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.     

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.     

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.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

Enhancement of 45Ca2+ Binding to Acidic Lipids by Barbiturates, Diphenylhydantoin, and Ethanol

Abstract: Effects of barbiturates, diphenylhydantoin, and ethanol on ⁴⁵Ca²⁺ binding to acidic lipids have been examined in an organic solvent-aqueous partition system. Hexobarbital, pentobarbital, and phenobarbital, at concentrations of 0.3 and/or 0.6 mM, enhanced the binding of ⁴⁵Ca²⁺ to phosphatidic acid, phosphatidylserine, and sulfatide but not to phosphatidylinositol or cardiolipin. Diphenylhydantoin, 0.3 mM, enhanced ⁴⁵Ca²⁺ binding to phosphatidic acid and phosphatidylserine but not to sulfatide. Ethanol at 80 mM did not enhance ⁴⁵Ca²⁺ binding to phosphatidic acid, but ethanol decreased the binding to cardiolipin and increased it to sulfatide.     

Rate of 59Fe Uptake into Brain and Cerebrospinal Fluid and the Influence Thereon of Antibodies Against the Transferrin Receptor

Abstract: Uptake of ⁵⁹Fe from blood into brains of anaesthetized rats and mice has been studied by intravenous infusion of [⁵⁹Fe]ferrous ascorbate or of ⁵⁹Fe-transferrin, the results not being significantly different. Uptakes in the rat were linear with time, but increased at longer times in the mouse. Transfer constants, K _in (in ml/g/h × 10³), for cerebral hemispheres were 5.2 in the adult rat and 5.6 in the mouse. These K _in values corresponded to ⁵⁹Fe influxes of 145 and 322 pmol/g/h, respectively. ⁵⁹Fe uptake into the mouse brain occurred in the following order: cerebellum > brainstem > frontal cerebral cortex > parietal cortex > occipital cortex > hippocampus > caudate nucleus. In genetically hypotransferrinaemic mice, ⁵⁹Fe uptake into brain was 80–95 times greater than in To strain mice. Pretreatment of young rats and mice with monoclonal antibodies to transferrin receptors, i.e., the anti-rat immunoglobulin G OX 26 and the anti-mouse immunoglobulin M RI7 208, inhibited ⁵⁹Fe uptake into spleen by 94% and 98%, respectively, indicating saturation of receptors. The antibodies reduced ⁵⁹Fe uptake into rat brain by 35–60% and that into mouse brain by 65–85%. Although a major portion of iron transport across the blood-brain barrier is normally transferrin-mediated, non-transferrin-bound iron readily crosses it at low serum transferrin levels.     

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.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

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.     

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.     

Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

Abstract: A ⁴⁵Ca²⁺ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N -methyl- d -aspartate (NMDA) receptors. Inhibition of glutamate-stimulated ⁴⁵Ca²⁺ influx by six glycine-site antagonists and inhibition of glycine-stimulated ⁴⁵Ca²⁺ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated ⁴⁵Ca²⁺ influx for recombinant NMDA receptors, inhibiting and stimulating ⁴⁵Ca²⁺ influx into cells expressing NR1a/NR2A receptors (IC₅₀ = 408 µ M ) and NR1a/NR2B receptors (EC₅₀ = 37.3 µ M ), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC₅₀ = 0.099 µ M ) compared with NR1a/NR2A receptors (IC₅₀ = 164 µ M ). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg²⁺ and Zn²⁺ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of ⁴⁵Ca²⁺ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.     

Synaptosomal Calcium Uptake Systems: Prostaglandins Are Probably Not Involved in the Regulation of Calcium Fluxes Into and Within the Nerve Endings

Abstract: ⁴⁵Ca²⁺ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca²⁺ movements into and within the nerve endings. The K⁺-induced ⁴⁵Ca²⁺ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca²⁺ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca²⁺ uptake, were able to induce Ca²⁺ efflux from preloaded intrasynaptosomal organelles. The PGs E₂, F_2α, D₂, and thromboxane B₂ were without effect on ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca²⁺ availability by modulating Ca²⁺ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca²⁺ uptake might be due to unspecific impairment of mitochondrial functions.     

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.     

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).     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Relationship between polar basipetal auxin transport and acropetal Ca2+ transport into tomato fruits

The dependence of acropetal Ca²⁺ transport on polar basipetal indoleacetic acid (IAA) transport was investigated in excised tomato fruits ( Lycopersicon esculentum L. Mill.) using an in vitro fruit system. Auxin transport inhibitors like triiodobenzoic acid (TIBA), chlorofluorenolmethyl ester (CME) and naphthylphthalamic acid (NPA) were used in order to investigate the effect of restricted polar basipetal auxin transport on the acropetal transport of ⁴⁵Ca²⁺, ⁸⁶Rb⁺ and ⁹⁸Sr²⁺ into the same fruits. TIBA and CME inhibited basipetal transport of IAA. particularly in 10- to 12-day-old tomato fruits, and simultaneously restricted the acropetal transport of ⁴⁵Ca²⁺. The auxin transport inhibitors failed to significantly reduce the upward transport of ⁸⁶Rb⁺ and the transport of ⁹⁶Sr²⁺ was less inhibited than that of ⁴⁵Ca²⁺. TIBA and CME did not significantly affect the acropetal transport of labelled water into the fruit, nor the cation-exchange capacity or K⁺ and Mg²⁺ concentrations in the tomato fruit. These results support the view that a part of the Ca²⁺-specific acropetal transport into tomato fruits is associated with the polar basipetal IAA transport. This Ca²⁺ transport is independent of the transpiration stream into the fruit and the cation exchange capacity of the fruit tissue.     

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.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.