S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin

Calcyclin is a homodimeric protein belonging to the S100 subfamily of EF-hand Ca(2+)-binding proteins, which function in Ca(2+) signal transduction processes. A refined high-resolution solution structure of Ca(2+)-bound rabbit calcyclin has been determined by heteronuclear solution NMR. In order to understand the Ca(2+)-induced structural changes in S100 proteins, in-depth comparative structural analyses were used to compare the apo and Ca(2+)-bound states of calcyclin, the closely related S100B, and the prototypical Ca(2+)-sensor protein calmodulin. Upon Ca(2+) binding, the position and orientation of helix III in the second EF-hand is altered, whereas the rest of the protein, including the dimer interface, remains virtually unchanged. This Ca(2+)-induced structural change is much less drastic than the "opening" of the globular EF-hand domains that occurs in classical Ca(2+) sensors, such as calmodulin. Using homology models of calcyclin based on S100B, a binding site in calcyclin has been proposed for the N-terminal domain of annexin XI and the C-terminal domain of the neuronal calcyclin-binding protein. The structural basis for the specificity of S100 proteins is discussed in terms of the variation in sequence of critical contact residues in the common S100 target-binding site.     

Hysteretic activation of the Ca pump revealed by calcium transients in human red cells

The enzymatic basis for the Ca²⁺ pump in human red cells is an ATPase with hysteretic properties. The Ca²⁺-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca²⁺ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133–143). In order to study whether the Ca²⁺ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Ca_i), induced by the divalent cation ionophore A23187. The time-dependent changes of Ca_i were measured by use of radioactive calcium (⁴⁵Ca²⁺) and analysed with the aid of a mathematical model, based partly on the Ca²⁺-dependent parameters obtained from Ca²⁺-ATPase experiments, partly on the A23187-induced Ca²⁺ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca²⁺ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca²⁺ pump in human red cells responds hysteretically. It is suggested that Ca²⁺ pumps in other types of cell also have hysteretic properties.     

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes

Abstract

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15?Hz, 50?Hz, 75?Hz and 100?Hz) and at a flux density of 2?mT. Intracellular calcium concentration ([Ca²⁺]_i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10?mM) was carried out to verify the function of the cardiac Na⁺/Ca²⁺ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca²⁺-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca²⁺]_i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

人类Cornulin蛋白S100结构域钙依赖的多聚化能有效减少细胞的损伤

在哺乳动物中发现一类新的能够抵制环境压力和保持组织完整性的应激蛋白.含有S100钙结合结构域的Cornulin(CRNN)是这类蛋白质之一,它在人类食管鳞状上皮细胞中高表达,而在食管鳞状上皮细胞癌中却低表达,它能抑制脱氧胆酸诱导的细胞损伤.S100结构域在CRNN的功能上具有重要作用.为了进一步探讨CRNN S100结构域的生物学特性,克隆、表达、纯化了该结构域,证明其折叠正确,适合用于生物物理和生物化学特性的研究.更为重要的是,通过核磁共振、凝胶过滤层析、超速离心、质谱和蛋白质交联分析,发现S100结构域具有钙依赖的多聚性质,而多聚体的形成更有利于保护细胞免受脱氧胆酸和乙醇的损伤.上述结果表明,S100结构域是CRNN发挥功能的关键结构域,它可以通过多聚化更好地保护细胞.该工作将进一步揭示S100结构域的生物学功能.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy     

Effects of calcium and bay K-8644 on calcium currents in adrenal medullary chromaffin cells

Summary The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca²⁺ currents, recorded in the presence of either 5.2 or 2.6mm Ca²⁺ exhibited a single, noninactivating component. To analyze the effects of Ca²⁺ and Bay K-8644 on the kinetics of the Ca²⁺ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca²⁺] (2.5mm) the midpoint of the steady-state Ca²⁺-channel activation curve lay at –6.9 mV. Increasing the [Ca²⁺] to 5.2mm shifted the midpoint by –4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2mm Ca²⁺) and 9.2 mV (for 2.5mm Ca²⁺) induced ane-fold change in the activation of the current. Increasing [Ca²⁺]₀ from 2.5 to 5.2mm induced a marked increase in the rate constant for turning on the Ca²⁺ permeability. Conductances were estimated from the slope of the linear part of the currentvoltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5mm Ca²⁺, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 m increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 m induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 m) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from –6.9 to –13 mV. At the midpoint potential of –13 mV, a change in potential of 6.9 mV caused ane-fold change in Ca²⁺ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca²⁺ permeability.     

Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.     

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

[³H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca²⁺-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [³H]purine release. Nimodipine only at the concentration of 10 M modified [³H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [³H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of⁴⁵Ca²⁺ by the cultures. The treatment of cells with the Ca²⁺ agonist Bay K 8644 did not affect [³H]purine release or the⁴⁵Ca²⁺ uptake. The drug did not either alter [Ca²⁺]_i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca²⁺ discharge, significantly reduced the evoked [³H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca²⁺ ATPase, was able to increase either the culture [³H]purine release or the [Ca²⁺]_i. Together, the findings indicate that voltage-sensitive calcium channels (VSCC_s) of the neuronal N and L-types are not involved in the modulation of [³H]purine release from rat cultured astrocytes whereas Ca²⁺ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC_s seem to be able to affect [³H]purine outflow with mechanisms other than VSCC gating.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

Ca2+-Binding S100 Proteins in the Central Nervous System

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca²⁺ homeostasis regulated by Ca²⁺-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Enhanced proliferation and altered calcium handling in RGS2-deficient vascular smooth muscle cells

Abstract

Context: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca²⁺ signalling in vascular smooth muscle cells (VSMC). Objective: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca²⁺ concentration [Ca²⁺]i. RGS2 involvement in VSMC proliferation had not been examined. Methods: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca²⁺]i before and after UTP and thapsigargin. [³H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca²⁺ transporters was performed in primary aortic VSMC. Results and discussion: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca²⁺ homeostasis, RGS2KO VSMC showed lower resting [Ca²⁺]i but higher thapsigargin-induced [Ca²⁺]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca²⁺ ATPase-4 (PMCA4) and Na⁺ Ca²⁺ Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca²⁺ ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca²⁺ efflux activity, ⁴⁵Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La³⁺, but not by replacing extracellular Na⁺ with choline, implicating differences in the activity of PMCA and not NCX. Conclusion: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca²⁺-handling mechanisms in response to PDGF stimulation.     

The role of S100A4 protein in anticancer cytotoxicity: its presence is required on the surface of CD4+CD25+PGRPs+S100A4+ lymphocyte and undesirable on the surface of target cells

S100A4 is a Ca²⁺-binding protein that performs an important role in metastasis. It is also known for its antitumor functions. S100A4 is expressed by a specialized subset of CD⁴⁺CD²⁵⁺ lymphocytes and is present on those cell's membranes along with peptidoglycan recognition proteins (PGRPs). There, by interacting with major heat shock protein Hsp70, S100A4 plays an important cytotoxic role. The resulting stably formed complex of PGRPs, S100A4 and Hsp70 is required for the identification and binding between a lymphocyte and a target cell. Here, we investigated the S100A4 functions in CD⁴⁺CD²⁵⁺PGRPs⁺S100A4⁺ lymphocyte cytotoxicity against target cells. We demonstrated that those lymphocytes do not form a stable complex with the tumor target cells that themselves have S1004A on their surface. That observation can be explained by our finding that S100A4 precludes the formation of a stable complex between PGRPs, S100A4 (on the lymphocytes’ surface), and Hsp70 (on the target cells’ surface). The decrease in S100A4 level in CD⁴⁺CD²⁵⁺PGRPs⁺S100A4⁺ lymphocytes inhibits their cytotoxic activity, while the addition of S100A4 in the medium restores it. Thus, the resistance of target cells to CD⁴⁺CD²⁵⁺PGRPs⁺ S100A4⁺ lymphocyte cytotoxicity depends on their S100A4 expression level and can be countered by S100A4 antibodies.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na⁺/Ca²⁺ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca²⁺ reabsorption in the kidney. (1) Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca²⁺ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca²⁺ reabsorption. (2) The vesicle preparation contained high, digitalis-sensitive (Na⁺+K⁺-ATPase activities indicating its origin from the basolateral portion of plasma membrane. (3) The operation of Na⁺/Ca²⁺ antiport device was excluded by the findings that steep Ca²⁺ gradients formed by ATP-dependent Ca²⁺ accumulation in the vesicles were not discharged by extravesicular Na⁺, and did not drive ⁴⁵Ca²⁺ uptake into the vesicles via a Ca²⁺-⁴⁵Ca²⁺ exchange. (4) The ATP-dependent Ca²⁺ uptake into the vesicles became increasingly depressed with time by extravesicular Na⁺. This was not due to an impairment of the Ca²⁺ pump itself, but caused by Na⁺/Ca²⁺ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca²⁺ accumulation in the vesicles. (5) Earlier observations on Na⁺-induced release of Ca²⁺ from vesicles pre-equilibrated with Ca²⁺, seemingly favoring the existence of a Na⁺/Ca²⁺ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na⁺/Ca²⁺ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca²⁺ reabsorption through tubule epithelium essentially depending on the operation of a Na⁺/Ca²⁺ antiport device.     

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Susceptibility of cerebellar granule neurons from GM2/GD2 synthase-null mice to apoptosis induced by glutamate excitotoxicity and elevated KCl: Rescue by GM1 and LIGA20

Our previous study showed an impaired regulation of Ca(2+) homeostasis in cultured cerebellar granule neurons (CGN) from neonatal mice lacking GM2, GD2 and all gangliotetraose gangliosides, due to disruption of the GM2/GD2 synthase (GalNAc-T) gene. In the presence of depolarizing concentration (55 mM) K(+), these cells showed persistent elevation of intracellular Ca(2+) ([Ca(2+)]( i )) leading to apoptosis and cell destruction. This was in contrast to CGN from normal littermates whose survival was enhanced by high K(+). In this study we demonstrate that glutamate has the same effect as K(+) on CGN from these ganglioside-deficient knockout (KO) mice and that apoptosis in both cases is averted by exogenous GM1. Even more effective rescue was obtained with LIGA20, a semi-synthetic derivative of GM1. LC(50) of glutamate in the KO cells was 3.1 microM, compared to 46 microM in normal CGN. [Ca(2+)]( i ) measurement with fura-2 revealed no difference in glutamate-stimulated Ca(2+) influx between the 2 cell types. However, reduction of [Ca(2+)]( i ) following application of Mg(2+) was significantly impaired in the mutant CGN. The rescuing effects of exogenous GM1 and LIGA20 corresponded to their ability to restore Ca(2+) homeostasis. The greater potency of LIGA20 is attributed to its greater membrane permeability with resultant ability to insert into both plasma and nuclear membranes at low concentration (相似文献