 0mg·L- 1作用最强 ,抑制率分别可达 59.6 % ,6 9.7%和 4 4 .7% ,且呈时间依赖性 ,于d 4达高峰 ;浓度依赖性 (50～ 2 0 0mg·L- 1)抑制CYP1A1,CYP2E1和CYP3A1mRNA表达水平 ,分别可达 4 4 .5% ,58.1%和 37.0 % .上述结果表明 18α GA在转录水平下调大鼠肝细胞CYP1A1,CYP2E1和CYP3A1表达     

金雀异黄素对低密度脂蛋白诱导的内皮细胞c-myc mRNA表达的影响(英文)

目的 :研究金雀异黄素对氧化型低密度脂蛋白诱导的人血管内皮细胞c mycmRNA表达的影响。方法 :低密度脂蛋白采用密度梯度超速离心法从健康人血浆中提取 ,经铜离子氧化制备成氧化型低密度脂蛋白。培养的人血管内皮细胞给予氧化型低密度脂蛋白 2 0 0mg·L- 1或同时给予金雀异黄素 10 0 μmol·L- 1诱导不同时间 (1,2 ,4h) ,提取细胞总RNA进行Northernblot杂交。结果 :2 0 0mg·L- 1氧化型低密度脂蛋白刺激内皮细胞c mycmR NA在 1h增加为对照水平的 3倍 ,2h增加为对照水平的 3.3倍 ,4h降到对照水平以下 ;给予氧化型低密度脂蛋白诱导的同时给予金雀异黄素 10 0 μmol·L- 1其c mycmRNA表达量在 1h为单独给予氧化型低密度脂蛋白诱导的 80 % ,2h为 6 0 %。结论 :金雀异黄素能有效抑制氧化型低密度脂蛋白诱导的人血管内皮细胞c mycmRNA表达增高     

Overexpression of cytochrome P450 CYP2J2 protects against hypoxia-reoxygenation injury in cultured bovine aortic endothelial cells.

CYP2J2 is abundant in human heart and its arachidonic acid metabolites, the epoxyeicosatrienoic acids (EETs), have potent vasodilatory, antiinflammatory and cardioprotective properties. This study was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Early passage BAECs were exposed to 24-h hypoxia followed by 4-h reoxygenation (HR). HR resulted in cell injury, as indicated by significant increases in lactate dehydrogenase (LDH) release and trypan blue stained cells (p < 0.01) and was associated with a decrease in CYP2J2 protein expression. Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs, as indicated by increases in LDH release and trypan blue-stained cells (p < 0.01); however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F(2alpha) (p < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F(2alpha) (p < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production. Treatment of BAECs with synthetic EETs and/or epoxide hydrolase inhibitors also showed protective effects against HR injury (p < 0.05). These observations suggest: (1) HR results in endothelial injury and decreased CYP2J2 expression; (2) transfection with the CYP2J2 cDNA protects against HR injury; and (3) the cytoprotective effects of CYP2J2 may be mediated, at least in part, by antioxidant effects.     

Increased CYP2J expression and epoxyeicosatrienoic acid formation in spontaneously hypertensive rat kidney

Epoxyeicosatrienoic acids (EETs) are major products of cytochrome P450 (CYP)-catalyzed metabolism of arachidonic acid in the kidney. The potent effect of EETs on renal vascular tone and tubular ion and water transport implicates their role in the regulation of renal function and blood pressure. The present study was designed to test the hypothesis that CYP-catalyzed EET formation was altered in the spontaneously hypertensive rat (SHR) kidney. The formation of 14,15- and 11,12-EET was approximately 2-fold higher in incubations of arachidonic acid with SHR renal cortical microsomes relative to microsomes from normotensive Wistar-Kyoto (WKY) rats. This was consistent with increased expression of a CYP2J2 immunoreactive protein in the SHR cortex and outer medulla. In contrast, there was no significant difference in the levels of the CYP2E and CYP2C epoxygenases in SHR and WKY kidneys. Protein and RNA analysis suggests that the CYP2J2 immunoreactive protein that is overexpressed in the SHR kidney is distinct from the known rat CYP2J isoforms. EET formation also was documented in vivo from measurements of urinary EET excretion. Importantly, the excretion rates of 14,15-, and 11,12-EETs were 2.5- and 1.8-fold higher, respectively, in SHR than WKY kidney. These studies provide both in vitro and in vivo evidence for increased EET formation in the SHR kidney and identify a novel CYP2J2 immunoreactive protein that is differentially expressed in the hypertensive kidney. In light of the known biological properties of the EETs, these findings may be important in elucidating the mechanisms that control renal vascular tone and tubular ion transport in the SHR.     

Cytochrome P450 eicosanoids are activators of peroxisome proliferator-activated receptor alpha.

Cytochrome P450 (P450) eicosanoids regulate vascular tone, renal tubular transport, cellular proliferation, and inflammation. Both the CYP4A omega-hydroxylases, which catalyze 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and soluble epoxide hydrolase (sEH), which catalyzes epoxyeicosatrienoic acid (EET) degradation to the dihydroxyeicosatrienoic acids (DHETs), are induced upon activation of peroxisome proliferator-activated receptor alpha (PPARalpha) by fatty acids and fibrates. In contrast, the CYP2C epoxygenases, which are responsible for EET formation, are repressed after fibrate treatment. We show here that P450 eicosanoids can bind to and activate PPARalpha and result in the modulation of PPARalpha target gene expression. In transactivation assays, 14,15-DHET, 11,2-EET, and 20-HETE were potent activators of PPARalpha. Gel shift assays showed that EETs, DHETs, and 20-HETE induced PPARalpha-specific binding to its cognate response element. Expression of apolipoprotein A-I was decreased 70% by 20-HETE, whereas apolipoprotein A-II expression was increased up to 3-fold by 11,12-EET, 14,15-DHET, and 20-HETE. In addition, P450 eicosanoids induced CYP4A1, sEH, and CYP2C11 expression, suggesting that they can regulate their own levels. Given that P450 eicosanoids have multiple cardiovascular effects, pharmacological modulation of their formation and/or degradation may yield therapeutic benefits.     

Humic acid induces the endothelial nitric oxide synthase phosphorylation at Ser1177 and Thr495 Via Hsp90α and Hsp90β upregulation in human umbilical vein endothelial cells

Humic acid (HA) has been implicated as a contributory factor for blackfoot disease, which is an endemic peripheral vascular disease. We investigated the effect of HA on the regulation of endothelial nitric oxide (NO) synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) to evaluate the involvement of eNOS and related factors in peripheral vascular impairment with HA exposure. Treatment of HUVECs with HA induced upregulation of eNOS. This result coincides with those of previous studies. Furthermore this is the first study to report that HA induces upregulation of heat shock protein (Hsp)90α, Hsp90β, eNOS phosphorylation at Ser1177, and eNOS phosphorylation at Thr495, as compared to that in the control. In contrast, treatment with BAPTA, an intracellular Ca²⁺ chelator, inhibited upregulation of these proteins induced by HA. This study demonstrates that HA treatment leads to increases in both Hsp90α and Hsp90β proteins and indicates that Hsp90α leads to eNOS phosphorylation at Ser1177 and that Hsp90β leads to eNOS phosphorylation at Thr495, respectively. Upregulation of eNOS, Hsp90α, and Hsp90β in HUVECs is regulated by intracellular Ca²⁺ accumulation induced by HA. These results suggest that upregulation of eNOS phosphorylation at Ser1177 and eNOS phosphorylation at Thr495 produce NO and superoxide anions, respectively, resulting in generation of peroxynitrite, which causes impairment of vascular endothelial cells. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 223–231, 2015.     

The roles of CYP450 epoxygenases and metabolites, epoxyeicosatrienoic acids, in cardiovascular and malignant diseases

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active eicosanoids. The primary epoxidation products are four regioisomers of cis-epoxyeicosatrienoic acid (EET): 5,6-, 8,9-, 11,12-, and 14,15-EET. CYP2J2, CYP2C8, and CYP2C9 are the predominant epoxygenase isoforms involved in EET formation. CYP2J and CYP2C gene families in humans are abundantly expressed in the endothelium, myocardium, and kidney. The cardiovascular effects of CYP epoxygenases and EETs range from vasodilation, anti-hypertension, pro-angiogenesis, anti-atherosclerosis, and anti-inflammation to anti-injury caused by ischemia-reperfusion. Using transgenic animals for in vivo analyses of CYP epoxygenases revealed comprehensive and marked cardiovascular protective effects. In contrast, CYP epoxygenases and their metabolites, EETs, are upregulated in human tumors and promote tumor progression and metastasis. These biological effects result from the anti-apoptosis, pro-mitogenesis, and anti-migration roles of CYP epoxygenases and EETs at the cellular level. Importantly, soluble epoxide hydrolase (sEH) inhibitors are anti-hypertensive and anti-inflammatory and, therefore, protect the heart from damage, whereas the terfenadine-related, specific inhibitors of CYP2J2 exhibit strong anti-tumor activity in vitro and in vivo. Thus, CYP2J2 and arachidonic acid-derived metabolites likely play important roles in regulating cardiovascular functions and malignancy under physiological and/or pathological conditions. Moreover, although challenges remain to improving the drug-like properties of sEH inhibitors and identifying efficient ways to deliver sEH inhibitors, sEH will likely become an important therapeutic target for cardiovascular diseases. In addition, CYP2J2 may be a therapeutic target for treating human cancers and leukemia.     

Enhancement of cardiac L-type Ca2+ currents in transgenic mice with cardiac-specific overexpression of CYP2J2

CYP2J2 is abundant in cardiomyocytes and is involved in the metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs), which affect multiple cell functions. In this study, we investigated the effect of overexpression of CYP2J2 on cardiac L-type Ca2+ currents (ICa) in adult transgenic mice. Cardiac-specific overexpression of CYP2J2 was achieved using the alpha-myosin heavy chain promoter. ICa was recorded from isolated ventricular cardiomyocytes. Compared with the wild-type cardiomyocytes (n = 60), the density of ICa was significantly increased by 40 +/- 9% in the CYP2J2 transgenic cardiomyocytes (n = 71; P < 0.001). N-Methylsulfonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH), a specific inhibitor of EET biosynthesis, and clotrimazole, a cytochrome P450 inhibitor, significantly reduced ICa in both wild-type and transgenic cardiomyocytes; however, MS-PPOH inhibited ICa to a greater extent in the CYP2J2 transgenic cells (n = 10) than in the wild-type cells (n = 10; P < 0.01). Addition of 11,12-EET significantly restored ICa in MS-PPOH-treated cells. Intracellular dialysis with either of two inhibitory monoclonal antibodies against CYP2J2 significantly reduced ICa in both wild-type and transgenic mice. Membrane-permeable 8-bromo-cAMP and the beta-adrenergic agonist isoproterenol significantly reversed the monoclonal antibody-induced inhibition of ICa. In addition, the total protein level of the alpha1 subunit of the Cav1.2 L-type Ca2+ channel was not altered in CYP2J2 transgenic hearts, but the phosphorylated portion was markedly increased. In conclusion, overexpression of CYP2J2 increases ICa in CYP2J2 transgenic cardiomyocytes via a mechanism that involves cAMP-protein kinase A-dependent phosphorylation of the L-type Ca2+ channel.     

Beneficial effect of the oligomerized polyphenol oligonol on high glucose-induced changes in eNOS phosphorylation and dephosphorylation in endothelial cells

Background and purpose:

Hyperglycaemia is known to reduce nitric oxide (NO) bioavailability by modulating endothelial NO synthase (eNOS) activity, and polyphenols are believed to have cardiovascular benefit. One possible mechanism could be through interaction with eNOS.

Experimental approach:

The effects of the oligomerized polyphenol oligonol on eNOS phosphorylation status and activity were examined in porcine aortic endothelial cells cultured in high glucose concentrations.

Key results:

Exposure to high glucose concentrations strongly inhibited eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in bradykinin (BK)-stimulated cells. These inhibitory effects of high glucose were significantly prevented by treatment with oligonol. Akt and p38 mitogen-activated protein kinase (MAPK) were activated in BK-stimulated cells. High glucose inhibited Akt activation but enhanced p38 MAPK activation, both of which were reversed by oligonol treatment. The phosphatidylinositol 3-kinase inhibitor wortmannin blocked the reversal by oligonol of phosphorylation at Ser-1177, but not dephosphorylation at Thr-495, in BK-stimulated cells exposed to high glucose. The effect of oligonol on BK dephosphorylation under high glucose was mimicked by protein kinase C (PKC) ε-neutralizing peptides. These data suggest that the effects of oligonol on high glucose-induced attenuation of eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation may be regulated by Akt activation and PKCε inhibition respectively. Oligonol also prevented high glucose-induced attenuation of BK-stimulated NO production.

Conclusions and implications:

Oligonol prevented the impairment of eNOS activity induced by high glucose through reversing altered eNOS phosphorylation status. This mechanism may underlie the beneficial cardiovascular health effects of this oligomerized polyphenol.     

Overexpression of CYP3A4, but not of CYP2D6, promotes hypoxic response and cell growth of Hep3B cells

Cytochrome P450s (P450s) contribute to carcinogenesis by activating procarcinogens and also metabolize anti-cancer drugs. The activity and protein levels of P450s are important in cancer risk and in cancer therapy. In this study, we found that overexpression of CYP3A4 induced growth of a human hepatoma cell line, Hep3B. Overexpression of CYP2D6, by comparison, decreased cell growth. An inhibitor of CYP3A4, ketoconazole, significantly suppressed the growth of Hep3B cells overexpressing CYP3A4, but an inhibitor of CYP2D6, quinidine, did not restore Hep3B cell growth to baseline levels. Overexpression of CYP3A4 increased the production of reactive oxygen species, but this was not the cause of the CYP3A4-induced growth. Previously, we showed that CYP3A4 can produce epoxyeicosatrienoic acids (EETs) from arachidonic acid. The CYP3A4-enhanced cell growth was attenuated by a putative EET receptor antagonist, 14,15-EEZE. CYP3A4 promoted progression of the cell cycle from the G1 to the S phase. CYP3A4 also induced a hypoxic response of Hep3B cells, detected as enhanced erythropoietin gene expression (a typical hypoxic response). The cell growth promoted by CYP3A4 was inhibited by PI3K inhibitor LY294002. These results suggest that CYP3A4 plays an important role in tumor progression, independent of the activation of carcinogens and metabolism of anti-cancer drugs.