Regulation of Ca<Superscript>2+</Superscript> influx in proliferating and differentiating murine myoblasts with participation of L-type Ca<Superscript>2+</Superscript> channels

The basic mechanisms of regulation of Ca²⁺ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca²⁺ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca²⁺ through these channels is regulated by the adrenergic system. The influx of Ca²⁺ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca²⁺ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca²⁺ influx is substantially lower than in proliferating cells, and maximal influx of Ca²⁺ may be reached only upon exhaustion of reticular stocks.     

Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca²⁺ in rooting medium is essential for root elongation, even in the absence of added toxicants. In the presence of rhizotoxic levels of Al³⁺, H⁺, or Na⁺ (or other cationic toxicants), supplementation of the medium with higher levels of Ca²⁺ alleviates growth inhibition. Experiments to determine the mechanisms of alleviation entailed measurements of root elongation in wheat (Triticum aestivum L. cv Scout 66) seedlings in controlled medium. A Gouy-Chapman-Stern model was used to compute the electrical potentials and the activities of ions at the root-cell plasma membrane surfaces. Analysis of root elongation relative to the computed surface activities of ions revealed three separate mechanisms of Ca²⁺ alleviation. Mechanism I is the displacement of cell-surface toxicant by the Ca²⁺-induced reduction in cell-surface negativity. Mechanism II is the restoration of Ca²⁺ at the cell surface if the surface Ca²⁺ has been reduced by the toxicant to growth-limiting levels. Mechanism III is the collective ameliorative effect of Ca²⁺ beyond mechanisms I and II, and may involve Ca²⁺-toxicant interactions at the cell surface other than the displacement interactions of mechanisms I and II. Mechanism I operated in the alleviation of all of the tested toxicities; mechanism II was generally a minor component of alleviation; and mechanism III was toxicant specific and operated strongly in the alleviation of Na⁺ toxicity, moderately in the alleviation of H⁺ toxicity, and not at all in the alleviation of Al³⁺ toxicity.     

Distinctive characteristics and functions of multiple mitochondrial Ca<Superscript>2+</Superscript> influx mechanisms

Intracellular Ca²⁺ is vital for cell physiology. Disruption of Ca²⁺ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca²⁺ dynamics, various Ca²⁺ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca²⁺ transport mechanisms in responding to physiological Ca²⁺ pulses in cytosol is to take up Ca²⁺ for regulating energy production and shaping the amplitude and duration of Ca²⁺ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca²⁺ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca²⁺ transport across the inner mitochondrial membrane. The mitochondrial Ca²⁺ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca²⁺ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca²⁺ influx. Since multiple Ca²⁺ influx mechanisms (e.g. L-, T-, and N-type Ca²⁺ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca²⁺ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca²⁺ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca²⁺/H⁺ exchanger), and MCU and its Ca²⁺ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca²⁺ influx pathways and their differences in kinetics, Ca²⁺ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.     

Product fermented by <Emphasis Type="Italic">Lactobacilli</Emphasis> induces changes in intracellular calcium dynamics in rat brain neurons

In this work we investigated the effect of concentrated metabolic products of lactobacilli (PP) on the dynamics of intracellular calcium concentration ([Ca²⁺]_i) in rat brain neurons. [Ca²⁺]_i was recorded using a fluorescent probe Fura-2 and a ratiometric Ca²⁺ imaging. It was found that PP increased [Ca²⁺]_i, stimulating the intracellular signaling mechanisms. In these processes the activation of ryanodine receptors and protein kinase C are involved at least partially. Continuous application of PP stimulated a sustained release of Ca²⁺ from the endoplasmic reticulum and subsequent entry of Ca²⁺ into the cell. Given that PP is able to stimulate circulation and neurogenesis and is involved in calcium homeostasis in nerve cells in the brain, PP can be regarded as a product for the improvement of psychological parameters and cognitive functions of the brain.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Studies on the activation of isocitrate dehydrogenase kinase/phosphatase (AceK) by Mn<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg²⁺. We have studied the interactions of Mn²⁺and Mg²⁺ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn²⁺ also activates the enzyme activities. However, Mn²⁺ and Mg²⁺ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?10⁵ and 0.98?×?10⁵ M^?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn²⁺ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn²⁺ and Mg²⁺.     

Evolution of mechanisms of Ca<Superscript>2+</Superscript>-signaling: Role of calcium ions in signal transduction in prokaryotes

The review considers current concepts of mechanisms of Ca²⁺-signaling in cells of prokaryotes. Problems of structural-functional organization of the Ca²⁺-messenger system, mechanisms of membrane transport, and Ca²⁺ homeostasis are discussed. A special attention is paid to analysis of role of calcium ions and Ca²⁺-receptor proteins in regulation of intracellular processes in bacteria.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 12–17.Original Russian Text Copyright © 2005 by Shemarova, Nesterov.     

Alterations of the resin canal system of <Emphasis Type="Italic">Pinus pinaster</Emphasis> seedlings after fertilization of a healthy and of a <Emphasis Type="Italic">Hylobius abietis</Emphasis> attacked stand

Changes in resource availability and biotic and abiotic stress may alter the defensive mechanisms of pine trees. The effect of fertilisation on the resin canal structure of Pinus pinaster seedlings established in two trials in NW Spain, one attacked by Hylobius abietis and the other non-attacked, was studied. The leaders of 50 plants were destructively sampled and the resin canal density, the canal area and its relative conductive area in the phloem and xylem were assessed. Experimentally increased nutrient availability significantly decreased resin canal density in the phloem of the seedlings in the two analysed trials, where unfertilised seedlings presented up to 30% more resin canal density than the fertilised seedlings (mean value ± SEM = 0.32 ± 0.02 resin canals mm⁻² in the fertilised plants versus 0.45 ± 0.04 resin canals mm⁻² in the control plants). Fertilisation had no effect on the resin canal system in the xylem, but significantly increased tracheid size. Significant differences of resin canals among sites were observed mainly in the xylem; the resin canal density was 1.7-fold greater in the attacked site than in the non-attacked site. The similar structure of phloem resin canals in both sites supports that phloem resin canals are constitutive mechanisms of defence in P. pinaster, whereas xylem resin canals would be constitutive mechanisms but also inducible mechanisms of resistance following the attack of pine weevils or bark beetles. XM and LS equally contributed to this paper.     

Comparison of Ca<Superscript>2+</Superscript> Currents of Chromaffin Cells from Normotensive Wistar Kyoto and Spontaneously Hypertensive Rats

Spontaneously hypertensive rats (SHR) are widely used as model to investigate the pathophysiological mechanisms of essential hypertension. Catecholamine plasma levels are elevated in SHR, suggesting alterations of the sympathoadrenal axis. The residual hypertension in sympathectomized SHR is reduced after demedullation, suggesting a dysfunction of the adrenal medulla. Intact adrenal glands exposed to acetylcholine or high K⁺ release more catecholamine in SHR than in normotensive Wistar Kyoto (WKY) rats, and adrenal chromaffin cells (CCs) from SHR secrete more catecholamines than CCs from WKY rats. Since Ca²⁺ entry through voltage-gated Ca²⁺ channels (VGCC) triggers exocytosis, alterations in the functional properties of these channels might underlie the enhanced catecholamine release in SHR. This study compares the electrophysiological properties of VGCC from CCs in acute adrenal slices from WKY rats and SHR at an early stage of hypertension. No significant differences were found in the macroscopic Ca²⁺ currents (current density, I–V curve, voltage dependence of activation and inactivation, kinetics) between CCs of SHR and WKY rats, suggesting that Ca²⁺ entry through VGCC is not significantly different between these strains, at least at early stages of hypertension. Ca²⁺ buffering, sequestration and extrusion mechanisms, as well as Ca²⁺ release from intracellular stores, must now be evaluated to determine if alterations in their function can explain the enhanced catecholamine secretion reported in CCs from SHR.     

Investigating the mechanisms of glyphosate resistance in <Emphasis Type="Italic">Lolium multiflorum</Emphasis>

Evolved resistance to the herbicide glyphosate has been reported in eleven weed species, including Lolium multiflorum. Two glyphosate-resistant L. multiflorum populations were collected, one from Chile (SF) and one from Oregon, USA (OR), and the mechanisms conferring glyphosate resistance were studied. Based on a Petri dish dose–response bioassay, the OR and the SF populations were two and fivefold more resistant to glyphosate when compared to the susceptible (S) population, respectively; however, based on a whole-plant dose–response bioassay, both OR and SF populations were fivefold more resistant to glyphosate than the S population, implying that different resistance mechanisms might be involved. The S population accumulated two and three times more shikimic acid in leaf tissue 96 h after glyphosate application than the resistant OR and SF populations, respectively. There were no differences between the S and the glyphosate-resistant OR and SF populations in ¹⁴C-glyphosate leaf uptake; however, the patterns of ¹⁴C-glyphosate translocation were significantly different. In the OR population, a greater percentage of ¹⁴C-glyphosate absorbed by the plant moved distal to the treated section and accumulated in the tip of the treated leaf. In contrast, in the S and in the SF populations, a greater percentage of ¹⁴C-glyphosate moved to non-treated leaves and the stem. cDNA sequence analysis of the EPSP synthase gene indicated that the glyphosate-resistant SF population has a proline 106 to serine amino acid substitution. Here, we report that glyphosate resistance in L. multiflorum is conferred by two different mechanisms, limited translocation (nontarget site-based) and mutation of the EPSP synthase gene (target site-based).     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Time-course development of the Cd<Superscript>2+</Superscript> hyper-accumulating phenotype in <Emphasis Type="Italic">Euglena gracilis</Emphasis>

To determine the onset of the Cd²⁺-hyperaccumulating phenotype in Euglena gracilis, induced by Hg²⁺ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd²⁺ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl₂, the Cd²⁺-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd²⁺ accumulated was similar for both control and Hg²⁺-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd²⁺. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd²⁺-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd²⁺ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd²⁺ compartmentation into organelles. César Avilés: In memoriam.     

Evolution of Ca<Superscript>2+</Superscript>-Signaling Mechanisms. Role of Calcium Ions in Signal Transduction in Lower Eukaryotes

This review summarizes current concepts of Ca²⁺-signaling mechanisms in unicellular eukaryotes. Pathways of activation and transduction of Ca²⁺-signal are analyzed and the role of Ca²⁺ in regulation of cellular physiological processes is considered. A special attention is paid to evolutionary aspects of participation of Ca²⁺ ions and Ca²⁺-receptor proteins in regulation of intracellular processes.     

Accumulation of sodium and potassium ions in oocytes of the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> at the prespawning period

Accumulation of Na⁺ and K⁺ ions in oocytes of the river lamprey Lampetra fluviatilis and their transport across the plasma membrane is realized by two main mechanisms-Na,K-pump and Na,K,Cl-cotransport. At the prespawning period from December to May, the intracellular Na⁺ concentration was observed to increase from 10 to 25 mM and the K⁺ concentration-from 28 to 45 mM. Results obtained on isolated oocytes with aid of radioactive labels ²²Na and ²⁰⁴Tl have shown that contributions of the Na,K-pump and Na,K,Cl-cotransport to potassium accumulations were close until March. In spring, the total K⁺ inflow almost doubled owing to activation of the Na,K-pump, whereas contribution of Na,K,Cl-cotransport did not change. It seems that an increase of the intracellular content of the main inorganic cations in oocytes resulted in parallel activation of the Na,K-pump and probably of Na/H-exchange. The biological significance of activation of these mechanisms of ion transport at the prespawning period might be due to a necessity of accumulation of Na⁺ and K⁺ ions in concentrations optimal for subsequent embryonic development.     

Functions of the adaptor protein p66<Superscript>Shc</Superscript> in solid tumors

p66^Shc is a 66 kDa Src homology 2 domain containing (Shc) adaptor protein homolog. Previous studies have demonstrated that p66^Shc is a proapoptotic protein involved in the cellular response to oxidative stress and in regulating mammalian lifespan. However, accumulating evidence also indicates its critical role in solid tumor progression. The expression of p66^Shc varies in different types of solid tumors, and it can paradoxically promote as well as suppress tumor progression, survival, and metastasis, depending on the cellular context. In this review, we discuss its functions in various solid tumors, the mechanisms by which it mediates the process of anoikis (detachment-induced cell death), and the epigenetic mechanisms that regulate its expression. These studies indicate the potential of p66^Shc as a novel prognostic marker and therapeutic target for the prevention of tumor progression and metastasis.     

Pivotal Role of Reduced Glutathione in Oxygen-induced Regulation of the Na<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>/K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Pump in Mouse Erythrocyte Membranes

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na⁺/K⁺ pump, K⁺-Cl^– and Na⁺-K⁺-2Cl^– cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na⁺/K⁺ pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na⁺-loaded cells, revealing no effect of O₂ on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na⁺/K⁺ pump activity.     

Regulation of Plant Plasma Membrane H<Superscript>+</Superscript>- and Ca<Superscript>2+</Superscript>-ATPases by Terminal Domains

In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H⁺-and Ca²⁺-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory mechanisms of these pumps. In plant plasma membrane H⁺- and Ca²⁺-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed.     

Analyses of erythropoiesis from embryonic stem cell‐CD34+ and cord blood‐CD34+ cells reveal mechanisms for defective expansion and enucleation of embryomic stem cell‐erythroid cells

Red blood cells (RBCs) generated ex vivo have the potential to be used for transfusion. Human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) possess unlimited self‐renewal capacity and are the preferred cell sources to be used for ex vivo RBC generation. However, their applications are hindered by the facts that the expansion of ES/iPS‐derived erythroid cells is limited and the enucleation of ES/iPS‐derived erythroblasts is low compared to that derived from cord blood (CB) or peripheral blood (PB). To address this, we sought to investigate the underlying mechanisms by comparing the in vitro erythropoiesis profiles of CB CD34⁺ and ES CD34⁺ cells. We found that the limited expansion of ES CD34⁺ cell‐derived erythroid cells was associated with defective cell cycle of erythroid progenitors. In exploring the cellular and molecular mechanisms for the impaired enucleation of ES CD34⁺ cell‐derived orthochromatic erythroblasts (ES‐ortho), we found the chromatin of ES‐ortho was less condensed than that of CB CD34⁺ cell‐derived orthochromatic erythroblasts (CB‐ortho). At the molecular level, both RNA‐seq and ATAC‐seq analyses revealed that pathways involved in chromatin modification were down‐regulated in ES‐ortho. Additionally, the expression levels of molecules known to play important role in chromatin condensation or/and enucleation were significantly lower in ES‐ortho compared to that in CB‐ortho. Together, our findings have uncovered mechanisms for the limited expansion and impaired enucleation of ES CD34⁺ cell‐derived erythroid cells and may help to improve ex vivo RBC production from stem cells.