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.     

Ca entry through the store-mediated pathway directly activates only the K current but the subsequent Ca release from the store activates both K and Cl currents in submandibular gland acinar cells of the rat

The store-mediated Ca²⁺ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca²⁺ activated ionic membrane currents. In the cells where intracellular Ca²⁺ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca²⁺-free extracellular condition, an employment of external Ca²⁺ in the absence of ACh caused a sustained increase of the K⁺ current without affecting the Cl⁻ current. A renewed ACh challenge without external Ca²⁺ caused repetitive spikes of both K⁺ and Cl⁻ currents due to the Ca²⁺ release. SK & F 96365 inhibited the generation of the sustained K⁺ current and refilling of the Ca²⁺ store following the Ca²⁺ readmission. It is suggested that the Ca²⁺ enters the cell through the store-mediated pathway near the K⁺ channels and is taken up by the store. Thus, only Ca²⁺ released from the store can activate both the K⁺ and Cl⁻ currents.     

Calcium ion control of platelet thrombosthenin ATPase activity

This study demonstrates that Ca²⁺ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10⁻⁴ M CaCl₂ (high ionized Ca²⁺) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) (low ionized Ca²⁺). In both high and low Ca²⁺, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca²⁺ (approx. 2.3 nmoles·mg⁻¹·min⁻¹) than in low Ca²⁺ (approx. 1.8 nmoles·mg⁻¹·min⁻¹). This difference, when analyzed by the Student's t-test for paired samples was highly significant (P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na⁺ + K⁺)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca²⁺, measured with the use of CaEGTA buffers, was studied. The Ca²⁺-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10⁻⁷ M Ca²⁺. This corresponds to an apparent binding constant of 2.2·10⁶ M⁻¹, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca²⁺ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet.     

A negative resistance region underlies the triggering property of membrane potential in human T-lymphocytes

Steady-state current-voltage relationships (SSCVRs) of the plasma membrane of human T-lymphocytes were studied at the physiological temperature of 37°C by using the whole-cell patch-clamp technique. SSCVRs displayed a characteristic N-like shape with a negative resistance region (NRR) in a voltage range of −45 to −35 mV. The majority of cells assayed revealed SSCVR patterns crossing the V-axis at three points (in mV): V₁ = −55 to −45, V₂ = −40 to −35, V₃ = −30 to −10. SSCVRs of T-cells activated by phytohaemagglutinin (48–96 h) also displayed NRR, but crossed the V-axis at one point only (V₁ = −55 to −60 mV). It implies the possibility of two stable levels of membrane potential (V₁ and V₃) for the resting T-cells, but only one (V₁) for activated T-cells. These data thus account for the triggering property of T-cell membrane potential previously reported. The NRR can be explained on the basis of the Hodgkin-Huxley type n⁴j model of K⁺ channel kinetics. According to the model the possibility for a membrane to have on or two stable levels of membrane potential depends on the ratio of selective K⁺ conductance to non-selective leaky conductance (G_k/G_leak). The steady-state level of K⁺ conductance in resting T-lymphocytes proved to be sensitive to Ca²⁺. Buffering Ca²⁺ ions from either external or internal solution resulted in an appreciable increase in K⁺ conductance. The possibility for membrane potential have two stable levels of membrane potential in connection with the Ca²⁺ dependence of K⁺ conductance was supposed to be important for Ca²⁺-signalling during T-cell activation.     

Effects of calcium on the thermal stability, stability in organic solvents and resistance to hydrogen peroxide of artichoke (Cynara scolymus L.) peroxidase: A potential method of enzyme control

The effects of calcium ions (Ca²⁺) on the stability of artichoke (Cynara scolymus L.) peroxidase (AKPC) have been studied. The thermal stability of AKPC was improved by the addition of Ca²⁺; the melting temperature increased by 20 °C and the deactivation energy by 26 kJ mol⁻¹. AKPC was stable in a selection of organic solvents but was less active with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) than under aqueous conditions. Ca²⁺-free AKPC retained more activity in the presence of organic solvents due to its better maintenance of the rate of compound I formation with hydrogen peroxide (H₂O₂) compared to AKPC-Ca²⁺. AKPC retained at least 75% activity over 24 h in the pH range 3.0–10.5 and about 50% over 1 month at pH 7.0 or 5.5, irrespective of the Ca²⁺ content. AKPC-Ca²⁺ was considerably more resistant to inactivation by H₂O₂ than Ca²⁺-free AKPC suggesting that the presence of Ca²⁺ boosts turnover under oxidizing conditions. AKPC has been applied as an alternative to horseradish peroxidase (HRP) in glucose concentration assays; the presence of Ca²⁺ or of the Ca²⁺ chelating agent ethylenediaminetetraacetic acid made no difference to the final result. The possibility is discussed that addition and removal of a labile Ca²⁺ from AKPC could be used to control enzyme activity both in vivo and in vitro.     

Functional expression of the plant K channel KAT1 in insect cells

Following the biophysical analysis of plant K⁺ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K⁺ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K⁺-selective (impermeable to Na⁺ and even NH₄⁺) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca²⁺ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.     

低氧及酸化胁迫对大黄鱼幼鱼离子调节与鳃组织结构的影响

为探讨大黄鱼幼鱼在低氧及酸化胁迫下机体离子调节情况,本研究探讨了低氧(溶解氧量DO 3.5 mg·L-1,pH 8.1)、酸化(DO 7.0 mg·L-1,pH 7.35)以及低氧酸化协同胁迫(DO3.5 mg·L-1,pH 7.35)对大黄鱼幼鱼鳃组织结构以及离子调节相关生理指标的影响.结果 表明:低氧胁迫下,大黄鱼...     

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.

Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat

Although ascorbic acid (AsA) is one of the most important and abundantly occurring water soluble antioxidants in plants, relatively little is known about its role in counteracting the adverse effects of salt stress on plant growth. To address this issue that whether exogenous application of ascorbic acid (AsA) through rooting medium could alleviate the adverse effects of salt stress on wheat plants, a hydroponic experiment was conducted under glasshouse conditions using two wheat cultivars, S-24 (salt tolerant) and MH-97 (moderately salt sensitive). Plants of both cultivars were subjected to 0 or 150 mM NaCl solution supplemented with 0, 50, or 150 mg L⁻¹ AsA for 58 days. Imposition of salt stress reduced the growth of both wheat cultivars by causing reduction in photosynthesis, and endogenous AsA level, and enhancing accumulation of Na⁺ and Cl⁻ coupled with a decrease in K⁺ and Ca²⁺ in the leaves and roots of both cultivars thereby decreasing tissue K⁺/Na⁺ ratio. However, root applied AsA counteracted the adverse effects of salt stress on the growth of cv. S-24 only, particularly at 100 mg L⁻¹ AsA level. AsA-induced enhancement in growth of salt-stressed plants of S-24 was associated with enhanced endogenous AsA level and CAT activity, and higher photosynthetic capacity, and accumulation of K⁺ and Ca²⁺ in the leaves. Although root applied AsA did not improve the growth of salt-stressed plants of MH-97, it enhanced endogenous level of AsA, CAT activity, photosynthetic capacity, and leaf K⁺ and Ca²⁺. These findings led us to conclude that root applied AsA counteracts the adverse effects of salt stress on growth of wheat by improving photosynthetic capacity of wheat plants against salt-induced oxidative stress and maintaining ion homeostasis, however, these effects were cultivar specific.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

A model of the guinea-pig ventricular cardiac myocyte incorporating a transverse-axial tubular system

A model of the guinea-pig cardiac ventricular myocyte has been developed that includes a representation of the transverse–axial tubular system (TATS), including heterogeneous distribution of ion flux pathways between the surface and tubular membranes. The model reproduces frequency-dependent changes of action potential shape and intracellular ion concentrations and can replicate experimental data showing ion diffusion between the tubular lumen and external solution in guinea-pig myocytes. The model is stable at rest and during activity and returns to rested state after perturbation. Theoretical analysis and model simulations show that, due to tight electrical coupling, tubular and surface membranes behave as a homogeneous whole during voltage and current clamp (maximum difference 0.9 mV at peak tubular I_Na of −38 nA). However, during action potentials, restricted diffusion and ionic currents in TATS cause depletion of tubular Ca²⁺ and accumulation of tubular K⁺ (up to −19.8% and +3.4%, respectively, of bulk extracellular values, at 6 Hz). These changes, in turn, decrease ion fluxes across the TATS membrane and decrease sarcoplasmic reticulum (SR) Ca²⁺ load. Thus, the TATS plays a potentially important role in modulating the function of guinea-pig ventricular myocyte in physiological conditions.     

Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes

Dimethyl adipimidate (DMA) reduces K⁺ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (P_sickle), the Ca²⁺-activated K⁺ channel (or Gardos channel) and the K⁺-Cl⁻ cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca²⁺ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of P_sickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K⁺ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.     

疏叶骆驼刺适应盐渍生境的离子分布、吸收和运输特征

以塔里木盆地南缘关键物种疏叶骆驼刺为材料,研究了不同盐渍土壤生境(轻度盐渍土、中度盐渍土、重度盐渍土)下其器官间Na⁺、K⁺、Ca²⁺、Mg²⁺的分布、吸收及运输特征,以探讨疏叶骆驼刺对自然盐渍生境的适应特性.结果表明: 在轻度和中度盐渍土生境,Na⁺在各器官中的分布规律为茎≈刺>叶>根,而在重度盐渍土生境,Na⁺分布规律为叶>茎≈刺>根;Ca²⁺和Mg²⁺在疏叶骆驼刺体内的分布规律为叶>刺>茎>根.随着土壤含盐量的增加,疏叶骆驼刺体内各器官Na⁺含量都增大,而叶片中K⁺含量呈下降趋势;根和叶器官中K⁺/Na⁺值明显降低,各器官中Ca²⁺/Na⁺、Mg²⁺/Na⁺值都降低.盐渍生境下,疏叶骆驼刺体内Ca²⁺选择性运输系数和Mg²⁺选择性运输系数均为茎-叶>茎-刺>根-茎.疏叶骆驼刺为适应盐渍生境,在土壤含盐量较低时,将Na⁺聚集于茎和刺;而在土壤含盐量较高时,则将Na⁺聚集于叶片.此外,Ca²⁺和Mg²⁺可能是疏叶骆驼适应盐渍生境的无机渗透调节物质.     

Improved salt tolerance of melon (Cucumis melo L.) by the addition of proline and potassium nitrate

A pot experiment was carried out under glasshouse conditions with melon (Cucumis melo) cv. “Tempo F1” in a mixture of peat, perlite and sand (1:1:1) to investigate the effects of external proline and potassium nitrate applications to salinity-treated (150 mM) plants with respect to fruit yield, plant growth, some physiological parameters and ion uptake. Treatments were—(i) control (C): plants receiving nutrient solution, (ii) salinity treatment, as for control plus 150 mM NaCl. Salinity treatment was combined with or without either 5 mM supplementary KNO₃ or 10 mM proline. The salt treatment (150 mM NaCl) led to significant decreases in plant growth, fruit yield, relative water content (RWC), stomatal density, uptake of Ca²⁺, K⁺ and N, and chlorophyll a and b contents, accompanied by significant increases in Na⁺ uptake, proline concentration and membrane permeability. Supplementary KNO₃ and proline treatments significantly ameliorated the adverse effects of salinity on plant growth, fruit yield and the physiological parameters examined. This could be attributed to the effects of all the external supplements in maintaining membrane permeability, and increasing concentrations of Ca²⁺, N and K⁺ in the leaves of plants subjected to salt stress.     

滨海滩涂地带乌哺鸡竹和淡竹离子含量变化及其与生长及光合作用的关系

在沿海滩涂防护林带低盐区(0.1%)、中盐区(0.2%)和重盐区(0.4%) 3个盐分梯度下,研究了栽植10年的乌哺鸡竹和淡竹Na⁺、K⁺、Ca²⁺、Mg²⁺含量变化及其与生长和光合作用的相关关系.结果表明: 从低盐区到重盐区,乌哺鸡竹的立竹密度和地径分别下降30.4%和28.8%,降幅低于淡竹的44.1%和31.2%;两竹种单株生物量下降,地上器官生物量降幅均显著高于地下器官;乌哺鸡竹和淡竹净光合速率(P_n)和PSⅡ最大光化学效率(F_v/F_m)分别下降57.6%和67.7%、6.1%和7.4%,乌哺鸡竹耐盐能力比淡竹强.随着土壤含盐量的增大,乌哺鸡竹和淡竹各器官Na⁺含量逐渐增加,K⁺、Ca²⁺、Mg²⁺含量逐渐降低.两竹种根Na⁺积累较多,而地上部分K⁺含量较高.盐胁迫环境导致乌哺鸡竹根Ca²⁺含量与淡竹叶片Mg²⁺含量明显下降.两竹种的生物量、P_n、F_v/F_m与Na⁺含量呈显著负相关,与K⁺、Ca²⁺含量呈显著正相关.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.     

磁化微咸水灌溉对欧美杨I-107离子稳态的影响

为探究微咸水磁化处理条件下植株的离子稳态特征,以欧美杨I-107一年生扦插苗为试材,于生长季节分别采用Hoagland营养液和4.0 g·L^-1 NaCl微咸水,经磁化处理后连续灌溉30 d.采用原子吸收分光光度法对叶片和根系中K⁺、Na⁺、Ca²⁺和Mg²⁺含量进行测定,分析离子平衡系数(K)和根-叶之间的离子选择性运输系数(S_Xi,Na).结果表明: 与非盐分胁迫处理相比,盐分胁迫处理根系和叶片中Na⁺和Ca²⁺含量及S_K,Na、S_Mg,Na升高,K⁺和Mg²⁺含量、K⁺/Na⁺及S_Ca,Na降低.与非磁化微咸水灌溉处理相比,磁化微咸水灌溉处理的根系和叶片中Na⁺含量降低、K⁺含量及K⁺/Na⁺提高;根系和叶片中Ca²⁺含量降低、Mg²⁺含量提高;磁化微咸水灌溉处理中K提高,且叶片中K值显著高于根系;S_K,Na和S_Mg,Na较非磁化微咸水灌溉提高,S_Ca,Na较其降低.磁化微咸水灌溉中根系和叶片Na⁺积累量减少,K⁺、Ca²⁺和Mg²⁺含量增加,且维持了较高水平的K⁺/Na⁺,这有利于植株整株水平生理代谢的调控.因此,盐分胁迫下磁化作用可通过调节离子的选择性吸收和运输来维持植株体内的离子平衡.     

Physiological blocking of the mechanisms of cold death: theoretical and experimental considerations

The cold inhibited functions of skin thermoreceptors, of the thermoregulation centre, and the respiration centre during deep hypothermia can be restored without rewarming the body. The methods used were developed to test the hypothesis that during deep hypothermia calcium ion concentration [Ca²⁺]_i in the cytoplasm increases. This causes a perturbation of cell metabolism, the impairment of cell membrane function that cause the inhibition of cell functioning, resulting in cell death. Such an increase in [Ca²⁺]_i most likely would result from an energy deficit in a deeply cooled cell, which would compromise the processes that maintain the [Ca²⁺]_i at about 10⁻⁷ M. These processes require large amounts of energy since they occur against a large concentration gradient. With the use of EDTA the extracellular concentration of Ca²⁺ has been lowered by 15–27%, so reducing the concentration gradient for Ca²⁺ between the cell and the medium and in consequence facilitated the process the extrusion of cell Ca²⁺.

During a period of cooling, sufficient to impair normal functioning, the experimental lowering of blood Ca²⁺ allowed the restoration of normal function without the need to rewarm. In such cases the animals survived after cooling the body to temperatures at which they would normally have succumbed. The data presented support the stated hypothesis that the impairment of cellular function in mammals by low temperatures is the result of an uncorrected rise in [Ca²⁺]_i.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K⁺-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74±14 and 65±16 pS for +60 and −60 mV, respectively (140 mM [K⁺]_out, 2 mM [Mg²⁺]_out and 0 mM [Ca²⁺]_out). The latency of the response to pressure steps was 50–100 ms and the time to peak 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20–30% of peak in 1 s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.