Effect of the ionic strength and prostaglandin E2 on the free Ca2+ concentration and the Ca2+ influx in human red blood cells

Human red blood cells (RBCs) were loaded with the Ca(2+)-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca(2+)-containing physiological (high) ionic strength (HIS) solution was 75.1 +/- 8.3 nM after 5 min incubation, increasing to 114.9 +/- 9.6 nM after 1 h. In Ca(2+)-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca(2+)-containing HIS solution (p = 0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca2+]i was seen after 1 h. In Ca(2+)-free (0 Ca2+ plus 15 microM EGTA) media, [Ca2+]i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10-45 min), both in HIS and LIS media, decreasing from 20.3 +/- 1.9 to 12.9 +/- 1.3 micromol/(lcells x h) in HIS, and from 36.7 +/- 5.3 to 8.6 +/- 1.2 micromol/(lcells x h) in LIS. Prostaglandin E2 (PGE2; 10(-7)-10(-11) M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca(2+)-containing HIS medium. Finally, the addition of carbachol (100 microM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca(2+)-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.     

Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 microm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 microm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 mirom id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (1/2 P(max)) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach 1/2 P(max) (6.9 +/- 0.2 s) than deformable RBCs (21.6 +/- 0.8 s). To verify that the increased amount of time to reach 1/2 P(max) is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher 1/2 P(max) value (26.0 s +/- 2.2 s) when compared to a 7% hematocrit (19.1 +/- 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.     

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.     

Red Blood Cell Stiffness and Adhesion Are Species-Specific Properties Strongly Affected by Temperature and Medium Changes in Single Cell Force Spectroscopy

Besides human red blood cells (RBC), a standard model used in AFM-single cell force spectroscopy (SCFS), little is known about apparent Young’s modulus (Ea) or adhesion of animal RBCs displaying distinct cellular features. To close this knowledge gap, we probed chicken, horse, camel, and human fetal RBCs and compared data with human adults serving as a repository for future studies. Additionally, we assessed how measurements are affected under physiological conditions (species-specific temperature in autologous plasma vs. 25 °C in aqueous NaCl solution). In all RBC types, Ea decreased with increasing temperature irrespective of the suspension medium. In mammalian RBCs, adhesion increased with elevated temperatures and scaled with reported membrane sialic acid concentrations. In chicken only adhesion decreased with higher temperature, which we attribute to the lower AE-1 concentration allowing more membrane undulations. Ea decreased further in plasma at every test temperature, and adhesion was completely abolished, pointing to functional cell enlargement by adsorption of plasma components. This halo elevated RBC size by several hundreds of nanometers, blunted the thermal input, and will affect the coupling of RBCs with the flowing plasma. The study evidences the presence of a RBC surface layer and discusses the tremendous effects when RBCs are probed at physiological conditions.     

Albumin-mediated selenium transfer by a selenotrisulfide relay mechanism

In this study, we demonstrated a human serum albumin (HSA)-mediated selenium transfer; the selenium exported from red blood cells (RBCs) was bound to HSA through the selenotrisulfide and then transferred into the hepatocyte. After the treatment of the RBCs with selenite, the selenium efflux from the RBCs occurred in an HSA concentration-dependent manner. Pretreatment of HSA with iodoacetamide almost completely inhibited the selenium efflux from the RBC to the HSA solution. The selenium efflux experiment was carried out in an HSA solution (45 mg/mL), and subsequently the HSA solution was subjected to gel permeation chromatographic separation. The peak fraction of the selenium content was consistent with that of the HSA. The selenium bound to HSA in this solution was completely eliminated by a treatment with penicillamine (Pen), which resulted in the generation of penicillamine selenotrisulfide, PenSSeSPen. The selenium efflux from the RBCs was also occurred in a Pen solution, and PenSSeSPen was observed in the resulting Pen solution. The selenium exported from the RBC was thought to bind to the HSA via a selenotrisulfide linkage with its single free thiol. A model of the selenium-bound HSA was prepared by the reaction of the HSA with PenSSeSPen. The selenium from PenSSeSPen can bind to HSA by a thiol exchange between Pen and the free thiol of HSA, which produces the selenotrisulfide-containing HSA (HSA-SSeSPen). When HSA-SSeSPen was incubated with isolated rat hepatocytes, the selenium content in the hepatocytes increased along with its decrease in the incubation medium. To verify the results from the model experiments using HSA-SSeSPen, we conducted the HSA-mediated selenium transfer experiment from RBC treated with selenite to the hepatocytes. The selenotrisulfide-containing HSA was able to transport the selenium into the hepatocyte. Overall, the selenium transfer from the RBC to the hepatocytes involves a relay mechanism of thiol exchange that occurs between the selenotrisulfide and thiol compounds (selenotrisulfide relay mechanism: R-SSeS-R + HSA-SH --> HSA-SSeS-R + R'-SH --> R-SSeS-R').     

Inhibition of superoxide generation and of increase in intracellular Ca2+ concentration by zinc in rat neutrophils stimulated with zymosan

The effect of Zn2+ on the O2- generation and change in intracellular Ca2+ concentration ([Ca2+]i) of rat peritoneal neutrophils was studied. Zymosan (serum-treated zymosan (STZ))-induced O2- generation was inhibited by Zn2+ at concentrations as low as 10 microM. A large amount of the inhibition was observed in the absence of extracellular Ca2+ but the inhibition could not be restored by increasing the extracellular Ca2+ concentration, indicating that Zn2+ does not necessarily inhibit the O2- generation competitively with extracellular Ca2+. In the absence of extracellular Ca2+, Zn2+ inhibited STZ-induced transient increase in [Ca2+]i in the concentration range that evoked a marked inhibition in the O2- generation. On the other hand, Zn2+ did not inhibit significantly STZ-induced uptake of 45Ca2+ from extracellular medium by the cells. From these results, it is suggested that Zn2+ inhibits STZ-induced release of Ca2+ from intracellular storage sites, resulting in the suppression of the activation mechanism of neutrophils.     

Calcium channels, potassium channels and membrane potential of smooth muscle cells of human allantochorial placental vessels.

The membrane potential (Um), the main factor of the excitation-contraction coupling, of human allantochorial placental vascular smooth muscle cells (VSMCs) has been previously shown to depend on voltage-sensitive K+ channels. These channels were blocked by high external K+. To characterize other channels which regulated Um, various constrictor or/and vasodilators and channel blockers were used. Serotonin depolarized VSMCs, in normal medium, but induced a more marked depolarization in VSMCs predepolarized by high external K+. This depolarization was inhibited by nifedipine, a blocker of voltage-gated Ca2+ channels. Acetylcholine, sodium nitroprusside (without effect on Um in normal medium), hyperpolarized the predepolarized-high K+ medium VSMCs. This hyperpolarization was inhibited after addition of charybotoxin (a blocker of Ca2+-activated K+ channels) or/and glibenclamide (a blocker of ATP-sensitive K+ channels). A similar effect was obtained with isoproterenol. These results indicated that membrane potential of human placental allantochorial VSMCs was regulated by voltage-gated, Ca2+- and ATP-sensitive K+ channels and by voltage-dependent Ca2+ channels.     

Monitoring erythrocytes in a microchip channel that narrows uniformly: towards an improved microfluidic-based mimic of the microcirculation

The release of adenosine triphosphate (ATP) from red blood cells (RBCs) flowing through PDMS microchannels has been determined as a function of channel cross-sectional area using a design containing a channel that narrows uniformly. ATP, released from the RBCs in response to the mechanical deformation of their cell membranes, increased as the channel cross-section decreased. One sample of rabbit RBCs released 1.16 +/- 0.11, 1.92 +/- 0.14 and 2.09 +/- 0.10 microM ATP as the median cross-sectional area decreased from 4314 to 3192 to 2052 microm(2), respectively. Numerous samples (n = 6) displayed the same trend. Incubating a sample of RBCs with diamide, a substance known to stiffen cell membranes without harming the cell cytosol, provided evidence that no cell lysis occurred in the microchip device. This novel use of lab-on-a-chip technology allows for channel designs that enable an in vitro study of physiological events that occur in the microcirculation.     

Single-cell imaging of the Ca2+ influx into bovine endothelial cells occurring in response to an alternating electric stimulus.

The electric control of cellular functions via Ca2+ was formerly suggested. From this viewpoint, the involvement of a Ca2+ channel was studied using bovine fetal arterial endothelial (BFAE) cells in which P2X4, an ATP-operated and fluid shear stress sensitive Ca2+ channel, exists predominantly. An electric stimulus (sine wave, 10 Hz, 10 VPP, 30 s) caused a marked influx of Ca2+ into BFAE cells from an extracellular solution. The magnitude of the [Ca2+]i change increased with a decrease in the frequency in the range from 100 Hz to 5 Hz. Regarding the pathway of this Ca2+ influx, single-cell imaging and an ATP depletion experiment strongly suggested the involvement of a pathway different from P2X4. This pathway was thought to be a non-specific one, because typical Ca2+ channel blockers, such as verapamil, Gd3+, and Co2+, could not inhibit the Ca2+ influx.     

Estimation of electrophoretic mobilities of red blood cells in 1-G and microgravity using a miniature capillary electrophoresis unit

Electrophoretic mobilities of red blood cells (RBCs) were measured in microgravity using a home-made capillary electrophoresis unit, which consisted of two small reservoirs of 0.6 mL and a fused-silica capillary tubing with 2 cm in length and 50 num in inner diameter. Migration of RBCs was observed by a microscope at 1000 times magnification and recorded on a videotape. The experiments were performed during stays in microgravity (about 0.01 G), which lasted 20 s and were attained by parabolic flights of an aircraft. On average, the electrophoretic mobilities of RBCs determined in microgravity were about 30% higher than those measured at 1-G condition irrespectively whether the cells were suspended in saline or serum during measurements. This difference might be explained as being mainly due to the cell floating in microgravity. Morphological changes of RBCs may contribute partly to the difference, while the variation in viscosity of the medium under microgravity could play only a minor role.     

Na~+/Ca~(2+) Exchange-mediated La~(3+) Entry in Human Lymphocytes

With extensive applications the rare earth elements to agriculture, medicine and animal husbandry, whether rare earth ions can cross cell membranes and participate in the metabolism of animals and plants is not clear until now. Otherwise, rare earth ions have different interaction fashions to different kinds of cells, therefore, in this study we determined whether La3+ enter human peripheral blood lymphocytes via Na+/Ca2+ exchanger (measured with fura-2).Relative sensitivity of fura-2 to Ca2+…     

在线预处理对乙二胺四乙酸二钠离子色谱分离的影响及其应用

1引言乙二胺四乙酸二钠(EDTA-2Na)作为食品添加剂,能够对原料中的微量金属离子进行络和,防止金属离子对产品质量的缓慢破坏作用[1],常用在果蔬罐头、八宝粥等食品中,同时也可作为蛋白洗涤液中的添加剂,既增加舒适度又提高溶液杀菌能力。但是含量过高,会刺激皮肤和粘膜,引起哮喘等疾病,国家标准规定食品中最高限量为     

脑梗塞后血清常量元素钙镁含量的动态研究

为探讨急性脑梗塞后血清钙镁含量变化及其对预防和治疗的临床意义,随机选取３２例患者采用不同时期晨起空腹采血,应用ＭＴＢ法６４００－Ａ型临床电解质分析仪测定血清中Ｃａ＾２＋和Ｍｇ＾２＋的含量。结果表明,病例组发病１周时血清Ｃａ＾２＋和Ｍｇ＾２＋含量均低于对照组（Ｐ〈０．０１）,发病后２４ｈ血清Ｃａ＾２＋,Ｍｇ＾２＋含量逐渐降低,到发病１周后血清Ｃａ＾２＋、Ｍｇ＾２＋含量降低最明显,２周后血清Ｃａ＾２＋     

Induction of intracellular Ca2+ and pH changes in Sf9 insect cells by rhodojaponin-III, a natural botanic insecticide isolated from Rhododendron molle

Many studies on intracellular calcium ([Ca2+](i)) and intracellular pH (pH(i)) have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III) on [Ca2+](i) and pH(i) and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+](i)) and intracellular pH (pH(i)) in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL) increase in [Ca2+](i) and pH(i) of Sf9 cells in presence of Ca2+-containing solution (Hanks) and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+](i), because completely treating Sf9 cells with CdCl(2) (5 mM), a Ca2+ channels blocker, R-III (100 μg/mL) induced a transient elevation of [Ca2+](i) in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pH(i) showed similar changes with that of [Ca2+](i) on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+](i), cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.     

Bestimmung der Oberflächenzusammensetzung von Calcit in Mg2+-haltigen Ca-45-markierten Lösungen

The Mg²⁺ ion content in the surface layer of calcite in relation to the Mg²⁺ ion content in Ca-45 labelled solution was determined by Ca²⁺-Mg²⁺ ion exchange process. It was found that the increase of the molar ratio Mg/Ca in the surface due to increase of Mg²⁺ concentration in solution is decreasing significantly when approaching ratios of Mg/Ca ～ 1 and ～ 3. The experimental exchange isotherm can be used to relate the Mg/Ca molar ratio of solutions to that of the surface layers of calcite.     

Lateral-driven continuous dielectrophoretic microseparators for blood cells suspended in a highly conductive medium

This paper presents lateral-driven continuous dielectrophoretic (DEP) microseparators for separating red and white blood cells suspended in highly conductive dilute whole blood. The continuous microseparators enable the separation of blood cells based on the lateral DEP force generated by a planar interdigitated electrode array placed at an angle to the direction of flow. The simplified line charge model that we developed for the theoretical analysis was verified by comparing it with simulated and measured results. Experimental results showed that the divergent type of microseparator can continuously separate out 87.0% of the red blood cells (RBCs) and 92.1% of the white blood cells (WBCs) from dilute whole blood within 5 min simply by using a 2 MHz, 3 Vp-p AC voltage to create a gradient electric field in a medium that conducts at 17 mS cm(-1). Under the same conditions, the convergent type of microseparator could separate out 93.6% of the RBCs and 76.9% of the WBCs. We have shown that our lateral-driven continuous DEP microseparator design is practical for the continuous separation of blood cells without the need to control the conductivity of the suspension medium, overcoming critical drawbacks of DEP microseparators.     

Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.     

新生儿缺氧缺血性脑病血清钙、钠的变化

为观察新生儿缺氧缺血性脑病血清钙,钠的谱化规律,对１４０例ＨＩＥ新生儿和１００例正常新生儿血清钙,钠进行了对比观察。提示ＨＩＥ新生儿要适当纠正血甭钙,钠。     

Growth of calcium hydroxyapatite (Ca-HAp) on cholesterol and cholestanol crystals from a simulated body fluid: A possible insight into the pathological calcifications associated with atherosclerosis

An experimental study into calcium phosphate (CP) nucleation and growth on cholesterol and cholestanol surfaces from a supersaturated simulated body fluid (SBF) is presented with the overall aim of gaining some fundamental insights into the pathological calcifications associated with atherosclerosis. Soaking of pressed cholesterol disks at physiological temperature in SBF solutions was found to lead to CP nucleation and growth if the disks were surface roughened and if an SBF with concentrations of the calcium and hydrogen phosphate ions at 2.25x physiological concentrations was used. The CP phase deposited was shown via SEM micrographs to possess a florette type morphology akin to that observed in earlier reported studies. The use of recrystallised cholesterol and cholestanol microcrystals as substrates for soaking in SBF facilitated the observation of CP deposition. In general, cholesterol recrystallised from polar solvents like 95% ethanol as a cholesterol monohydrate phase which was a better substrate for CP growth than cholesterol recrystallised from more non-polar solvents (e.g., benzene) which produced anhydrous cholesterol phases. CP was also observed to form on recrystallised cholestanol microcrystals, a molecule closely related to cholesterol. Inductively coupled plasma optical emission spectrometry (ICP-OES) data gave confirmation that Ca:P mole ratios of the grown CP were 1.3-1.5 suggesting a mixed phase of octacalcium phosphate (OCP) and Ca-deficient HAp and that the CP coating grows (with time of soaking) on the substrates after nucleation in the SBF growth medium. Infrared (IR) spectra of the extracted coatings from the cholesterol substrates confirmed that the CP phase deposited is a semi crystalline HAp with either carbonate substituted into its structure or else co-deposited as calcium carbonate. Soaking experiments involving modified cholesterol substrates in which the OH group in the molecule was replaced with the oleiyl or phosphonate group showed no CP nucleation and growth. This observation illustrates the importance of the known epitaxial relationship between cholesterol and HAp (which theoretically predicts favourable deposition of one phase upon the other) and the consequences of its destruction (by chemical modification of the cholesterol). In the case of the phosphorylated cholesterol, failure of this substrate to nucleate CP phases may have also been caused by the reduction in concentration of free solution Ca2+ in the SBF medium by complexation with the phosphonate groups on the phosphorylated cholesterol. This would have reduced the ion product of Ca2+ and inorganic phosphate and lowered the degree of supersaturation in the SBF medium.     

Association of (Ca + Mg)-ATPase activity with ATP-dependent Ca uptake in vesicles prepared from human erythrocytes.

Ghost membranes prepared from human erythrocytes exhibit 2 distinct (Ca + Mg)-ATPase1 activities (Quist and Roufogalis, Arch Biochem Biophys 168:240, 1975). (Ca + Mg)-ATPase activity dependent on a water soluble protein fraction is selectively lost from ghost membranes during preparation of vesicles under low ionic strength, slightly alkaline conditions. In this study, the Ca2+ dependence of the remaining membrane bound (Ca + Mg)-ATPase activity and ATP-dependent Ca uptake in vesicles were compared. The Ca2+ activation curves for (Ca + Mg)-ATPase activity and Ca uptake into vesicles were parallel over a Ca2+ range of 0.3-330 micrometer, and both curves have 2 apparent KA values for Ca2+ of 0.45 and 100 micrometer. Addition of a concentrated soluble protein fraction containing predominantly spectrin to the vesicles increased (Ca + Mg)-ATPase activity over twofold but did not affect the rate of Ca uptake. These findings suggest that the (Ca + Mg)-ATPase activity remaining in vesicles after extraction of the water soluble proteins is associated with the Ca pump whereas (Ca + Mg)-ATPase activity dependent on the soluble protein fraction is associated with some other function.