Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

Effects of interrupted K+ supply on growth and uptake of K+, Ca2+, Mg2+ and Na+ in spring wheat

Effects of interrupted K⁺ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K⁺ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K⁺ supply. Root structure was greatly affected by the length of the period in K⁺ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K⁺ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K⁺ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K⁺. In comparison, both higher and lower internal/external K⁺ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca²⁺, Mg²⁺ and Na⁺ uptake. Uptake of Ca²⁺ and especially Mg²⁺ significantly increased after K⁺ interruption, while Na⁺ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K⁺ in the cells and maintain electroneutrality down to a certain minimal range of K⁺ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)^?1] than in the root [20 to 30 μmol (g fresh weight)^?1]. It is suggested that the critical K⁺ values are a measure of the minimal amount of K⁺ that must be present for physiological activity in the cells. At the critical levels, K⁺ (⁸⁶Rb) influx and Ca²⁺ and Mg²⁺ concentrations were maximal. Below the critical K⁺ values, growth was reduced, and Ca²⁺ and Mg²⁺ could no longer substitute for K⁺ for electrostatic balance. In a short-term experiment, the ability of Ca²⁺ to compete with K⁺ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K⁺ status. The results indicate that K⁺, which was taken up actively and fastest at the external K⁺ concentration used (2.0 mM), partly determines the size of Ca²⁺ influx.     

Effects of cytoskeletal modifications on Ca2+ influx after cerebral ischemia

Summary.  The fungal toxin cytochalasin D as well as endogenous gelsolin depolymerize filamentous actin which may induce dynamic uncoupling of membrane ion channels. In vitro application of cytochalasin D reduced NMDA-induced [³H]noradrenaline release from mouse brain neocortical slices by 38%. In gsn deficient neocortical synaptosomes [Ca²⁺]_i increase in response to K⁺ (30 mM) depolarization was 33% higher than in wild-type. After transient focal cerebral ischemia K⁺-induced [Ca²⁺]_i increase in neocortical synaptosomes was 56% lower than in synaptosomes prepared from the non-ischemic contralateral hemisphere. After in vivo pretreatment with cytochalasin D 10 min before MCA occlusion K⁺-induced [Ca²⁺]_i increase in synaptosomes in vitro prepared 1 h after reperfusion from the ischemic hemisphere was only 25% lower than in contralateral synaptosomes, while cytochalasin D pretreatment in vivo did not reduce K⁺-induced [Ca²⁺]_i increase in vitro. Hence, presynaptic Ca²⁺ influx and subsequently neuronal vulnerability are attenuated by increased and are aggravated by decreased F-actin depolymerization. Received June 29, 2001 Accepted August 6, 2001 Published online August 9, 2002     

Appearance of Depolarization- and Maitotoxin-Induced [Ca2+]i Elevation in Single LAN-1 Human Neuroblastoma Cells on Exposure to Retinoic Acid

Abstract: LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca²⁺]_i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca²⁺]_i elevation elicited by 55 mM K⁺. Maitotoxin, a putative activator of voltage-sensitive Ca²⁺ channels, did not evoke an elevation of [Ca²⁺]_i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca²⁺]_i when superfused in retinoic acid-treated cells. Both high K⁺- and maitotoxin-induced [Ca²⁺]_i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd³⁺, an inorganic Ca²⁺-entry blocker, and by the snail toxin ω-conotoxin GVIA, which interacts with the N sub-type of voltage-sensitive Ca²⁺ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel sub-type, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca²⁺]_i due to Ca²⁺ influx elicited by membrane depolarization. K⁺-induced [Ca²⁺]_i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K⁺-induced increase of [Ca²⁺]_i was still present. In conclusion, the results of the present study demonstrated that retinoic acid-induced differentiation of LAN-1 cells, which lack a high K⁺-evoked [Ca²⁺]_i increase in the undifferentiated state, induces the functional expression of an ω-conotoxin GVIA-sensitive, dihydropyridine-insensitive N-type voltage-sensitive Ca²⁺ channel that can be activated by maitotoxin and negatively modulated by protein kinase C.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

Summary Using intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged –46.1±0.6 mV (sem). (2) Increasing extracellular K⁺ concentration ([K⁺] _o ) depolarizedV. Addition of Ba²⁺ could diminish this response. (3) Depolarization on doubling [K⁺] _o was increased at higher [K⁺] _o (or low voltage). (4) Removing extracellular Ca²⁺ decreasedV and reduced theV amplitude on increasing [K⁺] _o . (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K⁺] _o were reduced at acidic extracellular pH. (6) Removing K _o ⁺ depolarized, K _o ⁺ readdition after K⁺ depletion transiently hyperpolarizedV. These responses were insensitive to Ba²⁺ but were abolished in the presence of ouabain or in Na⁺-free medium. (7) Na⁺ readdition after Na⁺ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K⁺-free solution but unchanged by Ba²⁺. It is concluded that in cultured bovine pigmented ciliary epithelial cells K⁺ conductance depends on Ca²⁺, pH and [K⁺] _o (or voltage). An electrogenic Na⁺/K⁺-transport is present, which is stimulated during recovery from K⁺ or Na⁺ depletion. This transport is inhibited by ouabain and in K⁺-or Na⁺-free medium.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

The Extracellular K+ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na+ and Ca2+

It has been known for more than three decades that outward Kir currents (I_K1) increase with increasing extracellular K⁺ concentration ([K⁺]_o). Although this increase in I_K1 can have significant impacts under pathophysiological cardiac conditions, where [K⁺]_o can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]_o dependence of Kir2.1-mediated outward I_K1 was due to [K⁺]_o-dependent inhibition of outward I_K1 by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of I_K1 through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I_K1 in a similar [K⁺]_o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward I_K1 by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]_o dependence of Kir2.1-mediated outward I_K1.     

Light-induced cytosolic calcium transients in green plant cells. ll. The effect on a K+ channel as studied by a kinetic analysis in Chara corallina

The fluorescent dye chlorotetracycline was used to study the relationship between the light-induced decrease in cytosolic free calcium concentration, [Ca²⁺]_c, and its effect on ion transport at the plasma membrane in the giant cells of Chara corallina Klein ex Willd. A kinetic analysis of the simultaneously measured light-induced changes in membrane potential and in [Ca²⁺]_c led to the same time constant of about 40 s. The reversal potential of the light effect on membrane potential was in agreement with the dominant role of a K⁺ channel in the plasma membrane. Thus, the experiments reported here provide evidence for the following light-driven signal transduction chain from the chloroplasts to K⁺ transport of the plasma membrane: (i) light causes an uptake of Ca²⁺ into the chloroplasts, (ii) this causes a decrease in cytosolic [Ca²⁺]_c, (iii) this leads to a decrease in the activity of a K⁺ channel. The results also initiated a re-analysis of previously published data of the light effect on the velocity of cytosolic streaming and supported the hypothesis that Ca²⁺ fluxes coming out of the chloroplasts upon darkening cause a Ca²⁺-induced phosphorylation of myosin, which slows down cytoplasmic streaming. Received: 3 May 1997 / Accepted: 19 May 1998     

Influence of Abscisic Acid on K+ absorption by leaf tissues ofSolanum tuberosum L. (cv. Bintje). Relation with calcium fluxes and with calmodulin

Previous results with potato leaf tissues revealed that a treatment with ABA (10^-4 M) induced an increase of K⁺ uptake (Suleimanet al., 1990a). In this study, we investigate the relationship between increased K⁺ uptake, Ca²⁺ fluxes and calmodulin by treating potato leaf discs with Ca²⁺ channel blockers (La³⁺, verapamil and nifedipine) and with calmodulin inhibitors (chlorpromazine, W₇: N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and compound 4880). We found : a) all these inhibitors decreased K⁺ uptake in the same ratio in tissues treated or not with ABA; b) a synergistic effect between ABA and the Ca²⁺ ionophore A 23187 on K⁺ uptake. The results suggest that the hormone may act on K⁺ uptake like a Ca²⁺ agonist, in accord with HUDDART’s hypothesis.     

Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation

In guard cells, activation of anion channels (I_anion) is an early event leading to stomatal closure. Activation of I_anion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the dynamics of the action of [Ca²⁺]_i on I_anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca²⁺]_i dynamics of I_anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca²⁺]_i using Fura‐2 fluorescence imaging. We found that I_anion rises with [Ca²⁺]_i only at concentrations substantially above the mean resting value of 125 ± 13 nm , yielding an apparent K_d of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca²⁺ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I_anion activity, but without a dependence of the current on [Ca²⁺]_i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca²⁺]_i sensitivity of I_anion, displacing the apparent K_d for [Ca²⁺]_i to 573 ± 38 nm . These findings support previous evidence for different modes of regulation for I_anion, only one of which depends on [Ca²⁺]_i, and they underscore an independence of [Ca²⁺]_i from protein (de‐)phosphorylation in controlling I_anion. Most importantly, our results demonstrate a significant displacement of I_anion sensitivity to higher [Ca²⁺]_i compared with that of the guard cell K⁺ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Ciliary reversal inParamecium: Temperature dependence of K+-induced excitability decrease and of recovery

Summary The duration of K⁺-induced reversed swimming ofParamecium caudatum was used as a measure of excitability. The time course of excitability decrease following an exposure to high [K⁺]_o and of the subsequent recovery in a solution devoid of K⁺ was measured at various temperatures.When the temperature was increased during the exposure to K⁺ the loss of excitability increased with a Q₁₀ of ca. 1.3 (Fig. 2). The recovery of excitability was enhanced with a Q₁₀ of 2.0–3.0 (Fig. 4). The renormalization of ciliary beat in K⁺-rich medium showed Q₁₀-values between 2.1 and 2.5 (Fig. 5).The observed alterations in excitability are interpreted in terms of passive and active Ca²⁺ fluxes. Ca channels in the excitable membrane are assumed to inactivate as a result of increased [Ca²⁺]_i. Recovery of excitability is thought to be mediated by active Ca²⁺ extrusion.I would like to thank Mrs. A. Müller-Holtkamp, who carried out the experiments, for her invaluable help. This work is part of the research program SFB 160 of the Deutsche Forschungsgemeinschaft.     

Properties of Ca2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca²⁺ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)₂D₃ repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca²⁺ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg²⁺ and Sr²⁺, but not by Na⁺ or K⁺. Lowering the external [H⁺] or raising the internal [H⁺] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H⁺] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl₂ or SrCl₂ but not CaCl₂, NaCl, or KCl. Vesicles preloaded with K₂SO₄ failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl₂ was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca²⁺ uptake maintained at equilibrium. Therefore, the remainder of the Ca²⁺ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10^?6m was found for this binding. The bound Ca²⁺ could be rapidly released by external Mg²⁺ or Sr²⁺, but not Ca²⁺, Na⁺, or K⁺. Release of bound Ca²⁺ could also be induced by raising the [H⁺] of the external medium. Failure of external Ca²⁺ to release bound Ca²⁺ suggested that the release induced by external Mg²⁺, Sr²⁺, or H⁺ was not due to competitive displacement of Ca²⁺ from its binding sites. These results indicated that Ca²⁺ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca²⁺ to the vesicle. The effects of H⁺, Mg²⁺, and Sr²⁺ on Ca²⁺ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

Abstract: The effect of replacement of extracellular Na⁺ with N-methyl-d -glucamine (NMG) on P₂ receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) with an EC₅₀ value of 230 µM. Replacement of Na⁺ with NMG as well as removal of Mg²⁺ from the bathing buffer potentiated ethidium bromide uptake, [Ca²⁺]_i increase, and ⁴⁵Ca²⁺ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg²⁺ to limit the amount of ATP^4?, replacement of Na⁺ with NMG had no effect on the ATP-induced [Ca²⁺]_i increase but caused a markedly larger [Ca²⁺]_i increase when the calculated concentration of ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? stimulation of the [Ca²⁺]_i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca²⁺ release was the major pathway for the ATP-induced [Ca²⁺]_i increase; both removal of Mg²⁺ and replacement of Na⁺ with NMG did not affect the action of ATP. These data suggest that ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.