一氧化氮参与预处理心肌细胞早期保护作用

目的　明确一氧化氮 (NO)是否诱导预处理心肌细胞早期保护作用。方法　取体外培养的新生大鼠心肌细胞 ,分为如下各组 :①阴性对照组 (Normal组 ) ;②SNAP(5 0 0 μmol·L-1)预处理组 (NO组 ) ;③缺氧预处理组 (HP组 ) ;④NO合成抑制剂L NAME作用细胞后再行缺氧预处理组 (L NAME +HP组 ) ;⑤L 精氨酸 (L Arg)预处理心肌细胞组(L Arg组 ) ;⑥L NAME与L Arg共同作用于心肌细胞组(L NAME +L Arg组 ) ;⑦缺氧复氧损伤组 (H/R组 )。②～⑥组细胞在给予干预因素后使细胞经历 6h缺氧及 3h复氧 ,阳性对照组不予任何处理直接使其缺氧 6h复氧 3h。分别检测心肌细胞存活率及乳酸脱氢酶 (LDH)活性 ,以判定心肌细胞损伤程度。结果　NO预处理心肌细胞后使其缺氧复氧损伤减轻 ,表现为与单纯缺氧复氧组细胞比较其培养上清LDH活性降低 ,细胞存活率提高 (P <0 0 1) ;缺氧预处理和L Arg预处理细胞均可减轻心肌细胞缺氧复氧损伤 (P<0 0 1) ,但此作用可被NOS抑制剂L NAME所拮抗。结论　内源性及外源性NO均可诱导心肌细胞预处理早期保护作用     

阿魏酸钠对心肌细胞缺氧/复氧损伤的保护作用及其机制

目的研究阿魏酸钠(SF)预适应对心肌细胞缺氧/复氧损伤的保护作用及其机制。方法采用1～3 d新生SD大鼠,常规方法培养心肌细胞,给予模拟缺氧(缺血)液、模拟复氧(再灌注)液以及药理性预适应药物阿魏酸钠以分析K⁺_ATP通道、NO及PKC的影响。结果与对照组比,单纯缺氧/复氧使LDH,CK,MDA及LD水平显著升高,细胞搏动频率、细胞存活率和SOD,GSH-Px活力显著下降。SF预适应后明显减轻上述变化。Glib,L-NAME和Ploy B均部分取消SF预适应的减轻作用。结论SF预适应对心肌细胞缺氧/复氧损伤有显著的保护作用;此保护心肌过程是多种因素综合作用的结果。     

维司力农对缺血再灌注大鼠心肌NO含量及PKC活性的影响

目的　探讨维司力农对缺血再灌注心肌的保护作用机制。方法　采用NO和PKC测定试剂盒分别测定了两个对照组、心肌缺血预适应组、不同剂量维司力农组的大鼠心肌细胞内一氧化氮 (NO)含量和蛋白激酶C(PKC)活性。结果　高、低剂量维司力农组PKC活性高于两个对照组 ;低剂量组PKC活性与缺血预适应组相当 ,高剂量组PKC活性明显高于缺血预适应组。同时 ,NO含量也高于两个对照组和缺血预适应组。结论　维司力农可使大鼠心肌细胞内NO含量和PKC活性提高 ,由此推断 :维司力农对缺血再灌注心肌的保护作用很可能是通过NO激活PKC而实现的。     

ATP敏感性钾通道与心肌缺血预适应

ATP敏感性钾通道 (KATP通道 )作为心肌缺血预适应(早期与延迟效应 )细胞内信号转导途径中的一种“终末效应子” ,在缺血预适应中起重要作用。缺血预适应通过活化心肌细胞膜和 (或 )线粒体膜KATP通道而发挥保护作用。动物实验和临床研究均已证明KATP通道开放剂诱导预适应能有效地防治心肌缺血损伤。     

咪达唑仑对离体猪冠脉环舒张功能的影响及其机制研究

目的本实验观察咪达唑仑(midazolam)对离体猪冠状动脉环的作用,并探讨其作用机制。方法采用离体血管张力测定实验方法,记录咪达唑仑对KCl(30 mmol·L~(-1))预收缩猪冠状动脉环张力的变化及不同工具药的影响。结果各浓度组咪达唑仑(3×10~(-6)~1×10~(-4)mol·L~(-1))对预收缩猪冠脉环产生浓度依赖性舒张作用,内皮完整组舒张作用强于去内皮组(P<0.05);在KCl预收缩基础上,加入NOS抑制剂L-NAME、L-NAME+L-Arg后,咪达唑仑舒血管作用明显减弱(P<0.05),而加入外源性的NO合成底物L-Arg、i NOS抑制剂1400W、环氧合酶抑制剂Indo均不能抑制咪达唑仑的舒血管作用;Na~+/Ca~(2+)交换体特异性阻断剂KBR7943亦不能抑制咪达唑仑的舒血管作用;钾通道阻断剂K_(ATP)阻断剂Gli可以抑制咪达唑仑的舒张冠脉作用(P<0.05),BKCa阻断剂TEA、Kir阻断剂Ba Cl2、KV阻断剂4-AP则不能改变咪达唑仑的舒张冠脉作用。结论咪达唑仑可浓度和内皮依赖性地舒张KCl预收缩的猪冠脉环,内皮分泌的NO参与了其舒血管作用,外源性NO、i NOS以及PGI2的合成与其舒血管作用无关,咪达唑仑对猪冠脉的舒张可能与K_(ATP)通道有关,与Na~+/Ca~(2+)交换体、K_V通道、BK_(Ca)通道、K_(ir)通道无关。     

一氧化氮供体预处理对乳鼠心肌细胞的延迟保护作用及其机制

目的　探讨一氧化氮模拟预处理延迟保护作用的机制。方法　体外培养新生大鼠心肌细胞 ,实验分为 :①阴性对照组 (Normal组 ) ;②SNAP组 :一氧化氮 (nitricoxide ,NO)供体S 亚硝基 N 乙酰青霉胺 (S nitroso N acetyl 1 ,1 penicil lamine ,SNAP ,5 0 0 μmol·L-1 )与心肌细胞共育 2 4h ;③Che +SNAP组 :蛋白激酶C拮抗剂白屈菜季铵碱 (chelerythrinechlo ride ,Che ,1 0 μmol·L-1 )处理心肌细胞 30min后 ,加入SNAP与心肌细胞共育 2 4h ;④PDTC +SNAP组 :核因子κB(NF κB)特异性阻断剂PDTC(1 0 0 μmol·L-1 )处理心肌细胞 30min后 ,加入SNAP与心肌细胞共育 2 4h ;⑤缺氧复氧损伤组 (H/R组 ) :心肌细胞缺氧 6h ,复氧 3h。②～④组部分取细胞爬片以免疫组织化学法观察SNAP对热休克蛋白 70 (HSP70 )表达的影响 ;其余细胞经历H/R损伤后 ,检测心肌细胞存活率及乳酸脱氢酶 (LDH)活性。结果　在正常心肌细胞免疫组织化学法未检测到HSP70的表达 ,心肌细胞经过H/R损伤后 ,可检测到少量HSP70阳性细胞 ,其阳性染色A值为 94 6± 9 1 ,心肌细胞培养上清LDH活性为 (2 1 90 5± 1 5 1 7)U·L-1 ,细胞存活率为 5 1 7%± 4 6 % ,细胞损伤较正常组加重 (P <0 0 1 ) ;SNAP处理细胞后 2 4h ,HSP70阳性细胞数增多 ,     

丹参酮Ⅵ预处理对大鼠心肌细胞缺氧/复氧损伤的保护作用

目的 研究丹参酮Ⅵ(Tanshinone Ⅵ,Tan)预处理对心肌细胞缺氧/复氧(anoxia/reoxygenation,A/R)损伤的保护作用及其机制.方法 以细胞存活率、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量等为观察指标,用终浓度分别为0.1、1和10 μmoL/L的Tan预处理原代培养大鼠乳鼠心肌细胞1 h,观察其对A/R损伤的保护作用及NO合成酶抑制剂L-NAME(0.1 mmol/L)、K+-ATP通道阻断剂格列苯脲(12 μmol/L)对其保护作用的影响.结果 Tan预处理能显著提高细胞存活率,降低LDH活性,呈剂量依赖性,且显著能增加SOD及GSH-Px活性,降低MDA含量,能对抗A/R损伤;L-NAME和格列苯脲能部分取消Tan预处理的上述保护作用.结论 Tan预处理对心肌细胞A/R损伤有显著的心肌细胞保护作用,其机制可能与NO生成、K+-ATP通道开放有关.     

生物学靶标PKC在蒺藜皂苷抗心肌缺血中的作用

目的：探讨蛋白激酶C（PKCε）在蒺藜皂苷抗过氧化氢（H2O2）诱导新生大鼠心肌细胞凋亡中的作用及其机制。方法：新生大鼠心肌细胞原代培养，H2O2诱导建立心肌细胞缺氧模型，应用PKCε抑制剂Chelerythrine预处理，观察心肌细胞形态变化，MTT法检测蒺藜皂苷对缺氧心肌细胞存活力的影响，Hoechst33258染色观察凋亡心肌细胞核形态，     

内源性一氧化氮介导脂质胞壁酸预适应对人冠脉内皮细胞再复氧损伤的作用

目的探讨脂质胞壁酸(LTA)诱导的延迟预适应对内皮细胞再复氧(H/R)损伤的作用，以及内源性一氧化氮(NO)参与保护机制的作用。方法采用培养的人冠状动脉内皮细胞(HCAECs)在缺氧条件下培养2 h，然后在常氧条件下复氧培养4 h，模拟缺血/再灌注损伤的模型。HCAECs在缺氧前24 h预先在含LTA(30或300 μg·L^-1)的培养基中培养4 h。用台盼蓝排斥法和培养基中乳酸脱氢酶(LDH)含量来评价内皮细胞损伤程度，比色法检测培养基中一氧化氮(NO)含量。并用RT-PCR检测LTA预适应后(2-24) h HCAECs eNOS mRNA的表达。结果LTA预适应能显著减少台盼蓝排斥实验中的细胞死亡百分比，降低复氧末细胞培养液中LDH的含量。LTA预适应亦能显著增加HCAECs复氧末培养液中NO含量。LTA预适应的效应可被非选择性NOS抑制剂L-单甲基精氨酸(L-NMMA,100 μmol·L^-1)所取消。在LTA预适应后2和4 h，HCAECs的eNOS mRNA表达明显增加。结论 LTA诱导的延迟预适应能显著减少HCAECs 再复氧所致的细胞损伤和功能紊乱，eNOS产生的NO启动并介导了LTA保护内皮细胞的作用。     

内源性一氧化氮介导脂质胞壁酸预适应对人冠脉内皮细胞再复氧损伤的作用

目的　探讨脂质胞壁酸(LTA)诱导的延迟预适应对内皮细胞再复氧(H/R)损伤的作用,以及内源性一氧化氮(NO)参与保护机制的作用。方法　采用培养的人冠状动脉内皮细胞(HCAECs)在缺氧条件下培养 2h,然后在常氧条件下复氧培养 4h,模拟缺血 /再灌注损伤的模型。HCAECs在缺氧前 24h预先在含LTA(30或 300μg·L-1 )的培养基中培养 4h。用台盼蓝排斥法和培养基中乳酸脱氢酶(LDH)含量来评价内皮细胞损伤程度,比色法检测培养基中一氧化氮(NO)含量。并用RT PCR检测LTA预适应后(2-24)hHCAECseNOSmRNA的表达。结果　LTA预适应能显著减少台盼蓝排斥实验中的细胞死亡百分比,降低复氧末细胞培养液中LDH的含量。LTA预适应亦能显著增加HCAECs复氧末培养液中NO含量。LTA预适应的效应可被非选择性NOS抑制剂L 单甲基精氨酸(L NMMA, 100μmol·L-1 )所取消。在LTA预适应后 2和 4h,HCAECs的eNOSmRNA表达明显增加。结论LTA诱导的延迟预适应能显著减少HCAECs再复氧所致的细胞损伤和功能紊乱,eNOS产生的NO启动并介导了LTA保护内皮细胞的作用。     

Tumor suppressor p53 but not cGMP mediates NO-induced expression of p21(Waf1/Cip1/Sdi1) in vascular smooth muscle cells.

Cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascular smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-induced p21 expression, we investigated the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway. The induction of p21 by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) seemed to be due to transactivation because SNAP elevated the activity of p21 promoter but did not stabilize p21 mRNA and protein. Because SNAP did not stimulate the deletion mutant of p21 promoter that lacked p53 binding sites, we tested the involvement of p53. The expression level of p53 was down-regulated after mitogenic stimulation, whereas it was sustained in the presence of SNAP. SNAP markedly stimulated DNA binding activity of p53. Furthermore, SNAP failed to induce p21 in VSMCs obtained from p53-knock out mice and in A431 cells that contained mutated p53. The antiproliferative effect of SNAP also was attenuated in these cells. NO stimulates guanylate cyclase and its product cGMP has been shown to inhibit VSMC proliferation. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, did not prevent SNAP-induced p21 expression. 8-Bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination did not induce p21. Although 8-bromo-cGMP had a small antiproliferative effect, the elevation of cGMP concentration induced by SNAP was little throughout the G(1) phase. The antiproliferative effect of SNAP was not attenuated by Rp-8-bromoguanosine-3',5'-monophosphorothioate, an inhibitor of cGMP-dependent protein kinase. These results suggested that NO induces p21 through a p53-dependent but cGMP-independent pathway.     

Involvements of PHI-nitric oxide and PACAP-BK channel in the sustained relaxation of mouse gastric fundus

The roles of nitric oxide (NO) and K(+) channels in sustained relaxation induced by electrical field stimulation (EFS) in the presence of atropine and guanethidine were studied in circular muscle strips of mouse gastric fundus. In the wild-type mouse, N(G)-nitro-l-arginine (l-nitroarginine), a nitric oxide synthase inhibitor, significantly inhibited the sustained relaxation in addition to the rapid relaxation. The sustained relaxation in pituitary adenylate cyclase-activating peptide (PACAP)-knockout mouse, which was smaller than that of the wild-type mouse, was also inhibited by l-nitroarginine. l-Nitroarginine inhibited the relaxation induced by the peptide histidine isoleucine (PHI), but not that induced by PACAP. S-Nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, -induced relaxation was not affected by PACAP(6-38). EFS-induced sustained relaxation was inhibited by iberiotoxin, a big conductance calcium-activated K(+) (BK) channel inhibitor, but not by apamin, a small conductance calcium-activated K(+) (SK) channel inhibitor, and glibenclamide, an ATP-sensitive K(+) channel inhibitor. The relaxation that remained after the iberiotoxin-treatment was significantly inhibited by l-nitroarginine. Iberiotoxin inhibited PACAP-induced relaxation, while it had no effect on both PHI- and SNAP-induced relaxation. Immunoreactivities to anti-BK channel and anti-PHI antibodies were found in the circular muscle and the myenteric plexus layers, respectively. These results suggest interplay between PHI and NO in the sustained relaxation of the mouse gastric fundus, and that BK channels are involved in the PACAP-component of the sustained relaxation.     

Cellular and Molecular Mechanisms of Nitric Oxide–Induced Heart Muscle Relaxation

• 1.The nitric oxide (NO) donor S-nitro-N-acetyl-penicillamine (SNAP) inhibits Helix aspersa heart activity and relaxes muscles.
• 2.K-free saline and ouabain both depress SNAP-induced relaxation in most experiments, but in a few preparations they either had no effect or potentiated SNAP-induced relaxation.
• 3.Na-K pump reactivation following preincubation in K-free saline leads to the pronounced transient relaxation of heart muscle, the magnitude of which depends on the duration of preincubation.
• 4.0.1 mM SNAP inhibited the ouabain sensitive part of ⁸⁶Rb uptake, which reflects Na-K pump activity. This inhibition is potentiated by phospholipase C.
• 5.SNAP increased cGMP levels in the heart.
• 6.These results indicate that SNAP-induced relaxation depends on Na and Ca gradients across the membrane, which suggests that Na:Ca exchange is involved in the mechanisms of SNAP-induced relaxation. It is postulated that SNAP elicits its inhibitory effect on the heart through a cGMP-dependent Na:Ca exchange.

     

Effects of Kaempferia parviflora Wall. Ex. Baker and Sildenafil Citrate on cGMP Level, Cardiac Function, and Intracellular Ca2+ Regulation in Rat Hearts

ABSTRACT:: Although Kaempferia parviflora extract (KPE) and its flavonoids have positive effects on the nitric oxide (NO) signaling pathway, its mechanisms on the heart are still unclear. Because our previous studies demonstrated that KPE decreased defibrillation efficacy in swine similar to that of sildenafil citrate, the phosphodiesterase-5 inhibitor, it is possible that KPE may affect the cardiac NO signaling pathway. In the present study, the effects of KPE and sildenafil citrate on cyclic guanosine monophosphate (cGMP) level, modulation of cardiac function, and Ca transients in ventricular myocytes were investigated. In a rat model, cardiac cGMP level, cardiac function, and Ca transients were measured before and after treatment with KPE and sildenafil citrate. KPE significantly increased the cGMP level and decreased cardiac function and Ca transient. These effects were similar to those found in the sildenafil citrate-treated group. Furthermore, the nonspecific NOS inhibitor could abolish the effects of KPE and sildenafil citrate on Ca transient. KPE has positive effect on NO signaling in the heart, resulting in an increased cGMP level, similar to that of sildenafil citrate. This effect was found to influence the physiology of normal heart via the attenuation of cardiac function and the reduction of Ca transient in ventricular myocytes.     

rIL-2诱导一氧化氮产生对培养心肌细胞线粒体活性的影响

目的研究人类重组白介素2（rIL-2）对培养大鼠心肌细胞产生一氧化氮（NO）及线粒体活性的影响。方法在培养心肌细胞时分别或同时加入rIL-2、单甲基L-精氨酸（L-NMMA）以及L-精氨酸（L-Arg），并测定培养液中NO浓度，心肌细胞内的线粒体活性值。结果心肌细胞加rIL-2培养48h与对照组相比NO产生显著增加[（85±6.1）比（10±2.5）nmol·（106细胞）-1，P＜0.01]。rIL-2刺激NO产生呈时间（6～48h）和剂量（5×104～1×106IU·L-1）相关性。L-NMMA可抑制rIL-2诱导NO的产生而添加L-Arg可逆转这一作用。rIL-2组与对照组相比线粒体活性值明显受抑（0.397±0.03比0.599±0.02，P＜0.01），而添加L-NMMA可逆转（0.536±0.03，P＜0.01）。NO产生量与线粒体活性值呈负相关（P＜0.01）。结论rIL-2刺激培养大鼠心肌细胞产生NO可抑制线粒体活性。     

The relaxant action of nicorandil in bovine tracheal smooth muscle

The relaxant mechanisms of nicorandil were examined by comparing its effects with those of sodium nitroprusside and cromakalim in bovine tracheal smooth muscle. In preparations contracted with methacholine (0.3 μ mol/l) or high K(+)(40 mmol/l), nicorandil and sodium nitroprusside caused concentration-dependent relaxations. Their relaxant effects on high K(+) -contracted preparations were smaller than those on methacholine-contracted muscle. Cromakalim relaxed methacholine-contracted preparations, whereas it had no effect on high K(+) -contracted muscle. The inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 mol/l) completely prevented the relaxation induced by lower concentrations ( <30 μ mol/l) of nicorandil,whereas it partially attenuated relaxation caused by higher concentrations. The ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide only partially attenuated the relaxant responses to nicorandil (at 100 and 300 μ mol/l). Combination treatment with ODQ and glibenclamide almost completely prevented nicorandil-induced relaxations. The large-conductance Ca2(+) -activated K(+) channel (Maxi K(+) channel) inhibitor iberiotoxin significantly prevented the relaxations induced by lower concentrations (3 and 10 μ mol/l) of nicorandil. The preventive effect of iberiotoxin was markedly enhanced under the blockade of K(ATP) channels with glibenclamide. These results suggest that nicorandil relaxes bovine tracheal smooth muscle through 2 mechanisms: opening of K(ATP) channels and activation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Nicorandil may also activate Maxi K(+) channels, possibly through the NO-cGMP pathway, and the interaction of K ATP channels and Maxi K(+) channels may affect the relaxant effect of nicorandilin bovine tracheal smooth muscle.     

Sodium nitroprusside protects adult rat cardiac myocytes from cellular injury induced by simulated ischemia: role for a non-cGMP-dependent mechanism of nitric oxide protection

The cardioprotective actions of nitric oxide (NO) have largely been attributed to cGMP. NO may, however, elicit some biological actions independently of cGMP. We tested the hypothesis that the NO donor sodium nitroprusside specifically protects isolated cardiomyocytes from injury at least in part independently of its ability to elevate cGMP by using metabolic inhibition to simulate ischemia. Metabolic inhibition-induced injury of adult rat cardiomyocytes (increased activity of lactate dehydrogenase and creatine kinase) was significantly reduced by sodium nitroprusside by at least 30% at all concentrations studied (0.3-100 microM). Sodium nitroprusside (1 microM) increased cardiomyocyte cGMP content, but neither a stable analogue of cGMP (8-bromo-cGMP) nor a potent cGMP stimulus (atrial natriuretic peptide) mimicked the protective effects of sodium nitroprusside. Moreover, inhibition of soluble guanylyl cyclase failed to inhibit sodium nitroprusside cardiomyocyte protection. Conversely, inhibition of either ATP-sensitive potassium (K(ATP)) channels with glibenclamide (10 microM) or calcium-sensitive potassium (K(Ca)) channels with tetraethylammonium bromide (1 mM) or iberiotoxin (20 nM) markedly attenuated the cardioprotective actions of sodium nitroprusside. In conclusion, sodium nitroprusside protects isolated cardiomyocytes from metabolic inhibition independently of cGMP; rather, inhibition of K(Ca) and K(ATP) channels reverses the sodium nitroprusside actions, thus unmasking another mechanism for NO-mediated protection in cardiomyocytes.