抑制p38MAPK对大鼠缺血再灌注损伤心肌中TNF—α表达及细胞凋亡的影响

目的：探讨p38丝裂原活化蛋白激酶（p38MAPK）抑制剂对缺血再灌注损伤大鼠心肌细胞凋亡和肿瘤坏死因子-α（TNF-α）表达的影响。方法：将30只SD大鼠按随机数字法随机均分为空白对照组、缺血再灌注组和抑制剂组,各10只。检测各组p38MAPK mRNA表达,TNF—α水平及心肌细胞凋亡率,并进行比较分析。结果：与空白对照组比较,缺血再灌注组TNF-α[（3．68±0．16）μg／L比（5．02±0．09）μg／L3、p38MAPK mRNA的表达[（1．76±0．46）比（2．35±0．02）]和心肌细胞凋亡率[-（3．51±0．40）％比-（1．8±0．23）％]显著升高（P均=0．001）。抑制剂组p38MAPK mRNA的表达[（2．09±0．16）]、TNF-α水平[（4．15±0．11）μg／L]及心肌细胞凋亡[-（2．9±0．50）％]均较缺血再灌注组显著降低（P均=0．001）。结论：通过抑制大鼠心肌p38丝裂原活化蛋白激酶的表达能减少肿瘤坏死因子-α的生成,减少心肌细胞凋亡,进而减轻心肌细胞缺血再灌注损伤。     

雷帕霉素在肝脏缺血再灌注损伤中对自噬相关蛋白 ULK1、LC3表达的影响

目的研究雷帕霉素在肝脏缺血再灌注损伤中对自噬相关蛋白ULK1、LC3表达的影响及意义。方法建立3组大鼠肝脏缺血再灌注损伤模型,实验组(n=10)、对照组(n=10)、假手术组(n=10)。分别于术后24、72 h取材:检测血清ALT和AST水平、肝脏病理以及ULK1、LC3 mRNA水平、蛋白水平。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果肝脏缺血再灌注损伤24 h后血清ALT、AST水平升高,肝脏病理结构损伤(F值分别为1531. 83、1799. 97,P值均0. 05),实验组血清ALT[(354. 58±28. 40) U/L vs (556. 15±19. 32) U/L]、AST[(384. 37±8. 98) U/L vs (575. 96±30. 21) U/L]水平较对照组降低(P值均0. 05),肝脏病理结构损伤减轻;术后72 h后实验组血清ALT[(271. 81±8. 63) U/L vs (466. 33±30. 00) U/L]、AST[(358. 92±13. 20) U/L vs (497. 05±40. 14) U/L]水平低于对照组(P值均0. 05)。而术后72 h实验组、对照组血清ALT、AST水平均低于术后24 h(ALT:t=8. 87、7. 92; AST:t=5. 04、5. 34,P值均0. 05)。术后24 h和72 h实验组ULK1、LC3 mRNA水平、蛋白水平较假手术组升高(P值均0. 05),24 h实验组ULK1 mRNA水平(13. 23±6. 58 vs 4. 91±1. 64)、LC3 mRNA水平(7. 82±1. 65vs 3. 70±1. 10)、ULK1蛋白水平(1. 62±0. 19 vs 1. 17±0. 33)、LC3蛋白水平(1. 62±0. 19 vs 0. 84±0. 10)较对照组增加(P值均0. 05); 72 h实验组ULK1 mRNA水平(10. 58±3. 31 vs 4. 83±2. 66)、LC3 mRNA水平(6. 42±1. 13 vs 2. 71±0. 81)、ULK1蛋白水平(1. 29±0. 24 vs 0. 90±0. 29)、LC3蛋白水平(1. 40±0. 73 vs 0. 64±0. 08)较对照组增加(P值均0. 05)。肝脏缺血再灌注损伤72 h后实验组、对照组血清ALT、AST水平较术后24 h降低,并且肝脏病理结构损伤减轻,ULK1、LC3 mRNA水平、蛋白水平降低(P值均0. 05)。结论肝脏缺血再灌注损伤后ULK1、LC3表达增加,雷帕霉素可能在肝脏缺血再灌注损伤过程中通过上调细胞自噬保护肝脏功能。     

尾加压素Ⅱ诱导巨噬细胞分泌巨噬细胞集落刺激因子

目的探索尾加压素Ⅱ(UⅡ)能否促进小鼠单核巨噬细胞系RAW264.7细胞分泌巨噬细胞集落刺激因子(M-CSF),并探讨相关的信号机制。方法培养小鼠单核巨噬细胞系RAW264.7细胞,以Real-time PCR法和ELISA法分别检测细胞的M-CSF mRNA和蛋白表达水平,用细胞免疫印迹法检测p38MAPK和ERK的磷酸化水平。结果 UⅡ呈浓度依赖性和时间依赖性促进RAW264.7细胞中M-CSF mRNA和蛋白水平的表达,最大效应时间均为刺激12 h,M-CSF mRNA水平是对照组水平的2.73倍,蛋白表达水平显著高于对照组水平[(50.04±4.28)pg/ml比(20.39±2.75)pg/ml,P0.01],是对照组水平的2.45倍;最大效应浓度均为10-8mol/L和10-7mol/L,以10-8mol/L UⅡ刺激12 h,M-CSF的mRNA和蛋白表达水平较对照组分别增加了97.3%和80.8%。采用UⅡ受体(UT)阻断剂Urantide、ERK阻断剂PD98059和p38MAPK阻断剂SB203580分别预处理细胞后,M-CSF蛋白水平较UⅡ组显著降低,分别为[(45±4.04)pg/ml比(57.47±2.93)pg/ml]、[(42.13±4.28)pg/ml比(57.47±2.93)pg/ml]和[(44±5.34)pg/ml比(57.47±2.93)pg/ml],差异均有统计学意义(均P0.05)。10-8mol/L UⅡ刺激细胞3 min显著促进ERK和p38MAPK蛋白磷酸化,5 min时ERK磷酸化水平达到峰值,至15 min仍持续高值,而p38MAPK在15 min时磷酸化达到峰值;30 min时,两者的磷酸化水平均减弱。结论UⅡ可通过与受体结合活化ERK和p38MAPK信号通路促进RAW264.7细胞中M-CSF的表达。     

173例脓毒症患者血清CK-MB、ALT、AST水平与病情严重程度的关系分析

目的探讨磷酸肌酸激酶同工酶(CK-MB)、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基酶(AST)在脓毒症患者血清中的水平及意义。方法选取2016年1月至2018年9月在我院治疗的脓毒症患者173例,根据住院期间预后情况分为死亡组(n=61)和存活组(n=112),同时根据入院时APACHEⅡ评分分为轻度组(n=42)、中度组(n=73)和重度组(n=58),检测患者血清CK-MB、ALT和AST水平。结果重度患者血清CK-MB、ALT和AST分别为(190.22±23.32)U/L、(132.29±11.43)U/L和(140.22±15.50)U/L,明显高于中度和轻度患者(P0.05);中度患者血清CK-MB、ALT和AST分别为(150.92±20.05)U/L、(101.22±9.82)U/L和(110.28±12.21)U/L,明显高于轻度[(121.29±21.11)U/L、(86.93±10.02) U/L和(90.33±11.40) U/L]患者(P0.05);死亡患者血清CK-MB、ALT和AST分别为(201.11±25.58)U/L、(129.82±13.29)U/L和(134.40±14.33)U/L,明显高于存活患者[(143.39±30.22) U/L、(100.05±11.22)U/L和(109.28±13.32)U/L](P0.05);APACHEⅡ评分与CK-MB、ALT和AST呈正相关(r=0.483、0.434和0.455,P0.05)。结论脓毒症患者血清CK-MB、ALT和AST明显升高,与病情严重程度及预后有一定关系,CK-MB、ALT和AST水平越高,患者病情越严重,预后越差。     

缬沙坦对血管平滑肌细胞增殖与迁移及丝裂原活化蛋白激酶的影响

目的:观察缬沙坦对血管紧张素Ⅱ(AngⅡ)刺激下大鼠胸主动脉平滑肌细胞(VSMC)的增殖与迁移及磷酸化P42/44丝裂原活化蛋白激酶(MAPK)表达的影响。方法:组织贴块法培养大鼠胸主动脉平滑肌细胞,应用丝裂原活化蛋白激酶(MTT)分析检测细胞的增殖能力,transwell小室检测细胞的迁移能力,Western blot检测磷酸化P42/44MAPK蛋白表达的水平。结果:(1)AngII能明显促进血管平滑肌细胞的增殖与迁移,该作用可被缬沙坦和MAPK激酶的特异性抑制剂PD98059所抑制。(2)AngⅡ刺激血管平滑肌细胞5min时,磷酸化P42/44MAPK的表达量最大,该作用也可被缬沙坦和P     

雌激素膜快速效应对兔血管平滑肌细胞iNOS快速激活的影响

目的 探讨17β-雌二醇（17β-estradiol,E2）通过快速激活诱导型一氧化氮合成酶（iNOS）的活性,调节细胞外信号调节激酶（mitogen-activated protein kinase,p42/44 MAPK）表达抑制血管平滑肌细胞（vascular smooth muscle cells,VSMCs）增殖的机制。方法 在培养的VSMCs的基础上,采用放射免疫测定法(RIA)和Western blot检测E2预处理前后胎牛血清（fetal calf serum,FCS）对VSMCs中诱导型iNOS的活性及磷酸化p42/44 MAPK蛋白和p42/44 MAPK总蛋白表达的影响。采用比色法检测E2预处理后VSMCs中NO含量的变化。结果 E2预处理5 min,即可逆转FCS诱导的iNOS活性下降,此效应在E2预处理15 min时达到高峰,处理30 min后此效应减弱。基因转录抑制剂放线菌素D（actinomycin D,Act D）预处理后对此过程无影响,而三苯氧胺预处理可明显逆转E2诱导的iNOS活性增加。比色法检测上清液中NO的含量显示,E2预处理15 min,NO的含量明显增加。Western blot的结果显示,E2预处理15 min,可明显抑制磷酸化p42/44 MAPK蛋白的表达,而对总p42/44 MAPK蛋白的表达无影响。NO合酶(NOS)抑制剂L-硝基左旋精氨酸甲酯(L-NAME)预处理,可逆转E2诱导的磷酸化p42/44 MAPK的表达下降。结论 E2可通过快速激活iNOS增加NO释放,下调磷酸化p42/44 MAPK的活性,抑制VSMCs增殖。     

姜黄素对大鼠肝缺血再灌注早期肝组织一氧化氮表达的影响

目的:探讨姜黄素对大鼠肝脏缺血再灌注早期损伤微循环的影响.方法:将大鼠随机分为假手术组、对照组和实验组(姜黄素40 mg/kg,2次给药).通过检测再灌注早期1、3 h血清转氨酶水平、肝组织中一氧化氮(nitricoxide,NO)、一氧化氮合酶(nitricoxide synthase,NOS),诱导型一氧化氮合酶(inducible nitricoxide synthase,iNOS)mRNA及内皮型一氧化氮合酶(endothelium nitricoxide synthase,eNOS)mRNA水平,以及肝组织病理学检查来评价姜黄素对大鼠肝脏缺血再灌注早期损伤微循环的影响.结果:相对于对照组,姜黄素可降低大鼠肝脏缺血再灌注早期损伤1、3 h血清谷丙转氨酶(ALT)的水平(603.8 U/L±64.5 U/L vs 758.1 U/L±114.7U/L,837.1 U/L±33.3 U/L vs 1012.7 U/L±119.8 U/L,均P<0.01)和谷草转氨酶(AST)的水平(605.7 U/L±65.7 U/L vs 779.5 U/L±124.3 U/L,849.6 U/L±36.0 U/L vs 1027.8 U/L±139.8 U/L,均P<0.01);改善肝组织病理学损害;减少肝脏缺血再灌注早期损伤1、3 h肝组织由iNOS产生的NO蛋白水平(0.455±0.056 vs 0.594±0.087.0.492±0.040 vs 0.671±0.079,均P<0.01);降低肝脏缺血再灌注早期损伤1、3 h肝组织iNOS mRNA的表达强度(0.426±0.075 vs 0.569±0.073,0.527±0.066vs 0.702±0.089,均P<0.01).结论:姜黄素可通过减轻肝组织中由iNOS产生的NO生成,来改善肝缺血再灌注早期损伤中微循环的紊乱,从而减少对肝缺血再灌注肝实质细胞的损伤.     

丝裂素活化蛋白激酶参与心肌缺氧预处理的延迟保护作用

目的　探讨丝裂素活化蛋白激酶 (mitogen- activated protein kinase,MAPK)在心肌缺氧预处理延迟保护中的作用。方法　在培养乳鼠心肌细胞缺氧预处理的模型上 ,检测预处理后即刻、1h、6 h和 12 h的 MAPK活性变化 ,观察细胞缺氧 /复氧损伤后、延迟预处理后及蛋白激酶 C(PKC)抑制剂 chelerythrine(Ch)干预后的细胞存活率、L DH的释放、MDA含量和 SOD活性。结果　 MAPK活性在预处理后即刻明显增加 (P<0 .0 1) ,在 6 h后降至或接近对照水平。与未预处理组心肌细胞缺氧 /复氧损伤相比较 ,预处理后 2 4 h心肌细胞存活率增高 [(5 8.6 4± 5 .5 3) %vs (44 .2 9± 4 .2 7) % ,P<0 .0 1],L DH[(5 9.5 0± 11.0 8) U/ L vs(83.17± 13.6 9) U/ L,P<0 .0 1]和 MDA含量[(2 .33± 0 .4 9) nmol/ L vs(3.2 9± 0 .2 6 ) nmol/ L,P<0 .0 1]均降低 ,SOD活性增加 [(2 1.5 3± 3.6 3) n U/ m l vs(12 .86± 2 .6 8) n U/ ml,P<0 .0 1]。PKC抑制剂 chelerythrine可消除预处理的延迟保护作用。结论　预处理 2 4 h心肌细胞对再次缺氧 /复氧有保护作用 ,PKC、MAPK均参与心肌细胞预处理后的延迟保护作用     

p38MAPK信号转导通路与脑缺血

p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是一类重要的细胞内信号转导通路,其主要作用是参与炎症调节和细胞凋亡.脑缺血时,p38 MAPK被激活,其表达水平以及上游和下游蛋白磷酸化水平均发生改变.缺血预处理可能通过p38 MAPK信号转导通路介导脑缺血后神经细胞的存亡.     

p38丝裂原活化蛋白激酶在不可分型流感嗜血杆菌致人单核细胞炎症反应中的作用

目的 通过不可分型流感嗜血杆菌(NTHi)与单核细胞相互作用,研究丝裂原活化蛋白激酶(MAPK)信号传导通路在NTHi致人体免疫细胞炎症反应中的作用.方法 NTHi为临床分离株,经血清学方法和16S rRNA测序证实.外周血单核细胞来自健康成年人静脉血,分为4组:培养基组、NTHi刺激组、SB203580(p38 MAPK抑制剂)干预组和UO126(p44/42 MAPK抑制剂)干预组.NTHi与单核细胞共培养1 h、4 h后收集细胞,用Western blot法检测p38、p44/42 MAPK的磷酸化程度;16 h后用流式细胞仪检测细胞表面Toll样受体(TLR)4的表达.预先用SB203580或UO126与单核细胞共孵育1 h,然后加入NTHi(感染复数为200),分别在4 h、16 h后收集上清,用酶联免疫吸附法检测肿瘤坏死因子α(TNF-α)的水平.采用SPSS11.5统计软件,组间比较用t检验,单核细胞TNF-α的表达用单因素方差分析,组间比较用LSD检验.结果 NTHi可迅速诱导p38、p44/42 MAPK通路的磷酸化,并至少持续到刺激后4 h.与培养基组比较,NTHi刺激16 h后单核细胞表面TLR4的表达明显增加(11.8±1.6,4.8±0.6),差异具有统计学意义(t=4.08,P<0.05).NTHi刺激4 h和16 h后上清液中的TNF-α(16.4±5.3,30.2±10.7)较培养基组(0.6±0.6,1.4±1.1)显著增加,差异具有统计学意义(4 h时I-J值为15.78,16 h时I-J值为28.82,P均<0.01).与细菌组比较,SB203580干预组单核细胞TNF-α水平显著降低(4 h时I-J值为11.26,16 h时I-J值为21.32,P均<0.05),而UO126干预组TNF-α水平无明显变化(4 h时I-J值为6.32,16 h时I-J值为12.57,P均>0.05).结论 TLR4可能参与了NTHi诱导的单核细胞反应,p38 MAPK是该反应的关键信号分子.     

Protein kinase C-dependent activation of P44／42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning

AIM: To investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (MAPKs) and heat shock protein (HSP)70 signal transduction during hepatocyte ischemic preconditioning. METHODS: In this study we used an in vitro ischemic preconditioning (IP) model for hepatocytes and an in vivo model for rat liver to investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (P44/42 MAPKs) and heat shock protein 70 (HSP70) signal transduction in IP. Through a normal liver cell hypoxic preconditioning (HP) model in which cultured normal liver cells were subjected to 3 cycles of 5 rain of incubation under hypoxic conditions followed by 5 rain of reoxygenation and subsequently exposed to hypoxia and reoxygenation for 6 h and 9 h respectively. PKC inhibitor, activator and MEK inhibitor were utilized to analyze the phosphorylation of PKC, the expression of P44/42 MAPKs and HSP70. Viability and cellular ultrastructure were also observed. By using rat liver as an in vivo model of liver preconditioning (3 cycles of 10-min occlusion and 10-min reperfusion), in vivo phosphorylation of PKC and P44/42MAPKs, HSP70 expression were further analyzed. AST/ALT concentration, cellular structure and ultrastruture were also observed. All the data were statistically analyzed. RESULTS: Similar results were obtained in both in vivo and in vitro IP models. Compared with the control withouts IP (or HP), the phosphorylation of PKC and P44/42 MAPKs and the expression of HSP70 were obviously increased in IP (or HP) treated model in which cytoprotection could be found. The effects of preconditioning were mimicked by stimulating PKC with 4β phorobol-12-myristate 13-acetate (PMA). Conversely, inhibiting PKC with chelerythrine abolished the protection given by preconditioning. PD98059, inhibitor of MEK (the upstream kinase of P44/42MAPKs), also reverted the cytoprotection exerted by preconditioning. CONCLUSION: The results demonstrate that preconditioning induces a rapid activation of P44/421VlAPKs and PKC activation plays a pivotal role in the activation of P44/42 MAPKs pathway that participates in the preservation of liver cells. HSP expression is regulated by signals in PKC dependent P44/42 MAPKs pathway.     

Acute signaling by the LH receptor is independent of protein kinase C activation

LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC delta on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominantly PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.     

Role of phasic dynamism of p38 mitogen-activated protein kinase activation in ischemic preconditioning of the canine heart

Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 micromol/L) only during IP blunted the infarct size limitation by IP (37.3+/-6.3% versus 7.4+/-2.1% in the IP group, P:<0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3+/-9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8+/-3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.     

PI3 kinase and not p42/p44 appears to be implicated in the protection conferred by ischemic preconditioning

Ischemic preconditioning results in an immediate phase of protection against lethal ischemia/reperfusion injury that is comprised of both irreversible necrosis and programmed cell death, apoptosis. We hypothesized that preconditioning may activate putative anti-apoptotic pathways, through the induction of either phosphatidyl inositol 3-OH kinase (PI3 kinase) or p42/p44 extracellular receptor kinase, attenuating total cell death. Isolated perfused rat hearts were preconditioned with two cycles of 5 min ischemia and 10 min reperfusion. Then they were frozen for Western blot analysis or subjected to 35 min regional ischemia and 120 min reperfusion prior to infarct size assessment. Selective PI3 kinase inhibitors, wortmannin (W, 100 n M) and LY294002 (LY, 15 microM) and the p42/p44 inhibitor, PD 98059 (PD, 10 and 50 microM), were individually infused during the preconditioning protocol. One further group of hearts received both inhibitors (W and PD). The results were expressed as percentage of infarction within the risk zone. Inhibition of PI3 kinase by either W or LY partially abrogated the infarct sparing effect of ischemic preconditioning (I/R%: 44.6+/-2.7 in C, 17.6+/-2.0 in IP, vs 32.2+/-4.2 in W, and 30.9+/-2.6 in LY, P<0.05). Inhibition of ERK phosphorylation however, had no significant effect upon infarct size reduction (17.6+/-2.0 in ischemic preconditioning vs 21.4+/-3.0 in IP+10 microM PD and 15.2+/-1.4 in IP+50 microM PD, P>0.05). Western blot analysis confirmed that PD abrogated the phosphorylation of p42/p44 and LY the phosphorylation of AKT. Combined inhibition with PD+W failed to further attenuate protection (27.6+/-1.3%, P>0.1). These data appear to demonstrate that the PI3 kinase, but not the p42/p44 cascade, is implicated in early ischemic preconditioning.     

p38MAPK inhibition enhances basal and norepinephrine-stimulated p42/44MAPK phosphorylation in rat pinealocytes

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4beta phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.     

Diurnal variation in p42/44 mitogen-activated protein kinase in the rat pineal gland

In this study, we investigated whether there was a diurnal difference in mitogen-activated protein kinase (p42/44(MAPK)) phosphorylation in the rat pineal gland. Under a lighting regimen with 12h of darkness, there was a two- to four-fold increase in phosphorylated levels of MAPK kinase 1/2 (MEK1/2) and p42/44(MAPK) 2h after onset of darkness, an increase that was sustained for 8h. The increases in phosphorylated levels of MEK1/2 and p42/44(MAPK) occurred without increases in MEK1/2 and p42/44(MAPK) proteins. When rats were treated with propranolol 1h before onset of darkness or subjected to continuous light exposure during the dark phase, the nocturnal increase in MEK1/2 and p42/44(MAPK) phosphorylation was reduced. Acute light exposure during darkness caused a decline in MEK1/2 and p42/44(MAPK) phosphorylation within 30 min of light exposure. These results indicate the presence of a diurnal difference in MEK1/2 and p42/44(MAPK) phosphorylation in the rat pineal gland that is under adrenergic control.     

Role of epidermal growth factor in pathogenesis of uterine leiomyomas

Objective:To investigate the role of epidermal growth factor(EGF) in the pathogenesis of uterine leiomyomas.Methods:Human myometrial smooth muscle cells(HM-SMCs) and smooth muscle cells of human uterine leiomyomas(HL-SMCs) were separated from patients' specimens and cultured.After processed by EGF or PD98059(inhibitor of MKK/MEK) +EGF,the proliferation rate of both SMCs was detected by BrdU method and the phosphorylation level of p44/42 mitogen-activated protein kinase(MAPK) was determined by Western-blot.After different processing time by EGF,the phosphorylation levels of p44/42 MAPK and AKT and p27 expression level in both SMCs were detected by Western-blot.Results:EGF could significantly promote HL-SMCs proliferation and PD98059 could inhibit this effect(P0.05);besides,PD98059 could inhibit the increase of the phosphorylation level of p44/42 MAPK in both SMCs induced by EGF.When the processing time by EGF was over 15 min,the phosphorylation levels of p44/42 MAPK and AKT in both SMCs decreased sharply and were close to zero:p27 expression in HM-SMCs raised significantly while the upregulation in HL-SMCs was little.Conclusions:EGF could not cause activation of EGFR because of the dephosphorylation of p44/42 MAPK and AKT in HL-SMCs,which caused p27 expression insufficiently and cell cycle dysregulation.     

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.     

Protective role of an endothelin-converting enzyme inhibitor (FR901533) in hepatic ischemia/reperfusion injury.

INTRODUCTION: There is evidence that endothelin (ET) contributes to disturbances of the hepatic microcirculation after warm ischemia/reperfusion (I/R) by causing vasoconstriction and by enhancing leukocyte endothelium interactions. The aim of this study was to investigate a possible protective role of the endothelin converting enzyme (ECE) inhibitor FR901533 in this setting. METHODS: In an in vivo model (42 Wistar rats), hepatic ischemia was induced for 30 min by Pringle's maneuver. Sham operated (I), untreated ischemic (II), and treatment (III) groups with FR901533 (1 mg/kg bw iv) were investigated. The effect of FR901533 in I/R was assessed by in vivo microscopy (30-90 min after reperfusion), measurement of local tissue pO2 (30 and 60 min after reperfusion), and determination of AST/ALT levels (2 h, 6 h, and 2, 6, and 14 days after reperfusion). RESULTS: In the untreated ischemic group (II) sinusoidal constriction to 76.3 +/- 4.2% of basic diameters was observed, leading to significant decreases in perfusion rate (82.3 +/- 3.6% of sham group) and in liver tissue pO(2) (43.5 +/- 3.2% of sham group) (P < 0.05). In addition, we found an increased percentage of stagnant leukocytes in sinusoids (138.3 +/- 9.8) and sticking leukocytes in postsinusoidal venules (155.2 +/- 3.3% of sham group) (P < 0.05). Hepatocellular damage (AST/ALT increase to 430.6 +/- 47.7 U/L/200.2 +/- 23.8 U/L, pre: 27.4 +/- 2.7 U/L/28.1 +/- 2.7 U/L) was detected 6 h after reperfusion (P < 0.05). Administration of the ECE inhibitor before ischemia significantly reduced I/R injury. Sinusoidal diameters were maintained (102.2 +/- 1.7%), while perfusion rate (93.1 +/- 1.8%) and tissue pO2 (105.3 +/- 2.7%) increased significantly (P < 0.05). Hepatocellular damage was decreased (AST/ALT levels after 6 h of reperfusion: 166.6 +/- 26.3 U/L/132.4 +/- 22.5 U/L, P < 0.05) and leukocyte sticking and rolling were significantly reduced (P < 0.05). CONCLUSION: Our results provide evidence that the new therapeutic approach with an ECE inhibitor is effective in reducing hepatic I/R injury.     

Adenosine A(1) receptor induced delayed preconditioning in rabbits: induction of p38 mitogen-activated protein kinase activation and Hsp27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism

Transient adenosine A(1) receptor (A(1)R) activation in rabbits induces delayed preconditioning against myocardial infarction 24 to 72 hours later. The cellular mechanisms downstream of A(1)R mediating this delayed cardioprotection have not been elucidated. This study examined the role of protein kinase C (PKC) and tyrosine kinases (TKs) in the signaling cascade mediating A(1)R-induced late preconditioning in rabbits. The small heat shock protein Hsp27 has been shown to confer cytoskeletal protection when in the phosphorylated state. We therefore also evaluated the potential role of the p38 mitogen-activated protein kinase (p38 MAPK) and Hsp27 as distal mediators of A(1)R-induced delayed preconditioning. Pharmacological preconditioning of rabbits with the selective A(1) agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 100 microgram/kg) significantly reduced myocardial infarct size compared with control animals, after 30-minute regional ischemia/2-hour reperfusion in vivo 24 hours later (23.7+/-3.1 versus 43.0+/-4.1%; P<0.05). This delayed protection was abrogated by prior inhibition of either PKC with chelerythrine chloride (5 mg/kg) or of TKs with lavendustin A (1.3 mg/kg), suggesting that both PKC and TK are crucial for the development of delayed preconditioning after A(1) receptor activation in the rabbit. Myocardial tissue extracts obtained 24 hours after CCPA treatment were analyzed for p38 MAPK catalytic activity using an in vitro kinase assay. This showed an almost 7-fold increase in p38 MAPK activity in myocardial samples pretreated with CCPA compared with control hearts. Two-dimensional gel electrophoresis revealed an increase in the phosphorylated isoforms of Hsp27 in hearts pretreated with CCPA compared with control hearts. Prior inhibition of either PKC or TK prevented the CCPA-induced increase in p38 MAPK activity and phosphorylation of Hsp27. This study identifies new components of the signaling mechanism of A(1)R-induced delayed preconditioning. Our results suggest an important role for both PKC and TK as mediators of late preconditioning against infarction after A(1)R activation and, although correlative, point to the p38 MAPK/Hsp27 pathway as a potential distal effector of this protection.