Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes

Reactive oxygen species (ROS) are by-products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H(2)O(2) produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H(2)O(2) on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H(2)O(2) induced hypertrophy in control cells in a time-dependent manner. Pre-incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H(2)O(2) as compared to non-BSO pre-incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre-incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H(2)O(2), although total thiol levels were not markedly decreased. In the cells pre-incubated with BSO, hypertrophy was not observed by the addition of H(2)O(2) at any level of concentration. N-acetyl-L-cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H(2)O(2) but also induced hypertrophy in cells pre-incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H(2)O(2). On the other hand, the hypertrophied cells did not become larger by adding H(2)O(2), but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress.     

Oxygen pressure gradients in isolated cardiac myocytes

Intracellular oxygen pressure within intact isolated cardiac myocytes is studied as a function of steady state extracellular oxygen pressure. The fractional saturation of myoglobin with oxygen is used to report sarcoplasmic oxygen pressure. The fractional oxidation of cytochrome oxidase, the fractional oxidation of cytochrome c, the rate of respiratory oxygen uptake, and lactate accumulation are used to reflect the availability of oxygen at the inner mitochondrial membrane. These probes of mitochondrial function show no large change with decreasing extracellular oxygen pressure until that pressure is less than 2 torr and intracellular myoglobin is largely deoxygenated. Sarcoplasmic oxygen pressure in resting cells is nearly the same as extracellular oxygen pressure and is about 2 torr less in cells whose respiration has been increased 3.5-fold by mitochondrial uncoupling. Oxygen pressure at the mitochondrial inner membrane differs from sarcoplasmic oxygen pressure by no more than 0.2 torr and from extracellular oxygen pressure by no more than 2 torr. We conclude that differences of oxygen pressure within the cardiac myocyte are very small. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. We conclude also that mitochondria of the cardiac myocyte become oxygen limited only when sarcoplasmic myoglobin is almost entirely deoxygenated.     

Oxidative injury of isolated cardiomyocytes: dependence on free radical species

The contribution of lipid peroxidation to myocardial injury by free radicals (FR) is still unclear. Consequently, we examined the functional damages inflicted on cultured rat cardiomyocytes (CM) during FR stress provoked by the xanthine/xanthine oxidase system (X/XO) or by a hydroperoxidized fatty acid ((9 Z, 11 E, 13 (S), 15 Z)-13-hydroperoxyocta-decatrienoic acid; 13-HpOTrE), in order to simulate in vitro the initial phase and the propagation phase of the FR attack, respectively. Transmembrane potentials were recorded with glass microelectrodes and contractions were monitored photometrically. The EPR spectroscopy showed that X/XO produced superoxide and hydroxyl radicals during 10 min. The X/XO system altered sharply and irreversibly the spontaneous electrical and mechanical activities of the CM. However, the gas chromatographic analysis showed that these drastic functional damages were associated with comparatively moderate membrane PUFA degradation. Moreover, the EPR analysis did not reveal the production of lipid-derived FR. 13-HpOTrE induced a moderate and reversible decrease in electrical parameters, with no change in CM contractions. These results indicate that the functional consequences of FR attack are dependent on the radical species present and do not support the idea that the membrane lipid breakdown is a major factor of myocardial oxidant dysfunction.     

低压恒流灌流分离成年大鼠心肌细胞

目的:探寻合适的初始灌流压力与定量确定酶消化终止点,以提高恒流灌注分离成年大鼠心肌细胞的产量与质量.方法:采用Langendorff装置,行主动脉插管逆向灌流,0.08%胶原酶Ⅰ消化大鼠心脏,监测灌流压力的变化.结果:调节灌流流速以15 kPa初始压力恒流灌流时(n=4),灌流压力明显升高(压力峰值＞25 kPa),造成左心室在松弛状态下明显扩张,使分离的心肌细胞存活率降低,且收缩功能显著降低.以10 kPa初始压力行恒流灌流时,酶消化引起的压力升高均低于18.75 kPa,左心室扩张不明显;当酶消化过程中压力降至10 kPa(n=3)或5kPa(n=4)时终止消化,心肌细胞分别呈消化不足或过度的形态,且心肌细胞存活率均低于10%,恢复正常细胞外液钙离子浓度后,大部分心肌细胞死亡.当酶消化时灌流压降至7.5 kPa时终止消化(n=15),分离即刻心肌细胞存活率为82.6%±4.8%,复钙后心肌细胞存活率为30.4%±4.5%,复钙后4 h的存活率仍为24.8%±5.4%.细胞形态完好,边缘锐利、横纹清晰,无明显博动,收缩功能正常.结论:为获得存活率较高的成年大鼠心肌细胞,宜采用低压恒流灌流分离方法,即初始灌流压力保持在10 kPa,胶原酶Ⅰ消化的终止压力为7.5 kPa.     

Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis

High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.     

Oxygen partial pressure effects on metabolic rate and behavior of tethered flying locusts

Resting insects are extremely tolerant of hypoxia. However, oxygen requirements increase dramatically during flight. Does the critical atmospheric P (O)(2) (P(c)) increase strongly during flight, or does increased tracheal conductance allow even flying insects to possess large safety margins for oxygen delivery? We tested the effect of P(O)(2) on resting and flying CO(2) emission, as well as on flight behavior and vertical force production in flying locusts, Schistocerca americana. The P(c) for CO(2) emission of resting animals was less than 1 kPa, similar to prior studies. The P(c) for flight bout duration was between 10 and 21 kPa, the P(c) for vertical force production was between 3 and 5 kPa, and the P(c) for CO(2) emission was between 10 and 21 kPa. Our study suggests that the P(c) for steady-state oxygen consumption is between 10 and 21 kPa (much higher than for resting animals), and that tracheal oxygen stores allowed brief flights in 5 and 10 kPa P(O)(2) atmospheres to occur. Thus, P(c) values strongly increased during flight, consistent with the hypothesis that the excess oxygen delivery capacity observed in resting insects is substantially reduced during flight.     

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Summary Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Ca _f ) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Ca _f has been measured using the intracellular Casensitive fluorescent dyes Fura 2 and Quin. 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Ca _f was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Ca _f was 300–380 nM and, as dye content was increased, Ca _f progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na–Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Ca _f , prolonged treatment with ouabain had no effect on Ca _f despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na–Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Ca _f . However, exchange-mediated Ca efflux may play a role in Ca _f regulation when cytosolic calcium is elevated.     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

kappa-Opioid receptor stimulation increases intracellular free calcium in isolated rat ventricular myocytes.

The effect of two specific kappa-agonists, dynorphinA1-13 and U50,488H, on intracellular free calcium [Ca]i in isolated rat ventricular myocytes was studied. A spectrofluorimetric method using fura 2 as calcium indicator was employed. It was found that both agonists increased [Ca]i dose-dependently. The effect was attenuated by Mr 2266, a kappa-antagonist, indicating that the effect is a kappa-receptor mediated event. The effect was abolished by pretreatment with ryanodine, a drug that mobilizes calcium from the sarcoplasmic reticulum. It was, however, not affected by nifedipine, a calcium antagonist or removal of external calcium. The results indicate that the increase in [Ca]i due to kappa-opioid receptor stimulation results primarily from mobilization of calcium from an intracellular pool.     

Wave-like spontaneous contractions of isolated cardiomyocytes

Spontaneous wave-like contractions in single isolated rat cardiomyocytes are investigated by means of microcinematography and frame-by-frame analysis. Some basic wave patterns are described, as well as the effects of both solution components and preceding electrical stimulation. The typical wave velocity is found to be about 160 micron/s. Possible interrelation between the wave frequency and propagation velocity is discussed.     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Effect of methionine feeding on oxidative stress,intracellular calcium and contractility in cardiomyocytes isolated from male and female rats

Molecular and Cellular Biochemistry - Homocysteine (Hcy) is a breakdown product of methionine metabolism. The risk of cardiovascular disease (CVD) correlates with an increase in plasma Hcy levels....     

Use of a microfluorometric method for measuring free calcium in the cytoplasm of isolated cultured rat cardiomyocytes

Dual wavelength microfluorometry was used to measure the cytoplasmic free calcium concentration [( Ca2+]in) in single cultured cells from ventricular myocytes of neonatal rats loaded with the indicator fura-2. At 2.5 nmol/l extracellular Ca2+ in the resting cells [Ca2+]in was between 80 and 110 nmol/l. Sometimes, spontaneous low-frequency (approximately 0.1 Hz) [Ca2+]in oscillations were observed. High-potassium depolarization led to a Ca2+-antagonists-sensitive rise of [Ca2+]in. Both caffeine++ (5-10 mmol/l) and thymol (lmmol/l) initialized transient increase of [Ca2+]in. Mechanisms of [Ca2+]in homeostasis in heart muscle cells were discussed.     

Monoamine oxidase, an intracellular probe of oxygen pressure in isolated cardiac myocytes

The activity of monamine oxidase, an enzyme located almost exclusively at the outer mitochondrial membrane, toward the substrate phenylethylamine is used to report the oxygen pressure at the outer mitochondrial membrane of intact cardiac myocytes isolated from hearts of adult rats. The rate of substrate oxidation, under the conditions used, follows the Michaelis-Menten relation, and accordingly can be used as a measure of the local chemical activity of dissolved oxygen. The oxygen pressure at the outer mitochondrial membrane of myocytes, at rest and after 2- to 3-fold stimulation of respiratory oxygen consumption, differs from the extracellular oxygen pressure by at most 2 torr. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. At physiologically relevant concentrations of the substrates phenylethylamine and norepinephrine, monoamine oxidase activity is relatively insensitive to extracellular oxygen pressure in the range 155 to 8 torr, suggesting a limited role for regulation of biogenic amine oxidation by oxygen availability.     

Methodical approaches to obtaining of isolated cardiomyocytes

Isolated cardiac cells represent a unique model system for the study of many aspects of myocardial function. In this paper, several procedures for isolation ofmyocytes from adult animals and human hearts are described and discussed. They are based on successive treatments with Ca(2+)-free medium, disaggregating enzymes and/or mechanical agiatation.     

Specific limitations for intracellular diffusion of ADP in cardiomyocytes

Isolated cardiomyocytes and bundles of cardiac fibers were studied after lysis of their sarcolemma by saponin (40-50 micrograms/ml). 60-70% of cardiomyocytes were rod-like and Ca2(+)-tolerant. The kinetics of stimulation of oxidative phosphorylation by ADP and creatine via the mitochondrial creatine kinase reaction: MgATP + creatine----MgADP + phosphocreatine, was investigated after perforation of sarcolemma. The criterion for sarcolemmal perforation was an almost complete (80-100%) leakage of lactate dehydrogenase. It was shown that the Km values for ADP during stimulation of oxidative phosphorylation in cardiomyocytes are 250 +/- 39 microM (264 +/- 57 microM in cardiac bundles) which exceeds by one order of magnitude the Km value for ADP in isolated mitochondria (18 +/- 5 microM). On the contrary, Km for creatine is the same for all preparations studied (6-6.9 mM). The data obtained suggest the absence of diffusion difficulties for creatine inside the cells. In contrast, intracellular diffusion of ADP is restricted, most probably, dye to its binding to intracellular structures. These data emphasize the crucial role of the creatine kinase system in energy transfer processes. In the presence of 25 mM creatine Km for ADP is decreased to 36 +/- 6 mM due to a manyfold use of ADP in the coupled creatine kinase-oxidative phosphorylation reaction occurring in mitochondria.     

Biphasic increasing effect of angiotensin-II on intracellular free calcium in isolated rat early proximal tubule

In the freshly isolated early proximal tubule (S1), the effect of angiotensin II (ANG II) on cytosolic Ca++ concentration ([Ca++]i) was determined using the fluorescent indicator fura-2. In order to establish an adequate experimental system, we investigated firstly the relationship between cellular ATP and [Ca++]i under various conditions in late proximal tubule, the most fragile nephron segment, and cortical collecting tubule, a relatively stable one. We found out that cellular ATP depletion caused [Ca++]i to rise, and ANG II response to [Ca++]i under high ATP condition was higher than that under low ATP condition. ANG II-induced [Ca++]i rise in S1 was biphasic, demonstrating the two peaks corresponding to the 10(-11) and 10(-7) M ANG II. This study suggests for the first time 1) the necessity of high intracellular ATP to evaluate a high affinity ANG II actions and 2) the biphasic characteristics of [Ca++]i increase by ANG II in intact S1.     

Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios

Isolated rat myocytes incubated with the acetoxy methyl ester of fura 2 contained partially hydrolyzed esters, necessitating in vivo calibration of the signals obtained by fluorescence microscopy for calculation of pCa. Ionophores did not produce reliable R'max and R'min values in respiring myocytes, and elevated free calcium caused individual cells to hypercontract and burst. These difficulties were overcome by superfusion with a glucose-free buffer containing an inhibitor and an uncoupler of oxidative phosphorylation. R'max and R'min values obtained by ionophore treatment of deenergized myocytes were normalized to an in vitro calibration curve. Resting pCa derived from the individual curves averaged 6.9 for calcium-tolerant rod-shaped myocytes.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.