Mechanism of Ca2+ increase in myoblasts derived from chicken embryos

The mechanism of intracellular calcium ions (Ca(2+)) increase in chicken myoblasts was studied using histological, immunohistochemical, immunoblotting and Ca(2+) imaging techniques. Mononuclear myoblasts at embryonic day 12 (E12) contained myofibrils in the peripheral cytoplasm, and the sarcoplasmic reticulum was observed in the cytoplasm. Several Ca(2+)-related receptors, namely acetylcholine (ACh) receptors, dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), were detected in the tissue as early as E12. Western blotting analyses detected one band corresponding to RyR subtype 3 (RyR3) at E12 and two bands corresponding to RyR1 and RyR3 after E13. Ca(2+) imaging of mononuclear myoblasts in vitro revealed an intense Ca(2+)-increase response to ACh stimulation, and this effect was abolished after EGTA addition to the culture medium. Nifedipine treatment also led to a lack of Ca(2+) increase in response to ACh stimulation, while ryanodine treatment led to a weak Ca(2+)-increase response. On the other hand, multinuclear myoblasts showed a Ca(2+)-increase response to ACh stimulation in the presence of not only EGTA but also nifedipine, although ryanodine treatment led to a lack of Ca(2+) increase. These results suggest that the mechanism of Ca(2+) increase in mononuclear myoblasts involves extracellular Ca(2+) entry through DHPRs, which is amplified by Ca(2+) release from the cytoplasmic Ca(2+) store, while multinuclear myoblasts mainly depend on Ca(2+) release from the cytoplasmic Ca(2+) store.     

A dynamic action potential model analysis of shock-inducedaftereffects in ventricular muscle by reversible breakdown of cellmembrane

To elucidate the subcellular mechanism underlying the aftereffects of high-intensity dc shocks, a small pore, which mimics reversible breakdown of the cell membrane (electroporation), was incorporated into the phase-2 Luo-Rudy (L-R) model of ventricular action potentials. The pore size was set to occupy 0.15%-4.25% of the total cell membrane during the 10-ms shock. The pore was assumed to decrease after the shock exponentially with a time constant of 100-1,400 ms to simulate resealing process. In normal myocytes, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by prolonged depolarization and oscillation of membrane potential like early afterdepolarization (EAD). Time- and voltage-dependent changes in the delayed rectifier K+ currents (IKr, IKs) in combination with those of L-type Ca2+ current (ICa,(L)) and ion flux through the pore (I(pore)) are responsible for the potential changes. Spontaneous excitation from the oscillation depends on activation of ICa,(L). In myocytes overloaded with Na+ and Ca2+ secondary to 90% inhibition of Na+-K+ pump, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by slower cyclic depolarization in response to spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). This delayed afterdepolarization-type oscillation is abolished by complete block of Ca2+ release from the SR. These findings suggest that high-intensity electric field application will cause arrhythmogenic responses through a transient rupture of sarcolemma with different subcellular events in ventricular cells under normal and pathological conditions.     

Functional reentry's influence on intracellular calcium in the LRd membrane equations

This paper examines relationships between transmembrane potential (Vm), [Ca2+]i dependent membrane ionic currents, and [Ca2+]i handling by the sarcoplasmic reticulum (SR) in a two-dimensional model of cardiac tissue. Luo-Rudy dynamic (LRd) membrane equations were used because they include detailed formulations for triggered SR Ca2+ release dependent on membrane Ca2+ influx (CICR) and for spontaneous SR Ca2+ release following calsequestrin buffer overload (SCR). Reentry's rapid rate (110-ms cycle length) elevated [Ca2+]i and limited CICR, which in turn promoted SCR that occurred at intervals of 320-350 ms, was preferential at sites located inside the functional center, and destabilized the reentrant activation sequence. Although adjustment of LRd parameters for SR Ca2+ modified SCR interval and peak [Ca2+]i in voltage clamp simulations with a command waveform representing Vm time course within the functional center, SCR persisted. Using the same command waveform, SCR also occurred with an alternate SR Ca2+ formulation that represented subcellular details underlying CICR. LRd parameter adjustments to promote CICR and limit SCR in subsequent reentry simulations failed to eliminate SCR completely, as they modulated SCR intervals in a manner consistent with the voltage clamp simulations. Taken together, our findings support a destabilizing influence of functional reentry on [Ca2+]i handling. However, [Ca2+]i instabilities did not always fractionate depolarization wavefronts during reentry. Fractionation depended, in part, upon CICR and SCR parameters in the LRd formulation for SR Ca2+ release.     

Voltage-dependent effects of Ca(2+)/calmodulin on Cl(-) channel in cardiac sarcoplasmic reticulum

A new kind of chloride channels in the cardiac sarcoplasmic reticulum, 116 pS Cl(-) channel (500 mM Cl(-) in the cis and 50 mM Cl(-) in the trans chamber solutions), which is activated by protein-kinase-A-dependent phosphorylation, has been determined to conduct adenine nucleotide as a transporter between cytosol and SR lumen. We investigated the voltage-dependent gating of this Cl(-) channel by recording single-channel activities using the planar lipid bilayer-vesicle fusion technique. The channel activities did not change at different membrane potentials (-100 mV to +50 mV) or different Ca(2+) concentrations (1 nM to 1 mM) in cis solution. In the presence of calmodulin (CaM) (0.1 microM /microg SR vesicles), however, Ca(2+) added to the cis solution at 0 mV inhibited channel openings in a Ca(2+) -concentration-dependent manner. These effects were prevented by the addition of CaM inhibitors. The blocking effects of CaM differed depending on the membrane potentials at negative potentials below -20 mV. With CaM and 3 microM Ca(2+), the values of opening probability were 0 at -80 mV, 0.2 at -40 mV, 0.3 at -20 mV, 0.71 at 0 mV and 0.92 at +20 mV. These results may indicate the membrane potential affects the action of Ca(2+) /CaM complex     

Cytochemical and electron probe X-ray microanalysis studies on the distribution change of intracellular calcium in columella cells of soybean roots under simulated microgravity

The columella cells of soybean roots grown under gravity and simulated microgravity induced by a clinostat were examined using potassium pyroantimonate (PA) and quantitative X-ray microanalysis of cryosections to determine the role of Ca in the regulation of the gravitropic response. Amyloplasts in the columella cells were localized exclusively at the bottom under gravity, but diffusely distributed in the cytoplasmic matrix under simulated microgravity, thus supporting the statolith theory. In the columella cells, PA precipitates containing Ca were diffusely distributed in the cytoplasmic matrix under gravity. Under simulated microgravity, however, they decreased in number and size in the cytoplasmic matrix, whereas increased only in number in the vacuole, indicating that Ca moved from the cytoplasmic matrix into the vacuole. The vacuole of columella cells contained mostly electron-dense granular structures localized along the inner surface of tonoplasts, which closely resembled the tannin vacuole reported in Mimosa pulvinar motor cells. Under simulated microgravity, their configuration changed dramatically from a granular shape to a flat plate. The quantitative X-ray microanalysis of cryosections showed that the vacuolar electron-dense structures contained a large amount of Ca. Under simulated microgravity, the concentration of Ca increased conspicuously in these vacuolar electron-dense structures, concomitantly with a marked decrease of K in the vacuoles and an increase of K in the cell walls. These results suggest that the release of Ca(2+) from, and uptake by, the vacuolar electron-dense structures is closely related to the signal transmission in the gravitropic response and that Ca movement occurs opposite to that of K.     

Fast XYT imaging of elementary calcium release events in muscle with multifocal multiphoton microscopy and wavelet denoising and detection

We used multifocal multiphoton microscopy to image fast, localized elevations of the cytosolic Ca2+ concentration in two spatial dimensions plus time (XYT). This technique extends the common spatially 1-D XT imaging and allows the acquisition of more than ten times longer time series (>500 images) and ten times larger areas of interest than for previously used confocal XYT imaging techniques due to lower phototoxicity and fast multifocal scanning. We recorded spontaneously occurring elementary Ca2+ release events in chemically permeabilized adult mammalian skeletal muscle fibers using two-photon excitation of the fluorescent dye Fluo-4. The resulting time series were analyzed with an automated denoising and detection algorithm based on the à trous implementation of the discrete wavelet transform. Wavelet coefficient hard-thresholding is used for denoising and event detection is performed across several wavelet scales. The spatiotemporal characteristics of the detected Ca2+ release events are followed throughout the XYT stack and are parametrized using a biophysically valid anisotropic Gaussian event model. The proposed method allows a detailed spatiotemporal analysis of elementary Ca2+ release events underlying the excitation-contraction coupling process in muscle.     

小鼠精子附睾成熟过程中Ca^2＋及Ca^2＋—ATPase的研究

利用焦锑酸钾方法和电镜酶组织化学定位方法，研究了小鼠精子在附睾成熟过程中的Ｃａ＾２＋及Ｃａ＾２＋－ＡＴＰａｓｅ变化。结果发现：附睾头中的精子内元Ｃａ＾２＋或极低，而Ｃａ＾２＋－ＡＴＰａｓｅ活性极强，可能是由于Ｃａ＾２＋－ＡＴＰａｓｅ的作用将Ｃａ＾２＋从精子内排出了精子外；在附睾体中，精子Ｃａ＾２＋－ＡＴＰａｓｅ活性降低，而Ｃａ＾２＋浓度迅速上升，并且发现Ｃａ＾２＋来自于附睾管细胞；到了附睾尾部，精     

利用膜片钳-激光扫描共聚焦显微镜同步实时系统观察心肌细胞浆内钙离子的释放

目的:采用膜片钳和激光扫描共聚焦显微镜同步实时系统观察心肌细胞钙离子的释放.方法:在体外培养的单个心室肌细胞上进行全细胞模式膜片钳技术和共聚焦显微镜钙成像技术相结合,同步实时记录钙火花和钙离子浓度变化.结果:在全细胞模式膜片钳记录心肌细胞膜钙电流的同时,激光扫描共聚焦显微镜可准确记录到胞浆内出现的钙火花.此技术有可能对于明确钙火花的特征.准确理解兴奋一收缩偶联的微观机制有重要意义.     

Characterization of Ca(2+)-inhibited potassium channels in the LNCaP human prostate cancer cell line.

Potassium plasma membrane channels have been studied in the LNCaP androgen-sensitive human prostate cancer cell line, derived from a lymph node of a subject with metastatic carcinoma of the prostate. Membrane currents were recorded by the patchclamp technique, using the cell-attached, cell-free and whole-cell mode. A voltage-dependent, non-inactivating potassium channel (delayed rectifier) was the most commonly observed ion channel in LNCaP cells. The slope conductance of K+ channels in a symmetrical 140 mM K+ gradient was 78 pS. In excised inside-out patches, the channel was inhibited by increasing the cytoplasmic Ca2+ concentration (with half-block at 0.5 microM Ca2+) over a wide range of membrane potentials. The K+ channel had a high sensitivity to tetraethylammonium (TEA), that reduced the single channel conductance with Kd of 280 +/- 27 microM. The K+ channel open probability was inhibited by alpha-dendrotoxin (DTX) (with a half-blocking concentration of approximately 5 nM) and mast cell degranulating peptide (MCDP) (with half-blocking concentration of approximately 70 nM) at all membrane potentials and with very slow reversibility. In view of the biophysical and pharmacological properties of K+ channels in LNCaP cells, it is not possible to classify these channels as one of the previously characterized types of voltage- or ligand-gated K+ channels in other cell lines.     

植物细胞钙离子检测与成像技术研究进展

钙离子是一种重要的第二信使,参与调节植物多种生理和发育过程.胞内钙离子呈现复杂的时空动态变化,只有实时捕获钙离子在植株整体水平以及亚细胞水平的变化,才能深入理解钙信号的重要功能.过去几十年中,人们在钙离子检测与成像技术方面进行了大量探索,本文总结了近年来植物科学研究中常用的钙离子检测与成像技术,着重介绍了化学荧光探针和基于荧光蛋白钙离子探针的原理、特点、浓度计算方法、在细胞质以及细胞器钙离子检测与成像中的应用等,并总结了化学荧光探针的装载方法.     

Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system?

There is growing evidence for a nitric oxide/cyclic GMP pathway of signal transduction in the vestibular system. Recently, two isoforms of nitric oxide (NO) synthase (nNOS and eNOS) and NO itself have been identified at the light microscopic level in the vestibulocochlear system of mice using specific antibodies and a new fluorescence indicator. In order to acquire more information about signal transduction and tissue modulation in this neuroepithelium at the cellular and subcellular levels, ultrathin sections of London Resin White-embedded saccule maculae of the frog Rana pipiens were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labelled secondary antibody and the amount of the immunoreactions per microm2 was quantified for the different cell types and subcellular regions. Significant eNOS immunoreactivity was identified in the hair bundles, cuticular plates and the rest of the cytoplasm of the hair cells as well as in different subcellular regions of the supporting cells. Gold-labelled anti-nNOS antibodies stained mainly stereovilli and cuticular structures of hair cells and supporting cells, whereas the number of the immunoreactions in the remaining cytoplasm of both cell types was near the background level. The spatial co-localization of the two NOS isotypes in the same cell regions of hair cells and supporting cells was confirmed in double-labelling experiments. The immunocytochemical findings are suggestive of a redundant system in which one NOS isoform can (partially) replace the other. The different subcellular localization of the NOS isoforms may allow for isoform specific regulation of NOS activity by different Ca2+ currents at the subcellular level, underlining the importance of NO-regulated processes in neuroepithelia of the inner ear.     

A preliminary model of gastrointestinal electromechanical coupling

Gastrointestinal (GI) motility is coordinated by several cooperating mechanisms, including electrical slow wave activity, the enteric nervous system (ENS), and other factors. Slow waves generated in interstitial cells of Cajal (ICC) depolarize smooth muscle cells (SMC), generating basic GI contractions. This unique electrical coupling presents an added layer of complexity to GI electromechanical models, and a current barrier to further progress is the lack of a framework for ICC-SMC-contraction coupling. In this study, an initial framework for the electromechanical coupling was developed in a 2-D model. At each solution step, the slow wave propagation was solved first and [Ca(2+)](i) in the SMC model was related to a Ca(2+)-tension-extension relationship to simulate active contraction. With identification of more GI-specific constitutive laws and material parameters, the ICC-SMC-contraction approach may underpin future GI electromechanical models of health and disease states.     

心衰心肌细胞肌浆网钙泵对兴奋收缩偶联功能的影响

目的：研究钙泵介导的钙回摄对成年大鼠心室肌细胞兴奋收缩偶联功能的影响。方法：急性分离正常组和心衰组大鼠心室肌细胞,采用激光扫描共聚焦显微镜研究肌浆网的钙容量,然后利用异丙肾上腺素（ISO,1μmol/L）激活心肌细胞β-肾上腺素受体后采用激光扫描共焦显微镜记录其对心肌细胞介导的钙释放;采用Western-blot分析心肌细胞肌浆网钙泵表达水平。结果：心衰组的钙容量显著低于对照组（n=10,P〈0.01）;ISO胞外灌流后,心衰组心肌细胞钙释放水平显著降低（n=10,P〈0.05）;心衰组钙泵表达水平低于对照组（n=6,P〈0.01）。结论：钙泵介导的钙回摄能影响心肌细胞肌浆网钙容量,并进一步影响心肌细胞兴奋收缩偶联的功能。     

Automated scanning electron microscopy and x-ray microanalysis for in situ quantification of gadolinium deposits in skin

Gadolinium (Gd) has been identified as a possible causative agent of an emerging cutaneous and systemic fibrosing disorder, nephrogenic systemic fibrosis (NSF), which can cause serious disability and even death. To date, there are only two known associations with this disorder--renal insufficiency and Gd enhanced magnetic resonance imaging (MRI). We developed an automated quantitative scanning electron microscopy (SEM) and Energy dispersive x-ray spectroscopy (EDS) method for Gd in tissue of NSF patients. Freshly cut paraffin block surfaces examined using the variable pressure mode under standardized conditions and random search of the tissue area allow in situ detection and semiquantitative morphometric (volumetric) analysis of insoluble higher atomic number features using backscattered electron imaging. We detected Gd ranging from 1 to 2270 cps/mm2 in 57 cutaneous biopsies of NSF. Gd was associated with P, Ca, and usually Na in tissue deposits. Our method reproducibly determines the elemental composition, relative concentration, and spatial distribution of detected features within the tissue. However, we cannot detect features below our spatial resolution, nor concentrations below the detection limit of our SEM/EDS system. The findings confirm transmetallation and release of toxic Gd ions in NSF and allow dose-response analysis at the histologic level.     

PKA信号通路与ryanodine受体介导的心肌肌浆网钙泄漏关系的研究

目的：研究β肾上腺素激活的PKA信号通路与成年大鼠心肌细胞RyR2介导的肌浆网Ca^2＋泄漏之间的关系,探讨治疗心力衰竭的新方法。方法：NiCl2（5mmol/L）和毒胡萝卜素（TG,100nmol/L）分别阻断成年大鼠心室肌细胞的钠钙交换体和钙泵,胞外灌流异丙肾上腺素（ISO,1μmol/L）。采用激光扫描共聚焦显微镜记录灌流前后细胞内钙火花及[Ca2＋]i的变化,并使用Ca^2＋通道阻断剂钌红（RR,5μmol/L）对β肾上腺素刺激作用进行逆转,进而阻断RyR2介导的肌浆网内钙泄漏。结果：ISO胞外灌流后,心肌细胞钙火花发生率和[Ca^2＋]i与对照组相比明显升高（n=10,P〈0.05）;加入RR后,心肌细胞内钙火花发生率与对照组比较显著降低（n=20,P〈0.05）,[Ca^2＋]i两组没有明显差别（n=20,P〉0.05）。结论：ISO激活PKA信号通路后可诱导心肌细胞RyR2介导的钙泄漏,RR可有效地逆转这种钙泄漏。     

Isradipine interacts with the open state of the L-type calcium channel at high concentrations

Triple mutation of Tyr1485, Met1486 and Ile1493 in the IVS6 segment of alpha 1C-b subunit of the L-type calcium channel results in a loss of the high affinity inhibition by isradipine. The mutant channel (Ch30) yet exhibits a concentration-dependent inhibition by isradipine with a 110-fold lower affinity. The mechanisms underlying the remaining low affinity block were investigated. Isradipine accelerated the current decay in Ch30 but not in wild type channel in a concentration dependent manner. Dependence of the current amplitude inhibition on holding potential was parallel in Ch30 and in wild type channels, while the acceleration of current decay in Ch30 was independent of the membrane potential. The recovery from voltage-dependent inactivation was biphasic in both channels and was slowed down by isradipine in the wild type but not in the Ch30 channel. The change of the charge carrier (Ba2+ or Ca2+) and calcium chelator (EGTA or BAPTA) did not affect the acceleration of current decay indicating that isradipine did not interact with the Ca(2+)-inactivated state of the channel. These results demonstrate that the mutations of Ch30 affect selectively the high affinity inhibition of an inactivated channel and unmask a low affinity interaction of isradipine with an open state of the channel.     

Different populations of inositol 1,4,5-trisphosphate receptors expressed in the bovine adrenal cortex

The inositol 1,4,5-trisphosphate (InsP3) receptor forms a tetrameric channel responsible for the release of Ca2+ from intracellular stores. In the present study we showed that the experimental approach used to separate bound and free ligands may discriminate between two populations of InsP3 binding sites in bovine adrenal cortex microsomes. A large population of low affinity sites and a small population of high affinity sites were detected with centrifugation and filtration approaches, respectively. Both populations were found in the supernatant and the cytoskeleton fractions of Triton X-100 solubilized microsomes. After treatment of microsomes with thimerosal, an alkylating reagent known to increase InsP3 receptor affinity, the filtration and the centrifugation approaches yielded identical results. With selective anti-InsP3 receptor antibodies, we showed that types 1, 2 and 3 InsP3 receptors are present in intact microsomes and in the cytoskeleton fraction. Binding studies on immunoprecipitated receptors revealed that anti-type 1 antibody recognizes a large population of low affinity sites whereas anti-type 2 antibody recognizes a small population of high affinity sites. Our results indicate that the three types of InsP3 receptors are expressed at different levels in the bovine adrenal cortex. The presence of different types of InsP3 receptors with different ligand binding affinities and their association with the cytoskeleton offer a convenient way for the cell to simultaneously regulate its intracellular Ca2+ concentration and reorganize the spatial distribution of its Ca2+ stores.     

Stimulation of isolated ventricular myocytes within an open architecture microarray

This paper is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca2+ and cell shortening, a series of microfluidic chambers (50 microm wide, 180 microm long, 400 microm high, 500 microm pitch) and connecting channels (200 microm wide, 5000 microm long, 50 microm high, 500 microm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 microm long, 20 microm wide, 0.1-10 microm thick, 180 microm apart) patterned on a microscope coverslip base, thus defining cavities of approximately 4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and > or = 4 microL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca2+ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for approximately 3 min in the 4-nL volume and approximately 20 min for the 4 microL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.     

Characterization of hollow hydroxyapatite/copper microspheres prepared from the reduction of copper-modified hydroxyapatite by glucose

This study employed a co-precipitation method to synthesize copper-modified hydroxyapatite (HA) powders, where Cu(2+) ions had entered the structure of HA and occupied Ca(1) sites in the columns parallel to the c-axis. Through a hydrothermal treatment, hollow HA/copper (Cu(2)O and/or Cu) microspheres with core-shell structures were prepared in solutions containing glucose, sodium carbonate and sodium citrate. When prolonging the reduction time, Cu(2+) ions dissolved from copper-modified HA were reduced by glucose initially to Cu(+) ions and then to Cu atoms, which would precipitate as copper on the surface of HA. The formation of microspheres with hollow structures was explained by the Kirkendall effect which states that diffusion behaviors of ions were different for HA and copper precipitations. Hybrid HA/copper powders might find their applications in gas sensors, catalysts, electrodes and so on.