Na and K in outer segments of retinal rods

The amount of Na⁺ and K⁺ in isolated bovine retina outer segments and slices of outer segments obtained from frozen and freeze-dried bovine and frog retinas was established. It is shown that during the conventional procedure of isolation nearly 75% of the Na⁺ and K⁺ present in native structures was lost.

The average amount of K⁺ in bovine outer segments is 158 mmoles/kg dry wt.; Na⁺, 136 mmoles/kg dry wt. In frog outer segments there is: K⁺, 133 mmoles/kg dry wt.; Na⁺, 91 mmoles/kg dry wt.

With the help of the electron microscopic technique Na⁺ was shown to be located predominantly in the sacs of the outer segments. As for K⁺, it is, in all probability, in the extrasaccular space which agrees with some experimental biochemical data obtained.     

野皂荚对NaCl胁迫的生理响应及耐盐性

以盆栽野皂荚2年生实生苗为材料,设置土壤NaCl含量分别为0.053%（CK）、0.15%、0.3%、0.45%和0.6%的盐胁迫处理,研究不同浓度盐处理对苗木生长、细胞膜透性、细胞保护酶活性以及Na⁺和K⁺分布格局的影响,探讨了其耐盐阈值和机理.结果表明：随着NaCl浓度增加,苗木生长量逐渐降低,盐害指数逐渐升高;野皂荚可忍耐的土壤含盐量为0.42%.随着NaCl浓度增加,叶片相对电导率、氧自由基产生速率和丙二醛(MDA)含量均逐渐增大;超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性呈先上升后下降的变化趋势,在土壤含盐量0.3%或0.45%时达到峰值;高盐胁迫下,SOD、POD和CAT活性的增强可及时清除盐胁迫产生的氧自由基,进而缓解或避免膜脂过氧化作用对组织细胞的伤害.盐胁迫下根、茎、叶的Na⁺含量均逐渐增大,且呈现根>叶>茎的分布格局;K⁺含量和K⁺/Na⁺呈下降趋势,呈现叶>根>茎的分布格局;K⁺-Na⁺选择性运输系数(S_K⁺·Na⁺)随着土壤含盐量的增加逐渐升高,且叶S_K⁺·Na⁺高于茎S_K⁺·Na⁺.野皂荚耐盐机制是根系拒盐和叶片耐盐;盐胁迫下,根系Na⁺累积能力增强可控制Na⁺向地上运输以避免盐害发生,叶片K⁺选择性吸收和累积能力的显著提高可忍耐和补偿Na⁺对组织的伤害.     

Ion Channels and Membrane Potential in Stimulus-Secretion Coupling in Adrenal Medulla Cells

Abstract: The role of Na⁺ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)-sensitive Na⁺ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Locke's solution but has no effect on high concentrations of nicotine (20 μM). In Ca²⁺-sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na⁺ with Li⁺ in Locke's solution reduces the response to nicotine and to veratridine. Complete replacement of Na⁺ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM-100 mM-K⁺, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM-K⁺. Neither dibutyryl-cyclic AMP nor dibutyryl-cyclic GMP evokes secretion. α-Bungarotoxin (1 μM) did not inhibit nicotine-induced secretion. These studies indicate that Na⁺ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca²⁺. The membrane potential appears to affect nicotine- and veratridine-evoked secretion.     

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation–contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na⁺ current, and (ii) a decrease in an inwardly rectifying K⁺ current.

One cytotoxic effect of neutrophils appears to be initiated by enhanced Na⁺ entry into the myocytes. Thus, manoeuvres that precluded activation of Na⁺ channels, for example holding the membrane potential at −80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2–4% of the available Na⁺ channels on action potential duration and the corresponding intracellular Ca²⁺ transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.     

Amperometric glucose biosensor based on polymerized ionic liquid microparticles

A glucose amperometric biosensor based on the immobilization of glucose oxidase (GOx) in microparticles prepared by polymerization of the ionic liquid 1-vinyl-3-ethyl-imidazolium bromide (ViEtIm⁺Br⁻) using the concentrated emulsion polymerization method has been developed. The polymerization of the emulsion dispersed phase, in which the enzyme was dissolved together with the ionic liquid monomer, provides poly(ViEtIm⁺Br⁻) microparticles with entrapped GOx. An anion-exchange reaction was carried out for synthesizing new microparticles of poly(ViEtIm⁺(CF₃SO₂)₂N⁻) and poly(ViEtIm⁺BF₄⁻). The enzyme immobilization method was optimized for biosensor applications and the following optimal values were determined: pH 4.0 for the synthesis medium, 1.23 M monomer concentration and 3.2% (w/w) cross-linking content. The performance of the biosensor as a function of some analytical parameters such as pH and temperature of the measuring medium, and enzymatic load of the microparticles was also investigated. The effect of the substances which are present in serum samples such as uric and ascorbic acid was eliminated by using a thin Nafion layer covering the electrode surface. The biosensor thus prepared can be employed in aqueous and in non-aqueous media with satisfactory results for glucose determination in human serum samples. The useful lifetime of this biosensor was 150 days.     

Late sodium current in failing heart: Friend or foe?

Most cardiac Na⁺ channels open transiently upon membrane depolarization and then are quickly inactivated. However, some channels remain active, carrying the so-called persistent or late Na⁺ current (I_NaL) throughout the action potential (AP) plateau. Experimental data and the results of numerical modeling accumulated over the past decade show the emerging importance of this late current component for the function of both normal and failing myocardium. I_NaL is produced by special gating modes of the cardiac-specific Na⁺ channel isoform. Heart failure (HF) slows channel gating and increases I_NaL, but HF-specific Na⁺ channel isoform underlying these changes has not been found. Na⁺ channels represent a multi-protein complex and its activity is determined not only by the pore-forming subunit but also by its auxiliary β subunits, cytoskeleton, calmodulin, regulatory kinases and phosphatases, and trafficking proteins. Disruption of the integrity of this protein complex may lead to alterations of I_NaL in pathological conditions. Increased I_NaL and the corresponding Na⁺ flux in failing myocardium contribute to abnormal repolarization and an increased cell Ca²⁺ load. Interventions designed to correct I_NaL rescue normal repolarization and improve Ca²⁺ handling and contractility of the failing cardiomyocytes. This review considers (1) quantitative integration of I_NaL into the established electrophysiological and Ca²⁺ regulatory mechanisms in normal and failing cardiomyocytes and (2) a new therapeutic strategy utilizing a selective inhibition of I_NaL to target both arrhythmias and impaired contractility in HF.     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Channels as taste receptors in vertebrates

Taste reception is fundamental for proper selection of food and beverages. Chemicals detected as taste stimuli by vertebrates include a large variety of substances, ranging from inorganic ions (e.g., Na⁺, H⁺) to more complex molecules (e.g., sucrose, amino acids, alkaloids). Specialized epithelial cells, called taste receptor cells (TRCs), express specific membrane proteins that function as receptors for taste stimuli. Classical view of the early events in chemical detection was based on the assumption that taste substances bind to membrane receptors in TRCs without permeating the tissue. Although this model is still valid for some chemicals, such as sucrose, it does not hold for small ions, such as Na⁺, that actually diffuse inside the taste tissue through ion channels. Electrophysiological, pharmacological, biochemical, and molecular biological studies have provided evidence that indeed TRCs use ion channels to reveal the presence of certain substances in foodstuff. In this review, we focus on the functional and molecular properties of ion channels that serve as receptors in taste transduction.     

盐胁迫下外源褪黑素和Ca2+对甜瓜幼苗的缓解效应

以甜瓜品种‘羊角酥瓜’为试材,利用人工气候室控制环境条件(昼/夜25/18 ℃),研究盐胁迫条件下外源褪黑素(MT)和Ca²⁺对甜瓜幼苗根系和叶片中Cl^-、Na⁺、K⁺、Mg²⁺、Ca²⁺离子含量,Na⁺/K⁺、 Na⁺/Ca²⁺、Na⁺/Mg²⁺值,以及H⁺-ATP酶活性、渗透调节物质积累和细胞膜质过氧化的影响.结果表明: 与对照相比,盐胁迫处理显著抑制甜瓜幼苗生长,增加根系和叶片中Cl^-、Na⁺含量,降低K⁺、Mg²⁺、Ca²⁺含量.盐胁迫下,喷施外源MT或Ca²⁺处理均可以显著降低甜瓜根系和叶片中Cl^-、Na⁺含量,提高K⁺、Mg²⁺、Ca²⁺含量,植株体内Na⁺/K⁺、Na⁺/Ca²⁺和 Na⁺/Mg²⁺值下降;同时也提高了根系和叶片H⁺-ATP酶活性及叶片渗透调节物质的含量,降低盐胁迫对细胞膜的伤害,表现在甜瓜叶片相对电导率和丙二醛含量降低.总之,在盐胁迫条件下,外源MT、Ca²⁺单独和复配处理均可通过提高H⁺-ATP酶活性来降低盐害离子的含量,改善甜瓜幼苗中的离子平衡,同时增加渗透调节物质的含量,降低膜质过氧化水平,从而增强其对盐胁迫的适应性,其中MT和Ca²⁺复配处理时的效果更好.复配外施 MT 和Ca²⁺在诱导甜瓜幼苗提高耐盐方面具有协同增效作用.     

盐胁迫下外源褪黑素和Ca2+对甜瓜幼苗的缓解效应

以甜瓜品种‘羊角酥瓜’为试材,利用人工气候室控制环境条件(昼/夜25/18 ℃),研究盐胁迫条件下外源褪黑素(MT)和Ca²⁺对甜瓜幼苗根系和叶片中Cl^-、Na⁺、K⁺、Mg²⁺、Ca²⁺离子含量,Na⁺/K⁺、 Na⁺/Ca²⁺、Na⁺/Mg²⁺值,以及H⁺-ATP酶活性、渗透调节物质积累和细胞膜质过氧化的影响.结果表明: 与对照相比,盐胁迫处理显著抑制甜瓜幼苗生长,增加根系和叶片中Cl^-、Na⁺含量,降低K⁺、Mg²⁺、Ca²⁺含量.盐胁迫下,喷施外源MT或Ca²⁺处理均可以显著降低甜瓜根系和叶片中Cl^-、Na⁺含量,提高K⁺、Mg²⁺、Ca²⁺含量,植株体内Na⁺/K⁺、Na⁺/Ca²⁺和 Na⁺/Mg²⁺值下降;同时也提高了根系和叶片H⁺-ATP酶活性及叶片渗透调节物质的含量,降低盐胁迫对细胞膜的伤害,表现在甜瓜叶片相对电导率和丙二醛含量降低.总之,在盐胁迫条件下,外源MT、Ca²⁺单独和复配处理均可通过提高H⁺-ATP酶活性来降低盐害离子的含量,改善甜瓜幼苗中的离子平衡,同时增加渗透调节物质的含量,降低膜质过氧化水平,从而增强其对盐胁迫的适应性,其中MT和Ca²⁺复配处理时的效果更好.复配外施 MT 和Ca²⁺在诱导甜瓜幼苗提高耐盐方面具有协同增效作用.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Docosahexaenoic acid (omega-3) blocks voltage-gated sodium channel activity and migration of MDA-MB-231 human breast cancer cells

Omega − 3 polyunsaturated fatty acids have been suggested to play an important role in cancer prevention/progression, on the one hand, and in modulation of membrane ion channels on the other. We investigated whether docosahexaenoic acid would influence the in vitro migration of MDA-MB-231 human breast cancer cells. An important follow-up question was whether any effect would involve voltage-gated Na⁺ channels, shown previously to occur in human breast cancer in vitro and in vivo and to correlate with metastatic potential. Short-term (acute) and long-term (24–72 h) application of docosahexaenoic acid suppressed the activity of the channel activity in a dose-dependent manner. At the working concentrations of docosahexaenoic acid used (0.05–0.5 μM), there was no effect on proliferation. Long-term treatment with docosahexaenoic acid down-regulated mRNA and protein (total and plasma membrane) levels of neonatal Nav1.5 voltage-gated Na⁺ channel, known to be predominant in these cells. Docosahexaenoic acid suppressed migration of the MDA-MB-231 cells to the same extent as tetrodotoxin, a highly specific blocker of voltage-gated Na⁺ channels, but the two effects were not additive. It was concluded that the docosahexaenoic acid-induced suppression of cellular migration occurred primarily via down-regulation of voltage-gated Na⁺ channel (neonatal Nav1.5) mRNA and functional protein expression.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

盐胁迫下菊芋根系脱落酸对钠离子转运和光系统Ⅱ的影响

通过根系施加脱落酸(ABA)合成抑制剂钨酸钠,研究盐胁迫(150 mmol·L^-1 NaCl)下菊芋根系ABA信号对根系Na⁺转运、叶片Na⁺积累和光系统Ⅱ(PSⅡ)的影响。结果表明:钨酸钠抑制盐胁迫下根系ABA合成,降低根系Na⁺外排,提高根系Na⁺向叶片的转运系数。盐胁迫增加叶片Na⁺含量,没有影响叶片膜脂过氧化、PSⅡ反应中心蛋白合成和PSⅡ最大光化学效率(F_v/F_m)。根系ABA合成受抑制,显著增加盐胁迫下叶片Na⁺积累,加剧叶片膜脂过氧化,损伤PSⅡ反应中心蛋白,显著降低F_v/F_m,诱发PSⅡ光抑制。总之,盐胁迫下菊芋根系ABA信号诱导根系Na⁺外排,抑制Na⁺向地上部转运,有利于减少叶片Na⁺积累,防御PSⅡ氧化损伤。     

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

A significant fraction of calcium transients in intact Guinea Pig ventricular myocytes is mediated by Na-Ca exchange

Ca²⁺ mobilization elicited by simulation with brief pulses of high K + were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca²⁺ changes across the cell, suggesting the presence of significant spatial Ca²⁺ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca²⁺ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca²⁺]_i elicited by membrane depolarization. The overall spatial distribution of [Ca²⁺]_i changes appeared unchanged. Ca²⁺ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 μM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the exchange mechanism. Conversely, when the reversal potential of the exchange was shifted to negative potentials by lowering [Na⁺]₀ or by increasing [Na⁺]_i by treatment with 20 μM monensin, the amplitude of these Ca²⁺ transients increased. Ca²⁺ transients elicited by membrane depolarization and largely mediated by reverse operation of Na⁺-Ca²⁺ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca²⁺ influx through the exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Immobilization of horseradish peroxidase with a regenerated silk fibroin membrane and its application to a tetrathiafulvalene-mediating H2O2 sensor

Horseradish peroxidase (HRP) was immobilized onto a membrane of the regenerated silk fibroin (RSF) from waste milk. The structure of the blend membrane of RSF and HRP was characterized by the use of IR spectra. A second generation of H₂O₂ sensor on the basis of the immobilized HRP was fabricated, in which tetrathiafulvalene acts as mediating electron transfer between the immobilized enzyme and a glassy carbon electrode. Dependencies of pH and temperature on the H₂O₂ biosensor were checked by utilizing cyclic voltammetry. The sensor exhibits high sensitivity, good reproducibility and storage stability.     

Voltage-dependent L-type Ca channels and a novel type of non-selective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells

Undifferentiated P19 embryonal carcinoma cells (ECC P19), the P19-derived clonal cell lines END-2 (visceral endoderm-like), EPI-7 (epithelioid ectoderm-like), MES-1 (mesoderm-like) and a parietal yolk sac cell line (PYS-2) were used as cellular models to examine the functional expression of voltage-dependent Ca channels and other Ca-permeable cation channels at various stages of early embryonic development. Whole-cell currents were recorded by means of the patch clamp technique. Whereas more than 75% of MES-1 cells possessed Ca channel currents, neither P19, END-2, EPI-7 nor PYS-2 cells had detectable voltage-dependent inward currents. Ca channel currents of MES-1 cells were highly sensitive towards 1,4-dihydropyridines and blocked by cadmium. Adrenaline (10 μM) caused Ca channel stimulation in only 14% of MES-1 cells examined. However, in 62% of the cells adrenaline activated a linear current component which under physiological conditions reversed close to 0 mV. Removal of extracellular Na⁺ suppressed the adrenaline-induced inward current, while reducing extracellular Cl⁻ had no significant effect. These findings suggest that the adrenaline-induced current is carried through non-selective cation channels which were found to be permeable for Na⁺, K⁺, Cs⁺ å Ca²⁺. Remarkably, the intracellular signalling pathway for activation of the non-selective cation current involved the cascade of reactions leading to cAMP-dependent phosphorylation, a regulatory pathway well known for cardiac Ca channels. A possible functional role of adrenaline-induced non-selective cation currents and Ca channels in embryonal development is discussed.     

Detection of heavy metal toxicity using cardiac cell-based biosensor

Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner which offers insight into the physiological effect of an analyte. To investigate the potential applications of cell-based biosensors on heavy metal toxicity detection, a novel biosensor for monitoring electrophysiological activity was developed by light-addressable potentiometric sensor (LAPS). Extracellular field potentials of spontaneously beating cardiomyocytes could be recorded by LAPS in the range of 20 μV to nearly 40 μV with frequency of 0.5–3 Hz. After exposed to different heavy metal ions (Hg²⁺, Pb²⁺, Cd²⁺, Fe³⁺, Cu²⁺, Zn²⁺; in concentration of 10 μM), cardiomyocytes demonstrated characteristic changes in terms of beating frequency, amplitude and duration under the different toxic effects of ions in less than 15 min. This study suggests that, with the physiological monitoring, it is possible to use the cardiac cell-based biosensor to study acute and eventually chronic toxicities induced by heavy metal ions in a long-term and no-invasive way.