Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

Alkalinization Prolongs Recovery from Glutamate-Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µ M , for 15 s)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca²⁺ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca²⁺ recovery by affecting intraneuronal Ca²⁺ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca²⁺ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p -(trifluoromethoxyphenyl)hydrazone (FCCP; 750 n M ). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca²⁺ recovery was completely inhibited by the mitochondrial Na⁺/Ca²⁺ exchange inhibitor CGP-37157 (25 µ M ). This suggests that increased mitochondrial Ca²⁺ efflux via the mitochondrial Na²⁺/Ca²⁺ exchanger is responsible for the prolongation of [Ca²⁺]_i recovery caused by alkaline pH following glutamate exposure.     

N-System Amino Acid Transport at the Blood-CSF Barrier

Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[¹⁴C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na⁺-dependent mechanism. The Na⁺-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na⁺-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na⁺ with Li⁺ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V _max and K _max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

The Uptake of Carnitine by Slices of Rat Cerebral Cortex

Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 ± 3 for L-[¹⁴CH₃]carnitine and 27 ± 3 for D-[¹⁴CH₃]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of K _m for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of V _max (1 μmol/min/ml in-tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by γ-butyrobetaine ( K _i= 3.22 mM), acetylcarnitine ( K _i= 6.36 mM), and γ-aminobutyric acid ( K _i= 0.63 mM). The data suggest that carnitine and γ-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N₂ atmosphere, 2,4-dinitrophenol, carbonylcyanide- N -chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na⁺-dependent, but did not require K⁺ or Ca²⁺.     

CHARACTERISTICS OF D-GLUCOSAMINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of D-glucosamine by rat brain synaptosomes is studied as a function of time, temperature and synaptosomal protein and substrate concentrations. The rate of D-glucosamine uptake, after correcting for simple diffusion, obeys Michaelis-Menten kinetics. The apparent kinetic constants for the uptake process are K_m = 2.5 0.8 m m , V_max = 3.7 ± 1.2 nmol/mg protein/min. D-Glucose, D-mannose, 2-deoxy-D-glucose and 3-0-methyl-o-glucose are potent inhibitors of D-glucosamine uptake. 2-Deoxy-D-glucose and D-glucosamine inhibit the uptake of one another in a simple competitive manner, indicating their sharing of a common transport system. Cytochalasin B, phloretin and phloridzin are powerful competitive inhibitors of D-glucosamine uptake with apparent inhibitor constants ( K₁ ) of 7.0 × 10^-5, 2.3 × 10^-3 and 0.4 mM, respectively. The uptake is unaffected by Na⁺, Li⁺ and Mg²⁺, partially inhibited by NH₄⁺, Mn²⁺ and Ca²⁺, and slightly stimulated by PO₄-ions. D-Glucosamine uptake is also sensitive to inhibition by several sulfhydryl reagents, thus implying the involvement of sulfhydryl groups in the transport process. The apparent affinity constants for synaptosomal transport for both D-glucosamine and 2-deoxy-D-glucose are about 4 times greater in 7-day-old than in the adult rat brains.     

INHIBITION OF SYNAPTOSOMAL NORADRENALINE UPTAKE BY VERATRIDINE, GRAMICIDIN D AND VALINOMYCIN

Abstract— —The effects of brief exposures of rat brain synaptosomes to veratridine, gramicidin D and valinomycin on noradrenaline uptake were investigated. All three drugs inhibited the Na⁺-dependent component of noradrenaline uptake by synaptosomes. These effects were independent of extracellular Ca^{2 +} concentrations, indicating that the reductions were not due to the release of newly accumulated noradrenaline.
Gramicidin D reduced the V_max for noradrenaline uptake, whereas veratridine and valinomycin reduced the V^max and also increased the V^m for uptake.
Most of these findings can be explained on the basis of the effects that these drugs have on the inward-directed electrochemical gradients for Na⁺ across synaptosomal membranes, although, in the cases of veratridine and valinomycin, the elevated K_m's suggest that an impairment of noradrenaline binding to its carriers might also be involved.     

AMPA Receptor-Mediated Excitotoxicity in Human NT2-N Neurons Results from Loss of Intracellular Ca2+ Homeostasis Following Marked Elevation of Intracellular Na+

Abstract: Human NT2-N neurons express Ca²⁺-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors (AMPA-GluRs) and become vulnerable to excitotoxicity when AMPA-GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) was 1.9-fold greater in the presence than in the absence of cyclothiazide, Ca²⁺ entry via AMPA-GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40-fold rise in [Na⁺]_i, which induced a delayed [Ca²⁺]_i rise. Transfer of the neurons to a 5 m M Na⁺ medium after AMPA-GluR activation accelerated the delayed [Ca²⁺]_i rise and intensified excitotoxicity. Low-Na⁺ medium-enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK-801), and completely blocked by removal of extracellular Ca²⁺, suggesting that Ca²⁺ entry by reverse operation of Na⁺/Ca²⁺ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na⁺ loading. Our results serve to emphasize the central role of neuronal Na⁺ loading in AMPA-GluR-mediated excitotoxicity in human neurons.     

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.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

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.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Patterns of ion regulation in acidophilic fish native to the ion-poor, acidic Rio Negro

The Na⁺ uptake mechanism of cardinal tetras Paracheirodon axelrodi displayed specialization for operation in dilute waters of the amazonian Rio Negro. Kinetic analysis revealed low K_m and high J_max values which ensure high rates of uptake even in very dilute waters. In contrast, Na⁺ uptake of angelfish Pterophyllum scalare did not appear to be specialized for dilute waters at all, with much higher K_m and lower J_max values. Na⁺ uptake in cardinal tetras was high and completely unaffected down to pH 3·5, while uptake in angelfish was much lower and progressively inhibited by dropping pH; it was completely shut down at pH 3·5. During chronic exposure to pH 4·0 and 3·5, angelfish showed no ability to restore Na⁺ uptake and at pH 3·5 all individuals died between day 3 and 6 presumably due to small, but steady loss of Na⁺. At low pH, both species displayed a strong ability to prevent stimulation of diffusive Na+ losses. In angelfish, the ability to control diffusive ion losses at low pH was related to a high branchial affinity for Ca²⁺. For cardinal tetras, the rate of Na⁺ loss appeared to be independent of water Ca²⁺ concentration. The specializations of cardinal tetras are similar to other characid fishes tested, but the results for angelfish reveal a new pattern of ion regulation in acidophilic species from the Rio Negro. The differences between the species may be the result of their different distributions, cardinal tetras are found in the central Rio Negro region while angelfish are more peripheral, or they may reflect phylogenetic differences.     

K/Na selectivity at the plasmalemma of cortical root cells and preferential release of sodium to the xylem vessels in roots of Atriplex hortensis

Using excised roots of Atriplex hortensis L., cv. Gelbe Gartenmelde, the uptake, accumulation and xylem transport of K⁺ and Na⁺ have been measured. Influx as well as xylem transport proved to discriminate little between K⁺ and Na⁺, when considered in relation to the external solution. Both K⁺ and Na⁺ inhibited the uptake and xylem transport of each other to about the same degree. Measurements of intracel-lular Na⁺ fluxes by means of compartment analysis indicated that the low degree of K/Na discrimination during uptake was due to low influx selectivity. Moreover, K⁺/Na⁺ exchange at the plasmalemma was not very efficient in Atriplex roots. In order to establish the basis of the low K/Na discrimination in xylem transport, the rates of K⁺ and Na⁺ transport were related to the cytoplasmic K⁺ and Na⁺ concentrations to yield the selectivity ratio of transport, S(transport) = (φ_cx(K) × [Na⁺]_c)/(φ_cx(Na) × [K⁺]_c). Under all conditions this ratio was far below one indicating that Na⁺ was favoured during xylem release in excised roots of Atriplex at low external concentrations. The implications of this discrimination in favour of Na+ are discussed with respect to salt tolerance of A. hortensis .     

Changes in ion content and transport during development of embryonic rainbow trout

Wet mass and water content of four lots of whole eggs did not change throughout embryonic development of rainbow trout Oncorhynchus mykiss. Eggs in all four lots accumulated Na⁺. Eggs in lots 2 and 4 also accumulated Ca²⁺ and Cl^-, whereas eggs in lot 1 showed no significant change in Ca²⁺ or Cl^- and eggs in lot 3 showed no change in Cl^-and a small loss of Ca²⁺. Although the Na⁺ content of embryonic tissues increases in the later stages of development, the yolk sac content remained constant, indicating uptake of Na⁺ from the environment. Na+ uptake by whole eggs was non-saturable, consistent with diffusion of Na⁺ across the chorion into the perivitelline fluid. Na⁺ uptake in dechorionated embryos was saturable, as was Ca²⁺ uptake by both whole eggs and dechorionated embryos, consistent with active uptake or facilitated diffusion mechanisms at the surface of embryos. Very low Ca²⁺ uptake rates in dechorionated embryos suggest that the Ca²⁺ uptake mechanism is not fully developed until after hatching.     

Release of Neurotransmitter Amino Acids from Synaptosomes: Enhancement of Calcium-Independent Efflux by Oleic and Arachidonic Acids

Abstract: The release of preloaded [¹⁴C]neuroactive amino acids (glutamic acid, proline, γ-aminobutyric acid) from rat brain synaptosomes can occur via a time-dependent, Ca²⁺ -independent process. This Ca²⁺-independent efflux is increased by compounds that activate Na⁺ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [¹⁴C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on ²²Na⁺ permeability is observed. Since Na⁺ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na⁺ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na⁺ gradient.     

Mechanism Underlying the ATP-Induced Increase in the Cytosolic Ca2+ Concentration in Chick Ciliary Ganglion Neurons

Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca²⁺ concentration ([Ca]_in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]_in was dependent on external Ca²⁺, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca²⁺ channel blockers. ATP was effective in increasing [Ca]_in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]_in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na⁺ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na⁺ influx through P₂ purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, ATP still caused a small increase in [Ca]_in under Na⁺-free conditions, and this [Ca]_in increase was little affected by Ca²⁺ channel blockers. ATP also increased Mn²⁺ influx under Na⁺-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca²⁺ but also to Mn²⁺, in addition to monovalent cations.     

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.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.