腺苷对正常及缺血豚鼠心室肌细胞Na~+-K~+泵电流的调节
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摘要
Na~+-K~+泵(又称钠泵或Na~+,K~+-ATP酶)是P-ATP酶家族的成员,几乎存在于所有的动物细胞膜。Na~+-K~+泵通过将3个Na~+泵出膜外同时泵入2个K~+所建立的电化学势能为细胞内多种重要的生理功能(控制细胞容量和pH的稳定,营养物质的重吸收等)提供能量。在可兴奋组织(心脏和神经组织等),Na~+-K~+泵对建立和维持正常的电活性发挥关键作用。
研究发现,Na~+-K~+泵功能可被多种神经内分泌激素及病理状态所引发的生物化学变化调节。例如,儿茶酚胺、胰岛素、甲状腺激素、血管紧张素等通过激活细胞膜上的相应受体和下游的信号转导通路调节泵功能。此外,心肌缺血,做为造成发展中国家人群致死和致残的主要原因,可显著抑制Na~+-K~+泵功能。因此,详细阐明体内内源性信号分子和生物化学变化对泵功能的调节有助于更好的理解不同病理生理状态下Na~+-K~+泵的确切作用。
体内多种信号分子(缓激肽、腺苷、去甲肾上腺素等)对心肌缺血存在不同程度的保护作用,其中腺苷(adenosine,Ado)是研究最为广泛的“心脏保护者”。正常状态下细胞间隙中的腺苷浓度很低,但代谢应激(缺血和缺氧等)可刺激腺苷的大量释放,并与心血管细胞膜表达的腺苷受体(A_1R、A_(2A)R、A_(2B)R、A_3R)结合触发细胞内的一系列反应,发挥限制细胞死亡和失功能的作用。腺苷对组织的保护作用涉及不同的机制,包括增加氧供、缺血预适应、刺激血管生成、抑制炎症发生等。然而,Na~+-K~+泵作为维持细胞基本功能的主动转移体,其功能是否可被内源性分泌的腺苷调节呢?另外,腺苷能否阻断心肌缺血造成的Na~+-K~+泵功能抑制目前仍未见报道。
因此,本研究采用全细胞膜片钳的方法旨在观察:(一)腺苷对正常豚鼠心室肌细胞Na~+-K~+泵电流的影响,并探讨其可能机制;(二)腺苷对模拟缺血豚鼠心室肌细胞Na~+-K~+泵电流的影响;(三)心肌缺血抑制Na~+-K~+泵电流的确切机制。
第一部分腺苷对正常豚鼠心室肌细胞Na~+-K~+泵电流的调节
目的:研究腺苷对急性分离的豚鼠心室肌细胞Na~+-K~+泵电流的影响,并深入探讨可能参与这一调节过程的信号转导途径。
方法:(1)酶解法急性分离豚鼠心室肌细胞:使用Langendorff装置,行主动脉插管逆行灌流(温度、pH、水质恒定),用胶原酶NB8(Collagenase NB8,Serva公司)消化心脏,急性分离豚鼠心室肌细胞。选择胞膜光滑、横纹相对清晰、贴壁良好、稳定无收缩的细胞用于实验。(2)全细胞膜片钳技术(whole-cell patch-clamp technique)记录Na~+-K~+泵电流(Ip):Na~+-K~+泵经过一次主动转运过程将3个Na~+泵出膜外同时泵入2个K~+,从而产生一净外向电流。Ip的测定方法是:在特定灌流液和细胞内液成分阻断细胞膜上其它主要离子通道和交换体电流的条件下(K~+电流、Ca~(2+)电流、Na~+-Ca~(2+)交换体电流),灌流细胞特异性、可逆性Na~+-K~+泵阻断剂(即强心苷类),由于Na~+-K~+泵被阻断后膜电流产生内向性变化,此差别电流即反映Ip的大小。已有资料证实豚鼠心室肌细胞共表达两种α亚基的Na~+-K~+泵,即α1亚基Na~+-K~+泵和α2亚基Na~+-K~+泵。前者对强心苷的亲和力低,为低亲和力泵;后者对强心苷的亲和力高,为高亲和力泵。例如α1亚基Na~+-K~+泵对双氢哇巴因dihydroouabain(DHO,属于强心苷类)的解离常数为72μM,α2亚基Na~+-K~+泵对DHO的解离常数为0.75μM,两者亲和力相差100倍。故总泵电流(Ip)为α1亚基Na~+-K~+泵电流(Il)和α2亚基Na~+-K~+泵电流(Ih)之和:Ip=Il~+Ih。根据α1和α2亚基Na~+-K~+泵对DHO的不同亲和力(5μM DHO阻断89% Ih而仅阻断9% Il,1 mM DHO则阻断几乎全部的Ip),5μM DHO用于阻断Ih;在记录Il时,所有灌流液中均加入5μM DHO以阻断Ih,此时再灌流细胞1 mM DHO所造成的差别电流即Il。(3)腺苷对Ip影响的测定:实验采用自身对照的方法,即在同一细胞上分别测定对照和腺苷干预时的电流幅度,并以对照电流值标化给药后的电流值求得均数(至少5例细胞)。具体方法如下:破膜后至少稳定5 min使得电极内液和细胞内液充分交换。基线平稳后首先灌流细胞DHO测定对照Ip(Ip(Con)),用正常外液将DHO洗脱后膜电流恢复至初始水平;基线稳定后灌流腺苷,待腺苷引起的膜电流移动不再变化时(表示腺苷作用已经稳定),用同时含有腺苷和DHO的外液灌流细胞,此时测定的Ip反映腺苷干预后的Ip(Ip(Ado))。最后用Ip(Con)数值标化Ip(Ado)求得均数(Ip(Ado)/Ip(Con))。这种方法的优点在于避免了不同的细胞大小和泵蛋白表达量(即蛋白密度)造成的差异。(4)细胞内信号机制的鉴别:实验选用特异性腺苷A1R、A2AR、A3R激动剂(分别为CCPA、CGS21680、Cl-IB-MECA)和拮抗剂(分别为DPCPX、SCH58261、MRS1191),PKC拮抗剂(Staurosporine和bisindolylmaleimide I),PKA拮抗剂(H89)鉴别可能参与这一调节作用的腺苷受体亚型和信号通路。药物浓度的选择均参照文献报道。
结果:(1)腺苷特异性抑制Ih。生理浓度的腺苷(1 nM)显著抑制Ih,抑制率为39±0.04%(P<0.05)。洗脱腺苷和DHO后电流幅度能恢复到对照水平,这种“recontrol”的实验方法排除了泵电流衰减的可能性。相反,1 nM腺苷不影响Il,Il为对照水平的93±0.05%(P>0.05),且增加腺苷浓度至10-5 M也不影响Il,以上实验结果表明腺苷特异性调节Ih。(2)腺苷对Ih的抑制呈剂量依赖性。10-11-10-5 M腺苷剂量依赖性的抑制Ih(8-47%),腺苷浓度为10-8 M时产生最大抑制作用。(3)腺苷对Ih的抑制呈电压非依赖性。首先设置钳制电压为0 mV,然后在灌流DHO前后分别给予从+20至-100 mV的电压ramp protocol,两组数据相减即可得到泵电流的Ih-Vm关系曲线。之后在灌流细胞腺苷的情况下,再次于灌流DHO前后施加上述电压ramp protocol,两组数据相减即为腺苷作用后的Ih-Vm关系曲线。为便于比较灌流腺苷前后的Ih-Vm关系,特将对照和灌流腺苷后各电压下的电流值用0 mV的电流值进行标化。结果表明,在各电压下腺苷对Ih均产生约47%的抑制,即腺苷对Ih的抑制不具有电压依赖性。(4)腺苷通过激活A1R触发对Ih的抑制。同时灌流细胞腺苷和特异性A1R拮抗剂DPCPX(10 nM)时,Ih可恢复至对照电流值的96±0.06%(P>0.05)。另外,特异性A1R激动剂CCPA(10 nM)可模拟腺苷对Ih的抑制,抑制率为50±0.04%(P<0.05),以上结果提示腺苷通过激活A1R抑制Ih。此外,特异性A2AR和A3R激动剂CGS21680(0.2μM)和Cl-IB-MECA(0.5μM)不影响Ih,Ih分别为对照电流值的101±0.07%(P>0.05)和98±0.05%(P>0.05),并且腺苷和特异性A2AR和A3R拮抗剂SCH58261和MRS1191(均为0.1μM)同时灌流也不影响腺苷对Ih的抑制,抑制率为53±0.06%(P<0.05),与单独灌流腺苷时的抑制率(47±0.03%)相比无统计学差异(P>0.05)。结果提示A2AR和A3R不参与腺苷对Ih的抑制。(5)腺苷A1R激活后经PKC通路抑制Ih。PKC阻断剂Staurosporine(St,1.5μM)可拮抗腺苷对Ih的抑制,Ih可恢复至对照水平的95±0.05%(P>0.05)。另外,特异性更强的PKC阻断剂bisindolylmaleimide I(Bis- I,1μM)也产生与St相同的作用,Ih恢复至对照水平的91±0.04%(P>0.05)。相反,PKA阻断剂H89(1μM)不影响腺苷对Ih的抑制,抑制率为47±0.03%(P<0.05)。结果提示腺苷A1R激活后经PKC通路抑制Ih。(6)PKC-δ是介导腺苷作用的具体PKC亚型。经典PKC亚型(PKC-α,β,γ)阻断剂G?-6976(100 nM)不影响腺苷对Ih的抑制,抑制率为53±0.05%(P<0.05)。相反,特异性PKC-δ抑制剂rottleri(n10μM)可完全阻断腺苷的作用,Ih恢复至对照水平的91±0.05%(P>0.05)。另外,特异性PKC-δ转位激活肽PP114(200 nM)可显著抑制Ih,抑制率为52±0.04%(P<0.05),且其作用也可被Bis-1和rottlerin阻断。结果提示PKC-δ是介导腺苷作用的主要PKC亚型。
结论:腺苷对正常豚鼠心室肌细胞Na~+-K~+泵存在亚基特异性的调节作用,即腺苷特异性抑制高亲和力α2亚基Na~+-K~+泵电流。这一作用是经腺苷A1R和PKC通路介导,腺苷A2AR、A2BR、A3R和PKA通路不参与腺苷对Na~+-K~+泵的调节。
第二部分腺苷对缺血豚鼠心室肌细胞Na~+-K~+泵电流的调节
目的:已知心肌缺血可导致Na~+-K~+泵活性的显著降低,但在单细胞水平上鲜有文献研究缺血对Na~+-K~+泵电流的影响,因此本研究旨在详细描述缺血对心肌Na~+-K~+泵电流的抑制和电流特性的变化,以及观察腺苷处理能否拮抗心肌缺血对Na~+-K~+泵电流的抑制。
方法:(1)酶解法急性分离豚鼠心室肌细胞:同第一部分。(2)全细胞膜片钳方法记录Na~+-K~+泵电流(Ip):同第一部分。(3)使用代谢抑制剂模拟缺血状态:代谢抑制(metabolic inhibition,简称MI)是心肌缺血的主要特征,因此使用糖酵解抑制剂2-DG和线粒体解偶联剂FCCP可造成糖酵解和氧化磷酸化的联合抑制。
结果:(1)MI时间依赖性的抑制Ip。MI灌流2.5,4,6 min后,电流幅度分别被抑制了30±0.05%,49±0.02%,62±0.04% (r=0.82,P<0.05)。延长灌流时间至10 min时,MI对Ip的抑制程度并不产生进一步的增加(67%±0.02,P>0.05)。结果表明,MI对Ip的抑制呈时间依赖性,且MI灌流时间为6 min时对Ip产生最大抑制作用。(2)Antimycin A抑制Ip。为证实MI对Ip的抑制作用是代谢抑制的结果,实验选用另一种代谢抑制剂antimycin A,后者通过抑制呼吸链电子的传递过程造成代谢抑制。结果表明,10μM antimycin A也可显著抑制Ip,抑制率为68±0.02%(P<0.05)。(3)MI特异性抑制Il。MI灌流6 min后,Ih仍维持在对照水平的97±0.04%(P>0.05),提示MI对泵电流的抑制可能只涉及α1亚基Na~+-K~+泵。进而我们直接观察MI对Il的作用。结果表明,MI明显抑制Il,抑制率为60±0.03%(P<0.05),与MI对总泵电流的抑制率(62±0.04%)相比无统计学差异(P>0.05),提示MI特异性抑制α1亚基Na~+-K~+泵。(4)MI对Ip的抑制呈电压依赖性。Ip-Vm关系的测定方法同第一部分,仅以毒毛旋花子苷原strophanthidin(St,属于强心苷类)代替DHO作为Na~+-K~+泵阻断剂。结果表明,灌流MI前后V1/2数值分别是-107和-87 mV,即MI造成Ip-Vm曲线向正电压轴方向偏移了20 mV,两条曲线有统计学差异(P<0.05;two-way ANOVA)。(5)腺苷不影响MI对Ip的抑制。在10μM腺苷存在时,MI仍可对Ip产生66±0.05%(P<0.05)的抑制;与不存在腺苷时的抑制率(62±0.04%)相比无统计学差异(P>0.05)。甚至使用100μM的腺苷(临床治疗缺血性心脏疾病时的剂量)也不产生作用,提示腺苷不能阻断缺血对泵电流的抑制,即Na~+-K~+泵可能不参与腺苷对心肌缺血的保护作用。
结论:模拟心肌缺血显著抑制豚鼠心室肌细胞Na~+-K~+泵电流,呈时间依赖性。模拟心肌缺血对Na~+-K~+泵电流的抑制也具有亚基特异性,即缺血特异性抑制低亲和力α1亚基Na~+-K~+泵电流,且此作用呈电压依赖性。腺苷不影响模拟心肌缺血对泵电流的抑制,提示Na~+-K~+泵可能不参与腺苷对心肌缺血的保护作用。
第三部分心肌缺血抑制Na~+-K~+泵电流的机制研究
目的:阐明心肌缺血造成Na~+-K~+泵电流抑制的可能机制。
方法:(1)酶解法急性分离豚鼠心室肌细胞:同第一部分。(2)采用全细胞膜片钳技术测定Na~+-K~+泵电流(Ip):同第一部分。(3)使用代谢抑制剂模拟缺血状态:同第二部分。(4)细胞内pH(pHi)的检测:使用对pH敏感的双激发荧光染料carboxy-SNARF-1检测pHi。carboxy-SNARF-1负载细胞10 min后使用激光共聚焦显微镜检测荧光信号,激发波长514nm,并于580 nm和640 nm处同时检测荧光信号。pHi的变化由上述两种波长荧光强度的比值(F580/F640)所反映。580的荧光值代表细胞内已结合的H+,640的荧光值代表细胞内未结合的H+,其比值的变化可反应出pHi的变化。通过nigericin calibration technique将F580/F640比值转化为pHi。
结果:(1)心肌缺血造成的细胞内酸化可抑制Ip。心肌缺血引起细胞外环境的许多变化,如缺氧、葡萄糖耗竭及酸中毒等。但实验结果表明,若维持细胞外液pH稳定(pHo 7.4),仅单纯缺氧(灌流液充以100% N2)和耗竭葡萄糖(以等摩尔的蔗糖代替葡萄糖)时,Ip仅被抑制了7.5±0.04% (P>0.05);相反,若仅降低灌流液pH值而不改变葡萄糖和氧含量,即采用酸性灌流液(pHo 6.0,常氧,含糖)灌流心肌细胞,则可显著抑制Ip,且抑制程度与MI相似,为50±0.02%(P<0.05),提示细胞外酸中毒是造成Ip抑制的触发因素。由于细胞外酸中毒可迅速引起细胞内的酸化,且有资料报道Na~+-K~+泵对pHo不敏感,这些证据提示pHi可能是心肌缺血抑制Ip的主要介导因素。为验证这一设想,我们将电极内液中HEPES浓度增加至20 mM,从而提高HEPES对pH变化的缓冲能力。结果表明,在内液中含有高浓度HEPES的情况下,酸性灌流液(pHo 6.0)不引起明显的Ip抑制;Ip为对照水平的94±0.07%(P>0.05)。相反,通过灌流细胞酸性制剂造成细胞内酸化可模拟细胞外酸中毒对Ip的抑制。短暂灌流并洗脱NH4Cl是造成细胞内酸化的常规方法。结果表明,短暂灌流15 mM NH4Cl并洗脱后(3 min),Ip可被明显抑制,抑制率为60±0.03%(P<0.05)。同样,在内液中存在20 mM HEPES时,这一作用可被完全阻断,Ip恢复至对照水平的96±0.04%(P>0.05)。以上结果提示,细胞内酸化是心肌缺血抑制Ip的主要介导因素。(2)MI引起细胞内酸化,从而抑制Ip。在内液中存在20 mM HEPES时,MI对Ip的抑制作用被明显阻断,Ip恢复至对照水平的81±0.02%,但与内液中含有5 mM HEPES时的抑制率相比(62±0.04%)仍有统计学差异(P<0.05)。为明确pHi的作用,我们使用对pHi敏感的荧光染料在共聚焦显微镜下直接观察MI灌流前后pHi的变化。结果显示,MI灌流前pHi约为7.28±0.07,MI灌流后pHi呈时间依赖性的下降;MI灌流2.5,4,6 min时,pHi分别分别下降至7.02±0.06,6.81±0.06和6.65±0.08。以上结果提示,细胞内酸化是代谢抑制时Ip被抑制的主要介导因素。(3)氧化应激(ROS)、PKA和PKC不参与MI对Ip的抑制。电极内液中加入1 mM MPG不影响MI对Ip的抑制(68±0.05%),与内液中不含MPG的抑制率(62±0.04%)相比无统计学差异(P>0.05)。另外,特异性PKA阻断剂H89(1μM)或PKC阻断剂Bis-I(1μM)也不影响MI对Ip的抑制。
结论:模拟心肌缺血通过造成细胞内的酸化从而抑制Na~+-K~+泵电流。心肌缺血对泵电流的抑制可能不涉及缺血造成的其它细胞内变化,例如ROS,PKA和PKC的激活以及缺氧、葡萄糖耗竭等。
结论
1腺苷对正常豚鼠心室肌细胞Na~+-K~+泵存在亚基特异性的调节作用,即腺苷特异性抑制高亲和力α2亚基Na~+-K~+泵电流。这一作用是经腺苷A1R和PKC通路介导,腺苷A2AR、A2BR、A3R和PKA通路不参与腺苷对Na~+-K~+泵的调节。
2模拟心肌缺血显著抑制豚鼠心室肌细胞Na~+-K~+泵电流,呈时间依赖性。模拟心肌缺血对Na~+-K~+泵电流的抑制也具有亚基特异性,即缺血特异性抑制低亲和力α1亚基Na~+-K~+泵电流,且此作用呈电压依赖性。腺苷不影响模拟心肌缺血对泵电流的抑制,提示Na~+-K~+泵可能不参与腺苷对心肌缺血的保护作用。
3模拟心肌缺血通过造成细胞内酸化抑制Na~+-K~+泵电流。心肌缺血对泵电流的抑制可能不涉及缺血造成的其它细胞内变化,例如ROS,PKA和PKC的激活以及缺氧、葡萄糖耗竭等。
The Na~+-K~+ pump (also known as sodium pump or Na+, K+-ATPase) is a member of P-ATPase family and present in all animal cells. The Na~+-K~+ pump uses an ATP to transport 3 Na+ out of cells and 2 K+ into the cell and provides energy for several essential cellular functions (control of cell volume, pH homeostasis, reabsorption of nutrition). In excitable tissues (heart and nerve, etc), the Na~+-K~+ pump function is crucial for maintaining and establishing the normal electrical activity.
It has been demonstrated that the Na~+-K~+ pump function is subjected to regulation by a variety of hormones and biochemical changes associated with pathological conditions. For instance, catecholamines, insulin, angiotensin, and thyroid hormone mainly modulate the activity of Na~+-K~+ pump through interaction with receptors located in the plasma membrane and stimulation of downstream signaling pathways. In addition, myocardial ischemia, a leading cause of death and disability in deveoleped contries, leads to a marked inhibition of Na~+-K~+ pump. Thus, delineating detailed modulation of Na~+-K~+ pump by endogenous signaling molecules and biochemical changes is important for better understanding the exact and precise role of Na~+-K~+ pump play in various physiological and pathological conditions.
Many endogenous signaling molecules (bradykinin, adenosine, noradrenaline, etc) have been found to confer some degree of protection against myocardial ischemia. Of these, adenosine is perhaps the single most widely studied“cardioprotectnant”. Adenosine is present at low concentration and can be released as a result of metabolic stress (ischemia and hypoxia, etc). Adenosine exerts its effects through adenosine A1, A2A, A2B, and A3 receptor subtypes (A1R, A2AR, A2BR, and A3R), which are all expressed in myocardial and vascular cells, and coupled to G proteins to trigger a range of responses, with ultimate effects of limitation of cellular death and dysfunction. The nucleoside mediates tissue protection by different mechanisms including increased oxygen supply/demand, ischemic preconditioning and stimulation of angiogenesis, and also as a paracrine inhibitor of inflammation with effects in the lung, heart and brain. However, as an active transporter maintaining the essential cellular functions, it is not known whether Na~+-K~+ pump is subjected to modulation by endogenous adenosine. In addition, the possible involvement of Na~+-K~+ pump in the cardioprotection of adenosine during ischemia is also not known.
Therefore, using the whole-cell patch-clamp technique, the present study aimed to address the following questions: (1) whether there lies a possible modulatory effect of adenosine on the Na~+-K~+ pump under normal conditions; (2) whether adenosine could abolish the inhibition of Na~+-K~+ pump induced by ischemia; (3) the exact and presice mechaniam of ischemia-mediated Na~+-K~+ pump injury.
Part 1 The regulation of Na~+-K~+ pump current by adenosine in normal guinea pig ventricular myocytes
Objective: To study the effect of adenosine on Na~+-K~+ pump in ventricular myocytes acutely isolated from guinea pigs and to further elucidate the possible mechanisms involved.
Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes: guinea pig heart was digested by aorta retrograde perfusion with Collegnase NB8 (from Serva Chemical Co) using Langendorff apparatus. The temperature, pH and water quality should be properly controlled in the process of digestion. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip): the Na~+-K~+ pump exchanges 3 intracellular Na+ for 2 external K+ across the cell membrane during each active transport process. This excess positive charge movement generates a net outward current (Ip). With selected external and pipette solutions, membrane currents through K+ channel, Ca2+ channel, and Na+-Ca2+ exchanger were minimized. Under the experimental conditions, Ip was measured as the DHO-blocked current. Guinea pig ventricular myocytes coexpress two distinct Na~+-K~+ pumpαisoform,α1 andα2, which correspond to the low- and high-affinity isoforms for cardiac glycosides (dissociation constants for DHO are 72μM and 0.75μM, respectively). Thus, total Ip is the sum of current contributed by theα1-isoform (Il) plus that by theα2-isoform (Ih): Ip=Il+Ih. Based on the different affinities for cardiac glycosides (DHO at 1 mM blocked roughly 96% of total Ip, and that 5μM DHO blocked 89% of Ih while blocking only 9% of Il), we used 1 mM and 5μM DHO to identify Il and Ih, respectively. (3) Measuring of the adenosine effect on Ip: when we studied the adenosine effect on Ip, the standard protocol was to measure pump current in control and test conditions in the same cell, and then to calculate the ratio of test to control current and average this ratio from at least five cells. The detailed method is described as following: after whole-cell recording was initiated, a period of at least 5 min was required for the pipette and intracellualr solutions to come to steady state. When steady state was achieved, DHO was superfused to record the DHO-induced changes in holding current, which was reversed upon washout of DHO and was considered to reflect control Ip (Ip(Con)). Once the current had again stabilized, adenosine was applied to the myocytes. This caused a shift in holding current. After a new steady state was achieved, in the continous presence of adenosine, DHO was again applied and this inward shift reflect the changed Ip by adenosine (Ip(Ado)). Finally, Ip(Ado) was normalized to Ip(Con) to obtain the averaged ratio. This procedure uses each cell as its own control and removes the uncertainty due to cell to cell variation in size and pump density. (4) Mechanisms mediating the effect of adenosine on Ip: to clarify the cellular mechanisms responsible for the adenosine effect on Ip, specific adenosine A1R, A2AR, and A3R agonists (CCPA, CGS21680, Cl-IB-MECA, respectively), antagonists (DPCPX, SCH58261, MRS1191, respectively), PKC inhibitors(Staurosporine and bisindolylmaleimide I), and PKA inhibitor(H89)were used.
Results: (1) Adenosine specifically inhibits Ih. These results show that a physiological concentration of adenosine (1 nM) decreased Ih by 39±0.04%. The decrease in Ip was not due to pump“rundown”because the adenosine effect was reversible upon washout. Adenosine did not change Il significantly; Il remained unchanged at 93±0.05% of control values (P>0.05). Also, increasing adenosine to 10μM, adenosine had no effect on Il, suggesting that adenosine specifically inhibits Ih. (2) Adenosine concentration-dependently inhibites Ih. Perfusion of adenosine from 10-11-10-5 M induced an appreciable (8% to 47%) concentration-dependent inhibition of Ih, and the inhibition was maximal at 10-8 M. (3) The adenosine-induced inhibition of Ih is voltage independent. For measurement of the voltage dependence of Ip a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used in some experiments. The relationships were normalized to the Ih recorded at 0 mV to facilitate comparison of their slopes. The difference between these slopes was not statistically significant (P>0.05; two-way ANOVA). Thus, adenosine-induced Ih inhibition is voltage independent. (4) Adenosine binding to A1R triggers the inhibition of Ih. The specific A1R antangonist DPCPX (10 nM) abrogated the adenosine effect on Ih; Ih returned to 96±0.06% of control values (P>0.05). In a parallel study, we further tested A1R specificity by treating the cells with CCPA, a selective agonist for A1R. Perfusion of 10 nM CCPA produced a marked inhibition of Ih. Ih was inhibited by 50±0.04% in the presence of CCPA (P<0.05). Furthermore, treatment of cells with specific A2AR and A3R agonists, CGS21680 (0.2μM) and Cl-IB-MECA (0.5μM), respectively, did not affect Ih; Ih remained constant at 101±0.07% and 98±0.05% of control values (P>0.05), respectively. The specific A2AR and A3R antagonists, SCH58261 and MRS1191 (0.1μM each), did not alter the adenosine effect on Ih. Ih was decreased by 53±0.06% (P<0.05), which was not statistically different from 47±0.03% inhibition when compared to its absence (P>0.05). These data together suggest that the inhibitory effect of adenosine on Ih is mediated by adenosine A1R. (5) The activation of adenosine A1R stimulates the PKC pathway, thereby inhibiting Ih. The inhibitory effect of adenosine on Ih was abolished by PKC inhibitor staurosporine (St, 1.5μM). Current amplitudes returned to 95±0.05% of the initial control values (P>0.05). Again, 1μM Bis-I, a highly specific PKC blocker, clearly abrogated the inhibition of Ih by adenosine. The currents returned to 91±0.04% of control values after perfusion of Bis-I (P>0.05). In contrast, PKA inhibitor H89 did not affect the adenosine effect on Ih. Adenosine still induced 47±0.03% inhibition of Ih (P<0.05). Collectively, these results suggest that adenosine inhibits Ih through a PKC-dependent mechanism. (6) The activation of PKC-δinhibits Ih. Using the classical PKC isoforms (PKC-α,β,γ) inhibitor G?-6976 (100 nM), we show that the inhibitory effect of adenosine on Ih was not affected. Ih was still inhibited by 53±0.05% (P<0.05) in the presence of G?-6976. In addition, we tested the role of the novel PKC isoformδusing specific inhibitor rottlerin. Adenosine failed to inhibit Ih in the presence of 10μM rottlerin; Ih returned to 91±0.05% of control values (P>0.05), indicating that PKC-δis required for the adenosine effect on Ih. Like adenosine, PP114, a specific activator peptide of PKC-δ, also inhibited Ih. Ih was decreased by 52±0.04% on application of PP114 (P<0.05). Taken together, these results indicate that PKC-δplays a crucial role in the inhibition of Ih by adenosine.
Conclusion: Adenosine specifically inhibits theα2-isoform of Na~+-K~+ pump, but not theα1-isoform of Na~+-K~+ pump. The effect of adenosine on Na~+-K~+ pump is mediated by adenosine A1R and PKC pathway. However, the adenosine A2AR, A2BR, A3R, and PKA pathway are not involved.
Part 2 The regulation of Na~+-K~+ pump current by adenosine in ischemic guinea pig ventricular myocytes
Objective: To delineate the Na~+-K~+ pump current during myocardial ischemia and to determine whether adenosine treatment could prevent the inhibition of Na~+-K~+ pump current during myocardial ischemia.
Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip). (3) Simulation of myocardial ischemia using metabolic inhibitors: metabolic inhibition (MI) is a prominent feature of myocardial ischemia. Thus, we used metabolic inhibitors FCCP and 2-DG to produce combined inhibition of oxidative and glycolytic metabolism.
Results: (1) MI time-dependently inhibits Ip. MI reduced Ip in a time-dependent manner; its amplitudes were reduced by 30±0.05%, 49±0.02%, 62±0.04% (r=0.82, P<0.05) following 2.5, 4, and 6 min of MI, respectively. Increasing the time of MI to 10 min, however, did not produce any further reduction in Ip (67±0.02%, P>0.05), suggesting that a maximal effect was achieved at a MI time of 6 min. (2) Antimycin A inhibits Ip. To gain further evidence that the effect of FCCP and 2-DG was because of metabolic inhibition, we tested another metabolic inhibitor, antimycin A. After exposure of myocytes to 10μM antimycin A for 6 min, there was a 68±0.02% reduction in Ip (P<0.05). (3) MI specifically inhibits Il. After exposure of myocytes to MI for 6 min, Ih remained unchanged at 97±0.04% of control values (P>0.05). These results seem to suggest that only theα1-isoform of the Na~+-K~+ pump is involved in the inhibition of Ip induced by MI. We then proceeded to directly examine the effect of MI on Il. MI inhibited Il by 60±0.03% (P<0.05), which was not significantly different from 62±0.04% inhibition of total Ip produced by MI (P>0.05). Thus, MI-induced inhibition of Ip isα1-isoform specific. (4) Inhibition of Ip by MI is voltage-dependent. For measurement of the voltage dependence of Ip a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used. The relationships were normalized to the Ip recorded at 0 mV to facilitate comparison of their slopes. The difference between these slopes was statistically significant (P<0.05; two-way ANOVA). MI shifted the voltage dependence of Ip towards more positive potentials by approximately 20 mV. The V1/2 values in the absence and presence of MI were -107 and -87 mV, respectively. (5) Adenosine does not influence the inhibitory effect of MI on Ip. MI still reduced Ip by 66±0.05% (P<0.05) in the presence of 10μM adenosine and this was statistically insignificant when compared to its absence (P>0.05). Adenosine at 100μM, a therapeutic dose for ischemic myocardial injury, also did not affect MI-induced Ip inhibition. These results suggest that Na~+-K~+ pump was not involved in the cardioprotection of adenosine against myocardial ischemia.
Conclusion: Simulated myocardial ischemia markedly inhibits the Na~+-K~+ pump function in a time-dependent manner. Simulated myocardial ischemia specifically inhibits theα1-isoform of Na~+-K~+ pump, which is voltage-dependent. Adenosine does not affect the inhibition of Na~+-K~+ pump induced by myocardial ischemia, suggesting that the cardioprotection of adenosine against myocardial ischemia does not involves the Na~+-K~+ pump.
Part 3 The mechanism of myocardial ischemia-induced inhibition of Na~+-K~+ pump current
Objective:To delineate the characteristic of Na~+-K~+ pump current during myocardial ischemia and to further clarify the underlying mechanism involved.
Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip). (3) Simulation of myocardial ischemia using metabolic inhibitors. (4) Measuring of intracellular pH (pHi): The pHi of single isolated myocytes was measured using carboxy-SNARF-1, a dual-emission pH-sensitive fluoroprobe. The myocytes were loaded with the membrane-permeable acetoxy-methylester form of carboxy-SNARF-1 for 10 min and then pHi was measured using laser confocol microscope before and after MI superfusion. The fluorophore was excited by light at 514 nm, and the emitted fluorescence signals were measured simultaneously at 580 nm and 640 nm. The 580 nm/640 nm emission ratio was converted to a pHi value using the nigericin calibration technique
Results:(1) Myocardial ischemia-induced intracellular acidification inhibits Ip. No inhibition of Ip was observed when cells were exposed to the extracellular solution with anoxia (bubbling with 100% N2) and glucose deprivation but normal pHo (7.4); Ip was only decreased by 7.5±0.04% and this decrease was statistically insignificant (P>0.05). However, when treatment of myocytes with the extracellular solution containing normal oxygen and glucose but acidosis (pHo 6.0), Ip was markedly decreased by 50±0.02% (P<0.05) as under MI, demonstrating that the change in pHo itself was sufficient to inhibit Ip. Since extracellular acidosis leads to a rapid radification of the cytosol, and there is evidence suggest that Na~+-K~+ pump is not sensitive to pHo, we propose that pHi may be the key component triggering Ip inhibition during myocardial ischemia. To address this point, we increased the pHi buffering capability by elevating the concentration of HEPES in the pipette solution to 20 mM. Under these conditions, extracellular acidosis did not induce a significant inhibition of Ip; Ip returned to 94±0.07% (P>0.05) of control values. By contrast, perfusing myocytes with acidosic agent could mimic the effect of extracellular acidosis on Ip. Briefly perfusing myocytes with NH4Cl followed by washout, which is a common protocol that was used to induce an intracellular acidification. NH4Cl withdrawal drastically reduced Ip by 60±0.03% (P<0.05). Also, this inhibition was completely abolished with 20 mM HEPES in the patch pipette; Ip returned to 96±0.04% (P>0.05) of control values. (2) MI induces a rapid acidification of the cytosol, thus inhibiting Ip. With 20 mM HEPES in the pipette solution, the inhibitory effect of MI on Ip was markedly prevented; Ip return to 81±0.02% (P<0.05) of control values, which is not significantly different from that with 5 mM HEPES in the pipette solution (62±0.04%, P<0.05). We further monitored pHi with SNARF-1 under MI. It was found that resting pHi in these cells was 7.28±0.07. Perfusion of MI resulted in a sustained intracellular acidification. The mean pHi values corresponding to MI time of 2.5, 4, and 6 min were 7.02±0.06, 6.81±0.06, and 6.65±0.08, respectively. Therefore, MI leads to Ip inhibition in a pH-dependent manner in guinea pig ventricular myocytes. (3) ROS, PKA, and PKC are not involved in MI inhibition of Ip. Inclusion of 1 mM MPG in the patch pipette failed to block the MI-induced decrease in Ip and this was statistically insignificant when compared to its absence (68±0.05% for MI+MPG, P>0.05 vs. 62±0.04% for MI). In addition, the specific PKA and PKC inhibitors, H89 and Bis-I, respectively, did not significantly affect MI inhibition of Ip.
Conclusion: Simulated myocardial ischemia leads to a rapid acidification of the cytosol, thus causing the inhibition of Na~+-K~+ pump. However, oxidative stress, PKA, and PKC are less likely to be involved in the inhibition of Na~+-K~+ pump by myocardial ischemia.
SUMMARY
1 Adenosine specifically inhibits theα2-isoform of Na~+-K~+ pump, but not theα1-isoform of Na~+-K~+ pump. The effect of adenosine on Na~+-K~+ pump is mediated by adenosine A1R and PKC pathway. However, the adenosine A2AR, A2BR, A3R, and PKA pathway are not involved.
2 Simulated myocardial ischemia markedly inhibits the Na~+-K~+ pump function in a time-dependent manner. Simulated myocardial ischemia specifically inhibits theα1-isoform of Na~+-K~+ pump, which is voltage-dependent. Adenosine does not affect the inhibition of Na~+-K~+ pump induced by myocardial ischemia, suggesting that the cardioprotection of adenosine against myocardial ischemia does not involves the Na~+-K~+ pump.
3 Simulated myocardial ischemia leads to a rapid acidification of the cytosol, thus causing the inhibition of Na~+-K~+ pump. However, oxidative stress, PKA, and PKC are less likely to be involved in the inhibition of Na~+-K~+ pump by myocardial ischemia.
研究发现,Na~+-K~+泵功能可被多种神经内分泌激素及病理状态所引发的生物化学变化调节。例如,儿茶酚胺、胰岛素、甲状腺激素、血管紧张素等通过激活细胞膜上的相应受体和下游的信号转导通路调节泵功能。此外,心肌缺血,做为造成发展中国家人群致死和致残的主要原因,可显著抑制Na~+-K~+泵功能。因此,详细阐明体内内源性信号分子和生物化学变化对泵功能的调节有助于更好的理解不同病理生理状态下Na~+-K~+泵的确切作用。
体内多种信号分子(缓激肽、腺苷、去甲肾上腺素等)对心肌缺血存在不同程度的保护作用,其中腺苷(adenosine,Ado)是研究最为广泛的“心脏保护者”。正常状态下细胞间隙中的腺苷浓度很低,但代谢应激(缺血和缺氧等)可刺激腺苷的大量释放,并与心血管细胞膜表达的腺苷受体(A_1R、A_(2A)R、A_(2B)R、A_3R)结合触发细胞内的一系列反应,发挥限制细胞死亡和失功能的作用。腺苷对组织的保护作用涉及不同的机制,包括增加氧供、缺血预适应、刺激血管生成、抑制炎症发生等。然而,Na~+-K~+泵作为维持细胞基本功能的主动转移体,其功能是否可被内源性分泌的腺苷调节呢?另外,腺苷能否阻断心肌缺血造成的Na~+-K~+泵功能抑制目前仍未见报道。
因此,本研究采用全细胞膜片钳的方法旨在观察:(一)腺苷对正常豚鼠心室肌细胞Na~+-K~+泵电流的影响,并探讨其可能机制;(二)腺苷对模拟缺血豚鼠心室肌细胞Na~+-K~+泵电流的影响;(三)心肌缺血抑制Na~+-K~+泵电流的确切机制。
第一部分腺苷对正常豚鼠心室肌细胞Na~+-K~+泵电流的调节
目的:研究腺苷对急性分离的豚鼠心室肌细胞Na~+-K~+泵电流的影响,并深入探讨可能参与这一调节过程的信号转导途径。
方法:(1)酶解法急性分离豚鼠心室肌细胞:使用Langendorff装置,行主动脉插管逆行灌流(温度、pH、水质恒定),用胶原酶NB8(Collagenase NB8,Serva公司)消化心脏,急性分离豚鼠心室肌细胞。选择胞膜光滑、横纹相对清晰、贴壁良好、稳定无收缩的细胞用于实验。(2)全细胞膜片钳技术(whole-cell patch-clamp technique)记录Na~+-K~+泵电流(Ip):Na~+-K~+泵经过一次主动转运过程将3个Na~+泵出膜外同时泵入2个K~+,从而产生一净外向电流。Ip的测定方法是:在特定灌流液和细胞内液成分阻断细胞膜上其它主要离子通道和交换体电流的条件下(K~+电流、Ca~(2+)电流、Na~+-Ca~(2+)交换体电流),灌流细胞特异性、可逆性Na~+-K~+泵阻断剂(即强心苷类),由于Na~+-K~+泵被阻断后膜电流产生内向性变化,此差别电流即反映Ip的大小。已有资料证实豚鼠心室肌细胞共表达两种α亚基的Na~+-K~+泵,即α1亚基Na~+-K~+泵和α2亚基Na~+-K~+泵。前者对强心苷的亲和力低,为低亲和力泵;后者对强心苷的亲和力高,为高亲和力泵。例如α1亚基Na~+-K~+泵对双氢哇巴因dihydroouabain(DHO,属于强心苷类)的解离常数为72μM,α2亚基Na~+-K~+泵对DHO的解离常数为0.75μM,两者亲和力相差100倍。故总泵电流(Ip)为α1亚基Na~+-K~+泵电流(Il)和α2亚基Na~+-K~+泵电流(Ih)之和:Ip=Il~+Ih。根据α1和α2亚基Na~+-K~+泵对DHO的不同亲和力(5μM DHO阻断89% Ih而仅阻断9% Il,1 mM DHO则阻断几乎全部的Ip),5μM DHO用于阻断Ih;在记录Il时,所有灌流液中均加入5μM DHO以阻断Ih,此时再灌流细胞1 mM DHO所造成的差别电流即Il。(3)腺苷对Ip影响的测定:实验采用自身对照的方法,即在同一细胞上分别测定对照和腺苷干预时的电流幅度,并以对照电流值标化给药后的电流值求得均数(至少5例细胞)。具体方法如下:破膜后至少稳定5 min使得电极内液和细胞内液充分交换。基线平稳后首先灌流细胞DHO测定对照Ip(Ip(Con)),用正常外液将DHO洗脱后膜电流恢复至初始水平;基线稳定后灌流腺苷,待腺苷引起的膜电流移动不再变化时(表示腺苷作用已经稳定),用同时含有腺苷和DHO的外液灌流细胞,此时测定的Ip反映腺苷干预后的Ip(Ip(Ado))。最后用Ip(Con)数值标化Ip(Ado)求得均数(Ip(Ado)/Ip(Con))。这种方法的优点在于避免了不同的细胞大小和泵蛋白表达量(即蛋白密度)造成的差异。(4)细胞内信号机制的鉴别:实验选用特异性腺苷A1R、A2AR、A3R激动剂(分别为CCPA、CGS21680、Cl-IB-MECA)和拮抗剂(分别为DPCPX、SCH58261、MRS1191),PKC拮抗剂(Staurosporine和bisindolylmaleimide I),PKA拮抗剂(H89)鉴别可能参与这一调节作用的腺苷受体亚型和信号通路。药物浓度的选择均参照文献报道。
结果:(1)腺苷特异性抑制Ih。生理浓度的腺苷(1 nM)显著抑制Ih,抑制率为39±0.04%(P<0.05)。洗脱腺苷和DHO后电流幅度能恢复到对照水平,这种“recontrol”的实验方法排除了泵电流衰减的可能性。相反,1 nM腺苷不影响Il,Il为对照水平的93±0.05%(P>0.05),且增加腺苷浓度至10-5 M也不影响Il,以上实验结果表明腺苷特异性调节Ih。(2)腺苷对Ih的抑制呈剂量依赖性。10-11-10-5 M腺苷剂量依赖性的抑制Ih(8-47%),腺苷浓度为10-8 M时产生最大抑制作用。(3)腺苷对Ih的抑制呈电压非依赖性。首先设置钳制电压为0 mV,然后在灌流DHO前后分别给予从+20至-100 mV的电压ramp protocol,两组数据相减即可得到泵电流的Ih-Vm关系曲线。之后在灌流细胞腺苷的情况下,再次于灌流DHO前后施加上述电压ramp protocol,两组数据相减即为腺苷作用后的Ih-Vm关系曲线。为便于比较灌流腺苷前后的Ih-Vm关系,特将对照和灌流腺苷后各电压下的电流值用0 mV的电流值进行标化。结果表明,在各电压下腺苷对Ih均产生约47%的抑制,即腺苷对Ih的抑制不具有电压依赖性。(4)腺苷通过激活A1R触发对Ih的抑制。同时灌流细胞腺苷和特异性A1R拮抗剂DPCPX(10 nM)时,Ih可恢复至对照电流值的96±0.06%(P>0.05)。另外,特异性A1R激动剂CCPA(10 nM)可模拟腺苷对Ih的抑制,抑制率为50±0.04%(P<0.05),以上结果提示腺苷通过激活A1R抑制Ih。此外,特异性A2AR和A3R激动剂CGS21680(0.2μM)和Cl-IB-MECA(0.5μM)不影响Ih,Ih分别为对照电流值的101±0.07%(P>0.05)和98±0.05%(P>0.05),并且腺苷和特异性A2AR和A3R拮抗剂SCH58261和MRS1191(均为0.1μM)同时灌流也不影响腺苷对Ih的抑制,抑制率为53±0.06%(P<0.05),与单独灌流腺苷时的抑制率(47±0.03%)相比无统计学差异(P>0.05)。结果提示A2AR和A3R不参与腺苷对Ih的抑制。(5)腺苷A1R激活后经PKC通路抑制Ih。PKC阻断剂Staurosporine(St,1.5μM)可拮抗腺苷对Ih的抑制,Ih可恢复至对照水平的95±0.05%(P>0.05)。另外,特异性更强的PKC阻断剂bisindolylmaleimide I(Bis- I,1μM)也产生与St相同的作用,Ih恢复至对照水平的91±0.04%(P>0.05)。相反,PKA阻断剂H89(1μM)不影响腺苷对Ih的抑制,抑制率为47±0.03%(P<0.05)。结果提示腺苷A1R激活后经PKC通路抑制Ih。(6)PKC-δ是介导腺苷作用的具体PKC亚型。经典PKC亚型(PKC-α,β,γ)阻断剂G?-6976(100 nM)不影响腺苷对Ih的抑制,抑制率为53±0.05%(P<0.05)。相反,特异性PKC-δ抑制剂rottleri(n10μM)可完全阻断腺苷的作用,Ih恢复至对照水平的91±0.05%(P>0.05)。另外,特异性PKC-δ转位激活肽PP114(200 nM)可显著抑制Ih,抑制率为52±0.04%(P<0.05),且其作用也可被Bis-1和rottlerin阻断。结果提示PKC-δ是介导腺苷作用的主要PKC亚型。
结论:腺苷对正常豚鼠心室肌细胞Na~+-K~+泵存在亚基特异性的调节作用,即腺苷特异性抑制高亲和力α2亚基Na~+-K~+泵电流。这一作用是经腺苷A1R和PKC通路介导,腺苷A2AR、A2BR、A3R和PKA通路不参与腺苷对Na~+-K~+泵的调节。
第二部分腺苷对缺血豚鼠心室肌细胞Na~+-K~+泵电流的调节
目的:已知心肌缺血可导致Na~+-K~+泵活性的显著降低,但在单细胞水平上鲜有文献研究缺血对Na~+-K~+泵电流的影响,因此本研究旨在详细描述缺血对心肌Na~+-K~+泵电流的抑制和电流特性的变化,以及观察腺苷处理能否拮抗心肌缺血对Na~+-K~+泵电流的抑制。
方法:(1)酶解法急性分离豚鼠心室肌细胞:同第一部分。(2)全细胞膜片钳方法记录Na~+-K~+泵电流(Ip):同第一部分。(3)使用代谢抑制剂模拟缺血状态:代谢抑制(metabolic inhibition,简称MI)是心肌缺血的主要特征,因此使用糖酵解抑制剂2-DG和线粒体解偶联剂FCCP可造成糖酵解和氧化磷酸化的联合抑制。
结果:(1)MI时间依赖性的抑制Ip。MI灌流2.5,4,6 min后,电流幅度分别被抑制了30±0.05%,49±0.02%,62±0.04% (r=0.82,P<0.05)。延长灌流时间至10 min时,MI对Ip的抑制程度并不产生进一步的增加(67%±0.02,P>0.05)。结果表明,MI对Ip的抑制呈时间依赖性,且MI灌流时间为6 min时对Ip产生最大抑制作用。(2)Antimycin A抑制Ip。为证实MI对Ip的抑制作用是代谢抑制的结果,实验选用另一种代谢抑制剂antimycin A,后者通过抑制呼吸链电子的传递过程造成代谢抑制。结果表明,10μM antimycin A也可显著抑制Ip,抑制率为68±0.02%(P<0.05)。(3)MI特异性抑制Il。MI灌流6 min后,Ih仍维持在对照水平的97±0.04%(P>0.05),提示MI对泵电流的抑制可能只涉及α1亚基Na~+-K~+泵。进而我们直接观察MI对Il的作用。结果表明,MI明显抑制Il,抑制率为60±0.03%(P<0.05),与MI对总泵电流的抑制率(62±0.04%)相比无统计学差异(P>0.05),提示MI特异性抑制α1亚基Na~+-K~+泵。(4)MI对Ip的抑制呈电压依赖性。Ip-Vm关系的测定方法同第一部分,仅以毒毛旋花子苷原strophanthidin(St,属于强心苷类)代替DHO作为Na~+-K~+泵阻断剂。结果表明,灌流MI前后V1/2数值分别是-107和-87 mV,即MI造成Ip-Vm曲线向正电压轴方向偏移了20 mV,两条曲线有统计学差异(P<0.05;two-way ANOVA)。(5)腺苷不影响MI对Ip的抑制。在10μM腺苷存在时,MI仍可对Ip产生66±0.05%(P<0.05)的抑制;与不存在腺苷时的抑制率(62±0.04%)相比无统计学差异(P>0.05)。甚至使用100μM的腺苷(临床治疗缺血性心脏疾病时的剂量)也不产生作用,提示腺苷不能阻断缺血对泵电流的抑制,即Na~+-K~+泵可能不参与腺苷对心肌缺血的保护作用。
结论:模拟心肌缺血显著抑制豚鼠心室肌细胞Na~+-K~+泵电流,呈时间依赖性。模拟心肌缺血对Na~+-K~+泵电流的抑制也具有亚基特异性,即缺血特异性抑制低亲和力α1亚基Na~+-K~+泵电流,且此作用呈电压依赖性。腺苷不影响模拟心肌缺血对泵电流的抑制,提示Na~+-K~+泵可能不参与腺苷对心肌缺血的保护作用。
第三部分心肌缺血抑制Na~+-K~+泵电流的机制研究
目的:阐明心肌缺血造成Na~+-K~+泵电流抑制的可能机制。
方法:(1)酶解法急性分离豚鼠心室肌细胞:同第一部分。(2)采用全细胞膜片钳技术测定Na~+-K~+泵电流(Ip):同第一部分。(3)使用代谢抑制剂模拟缺血状态:同第二部分。(4)细胞内pH(pHi)的检测:使用对pH敏感的双激发荧光染料carboxy-SNARF-1检测pHi。carboxy-SNARF-1负载细胞10 min后使用激光共聚焦显微镜检测荧光信号,激发波长514nm,并于580 nm和640 nm处同时检测荧光信号。pHi的变化由上述两种波长荧光强度的比值(F580/F640)所反映。580的荧光值代表细胞内已结合的H+,640的荧光值代表细胞内未结合的H+,其比值的变化可反应出pHi的变化。通过nigericin calibration technique将F580/F640比值转化为pHi。
结果:(1)心肌缺血造成的细胞内酸化可抑制Ip。心肌缺血引起细胞外环境的许多变化,如缺氧、葡萄糖耗竭及酸中毒等。但实验结果表明,若维持细胞外液pH稳定(pHo 7.4),仅单纯缺氧(灌流液充以100% N2)和耗竭葡萄糖(以等摩尔的蔗糖代替葡萄糖)时,Ip仅被抑制了7.5±0.04% (P>0.05);相反,若仅降低灌流液pH值而不改变葡萄糖和氧含量,即采用酸性灌流液(pHo 6.0,常氧,含糖)灌流心肌细胞,则可显著抑制Ip,且抑制程度与MI相似,为50±0.02%(P<0.05),提示细胞外酸中毒是造成Ip抑制的触发因素。由于细胞外酸中毒可迅速引起细胞内的酸化,且有资料报道Na~+-K~+泵对pHo不敏感,这些证据提示pHi可能是心肌缺血抑制Ip的主要介导因素。为验证这一设想,我们将电极内液中HEPES浓度增加至20 mM,从而提高HEPES对pH变化的缓冲能力。结果表明,在内液中含有高浓度HEPES的情况下,酸性灌流液(pHo 6.0)不引起明显的Ip抑制;Ip为对照水平的94±0.07%(P>0.05)。相反,通过灌流细胞酸性制剂造成细胞内酸化可模拟细胞外酸中毒对Ip的抑制。短暂灌流并洗脱NH4Cl是造成细胞内酸化的常规方法。结果表明,短暂灌流15 mM NH4Cl并洗脱后(3 min),Ip可被明显抑制,抑制率为60±0.03%(P<0.05)。同样,在内液中存在20 mM HEPES时,这一作用可被完全阻断,Ip恢复至对照水平的96±0.04%(P>0.05)。以上结果提示,细胞内酸化是心肌缺血抑制Ip的主要介导因素。(2)MI引起细胞内酸化,从而抑制Ip。在内液中存在20 mM HEPES时,MI对Ip的抑制作用被明显阻断,Ip恢复至对照水平的81±0.02%,但与内液中含有5 mM HEPES时的抑制率相比(62±0.04%)仍有统计学差异(P<0.05)。为明确pHi的作用,我们使用对pHi敏感的荧光染料在共聚焦显微镜下直接观察MI灌流前后pHi的变化。结果显示,MI灌流前pHi约为7.28±0.07,MI灌流后pHi呈时间依赖性的下降;MI灌流2.5,4,6 min时,pHi分别分别下降至7.02±0.06,6.81±0.06和6.65±0.08。以上结果提示,细胞内酸化是代谢抑制时Ip被抑制的主要介导因素。(3)氧化应激(ROS)、PKA和PKC不参与MI对Ip的抑制。电极内液中加入1 mM MPG不影响MI对Ip的抑制(68±0.05%),与内液中不含MPG的抑制率(62±0.04%)相比无统计学差异(P>0.05)。另外,特异性PKA阻断剂H89(1μM)或PKC阻断剂Bis-I(1μM)也不影响MI对Ip的抑制。
结论:模拟心肌缺血通过造成细胞内的酸化从而抑制Na~+-K~+泵电流。心肌缺血对泵电流的抑制可能不涉及缺血造成的其它细胞内变化,例如ROS,PKA和PKC的激活以及缺氧、葡萄糖耗竭等。
结论
1腺苷对正常豚鼠心室肌细胞Na~+-K~+泵存在亚基特异性的调节作用,即腺苷特异性抑制高亲和力α2亚基Na~+-K~+泵电流。这一作用是经腺苷A1R和PKC通路介导,腺苷A2AR、A2BR、A3R和PKA通路不参与腺苷对Na~+-K~+泵的调节。
2模拟心肌缺血显著抑制豚鼠心室肌细胞Na~+-K~+泵电流,呈时间依赖性。模拟心肌缺血对Na~+-K~+泵电流的抑制也具有亚基特异性,即缺血特异性抑制低亲和力α1亚基Na~+-K~+泵电流,且此作用呈电压依赖性。腺苷不影响模拟心肌缺血对泵电流的抑制,提示Na~+-K~+泵可能不参与腺苷对心肌缺血的保护作用。
3模拟心肌缺血通过造成细胞内酸化抑制Na~+-K~+泵电流。心肌缺血对泵电流的抑制可能不涉及缺血造成的其它细胞内变化,例如ROS,PKA和PKC的激活以及缺氧、葡萄糖耗竭等。
The Na~+-K~+ pump (also known as sodium pump or Na+, K+-ATPase) is a member of P-ATPase family and present in all animal cells. The Na~+-K~+ pump uses an ATP to transport 3 Na+ out of cells and 2 K+ into the cell and provides energy for several essential cellular functions (control of cell volume, pH homeostasis, reabsorption of nutrition). In excitable tissues (heart and nerve, etc), the Na~+-K~+ pump function is crucial for maintaining and establishing the normal electrical activity.
It has been demonstrated that the Na~+-K~+ pump function is subjected to regulation by a variety of hormones and biochemical changes associated with pathological conditions. For instance, catecholamines, insulin, angiotensin, and thyroid hormone mainly modulate the activity of Na~+-K~+ pump through interaction with receptors located in the plasma membrane and stimulation of downstream signaling pathways. In addition, myocardial ischemia, a leading cause of death and disability in deveoleped contries, leads to a marked inhibition of Na~+-K~+ pump. Thus, delineating detailed modulation of Na~+-K~+ pump by endogenous signaling molecules and biochemical changes is important for better understanding the exact and precise role of Na~+-K~+ pump play in various physiological and pathological conditions.
Many endogenous signaling molecules (bradykinin, adenosine, noradrenaline, etc) have been found to confer some degree of protection against myocardial ischemia. Of these, adenosine is perhaps the single most widely studied“cardioprotectnant”. Adenosine is present at low concentration and can be released as a result of metabolic stress (ischemia and hypoxia, etc). Adenosine exerts its effects through adenosine A1, A2A, A2B, and A3 receptor subtypes (A1R, A2AR, A2BR, and A3R), which are all expressed in myocardial and vascular cells, and coupled to G proteins to trigger a range of responses, with ultimate effects of limitation of cellular death and dysfunction. The nucleoside mediates tissue protection by different mechanisms including increased oxygen supply/demand, ischemic preconditioning and stimulation of angiogenesis, and also as a paracrine inhibitor of inflammation with effects in the lung, heart and brain. However, as an active transporter maintaining the essential cellular functions, it is not known whether Na~+-K~+ pump is subjected to modulation by endogenous adenosine. In addition, the possible involvement of Na~+-K~+ pump in the cardioprotection of adenosine during ischemia is also not known.
Therefore, using the whole-cell patch-clamp technique, the present study aimed to address the following questions: (1) whether there lies a possible modulatory effect of adenosine on the Na~+-K~+ pump under normal conditions; (2) whether adenosine could abolish the inhibition of Na~+-K~+ pump induced by ischemia; (3) the exact and presice mechaniam of ischemia-mediated Na~+-K~+ pump injury.
Part 1 The regulation of Na~+-K~+ pump current by adenosine in normal guinea pig ventricular myocytes
Objective: To study the effect of adenosine on Na~+-K~+ pump in ventricular myocytes acutely isolated from guinea pigs and to further elucidate the possible mechanisms involved.
Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes: guinea pig heart was digested by aorta retrograde perfusion with Collegnase NB8 (from Serva Chemical Co) using Langendorff apparatus. The temperature, pH and water quality should be properly controlled in the process of digestion. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip): the Na~+-K~+ pump exchanges 3 intracellular Na+ for 2 external K+ across the cell membrane during each active transport process. This excess positive charge movement generates a net outward current (Ip). With selected external and pipette solutions, membrane currents through K+ channel, Ca2+ channel, and Na+-Ca2+ exchanger were minimized. Under the experimental conditions, Ip was measured as the DHO-blocked current. Guinea pig ventricular myocytes coexpress two distinct Na~+-K~+ pumpαisoform,α1 andα2, which correspond to the low- and high-affinity isoforms for cardiac glycosides (dissociation constants for DHO are 72μM and 0.75μM, respectively). Thus, total Ip is the sum of current contributed by theα1-isoform (Il) plus that by theα2-isoform (Ih): Ip=Il+Ih. Based on the different affinities for cardiac glycosides (DHO at 1 mM blocked roughly 96% of total Ip, and that 5μM DHO blocked 89% of Ih while blocking only 9% of Il), we used 1 mM and 5μM DHO to identify Il and Ih, respectively. (3) Measuring of the adenosine effect on Ip: when we studied the adenosine effect on Ip, the standard protocol was to measure pump current in control and test conditions in the same cell, and then to calculate the ratio of test to control current and average this ratio from at least five cells. The detailed method is described as following: after whole-cell recording was initiated, a period of at least 5 min was required for the pipette and intracellualr solutions to come to steady state. When steady state was achieved, DHO was superfused to record the DHO-induced changes in holding current, which was reversed upon washout of DHO and was considered to reflect control Ip (Ip(Con)). Once the current had again stabilized, adenosine was applied to the myocytes. This caused a shift in holding current. After a new steady state was achieved, in the continous presence of adenosine, DHO was again applied and this inward shift reflect the changed Ip by adenosine (Ip(Ado)). Finally, Ip(Ado) was normalized to Ip(Con) to obtain the averaged ratio. This procedure uses each cell as its own control and removes the uncertainty due to cell to cell variation in size and pump density. (4) Mechanisms mediating the effect of adenosine on Ip: to clarify the cellular mechanisms responsible for the adenosine effect on Ip, specific adenosine A1R, A2AR, and A3R agonists (CCPA, CGS21680, Cl-IB-MECA, respectively), antagonists (DPCPX, SCH58261, MRS1191, respectively), PKC inhibitors(Staurosporine and bisindolylmaleimide I), and PKA inhibitor(H89)were used.
Results: (1) Adenosine specifically inhibits Ih. These results show that a physiological concentration of adenosine (1 nM) decreased Ih by 39±0.04%. The decrease in Ip was not due to pump“rundown”because the adenosine effect was reversible upon washout. Adenosine did not change Il significantly; Il remained unchanged at 93±0.05% of control values (P>0.05). Also, increasing adenosine to 10μM, adenosine had no effect on Il, suggesting that adenosine specifically inhibits Ih. (2) Adenosine concentration-dependently inhibites Ih. Perfusion of adenosine from 10-11-10-5 M induced an appreciable (8% to 47%) concentration-dependent inhibition of Ih, and the inhibition was maximal at 10-8 M. (3) The adenosine-induced inhibition of Ih is voltage independent. For measurement of the voltage dependence of Ip a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used in some experiments. The relationships were normalized to the Ih recorded at 0 mV to facilitate comparison of their slopes. The difference between these slopes was not statistically significant (P>0.05; two-way ANOVA). Thus, adenosine-induced Ih inhibition is voltage independent. (4) Adenosine binding to A1R triggers the inhibition of Ih. The specific A1R antangonist DPCPX (10 nM) abrogated the adenosine effect on Ih; Ih returned to 96±0.06% of control values (P>0.05). In a parallel study, we further tested A1R specificity by treating the cells with CCPA, a selective agonist for A1R. Perfusion of 10 nM CCPA produced a marked inhibition of Ih. Ih was inhibited by 50±0.04% in the presence of CCPA (P<0.05). Furthermore, treatment of cells with specific A2AR and A3R agonists, CGS21680 (0.2μM) and Cl-IB-MECA (0.5μM), respectively, did not affect Ih; Ih remained constant at 101±0.07% and 98±0.05% of control values (P>0.05), respectively. The specific A2AR and A3R antagonists, SCH58261 and MRS1191 (0.1μM each), did not alter the adenosine effect on Ih. Ih was decreased by 53±0.06% (P<0.05), which was not statistically different from 47±0.03% inhibition when compared to its absence (P>0.05). These data together suggest that the inhibitory effect of adenosine on Ih is mediated by adenosine A1R. (5) The activation of adenosine A1R stimulates the PKC pathway, thereby inhibiting Ih. The inhibitory effect of adenosine on Ih was abolished by PKC inhibitor staurosporine (St, 1.5μM). Current amplitudes returned to 95±0.05% of the initial control values (P>0.05). Again, 1μM Bis-I, a highly specific PKC blocker, clearly abrogated the inhibition of Ih by adenosine. The currents returned to 91±0.04% of control values after perfusion of Bis-I (P>0.05). In contrast, PKA inhibitor H89 did not affect the adenosine effect on Ih. Adenosine still induced 47±0.03% inhibition of Ih (P<0.05). Collectively, these results suggest that adenosine inhibits Ih through a PKC-dependent mechanism. (6) The activation of PKC-δinhibits Ih. Using the classical PKC isoforms (PKC-α,β,γ) inhibitor G?-6976 (100 nM), we show that the inhibitory effect of adenosine on Ih was not affected. Ih was still inhibited by 53±0.05% (P<0.05) in the presence of G?-6976. In addition, we tested the role of the novel PKC isoformδusing specific inhibitor rottlerin. Adenosine failed to inhibit Ih in the presence of 10μM rottlerin; Ih returned to 91±0.05% of control values (P>0.05), indicating that PKC-δis required for the adenosine effect on Ih. Like adenosine, PP114, a specific activator peptide of PKC-δ, also inhibited Ih. Ih was decreased by 52±0.04% on application of PP114 (P<0.05). Taken together, these results indicate that PKC-δplays a crucial role in the inhibition of Ih by adenosine.
Conclusion: Adenosine specifically inhibits theα2-isoform of Na~+-K~+ pump, but not theα1-isoform of Na~+-K~+ pump. The effect of adenosine on Na~+-K~+ pump is mediated by adenosine A1R and PKC pathway. However, the adenosine A2AR, A2BR, A3R, and PKA pathway are not involved.
Part 2 The regulation of Na~+-K~+ pump current by adenosine in ischemic guinea pig ventricular myocytes
Objective: To delineate the Na~+-K~+ pump current during myocardial ischemia and to determine whether adenosine treatment could prevent the inhibition of Na~+-K~+ pump current during myocardial ischemia.
Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip). (3) Simulation of myocardial ischemia using metabolic inhibitors: metabolic inhibition (MI) is a prominent feature of myocardial ischemia. Thus, we used metabolic inhibitors FCCP and 2-DG to produce combined inhibition of oxidative and glycolytic metabolism.
Results: (1) MI time-dependently inhibits Ip. MI reduced Ip in a time-dependent manner; its amplitudes were reduced by 30±0.05%, 49±0.02%, 62±0.04% (r=0.82, P<0.05) following 2.5, 4, and 6 min of MI, respectively. Increasing the time of MI to 10 min, however, did not produce any further reduction in Ip (67±0.02%, P>0.05), suggesting that a maximal effect was achieved at a MI time of 6 min. (2) Antimycin A inhibits Ip. To gain further evidence that the effect of FCCP and 2-DG was because of metabolic inhibition, we tested another metabolic inhibitor, antimycin A. After exposure of myocytes to 10μM antimycin A for 6 min, there was a 68±0.02% reduction in Ip (P<0.05). (3) MI specifically inhibits Il. After exposure of myocytes to MI for 6 min, Ih remained unchanged at 97±0.04% of control values (P>0.05). These results seem to suggest that only theα1-isoform of the Na~+-K~+ pump is involved in the inhibition of Ip induced by MI. We then proceeded to directly examine the effect of MI on Il. MI inhibited Il by 60±0.03% (P<0.05), which was not significantly different from 62±0.04% inhibition of total Ip produced by MI (P>0.05). Thus, MI-induced inhibition of Ip isα1-isoform specific. (4) Inhibition of Ip by MI is voltage-dependent. For measurement of the voltage dependence of Ip a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used. The relationships were normalized to the Ip recorded at 0 mV to facilitate comparison of their slopes. The difference between these slopes was statistically significant (P<0.05; two-way ANOVA). MI shifted the voltage dependence of Ip towards more positive potentials by approximately 20 mV. The V1/2 values in the absence and presence of MI were -107 and -87 mV, respectively. (5) Adenosine does not influence the inhibitory effect of MI on Ip. MI still reduced Ip by 66±0.05% (P<0.05) in the presence of 10μM adenosine and this was statistically insignificant when compared to its absence (P>0.05). Adenosine at 100μM, a therapeutic dose for ischemic myocardial injury, also did not affect MI-induced Ip inhibition. These results suggest that Na~+-K~+ pump was not involved in the cardioprotection of adenosine against myocardial ischemia.
Conclusion: Simulated myocardial ischemia markedly inhibits the Na~+-K~+ pump function in a time-dependent manner. Simulated myocardial ischemia specifically inhibits theα1-isoform of Na~+-K~+ pump, which is voltage-dependent. Adenosine does not affect the inhibition of Na~+-K~+ pump induced by myocardial ischemia, suggesting that the cardioprotection of adenosine against myocardial ischemia does not involves the Na~+-K~+ pump.
Part 3 The mechanism of myocardial ischemia-induced inhibition of Na~+-K~+ pump current
Objective:To delineate the characteristic of Na~+-K~+ pump current during myocardial ischemia and to further clarify the underlying mechanism involved.
Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip). (3) Simulation of myocardial ischemia using metabolic inhibitors. (4) Measuring of intracellular pH (pHi): The pHi of single isolated myocytes was measured using carboxy-SNARF-1, a dual-emission pH-sensitive fluoroprobe. The myocytes were loaded with the membrane-permeable acetoxy-methylester form of carboxy-SNARF-1 for 10 min and then pHi was measured using laser confocol microscope before and after MI superfusion. The fluorophore was excited by light at 514 nm, and the emitted fluorescence signals were measured simultaneously at 580 nm and 640 nm. The 580 nm/640 nm emission ratio was converted to a pHi value using the nigericin calibration technique
Results:(1) Myocardial ischemia-induced intracellular acidification inhibits Ip. No inhibition of Ip was observed when cells were exposed to the extracellular solution with anoxia (bubbling with 100% N2) and glucose deprivation but normal pHo (7.4); Ip was only decreased by 7.5±0.04% and this decrease was statistically insignificant (P>0.05). However, when treatment of myocytes with the extracellular solution containing normal oxygen and glucose but acidosis (pHo 6.0), Ip was markedly decreased by 50±0.02% (P<0.05) as under MI, demonstrating that the change in pHo itself was sufficient to inhibit Ip. Since extracellular acidosis leads to a rapid radification of the cytosol, and there is evidence suggest that Na~+-K~+ pump is not sensitive to pHo, we propose that pHi may be the key component triggering Ip inhibition during myocardial ischemia. To address this point, we increased the pHi buffering capability by elevating the concentration of HEPES in the pipette solution to 20 mM. Under these conditions, extracellular acidosis did not induce a significant inhibition of Ip; Ip returned to 94±0.07% (P>0.05) of control values. By contrast, perfusing myocytes with acidosic agent could mimic the effect of extracellular acidosis on Ip. Briefly perfusing myocytes with NH4Cl followed by washout, which is a common protocol that was used to induce an intracellular acidification. NH4Cl withdrawal drastically reduced Ip by 60±0.03% (P<0.05). Also, this inhibition was completely abolished with 20 mM HEPES in the patch pipette; Ip returned to 96±0.04% (P>0.05) of control values. (2) MI induces a rapid acidification of the cytosol, thus inhibiting Ip. With 20 mM HEPES in the pipette solution, the inhibitory effect of MI on Ip was markedly prevented; Ip return to 81±0.02% (P<0.05) of control values, which is not significantly different from that with 5 mM HEPES in the pipette solution (62±0.04%, P<0.05). We further monitored pHi with SNARF-1 under MI. It was found that resting pHi in these cells was 7.28±0.07. Perfusion of MI resulted in a sustained intracellular acidification. The mean pHi values corresponding to MI time of 2.5, 4, and 6 min were 7.02±0.06, 6.81±0.06, and 6.65±0.08, respectively. Therefore, MI leads to Ip inhibition in a pH-dependent manner in guinea pig ventricular myocytes. (3) ROS, PKA, and PKC are not involved in MI inhibition of Ip. Inclusion of 1 mM MPG in the patch pipette failed to block the MI-induced decrease in Ip and this was statistically insignificant when compared to its absence (68±0.05% for MI+MPG, P>0.05 vs. 62±0.04% for MI). In addition, the specific PKA and PKC inhibitors, H89 and Bis-I, respectively, did not significantly affect MI inhibition of Ip.
Conclusion: Simulated myocardial ischemia leads to a rapid acidification of the cytosol, thus causing the inhibition of Na~+-K~+ pump. However, oxidative stress, PKA, and PKC are less likely to be involved in the inhibition of Na~+-K~+ pump by myocardial ischemia.
SUMMARY
1 Adenosine specifically inhibits theα2-isoform of Na~+-K~+ pump, but not theα1-isoform of Na~+-K~+ pump. The effect of adenosine on Na~+-K~+ pump is mediated by adenosine A1R and PKC pathway. However, the adenosine A2AR, A2BR, A3R, and PKA pathway are not involved.
2 Simulated myocardial ischemia markedly inhibits the Na~+-K~+ pump function in a time-dependent manner. Simulated myocardial ischemia specifically inhibits theα1-isoform of Na~+-K~+ pump, which is voltage-dependent. Adenosine does not affect the inhibition of Na~+-K~+ pump induced by myocardial ischemia, suggesting that the cardioprotection of adenosine against myocardial ischemia does not involves the Na~+-K~+ pump.
3 Simulated myocardial ischemia leads to a rapid acidification of the cytosol, thus causing the inhibition of Na~+-K~+ pump. However, oxidative stress, PKA, and PKC are less likely to be involved in the inhibition of Na~+-K~+ pump by myocardial ischemia.
引文
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