氟烷和七氟醚对缺血再灌注心肌功能和氧自由基的影响

目的：研究１５肺泡最小浓度（ＭＡＣ）的氟烷和七氟醚对缺血再灌注心肌功能和氧自由基的影响。方法：应用离体大鼠心脏Ｌａｎｇｅｎｄｏｒｆ逆行灌注模型研究１５ＭＡＣ的氟烷、七氟醚对心肌缺血前后心功能的影响,测定缺血前、缺血１０ｍｉｎ、复灌３０ｍｉｎ３个不同时间的心肌超氧化物歧化酶（ＳＯＤ）活性和丙二醛（ＭＤＡ）含量。结果：七氟醚不同程度地抑制心肌收缩功能。缺血１０ｍｉｎ时,七氟醚组ＳＯＤ酶活性明显下降,ＭＤＡ含量显著升高。缺血２５ｍｉｎ复灌３０ｍｉｎ后,二药均能促进心肌功能和ＳＯＤ酶活性恢复,抑制ＭＤＡ生成,其中七氟醚的作用较为明显。结论：二药对缺血再灌注心肌具有一定的保护作用,七氟醚优于氟烷。     

PTSD样情感行为异常大鼠海马ATP酶活性与Ca2+/CaM改变

目的:探讨创伤后应激障碍(PTSD)精神与行为异常的病理生理基础。方法:通过频率25Hz、波宽1ms、串长10s、串隔7min、强度100μA的恒流、单向方波, 建立海马惊厥阈下电刺激PTSD动物模型;采用神经生化、流式细胞仪、荧光标记术及Westernblotting等方法, 定量观测了实验动物海马Na⁺-K⁺-ATP酶、Ca²⁺-ATP酶活性, 细胞内Ca²⁺含量与钙调素(CaM)相对活性平均通道荧光及海马组织总CaM表达的动态变化规律。结果:电刺激停止后48h内实验动物海马细胞线粒体Na⁺-K⁺-ATP酶活性明显下降, 72h内Ca²⁺-ATP酶活性显著降低;海马细胞[Ca²⁺]i于电刺激停止后72h内明显增高, 游离CaM平均通道荧光则同步降低, 而海马组织总CaM表达则于电刺激停止后48h内明显增多。结论:海马细胞[Ca²⁺]i持续增高、结合CaM含量明显增加及线粒体钠钾泵与钙泵功能受损, 可能是实验动物长时程PTSD样情感行为异常的重要病理生理基础之一。     

牛磺酸对缺血大鼠心肌线粒体Ca2+-Mg2+-ATP酶活性与MDA含量影响

目的和方法：用Ｗｉｓｔａｒ大鼠皮下注射异丙肾上腺素（ＩＳＰ,５ｍｇ／ｋｇ）诱导心肌缺血模型。观测心肌线粒体（Ｍｉｔ）中丙二醛（ＭＤＡ）含量、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性及牛磺酸（Ｔａｕ）的影响。结果：缺血组大鼠心肌Ｍｉｔ中ＭＤＡ升高８７８３％、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性分别降低３７５６％和５０２０％（Ｐ＜０．０１）。Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶活性与ＭＤＡ含量也呈显著负相关（ｒ＝－０．８７,Ｐ＜０．０１）。Ｃａ２＋－ＡＴＰ酶活性与ＭＤＡ含量也呈显著负相关（ｒ＝－０７９,Ｐ＜０．０１）。在注射异丙肾上腺素（ＩＳＰ）前３０ｍｉｎ腹腔注射Ｔａｕ（２００ｍｇ／ｋｇ）则Ｍｉｔ中ＭＤＡ含量、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性均未见显著异常改变。结论：Ｔａｕ可能通过抑制ＭＤＡ的生成实现其保护Ｃａ２＋－ＡＴＰ酶和Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶活性的作用。     

预缺氧对急性缺氧大鼠心肌线粒体功能及ATP含量的影响

目的：观察预缺氧对急性缺氧大鼠心肌线粒体功能及ＡＴＰ含量的影响。方法：实验大鼠分三组。１常氧对照组;２急性缺氧组;３预缺氧组。测定了心肌ＡＴＰ含量及线粒体呼吸功能,以荧光偏振法测定线粒体膜流动性。结果：经预缺氧处理的大鼠遭受急性缺氧后ＡＴＰ含量从（３１８±２４２）ｍｇ１·ｇ－１增加到（６０５５±３．５２）ｍｇ－１·ｇ－１（Ｐ＜００１）;线粒体呼吸控制率（ＲＣＲ）从１８４±０５８上升到４５５±０３２（Ｐ＜００１）;线粒体膜流动性（ＭＭＦ）明显增加（Ｐ＜００５）,Ｆ０Ｆ１－ＡＴＰ酶及Ｎａ＋－Ｋ＋－ＡＴＰ酶活性分别提高６６％和２５％。结论：预缺氧可有效改善缺氧大鼠心肌能量代谢,其作用环节可能和提高线粒体膜流动性,改善线粒体呼吸功能有关。     

1,6-二磷酸果糖对阿霉素引起大鼠心肌细胞内游离钙和肌浆网Ca2+-ATP酶活性改变的影响

目的:探讨1,6-二磷酸果糖(FDP)对阿霉素(ADR)所引起的大鼠心肌细胞内游离钙及心肌肌浆网钙-ATP酶(SRCa²⁺-ATPase)活性变化的影响。方法: 给大鼠腹腔注射ADR(2.5 mg·kg^-1,隔日1次,共6次),给ADR处理的大鼠腹腔注射不同剂量的FDP(隔日1次,共21次)进行干预。采用双抗体双夹心ELISA法定量测定血清肌钙蛋白I(CTnI);采用抗肌酸激酶同工酶(CK-MB)单克隆抗体包被小球测定血清CK-MB;用荧光分光光密度计测定心肌细胞内游离钙(MyoCa²⁺)浓度;用无机磷酸根法测定心肌肌浆网钙-ATP酶(SRCa²⁺-ATPase)活性。结果:FDP(300、600、1 200 mg·kg^-1)干预ADR处理的大鼠后,可显著降低血清CtnI、CK-MB及心肌细胞内MyoCa²⁺值,显著增高SRCa²⁺-ATPase活性(P＜0.01)。结论: FDP能通过降低心肌细胞内游离钙和对SRCa²⁺-ATPase活性的改变,起到减轻ADR对心肌的毒性作用。     

缺血预处理对大鼠心肌Na^＋,K^＋—ATP酶和Ca^2＋,Mg^2＋—ATP酶活…

离体大鼠心脏经１０ｍｉｎ左冠状动脉缺血加３０ｍｉｎ再灌注后，心肌中Ｎａ＾＋，Ｋ＾＋－ＡＴＰ酶的活力显著降低，Ｃａ＾２＋，Ｍｇ＾２＋－ＡＴＰ酶的活力稍上升。离体心脏经３次３ｍｉｎ全心缺血加５ｍｉｎ再灌注的缺血预处理后，显著减轻随后缺血－再灌注引起的Ｎａ＾＋，Ｋ＾＋－ＡＴＰ酶活力降低，并有进一步增高Ｃａ＾２＋，Ｍｇ＾２＋－ＡＴＰ酶活力的趋势。缺血预处理也显著减轻缺血－再灌注时的心肌收缩力下降和心律失常     

缺血预处理对大鼠心肌Na~ ,K~ -ATP酶和Ca~(2 ),Mg~(2 )-ATP酶活性的影响

离体大鼠心脏经１０ｍｉｎ左冠状动脉缺血加３０ｍｉｎ再灌注后，心肌中Ｎａ＋，Ｋ＋－ＡＴＰ酶的活力显著降低，Ｃａ２＋，Ｍｇ２＋－ＡＴＰ酶的活力稍上升。离体心脏经３次３ｍｉｎ全心缺血加５ｍｉｎ再灌注的缺血预处理后，显著减轻随后缺血－再灌注引起的Ｎａ＋，Ｋ＋－ＡＴＰ酶活力降低，并有进一步增高Ｃａ２＋，Ｍｇ２＋－ＡＴＰ酶活力的趋势。缺血预处理也显著减轻缺血－再灌注时的心肌收缩力下降和心律失常的发生。在体大鼠心脏缺血预处理对这些酶活力的影响与离体缺血预处理的结果相似，表明缺血预处理对这些酶活力的影响是作用于心脏本身所致。     

一氧化碳对缺血脑组织神经细胞胞内Ca2+浓度的影响

目的:研究一氧化碳对局灶性缺血脑组织神经细胞胞内Ca²⁺浓度的影响,试图从离子水平阐明CO对脑组织保护作用的机制。方法:将SD大鼠随机分为3组(n=6), 使用HO诱导剂、HO抑制剂腹腔注射为实验组,等量生理盐水腹腔注射为对照组,12 h后制成MCAO模型。栓塞后24 h检测血浆CO浓度、神经细胞胞内Ca²⁺浓度。结果: HO诱导剂组CO浓度明显高于生理盐水组,而胞内Ca²⁺浓度低于生理盐水组(P<0.05);HO抑制剂组CO浓度明显低于生理盐水组, 胞内Ca²⁺浓度高于生理盐水组(P<0.05)。HO诱导剂、HO抑制剂对非栓塞侧神经细胞胞内Ca²⁺浓度没有影响(P>0.05)。结论: CO通过作用于细胞膜上的Ca²⁺-K⁺通道,引起缺血脑组织神经细胞胞内Ca²⁺浓度的变化可能是CO脑保护作用的机制之一。     

闭塞大鼠单侧大脑中动脉后心肌酶活性的改变

实验性闭塞一侧大脑中动脉的大鼠２４ｈ后检测其心肌酶活性，结果表示：局部脑缺血２４ｈ的大鼠，与假手术对照组相比心肌组织Ｎａ＾＋－Ｋ＾＋－ＡＴＰａｓｅ，Ｃａ＾２＋－ＡＴＰａｓｅ活性均有明显降低，说明脑缺血可造成心肌酶活性不同程度改变。     

Effects of halothane on Ca2+-activated tension development in mechanically disrupted rabbit myocardial fibers

The effect of halothane on maximal and submaximal Ca²⁺-activated tension in mechanically disrupted right ventricular papillary muscle from rabbits was studied. Steady-state isometric tension generation was measured in the muscle bundle. The relaxing solution contained (in mM) [mg²⁺]=1, [K⁺]=70, [MgATP^¨-]=2, [creatine phosphate^¨-]=15, [EGTA total]=7 and imidazole proprionate. The contracting solution contained in addition Ca²⁺ in various concentrations. In all solutions ionic strength was maintained at 0.15 and pH at 7.00±0.02 at 20°C. Each fiber bundle was immersed in control solutions equilibrated with 100% N₂ and test solutions equilibrated with various concentrations of halothane-N₂ mixture. Increasing doses of halothane (1–4%) significantly shifted the relationship between Ca²⁺ and tension towards higher [Ca²⁺] and depressed the maximum Ca²⁺-activated tension. The maximum tension generated atpCa=3.8 was depressed 5% per 1% increase in halothane concentration. The percentage of maximum tension at submaximum Ca²⁺ concentrations (pCa=5.6–5.0) was not significantly decreased until halothane concentration was greater than 2%. It is concluded that halothane slightly but significantly depressed the interactions of contractile proteins and to a lesser degree Ca²⁺-activation of the regulatory proteins. The halothane-induced depression was completely reversible.     

Effects of diadenosine polyphosphates,ATP and angiotensin II on cytosolic Ca2+ activity and contraction of rat mesangial cells

Diadenosine polyphosphates (Ap _n A) are known to influence cellular Ca²⁺ activity ([Ca²⁺]_i) in several cells. Their vasoactive potency has been described in various systems including the kidney. We examined the effects of diadenosine polyphosphates, adenosine 5-triphosphate (ATP) and angiotensin II (Ang II) on cytosolic Ca²⁺ activity of mesangial cells (MC) in culture obtained from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. [Ca²⁺]_i was measured as a fluorescence ratio F ₃₄₀/F ₃₈₀ with the fura-2 technique using three excitation wavelengths (340 nm, 360 nm and 380 nm) and a photon counting tube. Resting [Ca²⁺]_i was not significantly different in MC from WKY and SHR rats and was measured as 132±9 nmol/l (n=65) and 114±12 nmol/l (n=36), respectively. Diadenosine polyphosphates (Ap₃A–Ap₆A) increased [Ca²⁺]_i transiently with an initial peak and a secondary plateau phase comparable to the effects of ATP or Ang II. Increases in [Ca²⁺]_i induced by all these agonists were not significantly different between MC of WKY and SHR rats. ATP, Ap₃A, Ap₄A, Ap₅A, Ap₆A (each 5 mol/l) increased the fura-2 fluorescence ratio initially by 0.66±0.09 (n=33), 0.52±0.08 (n=18), 0.25±0.05 (n=16), 0.09±0.06 (n=7), 0.09±0.04 (n=11), respectively. A half-maximal initial increase in the fura-2 fluorescence ratio was reached at 22 nmol/l, 0.9 mol/l, 2.0 mol/l and 4.0 mol/l with Ang II, Ap₃A, ATP and Ap₄A, respectively. Ap₄A (100 mol/l, n=18) led to a reversible contraction of MC. Diadenosine polyphosphates increase [Ca²⁺]_i in rat MC, in a similar manner to ATP or Ang II and lead to a contraction of MC, suggesting that these nucleotides are also involved in the control of glomerular haemodynamics.     

Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells

ATP and adenosine(5)tetraphospho(5)adenosine (Ap₄A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca²⁺]_i rises (EC₅₀=3.8 M) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca²⁺ concentration to around 100 nM suppressed the [Ca²⁺]_i transient evoked by ATP but not the [Ca²⁺]_i response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC₅₀= 12.5 M) and UTP, but did not respond to either adenosine 5-[-thio]diphosphate (ADP[S]), a P_2Y receptor agonist, adenosine 5-[,-methylene]triphosphate (pp[CH₂]pA), a P_2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca²⁺]_i responses when stimulated with ATP (EC₅₀=0.86 M), UTP (EC₅₀=1.6 M) and 2MeSATP (EC₅₀= 0.38 M). 2MeSATP behaved as a partial agonist. Ap₄A and ADP[S] also raised the [Ca²⁺]_i in endothelial cells, whereas AMP and pp[CH₂]pA were ineffective. Lowering extracellular free Ca²⁺ to around 100 nM did not affect the peak ATP-evoked [Ca²⁺]_i rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca²⁺-releasing P_2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P_2X, non-P_2Y subtype exclusively coupled to Ca²⁺ influx.     

睾酮对心肌梗塞大鼠肥大心肌肌球蛋白ATP酶活性及其同工酶分布的影响

本文观察了肌注睾酮对心肌梗塞(MI)雄鼠左室非梗塞区心肌肥大以及肥大心肌肌球蛋白ATP酶活性和同工酶分布的影响。术后第21日,经睾酮处理的MI组不仅心肌细胞直径明显大于对照MI组,且肌球蛋白ATP酶活性也比对照MI组明显提高(P<0.05)。电泳分析进一步表明,这种提高与同工酶V_1的含量增加有关(80% vs 74%,P<0.05)。提示MI后肌注睾酮在促进非梗塞心肌肥大的同时还能提高心肌的收缩性能,从而有助于左室功能的改善。     

缺血心肌蛋白质组初步研究

目的：研究缺血心肌相关蛋白表达谱。方法： 通过腹腔注射垂体后叶素造成心肌缺血模型，取左心室肌进行二维凝胶电泳，利用PDQuest7.1.1软件分析实验结果。结果： 心肌组织可得512±52个蛋白点，心肌缺血后有10个蛋白表达发生了显著变化(pI/Mr: 4.72/46.16 kD, 5.60/32.35 kD, 7.17/53.14 kD, 7.93/12.78 kD,6.59/35.72 kD,8.56/12.47 kD,8.68/37.49 kD,6.31/13.19 kD, 6.51/60.29 kD, 5.86/13.07 kD)。其中表达增强的有6个蛋白(pI/Mr: 4.72/46.16 kD,5.60/32.35 kD,7.17/53.14 kD, 6.59/35.72 kD, 8.68/37.49 kD, 6.51/60.29 kD)，表达降低的有4个蛋白(pI/Mr: 7.93/12.78 kD, 8.56/12.47 kD, 6.31/13.19 kD, 5.86/13.07 kD)。结论： 这些差异表达的蛋白可能在心肌缺血后的保护与损伤中发挥作用。     

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium

The enhanced diastolic Ca²⁺ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium- ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (V _max) was significantly reduced in failing heart preparations (NF crude membranes, 130±8; DCM crude membranes, 102±5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488±35; DCM crude membranes, 494±42; P=0.92), phospholamban (NF crude membranes, 497±51; DCM crude membranes, 496±45; P=0.98) and calsequestrin (NF crude membranes, 109±06; DCM crude membranes, 107±08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca²⁺ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca²⁺ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function. Received: 2 September 1997 / Accepted: 2 December 1997     

Role of the Ca2+-blocking effect of alcohol in the genesis of ischemic myocardial damage

Department of Pathophysiology, Novosibirsk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR E. D. Goldberg.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 2, pp. 168–170, February, 1991.