Isolation and characterization of membrane potential changes associated with release of calcium from intracellular stores in rat thymic lymphocytes

Membrane potential changes accompanying Ca²⁺ influx stimulated by release of Ca²⁺ from intracellular stores (store-regulated Ca²⁺ uptake) were monitored in BAPTA-loaded rat thymic lymphocytes using the fluorescent indicator bis(1,3-diethylthiobarbituric acid)trimethine oxonol. Depletion of [Ca²⁺] _i stores by the application of thapsigargin, ionomycin or cyclopiazonic acid induced a depolarization which was (i) dependent upon BAPTA-loading, (ii) dependent upon extracellular Ca²⁺, (iii) independent of extracellular Na⁺ and (iv) abolished by 5 mm extracellular Ni²⁺. This depolarization was followed by a charybdotoxin-sensitive repolarization and subsequent hyperpolarization to values approximating the K⁺ equilibrium potential, consistent with secondary activation of a K⁺ conductance. These membrane potential changes temporally correlated with Ca²⁺ influx from the extracellular medium as measured fluorimetrically with indo-1. The divalent cation permeability sequence was investigated by monitoring the magnitude of the depolarization observed following the addition of 4 mm Ca²⁺, Mn²⁺, Ba²⁺ or Sr²⁺ to cells pretreated with doses of thapsigargin or ionomycin known to activate the store-regulated calcium uptake pathway. On the basis of these experiments, we conclude that the store-regulated Ca²⁺ uptake pathway has the following permeability sequence: Ca²⁺ > Mn²⁺ Ba²⁺, Sr²⁺ with Mn²⁺ displaying significant permeability relative to Ca²⁺. This pathway is distinguishable from other divalent cation uptake pathways reported in other cells types on the basis of its activation by thapsigargin and its high Mn²⁺ permeability.This work is supported by grants from the American Heart Association, Louisiana Affiliate (LA-92-6-28), Louisiana Education Quality Support Fund (LEQSF(1993-96)-RD-A-31) and Tulane University Graduate Program in Molecular and Cellular Biology.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Solubilization of a divalent cation dependent ATPase from dog heart sarcolemma

Heart sarcolemma has been shown to contain an ATPase hydrolizing system which is activated by millimolar concentrations of divalent cations such as Ca²⁺ or Mg²⁺. Although Ca²⁺-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na⁺ + K⁺ ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca²⁺-stimulated Mg²⁺ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca^2– (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca²⁺ pump mechanism located in the plasma membrane of the cardiac cell.     

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     

A novel form of host defence: Membrane protection by Ca2+ and Zn2+

This review focusses on two questions: (1) How can the intracellular toxicity of ions such as Ca²⁺ or Zn²⁺ be reconciled with their extracellular benefit? (2) Why is the dietary requirement for Zn²⁺ so high when its documented biological role is that of a tightly-bound prosthetic group of certain enzymes? An answer to both questions is provided by the observation that extracellular cations such as Ca²⁺ and Zn²⁺ protect the plasma membrane of cells against non-specific leakage, including an influx of Ca²⁺ or Zn²⁺. It is suggested that such protection, against leakage induced by microbial and other toxins, may contribute to the high dietary requirement for zinc. These arguments lead to the proposal that a previously unrecognized form of host defence is one of protection of the cell plasma membrane by divalent cations against damage induced by cytotoxic agents of environmental origin.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

Stabilization of rat cardiac sacroplasmic reticulum Ca2+ uptake activity and isolation of vesicles with improved calcium uptake activity

Summmary The Ca²⁺ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca²⁺ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0° or 37°, but the Ca²⁺ uptake activity was relatively stable at 25°. The decay of Ca²⁺ uptake activity at 0° could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca²⁺ uptake activity of homogenates kept on ice but not of homogenates kept at 37°. We also found that release of the CSR from the cellular debris required homogenization in high KCI. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 mol/min-mg in the absence of CSR calcium channel blockers and 0.67 mol/min/mg in the presence of 10 M ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca²⁺-ATPase rates averaged 1.07 mol/min/mg and 0.88 mol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a crude preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca²⁺-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function.     

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.     

Insights into modulation of calcium signaling by magnesium in calmodulin, troponin C and related EF-hand proteins

The Ca²⁺-binding helix-loop-helix structural motif called “EF-hand” is a common building block of a large family of proteins that function as intracellular Ca²⁺-receptors. These proteins respond specifically to micromolar concentrations of Ca²⁺ in the presence of ~1000-fold excess of the chemically similar divalent cation Mg²⁺. The intracellular free Mg²⁺ concentration is tightly controlled in a narrow range of 0.5-1.0 mM, which at the resting Ca²⁺ levels is sufficient to fully or partially saturate the Ca²⁺-binding sites of many EF-hand proteins. Thus, to convey Ca²⁺ signals, EF-hand proteins must respond differently to Ca²⁺ than to Mg²⁺. In this review the structural aspects of Mg²⁺ binding to EF-hand proteins are considered and interpreted in light of the recently proposed two-step Ca²⁺-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-hand domain structure, the smaller Mg²⁺ ion cannot engage the ligands of an EF-hand in the same way as Ca²⁺ and defaults to stabilizing the apo-like conformation of the EF-hand. It is proposed that Mg²⁺ plays an active role in the Ca²⁺-dependent regulation of cellular processes by stabilizing the “off state” of some EF-hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca²⁺ levels. Therefore, some pathological conditions attributed to Mg²⁺ deficiency might be related to excessive activation of underlying Ca²⁺-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Effects of selenium deficiency on Ca transport function of sarcoplasmic reticulum and lipid peroxidation in rat myocardium

Two groups of weanling Sprague-Dawley rats were fed a low-selenium basal diet (Se 0.009 mg/kg) and the same diet supplemented with sodium selenite (Se 0.25 mg/kg), respectively, for 1, 2, and 3 months. At each feeding time, the Ca²⁺-ATPase activity, Ca²⁺ uptake rate and the capacity of Ca²⁺ uptake in isolated cardiac sacroplasmic reticulum from the Se-deficient rats were decreased significantly compared to those from the Se-supplemented rats, the contents of lipid peroxide in postmitochondrial supernatant and isolated sarcoplasmic reticulum from the Se-deficient rats were significantly higher than that from Se-supplemented rats. Compared to the Se-supplemented rats, the cytosolic glutathione peroxidase activity in Se-deficient rats decreased significantly. In addition, significant linear negative correlations of lipid peroxide in postmitochondrial supernatant to sarcoplasmic reticular Ca²⁺-ATPase activity, Ca²⁺ uptake rate and to whole blood selenium concentration were observed. The results suggest that the enhancement of lipid peroxidation via the depressed glutathione peroxidase activity might be responsible for the decrease of Ca²⁺-ATPase and Ca²⁺ uptake activities in sarcoplasmic reticulum in Se-deficient animals.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO⁻₃ together with a decrease in pCO₂, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of ⁴⁵Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca²⁺ and abolished at low extracellular Na⁺ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca²⁺. The removal of HCO⁻₃ and decrease in the pCO₂ also reduced the magnitude of both the secondary rise in ⁴⁵Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca²⁺ from islet cells as mediated by Na⁺-Ca²⁺ countertransport and the inflow of Ca²⁺ by gated Ca²⁺ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.     

Temperature-dependent modification of divalent cation flux in the rat parotid gland basolateral membrane

Divalent cation (Mn²⁺, Ca²⁺) entry into rat parotid acinar cells is stimulated by the release of Ca²⁺ from the internal agonist-sensitive Ca²⁺ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn²⁺ influx into internal Ca²⁺ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca²⁺-free medium for 10 min) and passive ⁴⁵Ca²⁺ influx in basolateral membrane vesicles (BLMV). Mn²⁺ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn²⁺ entry appeared to be inactivated since it was not increased by raising extracellular [Mn²⁺] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn²⁺ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q₁₀=1.7) at 21–37°C to 25 kcal/mol (Q₁₀=3.0) at 21-8°C. Under the same conditions, Mn²⁺ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E _a of 7.8 kcal/mol (Q₁₀=1.5) and 6.2 kcal/mol (Q₁₀ < 1.5), respectively. These data suggest that depletion-activated Mn²⁺ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn²⁺ entry into unstimulated acini.As in intact acini, Ca²⁺ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca²⁺ influx in BLMV at 37°C demonstrated the presence of two Ca²⁺ influx components: a saturable component, with K _Ca =279 ± 43 m, V_max = 3.38 ± 0.4 nmol Ca²⁺/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but V_max decreased by 61% to 1.3 ± 0.4 nmol Ca²⁺/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca²⁺ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca²⁺ uptake at 37°C there was no significant loss of Ca²⁺ influx. These data suggest that the temperature sensitive high affinity Ca²⁺ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca²⁺ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work.     

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.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells

Summary Using Ca²⁺- and K⁺-selective microelectrodes, the cytosolic free Ca²⁺ and K⁺ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca²⁺ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca²⁺ concentration were observed in the submicromolar ranges. During the Ca²⁺ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca²⁺ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca²⁺ concentration down to 10^–7 m or by applying a Ca²⁺ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca²⁺-activated K⁺ channel, sizable Ca²⁺ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K⁺ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K⁺ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca²⁺ level, which, in turn, periodically stimulate Ca²⁺-activated K⁺ channels.     

高锶离子亲和力传感蛋白介导CALYX突触囊泡异步和自发释放

突触囊泡在钙离子(Ca2+)触发下释放神经递质普遍存在着同步和异步两种形式.突触囊泡膜蛋白(synaptotagmin 2,Syt-2)已被证实是Calyx of Held突触囊泡同步释放的Ca2+传感蛋白,而相关的异步释放Ca2+传感蛋白还有待于探索.虽然锶离子(Sr2+)因其物理和化学性质都接近Ca2+,且能触发更多的囊泡异步释放成分而成为研究异步释放机制的常用工具,但有关Sr2+触发异步释放的机制存在着争议.本文在胞外以Sr2+替换Ca2+的条件下,通过对野生型(WT)和Syt-2敲除型(Z2B-/-)小鼠Calyx突触囊泡自发和诱发释放的电生理特性分析,发现Syt-2是介导Sr2+诱发的突触囊泡快速释放的传感蛋白,但不是介导Sr2+相关神经递质异步释放和自发释放的传感蛋白;而未知的触发囊泡异步释放的传感蛋白相比Syt-2对Sr2+具有更高的亲和力,同时也介导突触囊泡的自发释放.这一研究为探索并最终发现触发囊泡异步释放的未知传感蛋白提供了新的线索.     

Proportions of Ca2+ Channel Subtypes in Chick or Rat P2 Fraction and NG108-15 Cells Using Various Ca2+ Blockers

The proportions of calcium (Ca²⁺) channel subtypes in chick or rat P₂ fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca²⁺ channel blockers. KCl-stimulated ⁴⁵Ca²⁺ uptake by chick P₂ fraction was blocked by 40~50% using N-type Ca²⁺ channel blockers [-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1–13)], but was not inhibited by P- or P/Q-type blockers (-agatoxin IVA or -conotoxin MVIIC). On the other hand, KCl-stimulated ⁴⁵Ca²⁺ uptake by rat P₂ fraction was blocked by 30~40% using P- or P/Q-type Ca²⁺ channel blockers, but was not inhibited by N-type Ca²⁺ channel blockers. The L-type Ca²⁺ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated ⁴⁵Ca²⁺ uptake and veratridine-induced ²²Na⁺ uptake by chick or rat P₂ fraction with similar IC₅₀ values. CaS did not have any effect on ⁴⁵Ca²⁺ uptake by either chick or rat P₂ fraction. In NG108-15 cells, CaS, -agatoxin IVA and -conotoxin MVIIC, but not -conotoxin GVIA, inhibited KCl-stimulated ⁴⁵Ca²⁺ uptake by 30–40%. Various combinations of these Ca²⁺ channel blockers had no significant additional effects in chick or rat P₂ fraction or NG 108-15 cells. These findings suggest that KCl-stimulated ⁴⁵Ca²⁺ uptake by chick or rat P₂ fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca²⁺ channel antagonists or agonists.