45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Ca2+ UPTAKE BY SYNAPTOSOMES AND ITS EFFECT ON THE INHIBITION OF ACETYLCHOLINE RELEASE BY BOTULINUM TOXIN

Abstract— ⁴⁵Ca²⁺ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K⁺ at all times investigated. The accumulated ⁴⁵Ca²⁺ was bound within the synaptosome. ⁴⁵Ca²⁺-ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal ⁴⁵Ca²⁺ then returned to control values. A23187 also increased intra-synaptosomal Na⁺ and Cl⁻ contents. Botulinum toxin inhibits the K.⁺-stimulated release of [¹⁴C]ACh from synaptosomes but the ionophore released [¹⁴C]ACh from both normal and botulinum-treated preparations in a Ca²⁺-dependent manner. However, it also elicited Ca²⁺-dependent release of [choline. Increased extracellular Ca²⁺ (10 mM and 20 mM) released [¹⁴C]ACh (but not [¹⁴C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca²⁺ essential for the mechanism of ACh release.     

Calcium ion uptake by Methanobacterium thermoautotrophicum: Evidence for a carrier-mediated uptake system

Abstract Cell suspensions of Methanobacterium thermoautotrophicum took up ⁴⁵Ca²⁺ in a temperature-dependent, Ca²⁺-saturable and Co²⁺-sensitive process. The accumulation of ⁴⁵Ca²⁺ was lower in the cells energized by CO₂+ H₂ than in those under non-energized conditions. The accumulated Ca²⁺ were, in part, released by the divalent cations ionophore A23187 in the presence of EGTA while the uptake of Ca²⁺ was accelerated by the addition of A23187 to the medium containing Ca²⁺. The results indicate the presence of a carrier-mediated Ca²⁺ uptake in the Methanobacterium thermoautotrophicum membrane which is compensated by an energy-dependent and outward-directed Ca²⁺ transport.     

Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3-Ditolylguanidine

Abstract: The σ ligand 1,3-di- O -tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and blocked the depolarization-dependent increases in [Ca²⁺]_i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-( p -chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca²⁺]_i and the KCl-evoked rise in [Ca²⁺]_i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca²⁺]_i was not mediated via downstream regulation of cyclic GMP levels. DTG increased ⁴⁵Ca²⁺ uptake and efflux under basal conditions and inhibited the ⁴⁵Ca²⁺ uptake induced by depolarization with KCl. The KCl-evoked rise in [Ca²⁺]_i was inhibited by ω-conotoxin (ω-CgTx)-GVIA and -MVIIC but not nifedipine and ω-agatoxin-IVA. The effect of DTG on decreasing the KCl-evoked rise in [Ca²⁺]_i was additive with ω-CgTx-MVIIC but not with ω-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca²⁺ levels via inhibition of N-type Ca²⁺ channels.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

GM1 Ganglioside in the Nuclear Membrane Modulates Nuclear Calcium Homeostasis During Neurite Outgrowth

Abstract: GM1 in the nuclear membrane, previously shown to be up-regulated during neurite outgrowth, has been found to influence nuclear Ca²⁺ flux during differentiation of Neuro-2a cells. Nuclei were isolated from cultured Neuro-2a cells before and after neuraminidase-induced neuritogenesis and incubated with ⁴⁵Ca²⁺ for varying periods to determine uptake/efflux of Ca²⁺. At 5, 10, and 15 min ⁴⁵Ca²⁺ levels in nuclei from differentiated cells were significantly lower than those in nuclei from untreated cells. The same result was obtained when the GM1 level was elevated artificially by preincubation of the nuclei in 10 µ M GM1. In experiments designed to measure efflux specifically, isolated nuclei preincubated in GM1 released ⁴⁵Ca²⁺ more rapidly than untreated nuclei. We conclude that one role of GM1 in the nuclear membrane is to alter Ca²⁺ regulatory mechanisms in the nucleus following onset of neuronal process outgrowth.     

The Effects of Electroshock Convulsions on Calcium Transport within Synaptic Terminals

Abstract: Calcium transport was assessed within synaptic terminals isolated from cerebral cortices of rats which experienced one maximal electroshock (ES) convulsion daily. No significant change in calcium content [(Ca₁)] of synaptosomes was present after 2 consecutive days of maximal convulsions. After 4 and 6 days of maximal seizures, (Ca₁) rose 20% and 37%, respectively. ¹⁵Ca²⁺ influx within synaptosomes in vitro increased after 6 days of ES convulsions (1.94 ± 0.4 μmol/g protein/min in ES convulsions versus 1.54 ± .03 μmol/g protein/min in controls). The higher rate of ⁴⁵Ca²⁺ influx in convulsed animals was accounted for by elevated internal sodium [(Na₁)] values. Maximal ⁴⁵Ca²⁺ efflux decreased after ES convulsions (0.48 μmol/g protei/min in ES convulsions versus 0.8 μmol/g protei/min in controls). The slower rate of ⁴⁵Ca²⁺ efflux after convulsions was also accounted for by elevated (Na₁). Our results suggest that (Ca₁) increased within synapses after in vivo ES convulsions secondary to a primary ionic event, namely, elevated (Na₁).     

Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina

Abstract: The effect of melatonin on [³H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 n M ) significantly increased [³H]glutamate uptake, and this effect persisted in a Ca²⁺-free medium. On the other hand, melatonin significantly increased [³H]glutamate release in retinas excised at 2400 h, but this effect was Ca²⁺ sensitive. Melatonin significantly increased ⁴⁵Ca²⁺ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [³H]glutamate uptake, [³H]glutamate release, or ⁴⁵Ca²⁺ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'- O -dibutyrylguanosine 3',5'-cyclic monophosphate, significantly increased [³H]glutamate uptake, [³H]glutamate release, and ⁴⁵Ca²⁺ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

SODIUM AND THE FLUX OF CALCIUM IONS IN ELECTRICALLY-STIMULATED CEREBRAL TISSUE

Abstract— The rate of efflux of ⁴⁵Ca²⁺ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of ⁴⁵Ca²⁺ from the more stable pool and the time course for the redistribution of Na⁺ and K⁺ paralleled that for the increased efflux of Ca²⁺. This effect of stimulationwas dependent on the presence of Na⁺ in the incubation medium. Lack of Na⁺ in the medium during loading of the slices with ⁴⁵Ca²⁺ increased uptake but on subsequent transfer to a medium containing Na⁺, electrical pulses failed to increase the rate of efflux of ⁴⁵Ca²⁺. In unstimulated slices, the rate of efflux of ⁴⁵Ca²⁺ was dependent upon the concentration ratio of Na⁺ to Ca²⁺ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of ⁴⁵Ca²⁺ that occurred during electrical stimulation but exerted no effect on Ca²⁺-Ca²⁺ exchange. Our results suggest that there is a Na⁺-dependent turnover of Ca²⁺ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na⁺-Ca²⁺ interaction in the regulation of neurotransmitter function is discussed.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on ⁴⁵Ca²⁺ uptake and glutamate release. ⁴⁵Ca²⁺ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK-801. K⁺ depolarization-induced ⁴⁵Ca²⁺ uptake was also reduced by MK-801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the ⁴⁵Ca²⁺ uptake induced by depolarization. Opioid agonists, selective for μ-, κ-, and δ-receptors, inhibited the ⁴⁵Ca²⁺ uptake induced by K⁺ depolarization. The combination of low concentrations of MK-801 and opioid agonists resulted in additive inhibition of K⁺- induced ⁴⁵Ca²⁺ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca²⁺ uptake under acute, and perhaps also chronic, opiate treatment.     

MORPHOLOGICAL CHANGES OF CULTURED MYOCARDIAL CELLS DUE TO CHANGE IN EXTRACELLULAR CALCIUM ION CONCENTRATION

Increase in the extracellular Ca²⁺ concentration from low (≤ 10⁻⁷ M) to normal (10⁻³ M) caused morphological changes of cultured myocardial cells obtained from fetal mouse heart. The extracellular Na⁺ and K⁺ concentrations of the normal medium (10⁻³ M Ca²⁺) did not significantly affect the genesis of these morphological changes. Like Ca²⁺, Ba²⁺ and Sr²⁺, but not Mg²⁺, Co²⁺ or Ni²⁺, could induce morphological changes. Increase in the extracellular Ca²⁺ concentration from 10⁻⁸ M to 10⁻³M also caused excess uptake of ⁴⁵Ca²⁺ by cultured myocardial cells. B–16CW 1 cells, which did not show these morphological changes, did not take up excess ⁴⁵Ca²⁺ on this treatment. Treatments, such as addition of verapamil or incubation at pH 6.3, which reduced the genesis of morphological changes, reduced the rate of ⁴⁵Ca²⁺ uptake by myocardial cells. These facts show that the morphological changes of myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal are due to excess uptake of Ca²⁺ by the myocardial cells.
The morphological changes of cultured myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal were reversed on further incubation of the cells in medium with or without Ca²⁺.     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

ROLE OF MALATE TRANSPORT IN REGULATING METABOLISM IN MITOCHONDRIA ISOLATED FROM RABBIT BRAIN

Abstract— Partly purified chromaffin granules were incubated in vitro with Ca²⁺ (with trace amounts of ⁴⁵Ca²⁺) in concentrations ranging from 4 μm to 1 mm. After incubation the granules were washed with media containing EDTA and then subjected to density gradient centrifugation (1.3 to 2.0 m-sucrose solutions) in order to characterize the particles which had taken up ⁴⁵Ca²⁺. By using marker enzymes and various inhibitors of Ca²⁺ uptake into such cell particles as mitochondria it was established that under the conditions of the experiments chromaffin granules took up Ca²⁺ from the incubation medium. To characterize this uptake a simplified density gradient procedure was tested and found to be suitable. The uptake of Ca²⁺ into chromaffin granules was strongly dependent on temperature. It was not activated by ATP. The uptake was linear up to 10 min. At high calcium concentrations (above 200 μm) the rate of uptake levelled off. The uptake at 37°C was 1 nmol Ca²⁺/mg protein/min at a Ca²⁺ concentration of 500 μm. Mg²⁺ had no influence on Ca²⁺ uptake, whereas Sr²⁺ (1 mm) inhibited it. The methods established in this study should prove useful for a further characterization of this Ca²⁺ uptake into chromaffin granules which is likely to represent a useful model for the Ca²⁺ uptake occurring in the intact gland.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

The Quantity of Calcium that Appears to Induce Neuronal Death

Abstract: Excessive entry of Ca²⁺ through the NMDA receptor is thought to be the major cause of glutamate toxicity in brain neurons. However, actual quantitation of the calcium overload has not been achieved. Here we show that the absolute amount of ⁴⁵Ca²⁺ taken up via the NMDA receptor correlates quantitatively with the amount of acute cell death in cultured cerebellar granule cells of the rat. Analysis of 9-and 16-day cultures reveals that the NMDA-induced Ca²⁺ uptake is about the same at these ages, whereas the Ca-dependent lethal process is more developed in the older neurons. The calculated lethal concentration of ⁴⁵Ca taken up exceeds by ∼10,000 times the maximal concentration of [Ca²⁺], that can be measured by fluorescence imaging. It is suggested that the Ca²⁺ taken up induces the lethal process in a subcellular structure in which it has been segregated.