Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

Chronic inhibition of cortex microsomal Mg2+/Ca2+ ATPase-mediated Ca2+ uptake in the rat pilocarpine model following epileptogenesis

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.     

Characterization of a Ca2+-translocating ATPase from corn root microsomes

Brauer, D., Schubert C. and Tu, S,-I. 1990. Characterization of a Ca²⁺-translocating ATPase from corn root microsomes. - Physiol. Plant. 78: 335-344.
The existence of a Ca²⁺-translocating ATPase in microsomes from maize ( Zea mays L. cv, WF9 × Mo17) roots was evaluated using assays to follow Ca²⁺-stimulation of ATP hydrolysis and Ca²⁺ transport by changes in the fluorescence of chlorotetracycline, ATP hydrolysis by microsomes was stimulated by the addition of Ca²⁺ and further enhanced by the Ca ionophore A23187 and bovine brain calmodulin only in the presence of Ca²⁺, Stimulation by these agents was additive and sensitive to vanadate. These results were consistent with the presence of a Ca²⁺-translocating ATPase in microsomal membranes. The fluorescence of chlorotetracycline in the presence of microsomes and Ca²⁺ increased upon the addition of ATP, indicating the transport of Ca²⁺, The initial rate and extent of change in fluorescence were stimulated by calmodulin and quenched by the addition of either A23187 or EGTA, but not by protonophores. Changes in chlorotetracycline fluorescence were prevented by vanadate. Therefore, results using chlorotetracycline also indicated the presence of a Ca²⁺-translocating ATPase, Localization experiments indicated that the majority of the Ca²⁺-translocating ATPase was on the endoplasmic reticulum.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101, 401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69–75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca⁺⁺ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H⁺ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H⁺ ATPases.     

Temperature dependence of the barley root plasma membrane-bound Ca2+- and Mg2+-dependent ATPase

Arrhenius plots of the maximal velocities for the Ca²⁺-and Mg²⁺-dependent ATPase activities found in a plasma membrane-rich microsome fraction isolated from the roots of barley ( Hordeum vulgare L. cv. Conquest) were nonlinear. Arrhenius plot analyses using a relation which produced curvilinear Arrhenius plots accurately fit the data and allowed the calculation of the activation enthalpies and molar heat capacities of activation. The temperature dependence of the computed Km values for the Ca²⁺- and Mg²⁺-dependent ATPase activities was complex, with the highest enzyme-substrate affinities being obtained near the barley seedling growth temperature (16°C). Using electron paramagnetic resonance spectroscopy with amphiphilic cationic and anionic spin probes, it was possible to demonstrate that temperature changes and increasing Ca²⁺ concentrations could alter the mobility of the membrane lipid polar head groups. Inhibition of the ATPase activities by high levels of Ca²⁺ may result from a Ca2+-induced reduction in the lipid polar head group mobility. The possible role of lipid polar head group-protein interactions in the complex temperature dependence of the barley root ATPase kinetic constants is discussed.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Hormonal Regulation of Ca2+ Stimulated K+ Influx and Ca2+, K+- ATPase in Rice Roots: in vivo and in vitro Effects of Auxins and Reconstitution of the ATPase

The role of natural and synthetic auxins in regulation of ion transport and ATPase activity was studied in rice roots (Oryza sativa L. cv. Dunghan Shah). In vivo treatment of seedlings with 2,4-dichlorophenoxyacetic acid at 2 × 10^?6M for a short period enhanced subsequent Ca²⁺ stimulated K⁺ influx and ATPase activity, while a longer treatment diminished both K⁺ influx and ATPase activity. Indoleacetic acid at 10^?10–10^?8M induced ATPase activity. In in vitro experiments both 2,4-dichloro phenoxyacetic acid and indoleacetic acid (10^?10–10^?8M) stimulated Ca²⁺, K⁺-ATPase activity of a plasmalemma rich micro somal fraction from the roots. Acetone extracted ATPase preparations lost their activity. The enzyme regained its activity and its sensitivity towards ions (Ca²⁺+ K⁺) when reconstituted with phosphatidyl choline. Addition of auxins also indicated that the presence of the lipid was necessary in the interaction between the ATPase and auxins. Auxins and ions probably interact with the intact ATPase lipoprotein complex, which may possess a receptor site for the auxins, possibly as a sub unit.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

The effect of Ca2+-stress on fluxes of Ca2+ and K+ in wheat and cucumber

Betula papyrifera Marsh, seedlings adapted very poorly to flooding for up to 60 days. Responses to flooding included increased ethylene production; stomatal closure; leaf senescence; drastic inhibition of shoot growth, cambial growth, and root growth; decay of roots, and death of many seedlings. Flooding inhibited growth of leaves that formed prior to flooding, inhibited formation of new leaves, and induced abscission of old leaves. As a result of extensive leaf abscission, fewer leaves were present after flooding than before flooding was initiated. The drastic reduction in leaf area was associated with greatly decreased growth of the lower stem and roots. No evidence was found of adaptive morphological changes to flooding. The data indicate that intolerance of B. papyrifera seedlings to flooding is an important barrier to regeneration of the species on sites subject to periodic inundation.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Ca2+- or Mg2+-Stimulated ATPase Activity in Bullfrog Spinal Nerve: Relation to Ca2+ Requirements for Fast Axonal Transport

Adenosine triphosphatase (ATPase) activity stimulated by Ca2+ or Mg2+ was characterized in spinal nerve and spinal sensory ganglion of bullfrog. Enzyme activity of homogenates from both sources reached a maximum at a 1-2 mM concentration of either cation, although the level of maximal activity in nerve trunks was approximately twice that in ganglia. Enzyme activation was not observed with 2 mM-Sr2+ or Ba2+. Co2+ or Mn2+, at 2 mM, depressed Ca2+ activation of the enzyme by 50-60% in nerve but had no inhibitory effect on ganglia activity. In intact spinal ganglion/spinal nerve preparations, incubated for 20 h in medium containing 0.2 mM-Co2+, no effect was detected on Ca2+/Mg2+ ATPase activity in ganglia or nerve trunks whereas fast axonal transport was inhibited by 80%. Incubation in medium containing 0.02 mM-Hg2+ depressed enzyme activity in ganglia by 64% and in nerve trunks by 44%, whereas fast transport was again inhibited by 80%. When only nerve trunks were exposed to these ions, Hg2+ but not Co2+ was observed to slow the rate of fast axonal transport. The divalent cation specificity of the Ca2+/Mg2+ ATPase activity is distinct from the ion specificities, determined in previous work, of the Ca2+ requirement during initiation of fast axonal transport in the soma, and of the Ca2+ requirement during translocation in the axon. Thus, previous observations of Ca2+-dependent events in fast axonal transport cannot be taken per se to suggest the involvement of Ca2+/Mg+ ATPase in the transport process.     

Kinetic characterization of barley root plasma membrane-bound Ca2+- and Mg2+-dependent adenosine triphosphatase activities

A plasma membrane-rich microsome fraction isolated from barley (Hordeum vulgare L. cv. Conquest) roots contained considerable divalent cation-dependent ATPase activity when assayed at 16°C. The maximal divalent cation-stimulation of the apparent basal ATPase activity varied as Ca²⁺ > Mg²⁺ > Mn²⁺= Zn²⁺ > Co²⁺ > Ni²⁺, with all other divalent cations tested being inhibitory. Double reciprocal plots of the Ca²⁺- and Mg²⁺-dependent ATPase velocities as a function of substance concentration were nonlinear, suggesting the presence of multiple catalytic sites. Both MgATP^2- and CaATP^2- served as the true substrates and apparently bind to the same catalytic sites. Free ATP and Ca²⁺ could inhibitit the Ca²⁺- and Mg²⁺-dependent ATPase. Increasing free Mg²⁺ levels enhanced the affinity of the Mg²⁺-dependent ATPase for MgATP^2-, while slightly inhibiting the V_max values. Other divalent cation-nucleoside triphosphate complexes produced maximal enzyme velocities equal to or greater than those generated by CaATP^2- and MgATP^2-. However, the ATPase had significantly higher affinities for CaATP^2- and MgATP^2-, than for the alternative substrates. The high and low affinity components of the Ca²⁺- and Mg²⁺-dependent ATPase exhibited optimal V_max values at pH 5 and 6, respectively. Analysis of the pH-dependence of the enzyme K_m values indicated enzyme-substrate binding with charge neutralization at neutral and alkaline pH's. Nonlinear double reciprocal plots were obtained at all assay temperatures. However, the complexity of the enzyme kinetics became less apparent at the higher assay temperatures. The kinetics of the barley root divalent cation-dependent ATPase activities are discussed in terms of the kinetics of ATPases from other plants and the methods used to obtain them, and compared to the kinetics of ion transport ATPases from animal membranes.     

Relation of Acetylcholine Release to Ca2+ Uptake and Intraterminal Ca2+ Concentration in Guinea-Pig Cortex Synaptosomes

[14C]Acetylcholine (ACh) release and parallel alterations in 45Ca2+ uptake and intrasynaptosomal free CA2+ concentration ([Ca2+]i) were measured in guinea-pig brain cortex synaptosomes. Depolarization by high K+ concentrations caused a rapid transient increase in Ca2+ uptake, terminating within 60 s (rate constant = 0.060 s-1; t1/2 = 11.6 s). This resulted in a rapid increase (within 1 s) in [Ca2+1]i, which then fell to a maintained but still-elevated plateau level (t1/2 for the decline was 15 s). Peaks of [Ca2+]i showed a sigmoidal dependence on depolarization, contrasting with the simple linear dependence of plateau levels of [Ca2+]i. The K+-evoked ACh release also had two phases: a fast initial increase (t1/2 = 11.3 s), which terminated within 60 s, was followed by a slow additional increase during sustained depolarizations of up to 10 min. Depolarization by veratridine led to a slow gradual increase in Ca2+ uptake (t1/2 = 130 s) over a 10-min incubation period, whereas an elevated plateau level of [Ca2+]i was achieved within 2 min (without a rapid peak elevation). The Ca2+-dependent fraction of the veratridine-evoked ACh release correlated with the increase in [Ca2+]i rather than with Ca2+ uptake. Using two different methods of depolarization partially circumvented the time limitations imposed by a buffering Ca2+ indicator and we suggest that, in the main, ACh is released in bursts associated with [Ca2+]i transients.     

Allosteric Activation off Brain Mitochondrial Ca2+ Uptake by Spermine and by Ca2+: Developmental Changes

Kinetic analysis of 45Ca2+ uptake by rat brain mitochondria in Ca2+ - 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that spermine both increased the apparent affinity for Ca2+ and decreased the cooperativity of uptake. Both effects are consistent with an allosteric activation of uptake by spermine. The stimulating effect of spermine on 45Ca2+ uptake was maximal with mitochondria from postnatal day 10 animals and then steadily decreased with increasing age to reach adult values by approximately 30 postnatal days; this was observed independently of the substrates used to fuel mitochondria. Mitochondrial Ca2+ buffering was also analyzed by use of a Ca2+-selective electrode. Addition of a large bolus of Ca2+ produced a decrease in the subsequent equilibrium extramitochondrial Ca2+ concentration (or a "rebound overshoot") under some conditions. It is proposed that this effect is the result of an allosteric activation of Ca2+ uptake by Ca2+. This effect was slowly reversible, or hysteretic, and was blocked by spermine. The overshoot was increased in the presence of higher concentrations of Mg2+ and was absent when mitochondria were incubated with 0.3 mM Mg2+. It was maximal in mitochondria prepared from early postnatal brain, and changes in the magnitude of the effect during development paralleled those obtained with spermine stimulation of 45Ca2+ uptake. The data suggest that spermine produces an allosteric activation of Ca2+ uptake by binding to the same regulatory sites that are involved in the Ca2+-induced activation. The results as a whole suggest that spermine could modulate mitochondrial buffering of the intracellular Ca2+ concentration in brain, particularly during the early postnatal period.