Sodium–hydrogen inhibitor cariporide (HOE 642) improves in situ protection of hearts from non–heart-beating donors

BACKGROUND: Reperfusion injury is a vital problem in non-heart-beating donor (NHBD) organs. The sodium-hydrogen inhibitor cariporide is thought to improve cellular integrity after ischemia and reperfusion. Recently, we demonstrated the possibility of preserving hearts with in situ perfusion after circulatory death. The purpose of this study was to determine whether cariporide improves in situ heart protection. METHODS: We studied 20 pigs (18 +/- 2 kg). Hearts in the conventional group (CON, n = 6) underwent cardioplegic arrest with University of Wisconsin solution and then were explanted and stored for 150 minutes on ice. In the other groups, a catheter was placed in each ascending aorta and right atrium. After disconnecting the ventilator, hypoxia caused circulatory arrest within 7 +/- 2 minutes. The aorta was endoclamped, and continuous in situ perfusion of the aortic root was maintained for 60 minutes with University of Wisconsin solution (UW, n = 7) or with UW solution and cariporide (CAR, n = 7). After explantation, the hearts were stored on ice for 90 minutes. In all groups, hearts were reperfused with homologous, whole pig blood in an isolated working heart model for 45 minutes. We monitored stroke-work index on-line, intermittently measured troponin I and malondialdehyde, and compared light microscopic examinations among the groups. RESULTS: Stroke-work index was higher in the CAR group compared with the UW group during the last 20 minutes of reperfusion (10(3)dynes x cm x beats(-1)x gm(-1), 6.6 +/- 1.4 vs 4.5 +/- 2.0, p < 0.05), troponin I was lower in the CAR group compared with the UW group (161 +/- 32 ng/ml vs 277 +/- 35 ng/ml, p < 0.05). Results of malondialdehyde and light microscopic examinations were slightly better in the CAR group, without reaching statistical significance. CONCLUSION: Cariporide as an additive to UW solution improves functional recovery and decreases myocardial damage in hearts from NHBDs protected with an in situ perfusion technique.     

选择性钠氢通道拮抗剂HOE642对未成熟心肌的保护作用

目的比较选择性钠氢通道(NHE1)拮抗剂HOE642在缺血/再灌注不同阶段(缺血前、缺血中、再灌注期)对未成熟心肌的保护作用。方法建立Langendortt离体心脏灌注模型,32只新西兰幼兔随机分为对照组(组Ⅰ,n=8)和HOE642治疗组(n=24),根据给药时间不同, HOE642治疗组又分为:组Ⅱ(n=8)、组Ⅲ(n=8)和组Ⅳ(n=8)。所有心脏均37℃全心缺血60 min,再灌注60 min。组Ⅱ心脏停跳前采用HOE642预处理15 min;组Ⅲ在St.Thomas停跳液中加入HOE642;组Ⅳ于再灌注最初15 min内的KH缓冲液中加入HOE642。测定再灌注期左室发展压(LVDP)、最大收缩变化率( dp/dtmax)、最大舒张变化率(-dp/dtmax)及冠脉流量(CAF);测定心肌细胞内三磷酸腺苷(ATP)、丙二醛(MDA)及心肌含水量(WC);并用透射电镜观察心肌细胞超微结构改变。结果HOE642各治疗组INDP、 dp/dtmax、-dp/dtmax、CAF恢复率及ATP含量明显高于对照组(P<0.05),MDA、心肌含水量明显低于对照组(P<0.05)。HOE642治疗组之间,组Ⅳ再灌注前15 min时 dp/dtmax、-dp/dtmax恢复率低于组Ⅱ和组Ⅲ(P<0.05),而60 min时CAF恢复率高于组Ⅱ和组Ⅲ(P<0.05);组ⅡATP高于组Ⅲ和组Ⅳ(P<0.05),其余指标差异无统计学意义(P> 0.05)。超微结构显示,HOE642治疗能减轻心肌细胞损伤;而HOE642治疗组间,组Ⅳ损伤程度重于组Ⅱ和组Ⅲ。结论缺血/再灌注不同阶段使用HOE642都能减轻未成熟心肌缺血再灌注损伤,而缺血前使用更利于心脏功能恢复、减少心肌损伤。     

Cardioprotective effects of propofol in isolated ischemia-reperfused guinea pig hearts: role of KATP channels and GSK-3β

PURPOSE: Propofol exerts cardioprotective effects, but the involved mechanisms remain obscure. The present study examines the cardioprotective effects of propofol and its role in cardiac function, including its effect on K(ATP) channel opening and the inhibition of GSK-3beta activity in ischemia-reperfused hearts. METHODS: Ischemia-reperfusion (I/R) was produced in isolated guinea pig hearts by stopping coronary perfusion for 25 min, followed by reperfusion. The hearts were incubated for ten minutes, with or without propofol (25 or 50 microM), or for five minutes with 500 microM 5-hydroxydecanoate (a mitochondrial K(ATP) channel blocker) or 30 microM HMR1098 (sarcolemmal K(ATP) channel blocker), followed by five minutes with 50 microM propofol before ischemia. Action potentials on the anterior epicardial surface of the ventricle were monitored using a high-resolution charge-coupled device camera system, and at five minutes after reperfusion, GSK-3beta phosphorylation at the serine residue, Ser9, was examined. RESULTS: After 35 min of reperfusion, propofol (25 and 50 microM) blunted the adverse effects of I/R and reduced infarct size (P < 0.05). In addition, prior incubation with 5-hydroxydecanoate or HMR1098 had no effect on functional recovery improved by 50 microM propofol. At five minutes after reperfusion, propofol (25 and 50 microM) shortened the duration of the action potential and increased the levels of phospho-GSK-3beta (P < 0.05). CONCLUSIONS: Propofol enhanced mechanical cardiac recovery and reduced infarct size. The data further suggest that GSK-3beta play an important role in propofol cardioprotective actions during coronary reperfusion, but mitochondrial K(ATP) channels do not.     

The role of K(+) channels on the inhibitor effect of sevoflurane in pregnant rat myometrium.

Volatile anesthetics and K(+) channel openers inhibit spontaneous contractions in myometrial smooth muscle. Volatile anesthetics modulate K(+) channel activity. We investigated the role of two K(+) channel blockers on the effect of sevoflurane in pregnant rat myometrium. Term pregnant rat uteri were excised, and cross-sectional myometrial strips were mounted for isometric force recording. Sevoflurane inhibited the amplitude and frequency of spontaneous myometrial contractions in a concentration-dependent manner. The maximal inhibition measured in amplitude and frequency of spontaneous myometrial contractions with sevoflurane (at 3 minimum alveolar anesthetic concentration) was 44.32% and 33.32% of control contractions, respectively. Tetraethylammonium (TEA) and glibenclamide, K(+) channel blockers, increased spontaneous myometrial contractions in a concentration-dependent manner. Sevoflurane responses were repeated at concentrations with no effect on spontaneous contractility of TEA, a Ca(2+)-activated K(+) channel blocker, and glibenclamide, an adenosine triphosphate-sensitive K(+) channel blocker, in myometrial strips. TEA (3.10(-4) M) caused a significant reduction in sevoflurane-induced inhibitor responses, but glibenclamide (10(-6) M) did not. Sevoflurane-induced maximal inhibition (at 3 minimum alveolar anesthetic concentration) on amplitude and frequency of spontaneous myometrial contractions in the presence of TEA (3.10(-4) M) was 31.85% and 22.33% of control contractions, respectively (P < 0.05). These results suggest that the in vitroapplication of sevoflurane inhibited the amplitude and frequency of spontaneous myometrial contractions in pregnant rats in a concentration-dependent manner. Such inhibition was reduced by TEA. The inhibition of myometrial smooth muscle induced by sevoflurane seems to be mediated, at least in part, via activation of Ca(2+)-activated K(+) channels, because inhibition was reduced by TEA. IMPLICATIONS: In this study, we found that sevoflurane causes significantly decreased myometrial contractile activity in pregnant rats. The inhibition of myometrial smooth muscle induced by sevoflurane seems to be mediated, at least in part, via activation of Ca(2+)-activated K(+) channels, because inhibition was reduced by tetraethylammonium.     

The role of mitochondrial and sarcolemmal K(ATP) channels in canine ethanol-induced preconditioning in vivo

Chronic consumption of small doses of ethanol protects myocardium from ischemic injury. We tested the hypothesis that mitochondrial and sarcolemmal adenosine triphosphate-dependent potassium (K(ATP)) channels mediate these beneficial effects. Dogs (n = 76) were fed with ethanol (1.5 g/kg) or water mixed with dry food bid for 6 or 12 wk, fasted overnight before experimentation, and instrumented for measurement of hemodynamics. Dogs received intracoronary saline (vehicle), 5-hydroxydecanoate (a mitochondrial K(ATP) channel antagonist; 6.75 mg/kg over 45 min), or HMR-1098 (a sarcolemmal K(ATP) channel antagonist; 45 microg/kg over 45 min) and were subjected to a 60 min coronary artery occlusion followed by 3 h of reperfusion. A final group of dogs was pretreated with ethanol and chow for 6 wk before occlusion and reperfusion. Myocardial infarct size and transmural coronary collateral blood flow were measured with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. The area at risk of infarction was similar between groups. A 12-wk pretreatment with ethanol significantly reduced infarct size to 13% +/- 2% (mean +/- SEM; n = 8) of the area at risk compared with control experiments (25% +/- 2%; n = 8), but a 6-wk pretreatment did not (21% +/- 2%; n = 8). 5-hydroxydecanoate and HMR-1098 abolished the protective effects of 12-wk ethanol pretreatment (24% +/- 2% and 29% +/- 3%, respectively; n = 8 for each group) but had no effect in dogs that did not receive ethanol (22% +/- 2% and 23% +/- 4%, respectively; n = 8 for each group). No differences in hemodynamics or transmural coronary collateral blood flow were observed between the groups. The results indicate that mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning in dogs independent of alterations in systemic hemodynamics or coronary collateral blood flow. IMPLICATIONS: Mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning independent of alterations in systemic hemodynamics or coronary collateral perfusion in vivo.     

Morphine enhances pharmacological preconditioning by isoflurane: role of mitochondrial K(ATP) channels and opioid receptors

BACKGROUND: Adenosine triphosphate-regulated potassium channels mediate protection against myocardial infarction produced by volatile anesthetics and opioids. We tested the hypothesis that morphine enhances the protective effect of isoflurane by activating mitochondrial adenosine triphosphate-regulated potassium channels and opioid receptors. METHODS: Barbiturate-anesthetized rats (n = 131) were instrumented for measurement of hemodynamics and subjected to a 30 min coronary artery occlusion followed by 2 h of reperfusion. Myocardial infarct size was determined using triphenyltetrazolium staining. Rats were randomly assigned to receive 0.9% saline, isoflurane (0.5 and 1.0 minimum alveolar concentration [MAC]), morphine (0.1 and 0.3 mg/kg), or morphine (0.3 mg/kg) plus isoflurane (1.0 MAC). Isoflurane was administered for 30 min and discontinued 15 min before coronary occlusion. In eight additional groups of experiments, rats received 5-hydroxydecanoic acid (5-HD; 10 mg/kg) or naloxone (6 mg/kg) in the presence or absence of isoflurane, morphine, and morphine plus isoflurane. RESULTS: Isoflurane (1.0 MAC) and morphine (0.3 mg/kg) reduced infarct size (41 +/- 3%; n = 13 and 38 +/- 2% of the area at risk; n = 10, respectively) as compared to control experiments (59 +/- 2%; n = 10). Morphine plus isoflurane further decreased infarct size to 26 +/- 3% (n = 11). 5-HD and naloxone alone did not affect infarct size, but abolished cardioprotection produced by isoflurane, morphine, and morphine plus isoflurane. CONCLUSIONS: Combined administration of isoflurane and morphine enhances the protection against myocardial infarction to a greater extent than either drug alone. This beneficial effect is mediated by mitochondrial adenosine triphosphate-regulated potassium channels and opioid receptors in vivo.     

Functional and metabolic effects of nicardipine on ischemic rat hearts with multidose potassium cardioplegia

This study was undertaken to assess the effect of a calcium antagonist, nicardipine (N), added in a cardioplegic solution on the ischemic myocardium. Isolated rat hearts were perfused with oxygenated Krebs Ringer Bicarbonate (KRB) solution by Langendorff's perfusion method and were subjected to 2 hours of ischemic arrest at 30 degrees C with multidose cardioplegia (every 30 min, for 5 min) and a subsequent 60 min of reperfusion. HR, LVP, coronary flow and oxygen tension of coronary effluent were monitored. Oxygen saturation of intracellular myoglobin and redox state of mitochondrial cytochrome aa3 in the myocardial cell were continuously measured throughout studies by a spectrophotometer. Oxygenated crystalloid cardioplegic solution (KRB) containing 25 mM of potassium was used. 40 rats were divided into 4 groups (10 rats each) according to the concentration of N (none, 0.5, 1 and 2 mg/L) in fully oxygenated potassium cardioplegic solution (PO2: 601 +/- 31 mmHg). The percent recovery of pressure-rate product after reperfusion was compared in each group and the optimal concentration of N was found to be 1 mg per liter of cardioplegic solution. No significant difference was found between Group Ia (N = 0 mg/L) and Group Ib (N = 1 mg/L) in metabolic or hemodynamic recovery after reperfusion. In other experiments, 40 rats in Group IIa (N = 0 mg/L, n = 20) and Group IIb (N = 1 mg/L, n = 20) received 10 ml of poorly oxygenated cardioplegic solution (PO2: 215 +/- 10 mmHg) on each reinfusion followed by a 25 min interval of ischemic arrest. The index of oxygen utilization, MVO2/pressure-rate product after reperfusion was significantly lower in Group IIb than in Group IIa (p less than 0.05). The results show that the addition of N (1 mg/L) to the cardioplegic solution preserved a more aerobic state (higher intracellular oxygen level) in the myocardium by further suppressing myocardial oxygen demand during the ischemic period which resulted in better myocardial protection. Therefore, it is concluded that the addition of N to the cardioplegic solution enhances myocardial preservation during myocardial ischemia.     

HOE-642联合改良K/Mg冷血心麻液对犬供心的保护作用

目的探讨HOE-642联合改良K／Mg冷血心麻液提高犬供心的保存效果的作用。方法雄性成年杂种家犬32只，随机分为整合组和常规组（每组16只，供、受者各8只）。（1）整合组：供心保护采用HOE-642联合改良K／Mg冷血心麻液的“整合策略”；（2）常规组：供心保护采用临床上常规方法。所有动物的供心均冷浸浴保存4h，心脏移植用原位标准法。记录不同时段的心功能指标；实验结束后观察心肌超微结构并测定心肌含水量。结果每组各1例主动脉开放后因主动脉吻合口漏血，失血陛休克死亡，剔除出实验；其余动物均成功脱离体外循环。整合组的移植心脏左心室收缩功能和舒张功能恢复指标显著优于常规组（P〈0．05）；整合组心肌组织含水量显著小于常规组（P〈0．05）；整合组心肌细胞亚细胞结构的保存亦优于常规组。结论临床常规方法和HOE-642联合改良K／Mg冷血心麻液的“整合策略”，均可安全冷保存供心4h，但后者能进一步减轻心肌水肿、维持心肌细胞结构完整、促进缺血／再灌注损伤后心功能的恢复，具有更佳的心肌保护效果。     

PI3K、ERK1/2、mito-K_(ATP)通道及mPTP在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 评价磷脂酰肌醇-3-激酶(PI3K)及细胞外信号调节激酶1/2(ERK1/2)、线粒体ATP敏感性钾(mito-K_(ATP))通道及线粒体膜通透性转换孔(mPTP)在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 雄性清洁级SD大鼠,周龄7～10周,体重250～300 g,采用Langendorff法建立大鼠离体心脏灌注模型,取模型制备成功的心脏180个,随机分为12组(n=15),对照组(C组):持续灌注90 min;缺血再灌注组(IR组):停止灌注K-H液30 min,再灌注60 min;IR+LY组、IR+PD组、IR+ATR组、IR+5-HD组和IR+DMSO组:于再灌注即刻分别灌注PI3K特异性抑制剂LY294002(LV)15μmol/L、ERK1/2特异性抑制剂PD98059(PD)20 μmol/L、mPTP开放剂苍术甙(ATR)20 μmol/L、mito-K_(ATP)通道抑制剂5-羟癸酸(5-HD)100 μmol/L和溶剂二甲基亚砜(DMSO)0.02%15 min;七氟醚后处理组(S组):于再灌注即刻灌注经3%七氟醚饱和的K-H液15 min,随后更换正常K-H液再灌注45 min;S+LY组、S+PD组、S+ATR组和S+5-HD组:于再灌注即刻灌注经七氟醚饱和的K-H液同时分别灌注LY 15 μmol/L、PD 20 μmol/L、ATR 20 μmol/L、5-HD 100 μmol/L 15 min,随后更换为正常K-H液再灌注45 min.于平衡灌注20 min、停灌前即刻、再灌注15、30和60 min(T_(0～4))时测定冠状动脉流量(CF),记录心功能指标;C组、IR组和s组分别于T_0和T_4时收集冠状动脉流出液测定乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)活性和肌钙蛋白I(cTnI)浓度;于T_4时取左心室,测定心肌梗死面积,C组、IR组、IR+ATR组、IR+5.HD组、IR+DMSO组、S组、S+ATR组和S+5-HD组检测细胞凋亡情况,计算凋亡指数(AI),C组、IR组、IR+LY组、IR+PD组、IR+DMSO组、S组、S+LY组和S+PD组测定心肌烟酰胺腺嘌呤二核苷酸(NAD~+)含量.结果 与C组比较,其余各组再灌注时心功能降低,CF降低,心肌梗死面积增大,IR组、IR+ATR组、IR+5-HD组、IR+DMSO组、S组、S+ATR组和S+5-HD组AI升高,IR组、IR+LY组、IR+PD组、IR+DMSO组、S组、S+LY组和S+PD组NAD~+含量降低,IR组和S组LDH、CK-MB活性和cTnI浓度升高(P<0.05);与IR组比较,S组再灌注时心功能提高,CF升高,心肌梗死面积减小,AI降低,NAD~+含量升高,LDH、CK-MB活性和cTnI浓度降低(P<0.05),其余组上述指标差异无统计学意义(P>0.05).结论 七氟醚后处理可能通过激活PI3K及ERK1/2、促进mito-K_(ATP)通道开放、抑制mPTP开放,从而减轻大鼠离体心脏缺血再灌注损伤.     

Cardioprotective Effects of Propofol and Sevoflurane in Ischemic and Reperfused Rat Hearts: Role of KATP Channels and Interaction with the Sodium-Hydrogen Exchange Inhibitor HOE 642 (Cariporide)

Background: Sodium ion-hydrogen ion (Na+-H+) exchange inhibitors are effective cardioprotective agents. The N+-H+ exchange inhibitor HOE 642 (cariporide) has undergone clinical trials in acute coronary syndromes, including bypass surgery. Propofol and sevoflurane are also cardioprotective via unknown mechanisms. The authors investigated the interaction between propofol and HOE 642 in the ischemic reperfused rat heart and studied the role of adenosine triphosphate-sensitive potassium (KATP) channels in the myocardial protection associated with propofol and sevoflurane.

Methods: Isolated rat hearts were perfused by the Langendorff method at a constant flow rate, and left ventricular function and coronary pressures were assessed using standard methods. Energy metabolites were also determined. To assess the role of KATP channels, hearts were pretreated with the KATP blocker glyburide (10 [mu]M). Hearts were then exposed to either control buffer or buffer containing HOE 642 (5 [mu]M), propofol (35 [mu]M), sevoflurane (2.15 vol%), the KATP opener pinacidil (1 [mu]M), or the combination of propofol and HOE 642. Each heart was then subjected to 1 h of global ischemia followed by 1 h of reperfusion.

Results: Hearts treated with propofol, sevoflurane, pinacidil, or HOE 642 showed significantly higher recovery of left ventricular developed pressure and reduced end-diastolic pressures compared with controls. The combination of propofol and HOE 642 provided superior protection toward the end of the reperfusion period. Propofol, sevoflurane, and HOE 642 also attenuated the onset and magnitude of ischemic contracture and preserved high-energy phosphates (HEPs) compared with controls. Glyburide attenuated the cardioprotective effects of sevoflurane and abolished the protection observed with pinacidil. In contrast, glyburide had no effect on the cardioprotection associated with propofol treatment.     

HOE-642联合尼可地尔对无心跳猪供心的保护作用

目的 探讨无心跳供者(NHBD)的心脏进行移植的可行性,以及HOE-642联合尼可地尔对这种供心的保护效果.方法 将健康雄性家猪随机分为实验组和对照组,实验组供者采取主动脉快速完全放血法制成NHBD模型,取其心脏,冷保存4 h后进行移植;对照组供者经主动脉适量放血(仍有心跳),造成热缺血,然后取其心脏,冷保存4 h后进行移植.实验组于供心热缺血前5 min静脉给予HOE-642(2 mg/kg);以含HOE-642和尼可地尔的4 ℃ Stanford液灌洗和保存供心;供心吻合前经主动脉根部以含HOE-642和尼可地尔的4 ℃ 4:1血心停搏液(高钾)灌注1次;供心吻合期间以含HOE-642和尼可地尔的4 ℃ 4:1血心停搏液(低钾)间断灌注;心脏吻合完毕主动脉开放后初始5 min时段内,静脉给予HOE-642(2 mg/kg).对照组的供心处理除不用HOE-642和尼可地尔外,其余同实验组.采集供心主动脉根部放血前、移植心脏吻合完毕主动脉开放后1 h(停机)以及主动脉开放后2 h(实验结束)3个时点的心脏血流动力学指标,测定移植心脏的心肌含水量,观察心肌的组织学变化.结果 两组在主动脉开放后心肌红润,心脏搏动有力,均成功脱机.两组各检测时点的左心室舒张末压、左心室峰发展压及左心室压力变化速率的差异均无统计学意义(P＞0.05);实验组和对照组心肌组织含水量分别为(78.6±5.7)%和(76.4±4.2)%,其差异无统计学意义(P＞0.05);光镜下见两组的心肌纤维结构清楚,排列紧密,间质未见炎症细胞浸润,也未见变性、坏死,无排斥反应征象,电镜下见心肌细胞超微结构完整.结论 NHBD的供心可用于移植,其效果与有心跳者相近;HOE-642和尼可地尔联用可能对该类供心具有一定的保护作用.     

The role of nitric oxide, K(+)(ATP) channels, and cGMP in the preconditioning response of the rabbit

BACKGROUND: The role of nitric oxide (NO), K(+)(ATP) channels, and cyclic GMP (cGMP) in preconditioning is unknown. MATERIAL AND METHODS: Isolated rabbit hearts were pretreated with the NO precursor L-arginine (L-Arg), both alone and after infusion of the NO synthetase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Guanylate cyclase inhibitor methylene blue (MB) was infused prior to L-Arg in a separate group of hearts. To contrast the mechanisms of NO preconditioning and potassium channel opener (PCO) preconditioning, we infused the PCO pinacidil after L-NAME and the PCO blocker glibenclamide before L-Arg. Control hearts had no drug infused. The LAD coronary artery was occluded for 1 h and reperfused for 1 h in all hearts. Action potential duration (APD(50)), coronary flow (CF), and left ventricular developed pressure (DP) were measured, and infarct size (IS) was determined and expressed as a percentage of the area at risk. RESULTS: L-Arg prolonged APD(50) at 60 min of reperfusion (94 +/- 6 ms vs 69 +/- 2 ms (control) vs 70 +/- 2 ms (L-NAME) vs 74 +/- 3 ms (MB), P < 0.05). L-Arg reduced IS compared with control (24 +/- 2% vs 49 +/- 3%, P < 0.05); this was reversed by either L-NAME (53 +/- 4%, P < 0.05) or MB (43 +/- 3%, P < 0.05), but not by glibenclamide (20 +/- 4%), unlike the increase in CF during L-Arg infusion, which was blocked by glibenclamide. Pinacidil infusion decreased IS (26 +/- 2%), but this effect was blocked by L-NAME (53 +/- 7%, P < 0.05 vs pinacidil), although L-NAME did not blunt the increase in CF. There were no significant differences in DP among groups. CONCLUSION: L-Arginine preconditions the heart through NO generation, and this response is mediated through a cGMP-dependent mechanism, but is independent of the K(+)(ATP) channels. Coronary vasodilation is mediated through a mechanism different from that responsible for cardiomyocyte preconditioning.     

Differential effects of anesthetics on mitochondrial K(ATP) channel activity and cardiomyocyte protection

BACKGROUND: Mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels play a pivotal role in mediating cardiac preconditioning. The effects of intravenous anesthetics on this protective channel have not been investigated so far, but would be of importance with respect to experimental as well as clinical medicine. METHODS: Live cell microscopy was used to visualize and measure autofluorescence of flavoproteins, a direct reporter of mitoK(ATP) channel activity, in response to the direct and highly selective mitoK(ATP) channel opener diazoxide, or to diazoxide following exposure to various anesthetics commonly used in experimental and clinical medicine. A cellular model of ischemia with subsequent hypoosmolar trypan blue staining served to substantiate the effects of the anesthetics on mitoK(ATP) channels with respect to myocyte viability. RESULTS: Diazoxide-induced mitoK(ATP) channel opening was significantly inhibited by the anesthetics R-ketamine, and the barbiturates thiopental and pentobarbital. Conversely, urethane, 2,2,2-trichloroethanol (main metabolite of alpha-chloralose and chloral hydrate), and the opioid fentanyl potentiated the channel-opening effect of diazoxide, which was abrogated by coadministration of chelerythrine, a specific protein kinase C inhibitor. S-ketamine, propofol, xylazine, midazolam, and etomidate did not affect mitoK(ATP) channel activity. The significance of these modulatory effects of the anesthetics on mitoK(ATP) channel activity was substantiated in a cellular model of simulated ischemia, where diazoxide-induced cell protection was mitigated by R-ketamine and the barbiturates, while urethane, 2,2,2-trichloroethanol, and fentanyl potentiated myocyte protection. CONCLUSIONS: These results suggest distinctive actions of individual anesthetics on mitoK(ATP) channels and provide evidence that the choice of background anesthesia may play a role in cardiac protection in both experimental and clinical medicine.     

不同浓度七氟醚预处理对大鼠海马神经元缺氧复氧时细胞凋亡的影响及线粒体ATP敏感型钾通道在其中的作用

目的 探讨不同浓度七氟醚预处理对大鼠海马神经元缺氧复氧时细胞凋亡的影响及线粒体ATP敏感型钾通道(mito-KATP通道)在其中的作用.方法 新生(出生＜24 h)SD大鼠,雌雄不拘,体重5～6 g,原代培养海马神经元,接种于培养孔或培养皿中,采用随机数字表法,将其随机分为7组,每组48孔和12皿,正常对照组(C组):不予任何处理;缺氧复氧组(HR组):缺氧4 h复氧24 h;6%七氟醚预处理组(S1 组)、4%七氟醚预处理组(S2 组)、2%七氟醚预处理组(S3 组):分别经6%、4%、2%七氟醚预处理后行缺氧复氧;5-羟葵酸100 μmol/L预处理组(5-HD组):经mito-KATP通道阻断剂5-羟葵酸(终浓度100 μmol/L)预处理后进行缺氧复氧;5-羟葵酸100 μmol/L+6%七氟醚预处理组(5-HD+S组):同时行5-羟葵酸和6%七氟醚预处理后进行缺氧复氧.各组以上处理结束后,测定神经元活力、凋亡率、Bcl-2和Bax蛋白的表达水平.结果 与C组比较,其余6组海马神经元活力降低,细胞凋亡率升高,Bcl-2和Bax蛋白表达上调(P＜0.01);与HR组比较,S1组～S3组海马神经元活力增强,细胞凋亡率降低,Bcl-2蛋白表达上调,Bax蛋白表达下调(P＜0.01),5-HD组和5-HD+S组上述指标比较差异无统计学意义(P＞0.05);与S1组比较,S2组、S3组和5-HD+S组海马神经元活力降低,细胞凋亡率升高,Bcl-2蛋白表达下调,Bax蛋白表达上调(P＜0.01);与S2组比较,S3组海马神经元活力降低,细胞凋亡率升高,Bcl-2蛋白表达下调,Bax蛋白表达上调(P＜0.01).结论 七氟醚预处理可抑制大鼠海马神经元缺氧复氧时细胞凋亡,从而减轻神经元损伤,且呈浓度依赖性,机制可能与开放神经元mito-KATP通道,上调Bcl-2蛋白表达,下调Bax蛋白表达有关.

Abstract：

Objective To investigate the effect of preconditioning with different concentrations of sevoflurane on hypoxia-reoxygenation(H/R)-induced apoptosis in rat hippocampal neurons and the role of mitochondrial KATP(mito-KATP)channels.Methods Primary cultured hippocampal neurons isolated from newborn SD rats(＜24h)of both sexes,weighing 5-6 g,were randomly divided into 7 groups with 48 wells and 12 dishes in each one:control group(C group),H/R group,preconditioning with 6%,4%and 2% sevoflurane groups(S1-3 groups),5-hydroxydecanoate(5-HD,mito-KATP channel blocker)100 μmol/L preconditioning group(5-HD group)and preconditioning with 5-HD 100 μmol/L+6% sevoflurane group(5-HD+S group).The neurons were exposed to 4 h hypoxia followed by 24 h reoxygenation. In S1-3 groups, preconditioning was performed with 6% , 4% and 2% sevoflurane respectively before H/R. In 5-HD group, preconditioning was performed with 5-HD (final concentration 100 μmol/L) before H/R. In 5-HD + S group, preconditioning was performed with 5-HD 100 μmol/L and 6% sevoflurane before H/R. The neuronal viability, apoptosis rate and expression of Bcl-2 and Bax were determined after 24 h reoxygenation.Results The neuronal viability was significantly lower,while the apoptosis rate and expression of Bcl-2 and Bax were significantly higher in the other 6 groups than in group C(P＜0.01).The neuronal viability and expression of Bcl-2 were significantly higher,while the apoptosis rate and Bax expression were lower in S1-3 groups than in group H/R. There was no significant difference in the parameters mentioned above between 5-HD and 5-HD + S groups(P＞0.05).The neuronal viability and expression of Bcl-2 were significantly lower, while the apoptosis rate and Bax expression were higher in S2, S3 and 5-HD + S groups than in group S1, and in group S3 than in group S2(P＜0.0l) .Conclusion Sevoflurane preconditioning can inhibit H/R-induced apoptosis in rat hippocampal neurons and reduce the injury to neurons in a concentration-dependent manner, and the underlying mechanism may be related to activation of mito-KATP channels, up-regulation of Bcl-2 expression and down-regulation of Bax expression.     

The cardioprotective effect of sevoflurane depends on protein kinase C activation, opening of mitochondrial K(+)(ATP) channels, and the production of reactive oxygen species

Several studies suggest that the cardioprotective effect of sevoflurane depends on protein kinase C (PKC) activation, mitochondrial K(+)(ATP) channel (mitoK(+)(ATP)) opening, and reactive oxygen species (ROS). However, evidence for their involvement was obtained in separate experimental models. Here, we studied the relative roles of PKC, mitoK(+)(ATP), and ROS in sevoflurane-induced cardioprotection in one model. Rat trabeculae were subjected to simulated ischemia by applying metabolic inhibition (MI) through buffer containing NaCN, followed by 60-min reperfusion. Recovery of active force (F(a)) was assessed as percentage of pre-MI force. In time controls, F(a) amounted 60% +/- 5% at the end of the experiment. The recovery of F(a) after MI was reduced to 28% +/- 5% (P = 0.045 versus time control), whereas sevoflurane reversed the detrimental effect of MI (F(a) recovery, 67% +/- 8%; P = 0.01 versus MI). The PKC inhibitor chelerythrine, the mitoK(+)(ATP) inhibitor 5-hydroxy decanoic, and the ROS scavenger N-(2-mercaptopropionyl)-glycine all completely abolished the protective effect of sevoflurane (recovery of F(a), 31% +/- 8%, 33% +/- 8%, and 24% +/- 9% for chelerythrine, 5-hydroxy decanoic, and N-(2-mercaptopropionyl)-glycine, respectively). In conclusion, PKC activation, mitoK(+)(ATP) channel opening, and ROS production are all essential for sevoflurane-induced cardioprotection. These signaling events are arranged in series within a common signaling pathway, rather than in parallel cascades. Our findings implicate that the perioperative use of sevoflurane preserves cardiac function by preventing ischemia-reperfusion injury. IMPLICATIONS: Protein kinase C, mitochondrial K(+)(ATP) channels and reactive oxygen species act within one downstream signaling pathway in mediating the cardioprotective effect of sevoflurane.     

Effects of sevoflurane on the cAMP-induced short-circuit current in mouse tracheal epithelium and recombinant Cl- (CFTR) and K+ (KCNQ1) channels

Background: An optimal level of airway surface liquid is essential for mucociliaryclearance in lungs. The cAMP-activated cystic fibrosis transmembraneconductance regulator (CFTR) and KCNQ1 channels in trachealepithelium play key roles in luminal and basolateral membranes,respectively. The aim of this study was to examine the effectsof sevoflurane on cAMP-induced chloride secretion by the mousetracheal epithelium and the modulation of recombinant CFTR andKCNQ1 channels. Methods: The equivalent short-circuit current (I_sc) of the mouse trachealepithelium was measured using a flow-type Ussing chamber technique.Inhibition of Na⁺ absorption was achieved through the luminalapplication of amiloride. cAMP-dependent Cl^– secretionwas evoked by forskolin and isobutylmethylxanthine (Fsk/IBMX)applied to the basolateral side. The effect of sevoflurane onCFTR and KCNQ1 channels was assessed using a whole-cell patchclamp in human embryonic kidney 293T cells expressing CFTR andKCNQ1 channels. Results: Fsk/IBMX induced a sustained I_sc that was suppressed by theapplication of sevoflurane [decreased by 49 (4.5)% at 190 µM].The Fsk/IBMX-induced I_sc was also blocked by basolateral applicationof chromanol 293B, a blocker of the KCNQ1 K⁺ channel. In KCNQ1-expressingcells, sevoflurane 190 µM reduced the outward currentsto 59 (4.9)% at 80 mV. The CFTR current was not affected bysevoflurane (360 µM). Conclusions: These results suggest that the inhibition of KCNQ1 underliessevoflurane-induced decrease in airway secretion.     

Effect of hypothermia on the ischemic and reperfused rat skeletal muscle, monitored by in vivo (31)P-magnetic resonance spectroscopy.

The aim of this study was to compare the ischemic and postischemic energetic changes of rat skeletal muscle in response to hypothermia or room temperature, monitored noninvasively and continuously by in vivo (31)P-magnetic resonance spectroscopy ((31)P-MRS). A model of pedicled rat rectus femoris muscle was developed and analyzed by in vivo (31)P-MRS at a magnetic field strength of 2.35 T. Measurements were performed at three time points: before ischemia, after 4 hours of ischemia, and after 1 hour of reperfusion. Three groups were studied: (1) sham-operated rats (n = 6); (2) rats subjected to room temperature (24-26 degrees C, n = 6); and (3) rats subjected to hypothermia (9-12 degrees C, n = 6). Blood perfusion was measured by laser Doppler flowmetry (LDF). In the hypothermic group, phosphocreatine (PCr) recovered to 75% and adenosine triphosphate (ATP) to 86%; in the room temperature group, the recovery was 53% and 51%, respectively (P < 0.05). Skeletal muscle subjected to hypothermia (9-12 degrees C) was found to recover to a higher postischemic energetic level compared with skeletal muscle subjected to room temperature. Hypothermia appears to be a simple and effective method with which to reduce the damage related to ischemia and reperfusion.     

The role of K+ channels in vasorelaxation induced by hypoxia and the modulator effects of lidocaine in the rat carotid artery

Hypoxia induces vasodilation, partly via the activation of K(+) channels. Lidocaine impairs vasorelaxation mediated by a K(+) channel opener, suggesting that this antiarrhythmic drug may inhibit hypoxia-induced vasodilation mediated by K(+) channels. We designed the current study to determine whether, in the carotid artery, K(+) channels contribute to vasorelaxation in response to hypoxia and whether lidocaine modulates vasorelaxation induced by K(+) channels via pathophysiological and pharmacological stimuli. Rings of rat common carotid artery without endothelium were suspended for isometric force recording. During contraction to phenylephrine, hypoxia-induced vasorelaxation or concentration-response to an adenosine triphosphate-sensitive K(+) channel opener was obtained changing control gas to hypoxic gas and the cumulative addition of levcromakalim, respectively. Hypoxia-induced vasorelaxation was significantly reduced by glibenclamide (5 micro M) but not by iberiotoxin (0.1 micro M), apamin (0.1 micro M), BaCl(2) (10 micro M), or 4-aminopyridine (1 mM). Levcromakalim-induced vasorelaxation was completely abolished by glibenclamide. Lidocaine (10-100 micro M) concentration-dependently inhibited this vasodilation, whereas it did not affect hypoxia-induced vasodilation. These results suggest that adenosine triphosphate-sensitive K(+) channels play a role in hypoxia-induced vasodilation in the rat carotid artery and that lidocaine differentially modulates vasodilation via these channels activated by pathophysiological and pharmacological stimuli. IMPLICATIONS: In rat carotid artery, levcromakalim produced vasorelaxation mediated by adenosine triphosphate (ATP)-sensitive K(+) channels, whereas hypoxia induced it partly via these channels. Lidocaine inhibited vasorelaxation induced by an ATP-sensitive K(+) channel opener but not by hypoxia, indicating the differential mechanisms of modulatory effects of this antiarrhythmic drug on vasodilation via ATP-sensitive K(+) channels activated by pathophysiological and pharmacological stimuli.     

Antinociceptive action of epidural K+(ATP) channel openers via interaction with morphine and an alpha(2)- adrenergic agonist in rats

Potassium (K(+)) channels may play some role in the analgesic actions of mu-opioid agonists and alpha(2)-adrenergic agonists (alpha(2) agonists). We examined whether the adenosine triphosphate-sensitive K(+)(K(+)(ATP)) channel openers, levcromakalim and nicorandil, (given epidurally), might have antinociceptive effects in a tail flick test in adult male Sprague-Dawley rats implanted with a lumbar epidural catheter. The interactions with morphine and an alpha(2) agonist were also examined. The epidural administration of levcromakalim (10 microg, 100 microg) or nicorandil (10 microg, 100 microg) alone did not produce antinociception, but 100 microg levcromakalim or nicorandil did potentiate the antinociceptive effect induced by epidural morphine. Epidural glibenclamide (10 microg), a K(+)(ATP) channel blocker, or naloxone (10 microg) antagonized this potentiation. Systemic administration of levcromakalim or nicorandil (at the same dose as that given into the epidural space) did not potentiate the epidural morphine-induced analgesia. A combination of epidural dexmedetomidine (1 microg) and morphine (1 microg) (each at a subantinociceptive dose) had a significant antinociceptive effect, and epidural glibenclamide (10 microg) partly antagonized this antinociception. These data suggest that levcromakalim and nicorandil potentiate the analgesic action of both morphine and dexmedetomidine, probably via an activation of K(+)(ATP) channels at the spinal cord level.