巴豆醛抑制小鼠心肌收缩功能

目的研究香烟烟雾中α,β-不饱和醛-巴豆醛对小鼠心肌细胞收缩功能和心肌细胞内Ca~(2+)功能的影响。方法经Langendorff装置灌注消化分离出C57BL/6小鼠心肌细胞,与不同浓度(1、10、25和50μmol/L)巴豆醛孵育6 h,对照组不经巴豆醛处理,然后分别检测心肌细胞收缩峰值(PS)、最大收缩和舒张速率(±dL/dt)、心肌细胞收缩峰值时间(TPS)、心肌细胞舒张90%时间(TR_(90))、fura-2荧光强度(FFI)、细胞内Ca~(2+)衰退和SERCA~(45)Ca~(2+)摄取,Western blot分析Na~+-Ca~(2+)交换体的表达。结果与对照组相比,较高浓度(25和50μmol/L)的巴豆醛组能明显降低心肌细胞PS、±dL/dt、ΔFFI、SERCA活性及Ca~(2+)衰退速度(P0.05),并能延长TR_(90)(P0.05);而不同浓度巴豆醛对小鼠心肌细胞Na~+-Ca~(2+)交换体的表达无明显影响。结论巴豆醛可能是通过抑制小鼠心肌细胞内SERCA活性来影响心肌细胞内Ca~(2+)功能,从而抑制心肌细胞的收缩功能。     

细胞钠／钙交换可能参与高血压的发病机制

当前不论从组织水平,细胞水平还是亚细胞水平均证实血管平滑肌(VSM)细胞膜上存在着Na/Ca交换这一胞内Ca~(2+)调节机制。它可以利用Na~+的跨膜电化学梯度(g_(Na~+)来逆向转运Ca~(2+)。视Na~(2+)与Ca~(2+)电化学梯度的不同,它既可以介导Ca~(2+)进入细胞也可以将Ca~(2+)转运出细胞。 VSM细胞Ca~(2+)代谢的异常是高血压发病的一个重要原因。那么这一异常是否与Na/Ca交换有关呢?答案似乎是肯定的。在高血压病人的红细胞,自发性高血压大鼠(SHR)的红细胞或VSM细胞中Na~+与Ca~(2+)的含量均比正常对照明显增高。后者看来与Na~+,K~+-ATPase的抑制有关,因为不少证据显示高血压病人或动物血液中一种可以抑制Na~+,K~+-ATPase的内源性洋地黄类物质(endogenous digitalis)的水平升高。用高血压患者去蛋白     

心肌α肾上腺素受体耦联的多种亚细胞过程

心肌α受体所介导的正性肌力作用至少与胞内Ca~(2+)及肌原纤维对Ca~(2+)敏感性的增加均有关。此并非通过直接耦联L型钙通道,而可能经短暂K~+外向电流Ito的抑制实现。此外,磷酸肌醇代谢、Na~(a+)-H~+交换等也有涉及。     

钙对心肌ATP酶的损害及镁的保护作用

本工作在大鼠心肌肌膜观察到,Mg~(2+)为心肌Na~+-K~+-ATP酶激活所不可缺少的因素,当Mg~(2+)浓度在0～6mM时,Na~+-K~+-ATP酶及Mg~(2+)-ATP酶活性与其密切相关;当超过8 mM时,ATP酶总活性不变,但Na~+-K~+-ATP酶/Mg~(2+)-ATP酶之比值增高。高浓度Ca~(2+)对于Na~+-K~+-ATP酶及Mg~(2+)-ATP酶均具有抑制作用,而提高Mg~(2+)浓度可部分地拮抗高Ca~(2+)的损伤作用。     

复方中药注射液对小白鼠艾氏腹水癌细胞膜表面的(Na~+-K~+)-ATP酶和微绒毛影响的电镜观察

本实验应用电镜细胞化学和扫描电镜技术,观察到小白鼠艾氏腹水癌细胞在复方中药注射液的连续作用下,膜表面(Na~+-K~+)-ATP酶活性减弱,微绒毛减退等变化,同时观察到该复方中药注射液对癌细胞增殖的抑制作用,抑瘤率可达87%,癌细胞增殖和(Na~+-K~+)-ATP酶活性及微绒毛的多少有平行关系。讨论了该复方中药抑制癌细胞增殖与膜表面(Na~+-K~+)-ATP酶活性和微绒毛变化的关系。     

钠离子和洋地黄的心脏作用

多年来,已形成一个系统的假设来解释洋地黄甙如何与Na~ /K~ -ATP转运酶相互作用而增加心肌收缩力。在讨论细胞内Na~ 的增加如何刺激强心甙与Na~ 泵的进一步结合,减少泵的储备能力量,从而增强药物作用时,扩充了这种假设。假设中有缺陷的环节仍是Na~ -Ca~(2 )交换机制的确切作用。阐明这一环节,在发展更安全的强心药物的战略中是重要的。     

内毒素对兔骨骼肌细胞外液容积、钠钾离子及其Na~(+)-K~(+)-ATP酶活性的影响

本实验以氚水和Na_2~(35)SO_4为标记物,观察了内毒素对兔骨骼肌细胞外液容积(ECV)、Na~+、K~+含量以及(Na~+-K~+)-ATP酶活性的影响。实验结果显示,动物静注内毒素(0.5mg/kg)后10小时骨骼肌ECV、Na~+、K~+含量和(Na~+-K~+)-ATP酶活性均无显著变化;但血K~+浓度自内毒素注射后3小时即有显著下降。     

Klotho蛋白减轻低氧/复氧诱导的心肌细胞系H9C2损伤

目的研究Klotho蛋白对低氧-复氧诱导的心肌H9C2细胞系损伤的保护作用及其机制。方法将H9C2心肌细胞分为对照组、低氧/复氧组(1%O_2低氧6 h, 5%CO_2以及95%空气培养箱中复氧2 h)和Klotho蛋白(10μg/mL)干预组。通过酶标仪间接测定细胞内钙离子([Ca~(2+)]_i)水平;CCK8法和TUNEL法分别检测细胞活性及细胞凋亡;分光光度法检测细胞中钠ATP酶(Na~+/K~+-ATPase,NKA)和反向模式钠/钙交换体(Na~+/Ca~(2+)-exchange,NCX)的活性。结果体外培养的H9C2细胞低氧/复氧(6 h/2 h)处理后可明显降低H9C2细胞活性(P0.05);增加细胞内Ca~(2+)水平和凋亡率(P0.05);降低细胞中Na~+/K~+-ATPase的活性(P0.05);增加反向模式Na~+/Ca~(2+)交换体的活性(P0.05);10μg/mL Klotho蛋白处理可明显减轻上述损伤。结论 Klotho蛋白可减轻低氧/复氧诱导下的H9C2细胞内钙超载,最终减少该细胞系细胞的凋亡。     

离体灌流大鼠心脏Na^＋／H^＋交换的意义

离体大鼠等容收缩心脏预灌流30min,再换用含20mmol/L NH_4Cl的K-H液灌流10min后,随机分为两组:(1)对照组(n=5): 恢复常规K-H液冲洗15min;(2)给药组(n=5):在常规K-H液冲洗时,加入0.5mmol/L amiloride以抑制Na~+/H~+交换。结果如下:预灌及NH_4Cl负荷阶段,两组血液动力学指标无明显差别;在冲洗时,对照组血液动力学指标逐渐恢复,而给药组心肌一直处于挛缩状态。对照组心肌组织Na~+、Ca~(2+)、K~+含量高于给药组;而心肌组织MDA及冠脉流出液中LDH活性则低于给药组。因此,酸负荷状态下,Na~+/H~+交换是pHi及生理活动恢复的重要调节机制,抑制这—过程,将加重心肌组织的损伤,以至于死亡。     

食盐及钙对大鼠血压和红细胞膜ATP酶活性的影响

细胞膜各种离子在血压调节中的作用早被人们重视。有关红细胞膜Na~+、K~+、Ca~(2+)和Mg~(2+)等许多重要离子转运与高血压的关系,已成为80年代高血压研究的最新进展之一。     

Hypoxia delays the intracellular Ca2+ clearance by Na+-Ca2+ exchanger in human adult cardiac myocytes.

Transient myocardial ischemia during cardiac surgery causes a loss of energy sources, contractile depression, and accumulation of metabolites and H+ ion resulting in intracellular acidosis. The reperfusion following ischemic cardioplegia recovers intracellular pH, activates Na+-H+ exchange and Na+-Ca2+ exchange transports and consequently produces Ca2+ overload, which yields cell death. Among the various Ca2+ entry pathways, the Na+-Ca2+ exchanger is known to play one of the major roles during the ischemia/reperfusion of cardioplegia. Consequently, information on the changes in intracellular Ca2+ activities of human cardiac myocytes via the Na+-Ca2+ exchanger is imperative despite previous measurements of Ca2+ current of human single myocytes. In this study, human single myocytes were isolated from the cardiac tissues obtained during open-heart surgery and intracellular Ca2+ activity was measured with cellular imaging techniques employing fluorescent dyes. We report that the Na+-Ca2+ exchanger of adult cardiac myocytes is more susceptible to hypoxic insult than that of young patients.     

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by proteinase pretreatment

The Na+-Ca2+ exchange activity of purified canine cardiac sarcolemmal vesicles can be strikingly stimulated if the vesicles are pretreated with a serine or thiol proteinase. The Km (Ca2+) for Na+i-dependent Ca2+ influx is reduced from 22.2 +/- 2.3 to 8.1 +/- 0.3 microM while Vmax is increased from 15.1 +/- 3.6 to 18.9 +/- 5.2 nmol Ca2+ . mg protein-1 . s-1. Na+o-dependent Ca2+ efflux is also stimulated by proteinase pretreatment although passive (Na+-independent) Ca2+ efflux from the sarcolemmal vesicles is unaffected. Proteinase treatment reduces the sensitivity of Na+-Ca2+ exchange to the inhibitors chlorpromazine and polymyxin B, but not to the inhibitor, palmitylcarnitine. Using a newly developed technique we are able to demonstrate that the Na+-Ca2+ exchange of inside-out sarcolemmal vesicles is being stimulated by proteinase treatment (Philipson, K. D., and A. Y. Nishimoto, J. Biol. Chem: 257, 5111-5117, 1982; this technique uses the ATP-dependent Na+ pump to preload only inside-out vesicles with Na+ prior to Na+-Ca2+ exchange). Right-side-out vesicles may also be stimulated.     

Na(+)-Ca2+ exchange transport and pacemaker activity of the rabbit SA node.

Recent electrophysiological data have provided the evidences that background currents such as Na(+)-Ca2+ exchange can significantly modulate cardiac pacemaker activity. In this study, the effects of extracellular Na+ and Ca2+ concentrations on the pacemaker activity were investigated by measuring the intracellular Na+ activity (aiNa) with Na(+)-selective microelectrodes and the results are summarized as follows. 1) In the rabbit SA node, aiNa was 3.2 +/- 0.3 mM and mean MDP (maximal diastolic potential) was -63.3 +/- 1.4 mV. 2) Graded decreases of external Na+ concentration resulted in the loss of spontaneous beating, hyperpolarization and the decrease of aiNa. 3) An increase in extracellular Ca2+ concentration in low Na+ solution augmented the transient decrease of aiNa, about 3 minutes in low Na+ solution, until aiNa started to increase. 4) In low Na+ solution, which had extracellular Ca2+ concentration according to the calculation based on the equilibrium state of Na(+)-Ca2+ exchange, aiNa was continuously decreased. It was concluded that intracellular Na+ activity modulated by Na(+)-Ca2+ exchange could play an important role in the initiation of pacemaker potential.     

Na+-Ca2+ exchange induces low Na+contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca2+-ATPase

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.     

The effect of [Na+]i and [Na+]o on Ca2+ mobilization in the rat aortic smooth muscle cell line A7r5.

Measurements of intracellular calcium (Ca2+i) and sodium (Na+i) have been made in single smooth muscle cells in confluent cultures of the A7r5 cell line using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and sodium-binding benzofuran isophthalate (SBFI). Reversal of the Na+ gradient in control cells results in a small increase in Ca2+i and slows the rate of recovery in Ca2+i following agonist stimulation. This suggests that a Na(+)-Ca2+ exchange mechanism may be functioning in these cells. In ouabain-pretreated cells, Na+i is elevated and the recovery from agonist stimulation is significantly slowed. This suggests that the elevation of Na+i alters Ca2+ homeostasis. Reversal of the Na+ gradient in ouabain-pretreated cells results in a transient increase in Ca2+i which was larger than in control cells. This response is reduced during a second or third exposure to zero Na+o.NA+i, in Na(+)-loaded cells, falls in the absence of external Na+. This fall is slowed in the absence of external Ca2+ supporting the idea that the Na+ loss is via Na(+)-Ca2+ exchange. The possible modulation of the Na(+)-Ca2+ exchanger and Ca2+ mobilization by internal Na+ is discussed.     

Effect of cardiotoxic concentrations of catecholamines on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles

The mechanism by which the administration of large doses of catecholamines produces myocardial necrosis in experimental animals and humans has not yet been ascertained. One of the consequences of such administration is the accumulation of high concentrations of Ca in the heart and it has therefore been suggested that this is the factor that leads to cell injury. The elevated intracellular Ca appears to be due in part to enhanced Ca influx resulting from catecholamine-induced opening of membrane Ca channels and in part to increased membrane permeability caused by membrane damage. It is not clear, however, why the myocardial cells are unable to extrude their extra load of Ca, at least initially, so as to maintain normal Ca concentrations. The effects of high concentrations of epinephrine and isoproterenol on Na+-Ca2+ exchange transport in isolated sarcolemmal vesicles prepared from hearts of Sprague-Dawley rats were determined. It was found that catecholamine concentrations of 10(-4) to 10(-2) M inhibited the exchange in a dose-related manner while choline, in the same concentrations, had no effect. It is therefore possible that cardiotoxic concentrations of catecholamines also interfere with Na+-Ca2+ exchange transport across the myocardial sarcolemma in vivo and thereby inhibit the efflux of intracellular Ca.     

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.     

Evidence for Na(+)-Ca2+ exchange and Ca(2+)-induced Ca2+ release in a cultured vascular smooth muscle cell line from the rat.

Measurements of intracellular calcium (Cai2+) and sodium (Nai+) have been made in single smooth muscle cells from the rat aortic cell line (A10) using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Cai2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Cai2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na(+)-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca(2+)-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Cai2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca(2+)-induced Ca(2+)-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na(+)-Ca2+ exchanger operating in reverse. The possible modulation of the Na(+)-Ca2+ exchanger and Ca(2+)-induced Ca2+ release by internal Na+ is discussed.     

Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine-induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo-1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half-time, t 1/2 approximately 100-160 ms). During a caffeine-induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)-Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)-Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5-fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)-Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)-ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine-induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20-30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)-ATPase and Na(+)-Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)-ATPase, Na(+)-Ca2+ exchange, sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans-sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)-ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)-Ca2+ exchange in rat ventricular myocytes.     

Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)