摘要
APJ是1993年发现的7次跨膜的G蛋白偶联受体,其内源性配体由Tatemoto等于1998年发现并命名为Apelin (APJ endogenous ligand)。人APJ受体由380个氨基酸组成,与血管紧张素受体AT1高度相似,跨膜区有54%的氨基酸序列相同。Apelin由77个氨基酸的前体蛋白水解生成不同长度的活性肽段如Apelin-36,Apelin-31,Apelin-17,Apelin-13等。APJ受体及其内源性配体apelin在体内分布广泛,中枢与外周组织均有不同水平的表达,尤其心、脑、肺、血管、脾、肠道、乳腺等器官与组织有高水平表达。
有报道称apelin具有强的正性肌力作用,但国内尚无相关研究,其机制也尚不清楚。因此本实验从体外灌流整体心脏水平和单心肌细胞水平研究Apelin对大鼠心脏收缩功能的影响及其作用机制。
实验目的:
1.证实Apelin对大鼠心肌在整体心脏水平和单细胞水平均具有正性肌力作用。
2.证实Apelin对大鼠正性肌力作用的剂量效应关系。
3.比较Apelin与其他几种常见正性肌力作用药物之间的异同。
4.证明Apelin正性肌力作用的特异性。
5.寻找Apelin正性肌力作用的细胞内信号通路。
6.证明不同前负荷对Apelin正性肌力作用的影响。
7.发现PLC,PKC在Apelin正性肌力作用中的参与机制。
8.研究SERCA,RyR,NCX,NHE等蛋白在Apelin作用的信号通路中的位置和作用。
实验方法:
心脏灌流。断头法处死大鼠,迅速取下心脏行Langendorff逆行灌注Krebs-Henseleit碳酸氢盐缓冲液。
心脏收缩力测量。连接一个力位移换能器至心尖部,初始前负荷拉力为2g。60分钟稳定平衡后,记录5分钟的对照期,之后往灌流液中加入各种试剂记录30分钟(0.5ml/min)。首先测量Apelin-16的浓度效应曲线(0.01-1nmol/L)。之后比较Apelin与内皮素-1,肾上腺髓质素,以及β-肾上腺素能受体激动剂异丙肾上腺素作用效果之间的差异。
研究Apelin作用的特异性,灌流液中加入了多种受体的拮抗剂。所选浓度为所报道的离体心脏有效阻断浓度。
研究内源性NO在大鼠心脏正性肌力作用中的角色,灌流液中添加了L-NAME,300μmol/L的L-NAME能够有效地阻断心肌中的一氧化氮合酶。信号通路研究。灌流液中加入PLC抑制剂PKC抑制剂NHE抑制剂和NHE-1抑制剂,反向NCX抑制剂。
等容心脏灌流研究。为了研究Apelin对不同前负荷下的心脏的收缩力的影响,进行了离体等容心脏灌流实验。等容左室压力的测量是通过使用充液球囊置于左心室内并连接压力传感器从而完成。20分钟的平衡稳定后加入Apelin (1 nmol/L)或者对照液灌流30分钟。前10分钟内球囊是松的,之后球囊以10μL为单位逐渐膨胀使LVEDP达到1,5,10,15mmHg。当收缩数据稳定后记录。
心肌细胞的分离。以常规酶解法分离成年大鼠心肌细胞。
细胞内钙的测量。将Fura-2/AM (0.5 mmol/L)与心肌细胞避光孵育30 min (25°C),采用双激发荧光光电倍增系统检测荧光信号。将indo-1/AM(5μmol/L)与心肌细胞避光孵育30 min (25°C),用新鲜的Hepes溶液漂洗两次,静置15分钟。负载好的细胞置于灌注腔内,灌注腔内液可在100ms内更换。Indo-1荧光应用340nm激光激发,在410nm和516nm波长处测量发射光。
用可视化动缘探测系统检测心肌细胞收缩功能。该系统由倒置显微镜、Ion Optix细胞影像适配器、Ion Optix Myc Cam摄像系统及其控制器、Ion Optix光电倍增系统(PMT)、荧光系统控制器(FS1)、Ion Optix光源及其电源、监视器、电子刺激器、程控灌流泵和计算机等部分组成。
咖啡因挛缩的测量。咖啡因挛缩引起的钙瞬变C[Ca~(2+)]_i不同于电刺激引起的细胞内钙瞬变E[Ca~(2+)]_i,C[Ca~(2+)]_i的幅度常被用来作为SR Ca~(2+)含量的指标。
冷挛缩的测量。冷挛缩(Rapid cooling contracture,RCC)可导致心肌细胞内质网(SR)钙的完全释放,而释放出来的大量钙被限制在细胞质内。利用可控灌流器可以迅速更换冰的Tyrode’s溶液并在200ms内将温度降至0–1°C。
SERCA活性检测。心肌细胞肌浆网的制备参照经Kodavanti改良Jones等的差速离心方法。采用南京建成生物公司微量ATP酶试剂盒,以比色定磷法测定无机磷含量,以含Ca~(2+)与不含Ca~(2+)管之差计算SERCA活性。
实验结果:
1. Apelin(0.01-10nmol/L)可引起离体大鼠心脏剂量依赖性的心肌张力(developed tension,DT)的增高。最大效应剂量点出现在1nmol/L的浓度上,半效浓度(EC50)为33.9±1.8pmol/L。
2.与其他几种常见的正性肌力药物相比,Apelin的作用时程为逐渐起效并且保持较长的作用时间≥30min。
3. Apelin产生的正性肌力作用并不伴随最大张力产生时间(Time-to-peak tension)的明显变化。
4.当LVEDP升高到10及15mmHg后,Apelin能够明显增加dP/dtmax值。
5. AT1受体拮抗剂CV-11974对心肌活动张力(DT)无明显影响,并且对1nmol/L的Apelin的正性肌力作用亦无明显影响。灌流液中加入L-NAME对Apelin的正性肌力作用无明显影响。
6. PLC的抑制剂U-73122 100nmol/L单独使用对心肌收缩力无明显影响,但与Apelin(1nmol/L)合用时却可以显著降低Apelin的正性肌力作用效果,最多可以减弱其68%的正性肌力作用。
7.广谱的蛋白激酶抑制剂Staurosporine,以及特异性PKC抑制剂GF-109203X均可减弱Apelin的正性肌力作用。
8. NHE抑制剂MIA(1μmol/L)单独使用对心肌收缩力无影响(P=NS)。但MIA(1μmol/L)能够显著减弱Apelin(1nmol/L)的正性肌力作用最多可达55%(F=12.7,P<0.001)。
9.反向NCX的选择性抑制剂KB-R7934(250nmol/L)本身对心肌收缩的基线值无明显影响(P=NS),但却能够显著减弱Apelin(1nmol/L)作用的60%(F=18.5,P<0.001)。
10.在电刺激的情况下Apelin能够升高E[Ca~(2+)]_i transients的幅度。
11. Apelin能够增强细胞的收缩功能(FS)并且作用持续至少30分钟。
12.钙瞬变峰值时间(time to peak,TTP of E[Ca~(2+)]_i)被Apelin显著的降低了12.53±3.8%。
13.半效衰减时间(T50 of E[Ca~(2+)]_i)被Apelin降低了30.82±1.17%。
14. Apelin组咖啡因挛缩中测得的SR Ca~(2+) content轻微但却显著的下降了8.41±0.92%。
15. Apelin能够显著减少16.22±1.36%的咖啡因挛缩的半效衰减时间(T50 of C[Ca~(2+)]_i)。
16. Apelin降低了冷挛缩法测得的SR Ca~(2+) content值(n=5, P<0.05)。
17. Apelin增强了SR Ca~(2+)-ATPase的活性(n=7, P<0.05)。
18. PKC抑制剂CHE能够显著减弱Apelin对NCX、SERCA活性的提高作用。PKC抑制剂CHE能够显著减弱Apelin的正性肌力作用。
实验结论:
1.在大鼠整体心脏灌流水平,Apelin具有剂量依赖的正性肌力作用。
2.在大鼠单心肌细胞灌流水平,1nmol/L的Apelin具有正性肌力作用。
3.钙瞬变幅度的升高是Apelin正性肌力作用的原因之一。但仍然存在着不依赖钙瞬变幅度升高的可能机制,该机制与PKC的激活有关。
4.Apelin能够降低SR钙容量,该作用与PKC的激活有关,其中NCX和SERCA是两个关键的作用蛋白。
Regulation of myocardial contractility by endogenous peptides is important in physiological and pathophysiological conditions and may be a crucial therapeutic target.An autocrine or paracrine system which potentially regulates heart function is the recently discovered angiotensin receptor like-1 (APJ) with its endogenous ligand apelin.Apelin and APJ are widely expressed throughout the cardiovascular system. Apelin is actively synthesized by cardiac myocytes, vascular endothelial and smooth muscle cells, and has specific receptors in the heart. Ex vivo studies using isolated perfused rat hearts have identified apelin as one of the most potent inotropic substances yet recognised. Moreover, apelin has been reported to increase left ventricular contractility in vivo following acute as well as chronic infusion in rodents.
The APJ receptor and apelin have been implicated in the pathophysiology of human heart failure. Plasma concentration of apelin has been shown to decrease in patients with congestive heart failure and long-term cardiac resynchronization therapy could restore plasma levels of the peptide. Moreover, mechanical ventricular support increases APJ mRNA levels in patients with heart failure. Apelin has also been implicated in the pathophysiology of arrhythmias. Ellinor et al. demonstrated that plasma apelin levels decrease in patients with lone atrial fibrillation. Despite recent advances in our understanding of the cardiovascular effects of the apelin-APJ system in vivo, the direct effects of apelin on cardiomyocyte contractility remain unknown.
Aim:
1. To find Apelin’s effect on whole heart and isolated myocyte of rat.
2. To find the Dose-Effect relationship of Apelin’s positive inotropic action.
3. To find the differences between Apelin and other medicines.
4. To find the specificity of Apelin’s positive inotropic action.
5. To find the signal pathway of intracellular transduction of Apelin.
6. To find different pre-load’s effect on Apelin’s positive inotropic action.
7. To find the role of PLC and PKC in the mechanism of Apelin’s positive inotropic action.
8. To find the role of SERCA, RyR, NCX and NHE in the mechanism of Apelin’s positive inotropinc action.
Material and Methods:
Rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique as described previously.
Heart rate was maintained constant (304±1 beats/min) by atrial pacing using a Grass stimulator (model S88, 11 V, 0.5 ms). Contractile force (apicobasal displacement) was obtained by connecting a force displacement transducer (Grass Instruments, FT03) to the apex of the heart at an initial preload stretch of 2 g. A 60-minute equilibration period and a 5-minute control period was followed by addition of various drugs to the perfusate by an infusion pump at a rate of 0.5 mL/min for 30 minutes. Initially, we determined the concentration-dependent effect of apelin-16 (0.01 to 1 nmol/L) on cardiac contractility. Next, we compared the effect of apelin to the responses of endothelin-1, adrenomedullin, and theβ-adrenergic receptor agonist isoproterenol. To test the specificity of the effect of apelin, it was infused in the presence of various receptor antagonists.
To define the role of endogenous nitric oxide in apelin-induced inotropic response, the peptide was infused in the presence of L-NAME. L-NAME at a concentration of 300μmol/L effectively inhibited nitric oxide synthase in the myocardium.
For signal transduction studies, the concentration of U-73122 (100 nmol/L), staurosporine (10 nmol/L) and GF-109203X (90 nmol/L), MIA (1μmol/L) and zoniporide (1μmol/L), and KB-R7943 (250 nmol/L) were selected because these concentrations have been demonstrated to suppress phospholipase C and protein kinase C activity and inhibit Na+/H+ exchange and the reverse mode Ca~(2+)/Na+ exchange, respectively.
The perfusion technique and the composition of the Krebs-Henseleit bicarbonate buffer was similar to that described above, however, left ventricular contractility was assessed by measuring isovolumic left ventricular pressure. Isovolumic left ventricular pressure was measured using a fluid-filled balloon, which was placed in the left ventricular chamber and connected to a pressure transducer. The balloon was large enough so that negligible pressure resulted, when the balloon alone was filled up to the maximum volume used. The following parameters were obtained: peak systolic left ventricular pressure, left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (DP), maximum and minimum values of the first derivative of isovolumic pressure (dP/dtmax, dP/dtmin), time from peak systolic pressure to 60% relaxation (RT 60%), time from peak systolic pressure to 90% relaxation (RT 90%). A 20-minute equilibration was followed by addition of apelin (1 nmol/L) or vehicle to the perfusate for 30 minute. During the first 10 minutes of infusion the left ventricular balloon was flaccid, thereafter the balloon was inflated in 10μL steps to achieve a LVEDP of 1, 5, 10 and 15 mmHg. Parameters of left ventricular contractility were obtained when a new steady-state was reached.
Myocytes were isolated using standard procedures.
We used two different Ca~(2+) indicators and protocols to measure intracellular Ca~(2+). The first: Freshly isolated myocytes were placed on laminin-coated glass coverslips and allowed to attach for 30 minutes before they were loaded with fura 2-AM. The free [Ca~(2+)]_i of loaded cardiac myocytes were measured as the fluorescence ratio (360/380nm). The second: Cells were loaded during 30 minutes with 5μmol/l indo-1/AM and 0.01% pluronic befor each experiment. The field stimulation (0.5Hz) was elicited and indo-1 fluorescence was measured in dual emission mode, excited at 340nm with xenon lamp flashes (100 W). Dual wavelength emission was measured at 410 and 516 nm, respectively. Shortening of myocytes was simultaneously measured with E[Ca~(2+)]_i. Myocytes were perfused with Tyrode's solution at 22°C and 1 ml/min flow rate. Myocytes were equilibrated at 22°C for about 20 minutes before use. All experimental protocols were carried out at room temperature. The contractile
shortening of ventricular myocyte was measured by a video-based motion edge-detection system and an inverted microscope.
Caffeine-induced Ca~(2+) (C[Ca~(2+)]_i) transient amplitude was used as a measurement of SR Ca~(2+) content.
SR Ca~(2+) content was measured by another way since this was the most important observation in the current study. RC causes complete depletion of calcium from SR and calcium released remains confined to the cytoplasm.
The activity of Ca~(2+)-ATPase was determined with a kit (Jiancheng, Nanjing, China) by measuring the inorganic phosphate (Pi) liberated from ATP hydrolysis.
Results:
1. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1±1.5 pmol/L).
2. Moreover, preload-induced increase in dP/dtmax was significantly augmented (P<0.05) in the presence of apelin.
3. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P<0.001).
4. In addition, zoniporide, a selective inhibitor of NHE isoform-1, and KB-R7943, a potent inhibitor of the reverse mode NCX, significantly suppressed the response to apelin (P<0.001).
5. Compared with control, treatment with apelin caused a 55.7±13.9% increase in sarcomere fraction shortening (FS) and a 43.6±4.56% increase in amplitude of E[Ca~(2+)]_i transients (n=14,P<0.05).
6. SR Ca~(2+) content measured by caffeine-induced Ca~(2+) (C[Ca~(2+)]_i) transient was decreased 8.41±0.92% in response to apelin (n=14,P<0.05).
7. NCX function was increased since half-decay time (T50) of C[Ca~(2+)]_i was decreased 16.22±1.36% in response to apelin.
8. Sarcoplasmic reticulum Ca~(2+)-ATPase (SERCA) activity was also increased by apelin.
9. These responses can be partially or completely blocked by chelerythrine chloride (CHE), a PKC inhibitor.
10. In addition, to confirm our data, we used indo-1 as another Ca~(2+) indicator and rapid cooling(RC) as another way to measure SR Ca~(2+) content, and we observed the similar results.
Conclusions:
1. In the whole rat heart level, Apelin has dose-dependent positive inotropic action. PLC, PKC and NHE, NCX are involved.
2. In the isolated myocyte level, 1nmol/l Apelin has positive inotropic action.
3. The increase of amplitude of E[Ca~(2+)]_i is one of reasons of Apelin’s positive inotropic action. Some other reason is remained, and is PKC dependent.
4. Apelin decreases SR Ca~(2+) content, with a PKC dependent way, and NCX, SERCA are two key proteins.
引文
[1] O'Dowd B. F., Heiber M., Chan A., Heng H. H., Tsui L. C., Kennedy J. L., Shi X., Petronis A., George S. R., Nguyen T. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11[J]. Gene, 1993 136(1-2): 355-60.
[2] Tatemoto K., Hosoya M., Habata Y., Fujii R., Kakegawa T., Zou M. X., Kawamata Y., Fukusumi S., Hinuma S., Kitada C., Kurokawa T., Onda H., Fujino M. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor[J]. Biochem Biophys Res Commun, 1998 251(2): 471-6.
[3] 潘春水, 齐永芬, 唐朝枢. Apelin 及其生物学效应[J]. 生物科学进展, 2005 36(3): 223-226.
[4] O'Carroll A. M., Selby T. L., Palkovits M., Lolait S. J. Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues[J]. Biochim Biophys Acta, 2000 1492(1): 72-80.
[5] Foldes G., Horkay F., Szokodi I., Vuolteenaho O., Ilves M., Lindstedt K. A., Mayranpaa M., Sarman B., Seres L., Skoumal R., Lako-Futo Z., deChatel R., Ruskoaho H., Toth M. Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure[J]. Biochem Biophys Res Commun, 2003 308(3): 480-5.
[6] Szokodi I., Tavi P., Foldes G., Voutilainen-Myllyla S., Ilves M., Tokola H., Pikkarainen S., Piuhola J., Rysa J., Toth M., Ruskoaho H. Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiaccontractility[J]. Circ Res, 2002 91(5): 434-40.
[7] Berry M. F., Pirolli T. J., Jayasankar V., Burdick J., Morine K. J., Gardner T. J., Woo Y. J. Apelin has in vivo inotropic effects on normal and failing hearts[J]. Circulation, 2004 110(11 Suppl 1): II187-93.
[8] 吴小脉,龚永生. Apelin 的心血管效应[J]. 国外医学?生理、病理科学与临床分册, 2005 25(2): 123-126.
[9] Kagiyama S., Fukuhara M., Matsumura K., Lin Y., Fujii K., Iida M. Central and peripheral cardiovascular actions of apelin in conscious rats[J]. Regul Pept, 2005 125(1-3): 55-9.
[10] 钟久昌, 黄东阳. 新的心血管活性多肽[J]. 高血压杂志, 2004 12(5): 390-392.
[11] Masri B., Lahlou H., Mazarguil H., Knibiehler B., Audigier Y. Apelin (65-77) activates extracellular signal-regulated kinases via a PTX-sensitive G protein[J]. Biochem Biophys Res Commun, 2002 290(1): 539-45.
[12] Nisoli E., Clementi E., Paolucci C., Cozzi V., Tonello C., Sciorati C., Bracale R., Valerio A., Francolini M., Moncada S., Carruba M. O. Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide[J]. Science, 2003 299(5608): 896-9.
[13] Tatemoto K., Takayama K., Zou M. X., Kumaki I., Zhang W., Kumano K., Fujimiya M. The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism[J]. Regul Pept, 2001 99(2-3): 87-92.
[14] Katugampola S. D., Maguire J. J., Matthewson S. R., Davenport A. P. [(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man[J]. Br J Pharmacol, 2001 132(6): 1255-60.
[15] Kleinz M. J., Davenport A. P. Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells[J]. Regul Pept, 2004 118(3): 119-25.
[16] Ishida J., Hashimoto T., Hashimoto Y., Nishiwaki S., Iguchi T., Harada S., Sugaya T., Matsuzaki H., Yamamoto R., Shiota N., Okunishi H., Kihara M., Umemura S., Sugiyama F., Yagami K., Kasuya Y., Mochizuki N., Fukamizu A. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo[J]. J Biol Chem, 2004 279(25): 26274-9.
[17] 江海龙, 吴宏超, 唐朝枢. Apelin 在心血管系统中的作用[J]. 医学综述, 2005 11(7): 590-592.
[18] Cheng H., Wang S. Q. Calcium signaling between sarcolemmal calcium channels and ryanodine receptors in heart cells[J]. Front Biosci, 2002 7: d1867-78.
[19] Kamishima T., Quayle J. M. Ca2+-induced Ca2+ release in cardiac and smooth muscle cells[J]. Biochem Soc Trans, 2003 31(Pt 5): 943-6.
[20] Wang S. Q., Song L. S., Lakatta E. G., Cheng H. Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells[J]. Nature, 2001 410(6828): 592-6.
[21] Song L. S., Sham J. S., Stern M. D., Lakatta E. G., Cheng H. Direct measurement of SR release flux by tracking 'Ca2+ spikes' in rat cardiac myocytes[J]. J Physiol, 1998 512 ( Pt 3): 677-91.
[22] Wier W. G., Balke C. W. Ca(2+) release mechanisms, Ca(2+) sparks, and local control of excitation-contraction coupling in normal heart muscle[J]. Circ Res, 1999 85(9): 770-6.
[23] Sham J. S., Cleemann L., Morad M. Functional coupling of Ca2+ channelsand ryanodine receptors in cardiac myocytes[J]. Proc Natl Acad Sci U S A, 1995 92(1): 121-5.
[24] Sipido K. R., Carmeliet E., Van de Werf F. T-type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmic reticulum in guinea-pig ventricular myocytes[J]. J Physiol, 1998 508 ( Pt 2): 439-51.
[25] Cannell M. B., Soeller C. Mechanisms underlying calcium sparks in cardiac muscle[J]. J Gen Physiol, 1999 113(3): 373-6.
[26] Cannell M. B., Cheng H., Lederer W. J. The control of calcium release in heart muscle[J]. Science, 1995 268(5213): 1045-9.
[27] Lopez-Lopez J. R., Shacklock P. S., Balke C. W., Wier W. G. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells[J]. Science, 1995 268(5213): 1042-5.
[28] Guia A., Stern M. D., Lakatta E. G., Josephson I. R. Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance[J]. Biophys J, 2001 80(6): 2742-50.
[29] Sham J. S., Song L. S., Chen Y., Deng L. H., Stern M. D., Lakatta E. G., Cheng H. Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes[J]. Proc Natl Acad Sci U S A, 1998 95(25): 15096-101.
[30] DelPrincipe F., Egger M., Niggli E. Calcium signalling in cardiac muscle: refractoriness revealed by coherent activation[J]. Nat Cell Biol, 1999 1(6): 323-9.
[31] Shen J. X., Wang S., Song L. S., Han T., Cheng H. Polymorphism of Ca2+ sparks evoked from in-focus Ca2+ release units in cardiac myocytes[J]. Biophys J, 2004 86(1 Pt 1): 182-90.
[32] Berridge M. J., Bootman M. D., Lipp P. Calcium--a life and death signal[J].Nature, 1998 395(6703): 645-8.
[33] Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties[J]. J Biol Chem, 1985 260(6): 3440-50.
[34] B.Cannel M., V.Thomas M. Intracellular ion measurement with fluorescent indicators. Microelectrode Technique-The Plymouth Workshop handbook. Second edition. The Company of Biologists.[J].
[35] 徐涛, 瞿安连, 康华光, 周专等. 细胞膜离子通道电流及胞内游离钙离子浓度的同时定量检测[J]. 武汉大学学报, 1995 2(生物物理专刊): 24-28.
[36] Haugland R. P. Handbook of fluorescent probes and research chemicals,sixth edition. Molecular Probe.[J].
[37] Minta A., Kao J. P., Tsien R. Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores[J]. J Biol Chem, 1989 264(14): 8171-8.
[38] Miyawaki A., Llopis J., Heim R., McCaffery J. M., Adams J. A., Ikura M., Tsien R. Y. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin[J]. Nature, 1997 388(6645): 882-7.
[39] Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy[J]. Science, 1990 248(4951): 73-6.
[40] Xu C., Zipfel W., Shear J. B., Williams R. M., Webb W. W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy[J]. Proc Natl Acad Sci U S A, 1996 93(20): 10763-8.
[41] Rizzuto R., Carrington W., Tuft R. A. Digital imaging microscopy of living cells[J]. Trends Cell Biol, 1998 8(7): 288-92.
[42] Masri B., Knibiehler B., Audigier Y. Apelin signalling: a promisingpathway from cloning to pharmacology[J]. Cell Signal, 2005 17(4): 415-26.
[43] Kleinz M. J., Davenport A. P. Emerging roles of apelin in biology and medicine[J]. Pharmacol Ther, 2005 107(2): 198-211.
[44] Magga J., Vuolteenaho O., Marttila M., Ruskoaho H. Endothelin-1 is involved in stretch-induced early activation of B-type natriuretic peptide gene expression in atrial but not in ventricular myocytes: acute effects of mixed ET(A)/ET(B) and AT1 receptor antagonists in vivo and in vitro[J]. Circulation, 1997 96(9): 3053-62.
[45] Hautala N., Tenhunen O., Szokodi I., Ruskoaho H. Direct left ventricular wall stretch activates GATA4 binding in perfused rat heart: involvement of autocrine/paracrine pathways[J]. Pflugers Arch, 2002 443(3): 362-9.
[46] Kinnunen P., Szokodi I., Nicholls M. G., Ruskoaho H. Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration[J]. Am J Physiol Regul Integr Comp Physiol, 2000 279(2): R569-75.
[47] Talosi L., Kranias E. G. Effect of alpha-adrenergic stimulation on activation of protein kinase C and phosphorylation of proteins in intact rabbit hearts[J]. Circ Res, 1992 70(4): 670-8.
[48] Fulton D., McGiff J. C., Quilley J. Role of phospholipase C and phospholipase A2 in the nitric oxide-independent vasodilator effect of bradykinin in the rat perfused heart[J]. J Pharmacol Exp Ther, 1996 278(2): 518-26.
[49] Szokodi I., Kinnunen P., Tavi P., Weckstrom M., Toth M., Ruskoaho H. Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide[J]. Circulation, 1998 97(11):1062-70.
[50] Hu K., Nattel S. Mechanisms of ischemic preconditioning in rat hearts. Involvement of alpha 1B-adrenoceptors, pertussis toxin-sensitive G proteins, and protein kinase C[J]. Circulation, 1995 92(8): 2259-65.
[51] Moffat M. P., Karmazyn M. Protective effects of the potent Na/H exchange inhibitor methylisobutyl amiloride against post-ischemic contractile dysfunction in rat and guinea-pig hearts[J]. J Mol Cell Cardiol, 1993 25(8): 959-71.
[52] Knight D. R., Smith A. H., Flynn D. M., MacAndrew J. T., Ellery S. S., Kong J. X., Marala R. B., Wester R. T., Guzman-Perez A., Hill R. J., Magee W. P., Tracey W. R. A novel sodium-hydrogen exchanger isoform-1 inhibitor, zoniporide, reduces ischemic myocardial injury in vitro and in vivo[J]. J Pharmacol Exp Ther, 2001 297(1): 254-9.
[53] Iwamoto T., Watano T., Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1[J]. J Biol Chem, 1996 271(37): 22391-7.
[54] Yang H. T., Sakurai K., Sugawara H., Watanabe T., Norota I., Endoh M. Role of Na+/Ca2+ exchange in endothelin-1-induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943[J]. Br J Pharmacol, 1999 126(8): 1785-95.
[55] Kelly R. A., Eid H., Kramer B. K., O'Neill M., Liang B. T., Reers M., Smith T. W. Endothelin enhances the contractile responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway[J]. J Clin Invest, 1990 86(4): 1164-71.
[56] Kramer B. K., Smith T. W., Kelly R. A. Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellularalkalosis induced by activation of the protein kinase C-dependent Na(+)-H+ exchanger[J]. Circ Res, 1991 68(1): 269-79.
[57] Kojda G., Kottenberg K., Nix P., Schluter K. D., Piper H. M., Noack E. Low increase in cGMP induced by organic nitrates and nitrovasodilators improves contractile response of rat ventricular myocytes[J]. Circ Res, 1996 78(1): 91-101.
[58] Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses[J]. Faseb J, 1995 9(7): 484-96.
[59] Karmazyn M., Gan X. T., Humphreys R. A., Yoshida H., Kusumoto K. The myocardial Na(+)-H(+) exchange: structure, regulation, and its role in heart disease[J]. Circ Res, 1999 85(9): 777-86.
[60] Kentish J. C. A role for the sarcolemmal Na(+)/H(+) exchanger in the slow force response to myocardial stretch[J]. Circ Res, 1999 85(8): 658-60.
[61] Kawamata Y., Habata Y., Fukusumi S., Hosoya M., Fujii R., Hinuma S., Nishizawa N., Kitada C., Onda H., Nishimura O., Fujino M. Molecular properties of apelin: tissue distribution and receptor binding[J]. Biochim Biophys Acta, 2001 1538(2-3): 162-71.
[62] Lee D. K., Cheng R., Nguyen T., Fan T., Kariyawasam A. P., Liu Y., Osmond D. H., George S. R., O'Dowd B. F. Characterization of apelin, the ligand for the APJ receptor[J]. J Neurochem, 2000 74(1): 34-41.
[63] Hosoya M., Kawamata Y., Fukusumi S., Fujii R., Habata Y., Hinuma S., Kitada C., Honda S., Kurokawa T., Onda H., Nishimura O., Fujino M. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin[J]. J Biol Chem, 2000 275(28): 21061-7.
[64] Neer E. J. Heterotrimeric G proteins: organizers of transmembranesignals[J]. Cell, 1995 80(2): 249-57.
[65] Tavi P., Laine M., Weckstrom M., Ruskoaho H. Cardiac mechanotransduction: from sensing to disease and treatment[J]. Trends Pharmacol Sci, 2001 22(5): 254-60.
[66] Alvarez B. V., Perez N. G., Ennis I. L., Camilion de Hurtado M. C., Cingolani H. E. Mechanisms underlying the increase in force and Ca(2+) transient that follow stretch of cardiac muscle: a possible explanation of the Anrep effect[J]. Circ Res, 1999 85(8): 716-22.
[67] Perez N. G., de Hurtado M. C., Cingolani H. E. Reverse mode of the Na+-Ca2+ exchange after myocardial stretch: underlying mechanism of the slow force response[J]. Circ Res, 2001 88(4): 376-82.
[68] Xiao R. P., Spurgeon H. A., O'Connor F., Lakatta E. G. Age-associated changes in beta-adrenergic modulation on rat cardiac excitation-contraction coupling[J]. J Clin Invest, 1994 94(5): 2051-9.
[69] Sipido K. R., Callewaert G. How to measure intracellular [Ca2+] in single cardiac cells with fura-2 or indo-1[J]. Cardiovasc Res, 1995 29(5): 717-26.
[70] Cobbold P. H., Rink T. J. Fluorescence and bioluminescence measurement of cytoplasmic free calcium[J]. Biochem J, 1987 248(2): 313-28.
[71] Fiolet J. W., Baartscheer A., Schumacher C. A. Intracellular [Ca2+] and Vo2 after manipulation of the free-energy of the Na+/Ca(2+)-exchanger in isolated rat ventricular myocytes[J]. J Mol Cell Cardiol, 1995 27(8): 1513-25.
[72] Donoso P., Mill J. G., O'Neill S. C., Eisner D. A. Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes[J]. J Physiol, 1992 448: 493-509.
[73] duBell W. H., Boyett M. R., Spurgeon H. A., Talo A., Stern M. D., Lakatta E. G. The cytosolic calcium transient modulates the action potential of rat ventricular myocytes[J]. J Physiol, 1991 436: 347-69.
[74] Miyata H., Silverman H. S., Sollott S. J., Lakatta E. G., Stern M. D., Hansford R. G. Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes[J]. Am J Physiol, 1991 261(4 Pt 2): H1123-34.
[75] Hove-Madsen L., Bers D. M. Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes[J]. Am J Physiol, 1993 264(3 Pt 1): C677-86.
[76] B Z., F Z. H., Q F. Insulin improves cardiac myocytes contractile function recovery in simulated ischemia-reperfusion:key role of Akt.[J]. Chin Sci Bull, 2003 48(13): 1364-9.
[77] Yeung H. M., Kravtsov G. M., Ng K. M., Wong T. M., Fung M. L. Chronic intermittent hypoxia alters Ca2+ handling in rat cardiomyocytes by augmented Na+/Ca2+ exchange and ryanodine receptor activities in ischemia-reperfusion[J]. Am J Physiol Cell Physiol, 2007 292(6): C2046-56.
[78] Ho J. C., Wu S., Kam K. W., Sham J. S., Wong T. M. Effects of pharmacological preconditioning with U50488H on calcium homeostasis in rat ventricular myocytes subjected to metabolic inhibition and anoxia[J]. Br J Pharmacol, 2002 137(6): 739-48.
[79] Terracciano C. M., MacLeod K. T. Effects of acidosis on Na+/Ca2+ exchange and consequences for relaxation in guinea pig cardiac myocytes[J]. Am J Physiol, 1994 267(2 Pt 2): H477-87.
[80] Pieske B., Maier L. S., Bers D. M., Hasenfuss G. Ca2+ handling andsarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium[J]. Circ Res, 1999 85(1): 38-46.
[81] Quinn F. R., Currie S., Duncan A. M., Miller S., Sayeed R., Cobbe S. M., Smith G. L. Myocardial infarction causes increased expression but decreased activity of the myocardial Na+-Ca2+ exchanger in the rabbit[J]. J Physiol, 2003 553(Pt 1): 229-42.
[82] Bers D. M., Bridge J H, and Spitzer K W Intracellular Ca2+ transients during rapid cooling contractures in guinea-pig ventricular myocytes.[J]. J. Physiol., 1989 417(417): 537-553.
[83] Bers D. M. Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contractures[J]. Am J Physiol, 1987 253(3 Pt 1): C408-15.
[84] Jones L. R. Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum[J]. Biochim Biophys Acta, 1979 557(1): 230-42.
[85] Kodavanti P. R., Cameron J. A., Yallapragada P. R., Desaiah D. Effect of chlordecone (Kepone) on calcium transport mechanisms in rat heart sarcoplasmic reticulum[J]. Pharmacol Toxicol, 1990 67(3): 227-34.
[86] Pande M., Cameron J. A., Vig P. J., Desaiah D. Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum[J]. Toxicology, 1998 129(2-3): 95-102.
[87] Lindemann J. P., Jones L. R., Hathaway D. R., Henry B. G., Watanabe A. M. beta-Adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles[J]. J Biol Chem, 1983 258(1): 464-71.
[88] Pei J. M., Yu X. C., Fung M. L., Zhou J. J., Cheung C. S., Wong N. S.,Leung M. P., Wong T. M. Impaired G(s)alpha and adenylyl cyclase cause beta-adrenoceptor desensitization in chronically hypoxic rat hearts[J]. Am J Physiol Cell Physiol, 2000 279(5): C1455-63.
[89] Baker D. L., Hashimoto K., Grupp I. L., Ji Y., Reed T., Loukianov E., Grupp G., Bhagwhat A., Hoit B., Walsh R., Marban E., Periasamy M. Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts[J]. Circ Res, 1998 83(12): 1205-14.
[90] Loukianov E., Ji Y., Grupp I. L., Kirkpatrick D. L., Baker D. L., Loukianova T., Grupp G., Lytton J., Walsh R. A., Periasamy M. Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase[J]. Circ Res, 1998 83(9): 889-97.
[91] Bers D. M. Calcium fluxes involved in control of cardiac myocyte contraction[J]. Circ Res, 2000 87(4): 275-81.
[92] Shigekawa M., Iwamoto T. Cardiac Na(+)-Ca(2+) exchange: molecular and pharmacological aspects[J]. Circ Res, 2001 88(9): 864-76.
[93] Dai T., Ramirez-Correa G., Gao W. D. Apelin increases contractility in failing cardiac muscle[J]. Eur J Pharmacol, 2006 553(1-3): 222-8.
[94] Farkasfalvi K., Stagg M. A., Coppen S. R., Siedlecka U., Lee J., Soppa G. K., Marczin N., Szokodi I., Yacoub M. H., Terracciano C. M. Direct effects of apelin on cardiomyocyte contractility and electrophysiology[J]. Biochem Biophys Res Commun, 2007 357(4): 889-95.
[95] Carmeliet E. Intracellular Ca(2+) concentration and rate adaptation of the cardiac action potential[J]. Cell Calcium, 2004 35(6): 557-73.
[96] Yamamura K., Steenbergen C., Murphy E. Protein kinase C andpreconditioning: role of the sarcoplasmic reticulum[J]. Am J Physiol Heart Circ Physiol, 2005 289(6): H2484-90.
[97] Chen W., London R., Murphy E., Steenbergen C. Regulation of the Ca2+ gradient across the sarcoplasmic reticulum in perfused rabbit heart. A 19F nuclear magnetic resonance study[J]. Circ Res, 1998 83(9): 898-907.
[98] Osada M., Netticadan T., Tamura K., Dhalla N. S. Modification of ischemia-reperfusion-induced changes in cardiac sarcoplasmic reticulum by preconditioning[J]. Am J Physiol, 1998 274(6 Pt 2): H2025-34.
[99] Tani M., Asakura Y., Hasegawa H., Shinmura K., Ebihara Y., Nakamura Y. Effect of preconditioning on ryanodine-sensitive Ca2+ release from sarcoplasmic reticulum of rat heart[J]. Am J Physiol, 1996 271(3 Pt 2): H876-81.
[100] Zucchi R., Ronca-Testoni S., Yu G., Galbani P., Ronca G., Mariani M. Postischemic changes in cardiac sarcoplasmic reticulum Ca2+ channels. A possible mechanism of ischemic preconditioning[J]. Circ Res, 1995 76(6): 1049-56.