Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca²⁺-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with K_m for Ca ions of 0.4 M and V_max=12.5±2.4 mol P_i.mg^–1h^–1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive³H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with K_i=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >ZnCo>Ni. In contrast to low affinity (high K_m) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 M) and N-ethylmaleinimide (100 M). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.Abbreviations used BSA bovine serum albumin - HA Ca-ATPase high-affinity Ca²⁺-ATPase - K-pNPPase onabain-sensitive, K⁺-stimulated p-nitrophenyl phosphatase - NEM N-ethylmaleinimide - SIM 250 mM sucrose, 10 mM imidazole-HCl pH 7.4 - TRIS tris (hydroxymethyl) aminomethane     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca²⁺ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca²⁺) and high affinity (50 µM Ca²⁺) Ca²⁺ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca²⁺ ATPase activities measured at 1.25 mM and 4 mM Ca²⁺ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca²⁺ binding and Ca²⁺ ATPase activities, whereas the methylation-induced effect on the high affinity Ca²⁺ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca²⁺ binding and Ca²⁺ ATPase. A further decrease in Ca²⁺ binding and Ca²⁺ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na⁺-K⁺ ATPase and Mg²⁺ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca²⁺ ATPase and low affinity ATP-independent Ca²⁺ binding.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

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.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

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.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Participation of Intracellular Ca2+ Stores in Ca2+ Signalling in Neurons of the Thalamic Laterodorsal Nucleus of Rats

Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca²⁺ ions in the cytoplasm ([Ca²⁺] _i ) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca²⁺] _i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca²⁺] _i was observed in all tested neurons. To study the contribution of Ca²⁺-induced Ca²⁺ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Unitary Ca2+ Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca²⁺ currents were monitored when 2–30 mM Ca²⁺ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca²⁺ current (at 0 mV holding potential) and lumenal [Ca²⁺] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH₃SO₃ concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca²⁺; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca²⁺ (n > 6). Unitary Ca²⁺ current was also defined in the presence of symmetrical [Mg²⁺] (1 mM) and low [Cs⁺] (5 mM). Under these conditions, unitary Ca²⁺ current in 2 and 10 mM lumenal Ca²⁺ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs⁺] (50 mM), Mg²⁺ (1 mM), and lumenal [Ca²⁺] (10 mM), unitary Ca²⁺ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs⁺ and Mg²⁺ on unitary Ca²⁺ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg²⁺ effectively compete with Ca²⁺ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca²⁺ is 2 mM, then our results indicate that unitary Ca²⁺ current under physiological conditions should be <0.6 pA.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Ca2+ transport and chemoreception in Paramecium

Intracellular Ca²⁺ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca²⁺ pump and that a Ca²⁺-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca²⁺ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca²⁺ homeostasis and pump activity.Abbreviations EGTA ethyleneglycol tetra-acetate - ER endoplasmic reticulum - IBMX isobutyl methylxanthine - I _che index of chemokinesis - Mops 3-[N-morpholino] propanesulfonic acid - PEI phosphoenzyme intermediate - STEN sucrose, TRIS, EDTA, sodium chloride - TCA trichloroacetic acid - TRIS tris[hydroxymethyl] aminomethane