NMR studies of intracellular sodium ions in amphibian oocytes, ovulated eggs, and early embryos

23Na NMR, in combination with an anionic paramagnetic shift reagent dysprosium bis(tripolyphosphate), has been used to study intracellular Na+ in Rana oocytes, ovulated eggs, and early cleavage embryos. The technique allows accurate and simultaneous determination of both extracellular space and intracellular Na+ concentration. In prophase-arrested, follicle-enclosed oocytes, only about 17% of the total oocyte Na+ (approximately 40 mmol/kg of cells) was NMR-visible. Homogenizing oocytes in 0.24 M sucrose did not significantly affect the 23Na resonance. About 30% of the total oocyte Na+ was associated with the yolk platelets isolated at room temperature by differential centrifugation. NMR analysis, however, did not yield a detectable 23Na signal from these intact platelets. Thus, while yolk platelets are rich in Na+, this Na+ does not contribute to the oocyte 23Na NMR signal. Denuded oocytes, obtained by removing the follicular epithelium, gained about 10 mmol of total Na+/kg of cells and exhibited a comparable increase in NMR-visible Na+, suggesting the existence of compartments with varying degree of NMR visibility within the oocyte. Partially relaxed 23Na Fourier transform NMR spectra revealed the existence of at least two major intracellular compartments of NMR-visible Na+ with different magnetic environments and relaxation behavior in denuded oocytes. Since platelet Na+ appears to be NMR-invisible, one of the two observed compartments may be the nucleus. Progesterone action on the amphibian oocyte caused measurable changes in NMR-visible Na+. By ovulation (second metaphase), there is a gain in total egg Na+, and the NMR-visible Na+ is also increased. Following fertilization, however, there is some loss of total cell Na+ but, by the 2-4 cell stage, about 70% of the total Na+ becomes NMR-visible. These results indicate that a sizable fraction of the Na+ in follicle-enclosed, prophase oocyte is sequestered and located in NMR-invisible compartments and that changes in NMR-visible intracellular Na+ occur following hormonal and developmental stimuli.     

23Na NMR study of intracellular sodium ions in Dictyostelium discoideum amoeba

The intracellular sodium concentration in the amoebae from the slime mold Dictyostelium discoideum has been studied using 23Na NMR. The 23Na resonances from intracellular and extracellular compartments could be observed separately in the presence of the anionic shift reagent Dy(PPPi)7-2 which does not enter into the amoebae and thus selectively affects Na+ in the extracellular space. 31P NMR was used to control the absence of cellular toxicity of the shift reagent. The intracellular Na+ content was calculated by comparison of the intensities of the two distinct peaks arising from the intra- and extracellular spaces. It remained low (0.6 to 3 mM) in the presence of external Na+ (20 to 70 mM), and a large Na+ gradient (20- to 40-fold) was maintained. A rapid reloading of cells previously depleted of Na+ was readily measured by 23Na NMR. Nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular Na+ concentration. The time dependence of the 23Na and 31P NMR spectra showed a rapid degradation of Dy(PPPi)7-2 which may be catalyzed by an acid phosphatase.     

Competitive blockage of the sodium channel by intracellular magnesium ions in central mammalian neurones

The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mg _i ²⁺ , interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mg _i ²⁺ . This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mg _i ²⁺ leads to a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mg _i ²⁺ is decreased upon increasing intracellular or extracellular Na⁺ concentration, which suggests that Mg?' interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mg _i ²⁺ for the sodium channel has been calculated to be 8.65 ± 1.51 mM, with its binding site located at 0.26 ± 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Na _i ⁺, which is 83.76 ± 7.60 mM with its binding site located at 0.75 ± 0.23. The low dissociation constant of Mg _i ²⁺ reflects its high affinity for the sodium channel.     

Criticality in intracellular calcium signaling in cardiac myocytes

Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.     

Free and bound intracellular calmodulin measurements in cardiac myocytes

Calmodulin (CaM) is a ubiquitous Ca2+ binding protein and Ca2+-CaM activates many cellular targets and functions. While much of CaM is thought to be protein bound, quantitative data in cardiac myocytes is lacking regarding CaM location, [CaM]free and CaM redistribution during changes in [Ca2+]i. Here, we demonstrated that in adult rabbit cardiac myocytes, CaM is highly concentrated at Z-lines (confirmed by Di-8-ANEPPS staining of transverse tubules) using three different approaches: immunocytochemistry (endogenous CaM), Alexa Fluor 488 conjugate CaM (F-CaM) in both permeabilized cells (exogenous CaM) and in patch clamped intact cells (via pipette dialysis). Using 100 nM [CaM]free we washed F-CaM into permeabilized myocytes and saw a two-phase (fast and slow) CaM binding curve with a plateau after 40 min of F-CaM wash-in. We also measured myocyte [CaM]free using two modified null-point titration methods, finding [CaM]free to be 50-75 nM (which is only 1% of total [CaM]). Higher [Ca2+]i increased CaM binding especially in the nucleus and at Z-lines and significantly slowed F-CaM dissociation rate when F-CaM was washed out of permeabilized myocytes. Additionally, in both permeabilized and intact myocytes, CaM moved into the nucleus when [Ca2+]i was elevated, and this was reversible. We conclude that [CaM]free is very low in myocytes even at resting [Ca2+]i, indicating intense competition of CaM targets for free CaM. Bound CaM is relatively concentrated at Z-lines at rest but translocates significantly to the nucleus upon elevation of [Ca2+]i, which may influence activation of different targets and cellular functions.     

心肌细胞发育过程中胞浆内钙稳态的调控

Ca^2＋信号是细胞和各器官生长发育、行使其生理功能的基础,维持心肌细胞的钙稳态是保持正常心脏功能的先决条件。作为在胚胎发育过程中最早出现并行使功能的器官,胚胎期心脏的形态结构发生了明显的变化,泵血功能不断增强,以适应不断增强的机体的生理需求。从胚胎到成年,心肌细胞的功能有非常大的改变,各钙离子通道的表达也发生明显变化。因此,发育早期心肌细胞的钙稳态调控与成熟心肌细胞有明显的不同,在发育过程中引起细胞收缩的Ca^2＋来源也有明显的变化。随着分子和细胞生物学研究的发展,以及胚胎干细胞体外分化模型的应用,人们对心肌细胞发育过程中钙稳态的调控有了进一步的认识。本文综述了早期心肌细胞发育过程中胞浆内钙稳态的变化,总结了早期心肌细胞钙稳态调控机制的最新研究进展。     

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.     

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.     

Experimental generation and computational modeling of intracellular pH gradients in cardiac myocytes

It is often assumed that pH(i) is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO(2)) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH(i) gradient (up to 1 pH(i) unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH(i). The model assumes that H(i)(+) movement occurs via diffusive shuttling on mobile buffers, with little free H(+) diffusion. The average diffusion constant for mobile buffer was estimated as 33 x 10(-7) cm(2)/s, consistent with an apparent H(i)(+) diffusion coefficient, D(H)(app), of 14.4 x 10(-7) cm(2)/s (at pH(i) 7.07), a value two orders of magnitude lower than for H(+) ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H(i)(+) mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH(i) nonuniformity. Similar concentration gradients for weak acid (e.g., CO(2)) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH(i) gradients within the heart under some pathophysiological conditions.     

牛磺酸对大鼠心肌细胞内钙浓度的影响

牛磺酸 (Ｔａｕｒｉｎｅ ,Ｔａｕ)是可兴奋组织中含量最为丰富的游离氨基酸 ,是细胞自稳态的重要调节物质。在多种心血管疾病的临床与实验研究中具有明显的细胞保护作用。其作用机制与调节心肌细胞的钙浓度有关。用同位素示踪技术已证实Ｔａｕ在细胞内“高钙”状态下能抑制钙的跨膜内流。本文采用Ｆｕｒａ 2荧光技术测定Ｔａｕ对成年大鼠分离心肌细胞在静息、高钾去极化以及缺氧 /复氧条件下游离 [Ｃａ ]ｉ,旨在进一步探讨Ｔａｕ的作用机制。1　材料与方法(1)动物实验　雄性Ｗｉｓｔａｒ大鼠 (军事医学科学院四所提供 )。 2 0 %乌拉坦ｉｐ…     

Gene expression studies on bio-electrosprayed primary cardiac myocytes

Cardiovascular pathology accounts for the greatest number of mortalities in the western world and thus the development of ex vivo cardiac tissue has vast potential in cardiac therapy. Bio-electrosprays (BES), a recently discovered direct cell engineering protocol, has demonstrated tremendous applicability for regenerative and therapeutic medicine. For bio-electrospraying to be carried forward as a novel method of cardiac tissue engineering, it is important that the process does not adversely affect cellular physiology. Our previous work has shown that bio-electrospraying does not induce cell death, activate intracellular stress pathways or induce DNA damage in primary cardiac myocytes. Here we show for the first time using genome-wide microarray analysis, that bio-electrospraying has no negative effects on global gene expression in cardiac myocytes. Moreover, we show that bio-electrospraying does not lead to endothelial cell activation. These data suggest that BES has minimal effect upon the physiology of cardiac myocytes and endothelial cells and thus paves the way for the development of BES in cardiac tissue engineering.     

High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes

The prevailing paradigm is that cardiac ANG II is synthesized in the extracellular space from components of the circulating and/or local renin-angiotensin system. The recent discovery of intracrine effects of ANG II led us to determine whether ANG II is synthesized intracellularly in neonatal rat ventricular myocytes (NRVM). NRVM, incubated in serum-free medium, were exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent angiotensin type 1 (AT(1)) receptor-mediated internalization of ANG II. ANG II was measured in cell lysates and the culture medium, which represented intra- and extracellularly synthesized ANG II, respectively. Isoproterenol increased ANG II concentration in cell lysates and medium of NRVM in the absence or presence of candesartan. High glucose markedly increased ANG II synthesis only in cell lysates in the absence and presence of candesartan. Western analysis showed increased intracellular levels of angiotensinogen, renin, and chymase in high-glucose-exposed cells. Confocal immunofluorocytometry confirmed the presence of ANG II in the cytoplasm and nucleus of high-glucose-exposed NRVM and along the actin filaments in isoproterenol-exposed cells. ANG II synthesis was dependent on renin and chymase in high-glucose-exposed cells and on renin and angiotensin-converting enzyme in isoproterenol-exposed cells. In summary, the site of ANG II synthesis, intracellular localization, and the synthetic pathway in NRVM are stimulus dependent. Significantly, NRVM synthesized and retained ANG II intracellularly, which redistributed to the nucleus under high-glucose conditions, suggesting a role for an intracrine mechanism in diabetic conditions.     

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.     

Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system

This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。