Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

Accumulation of noradrenaline and its oxidation products by cultured rodent astrocytes

The accumulation of [³H]noradrenaline ([³H]NA) and its oxidation products was studied in primary cultures of cerebral astrocytes. Astroglial accumulation of radiolabeled catecholamine ([³H] NA and oxidation products) was enhanced by manganese or iron, but it was inhibited by unlabeled NA, dopamine or ascorbate. Tissue:medium ratios of radioactivity increased as extracellular [³H]NA was oxidized. When extracellular oxidation was prevented by ascorbate, as confirmed by high performance liquid chromatography with electrochemical detection, either ouabain pretreatment or nominally Na⁺-free incubation medium inhibited approximately one-half of specific [³H]NA accumulation by rat (but not mouse) astrocytes. These observations suggest that neurological responses to trace metals and ascorbate may arise from the effects of these agents on the clearance of extracellular catecholamines. Astrocytes can accumulate oxidation products of NA more rapidly than they take up NA itself, but ascorbate at physiological concentrations prevents the oxidation process in extracellular fluid. Furthermore, in the presence of ascorbate, Na⁺-dependent transport mediates a significant component of NA accumulation in rat astrocytes.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca²⁺ further increased the release rates of [³H]DA and [³H]NA induced by ischemic conditions. This finding indicated that the Na⁺/Ca²⁺ exchanger (NCX), working in reverse in the absence of extracellular Ca²⁺, fails to trigger the influx of Ca²⁺ in exchange for Na⁺ and fails to counteract ischemia by further increasing the intracellular Na⁺ concentration ([Na⁺]_i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca²⁺ was removed. Hypothermia inhibited the excessive release of [³H]DA in response to ischemia, even in the absence of Ca²⁺. These findings further indicate that the NCX plays an important role in maintaining a high [Na⁺]_i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [³H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H₂O₂), a mediator of ischemic brain injury enhanced the striatal resting release of [³H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca²⁺]_o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na⁺/Cl^?-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

     

Inhibitory effect of antidepressants on the NMDA-evoked [H]noradrenaline release from rat hippocampal slices

We have previously shown that monoamine uptake blocker-type antidepressants with different chemical structure and selectivity are able to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) in concentrations observed during antidepressant treatment. The mechanism of action of these drugs is similar to that of mecamylamine, a channel blocker-type antagonist of nAChRs. Since mecamylamine has been shown to block also NMDA receptors, our aim was to investigate whether the monoamine uptake blockers may affect the function of these ionotropic glutamate receptors.We studied, therefore the effect of the two most potent nicotinic antagonist antidepressants, the tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine on the NMDA-induced [³H]noradrenaline ([³H]NA) release from rat hippocampal slices. The NMDA-induced hippocampal [³H]NA release was effectively blocked by the selective, non-competitive NMDA antagonist MK-801 (IC₅₀ = 0.54 μM), indicating that the [³H]NA release was mediated through NMDA receptors. This response was also dose-dependently inhibited by desipramine (IC₅₀ = 14.57 μM) and fluoxetine (IC₅₀ = 41.06 μM). The Na⁺-channel blocker TTX equally inhibited both the electrical stimulation- and the NMDA-evoked [³H]NA release (the IC₅₀ was 55 nM and 66 nM, respectively), whereas the antidepressants inhibited only the NMDA-evoked response. These data suggest that the inhibitory effect of fluoxetine and desipramine on the NMDA-evoked [³H]NA release is exerted directly on NMDA receptors rather than indirectly on Na⁺-channels.Due to accumulation processes the concentration of desipramine and fluoxetine in the brain might be in the same range as the observed IC₅₀ values, thus our data indicate that monoamine uptake blocker-type antidepressants are able to influence the function of NMDA receptors during antidepressant treatment, and the inhibitory effect on NMDA receptors might contribute to the therapeutic effects of these drugs.     

The Action of Black Widow Spider Venom on Cholinergic Mechanisms in Synaptosomes

Abstract: Black widow spider venom (BWSV) promoted the massive release of labeled acetylcholine from synaptosomes and in addition, inhibited high-affinity choline uptake into the preparation. Both activities occurred in the absence of [Ca²⁺]₀. When Na⁺ in Krebs-Ringer was replaced isotonically by sucrose, BWSV did not cause any release of [³H]ACh. On the other hand, BWSV was still effective if Na⁺ was replaced by lithium, glucosamine, or Tris. Tetrodotoxin (10^?5 M) failed to prevent the ACh-releasing action of the venom. The uptake of [³H]norepinephrine and [³H]tyrosine into the P₂ fraction was significantly inhibited by BWSV pretreatment. However, the effect of the venom on the uptake of [³H]deoxyglucose was slight. In addition, the venom-induced release of [³H]norepinephrine was much higher than that of [³H]deoxyglucose. The change in membrane potential of the preparation in duced by BWSV as examined using the voltage-sensitive fluorescence probe, 3, 3′-dipentyl-2, 2′-oxacarbocyanine. BWSV pretreatment markedly increased the synaptosomal fluorescence, indicating a depolarization of the preparation. This action of the venom was also observed in a Ca²⁺ -free or K⁺ -free medium, but could be blocked by pretreatment with antivenom. Pretreatment of the P₂ fraction with concanavalin A completely blocked the action of BWSV. Also, the BWSV failed to promote the release of transmitter if the venom was prein-cubated with a low concentration of purified gangliosides. Even after prolonged treatment with high concentrations of BWSV, an electron microscopic study showed no depletion of the synaptic vesicles in presynaptic terminals of the cortical P₂ preparations or striatal slices. It is suggested that the venom expresses its activity by binding to glycoproteins and/or gangliosides on the synaptic membrane, opening a cation channel. The subsequent depolarization then inhibits uptake processes and promotes transmitter release that is independent of external calcium.     

High Affinity Stereospecific Binding of [3H] Cocaine in Striatum and its Relationship to the Dopamine Transporter

A high affinity (K_D 35 nM) binding site for [³H]cocaine is detected in rat brain Striatum present at 2–3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [³H]dopamine ([³H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [³H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [³H] cocaine binding stereos-pecifically, but with lower potency (IC₅₀ ～ 1μM) than does cocaine. It is suggested that the DA transporter in Striatum is the putative “cocaine receptor.

Binding of [³H] cocaine, measured in 10 mM Na₂HPO₄-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na⁺ and K⁺ and by biogenic amines. DA and Na⁺ reduce the affinity of the putative “cocaine receptor” for [³H]cocaine without changing the B_max, suggesting that inhibition may be competitive. However, TRIS reduces [³H]cocaine binding non-competitively while Na⁺ potentiates it in TRIS buffer. Binding of [³H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [³H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na⁺ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Reverse Na+/Ca2+-exchange mediated Ca2+-entry and noradrenaline release in Na+-loaded peripheral sympathetic nerves

[³H]noradrenaline ([³H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2 Hz, 1 ms, 60 V for 3 min) in the presence of uptake blockers (cocaine, 3 × 10⁻⁵ M and corticosterone, 5 × 10⁻⁵ M). The [³H]NA-release was fully blocked by the combined application of the selective and irreversible ‘N-type’ voltage-sensitive Ca²⁺-channel (VSCC)-blocker ω-conotoxin (ω-CgTx) GVIA (10⁻⁸ M) and the ‘non-selective’ VSCC-blocker aminoglycoside antibiotic neomycin (3 × 10⁻³ M). Na⁺-loading (Na⁺-pump inhibition by K⁺-free perfusion) was required to elicit further NA-release after blockade of VSCCs (ω-CgTx GVIA + neomycin). In K⁺-free solution, in the absence of functioning VSCCs (ω-CgTx GVIA + neomycin), the fast Na⁺-channel activator veratridine (10⁻⁵ M) further potentiated the nerve-evoked release of [³H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by $\text{C}{\text{a}}_{\text{o}}^{2+}-\text{removal}$. However, Li⁺-substitution was surprisingly ineffective. The non-selective K⁺-channel blocker 4-aminopyridine (4-AP, 10⁻⁴ M) also further potentiated the nerve-evoked release of NA in K⁺-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li⁺-substitution and abolished by $\text{C}{\text{a}}_{\text{o}}^{2+}-\text{removal}$.It is concluded that in Na⁺-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na⁺/Ca²⁺-exchange-mediated Ca²⁺-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na⁺-channel and the exchanger proteins are close to the vesicle docking sites.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

Modifications of Ca2+ mobilization and noradrenaline release by S-nitroso-cysteine in PC12 cells

The effects of nitrogen monoxide (NO)-related compounds on cytosolic free Ca2+ concentrations ([Ca2+]i) and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. The addition of S-nitroso-cysteine (SNC) stimulated [Ca2+]i increases from an intracellular Ca2+ pool continuously in a concentration-dependent manner. Other NO donors, which stimulate cyclic GMP accumulation, did not cause [Ca2+]i increases. After treatment with 0.2 mM SNC, transient increases in [Ca2+]i from the Ca2+ pool induced by caffeine were completely abolished. The addition of N-ethylmaleimide (NEM) caused sustained [Ca2+]i increases from the intracellular Ca2+ pool. Furthermore, caffeine did not stimulate further [Ca2+]i increases in PC12 cells pretreated with NEM. These findings suggest that SNC and NEM predominantly interact with a caffeine-sensitive Ca2+ pool. The addition of dithiothreitol (DTT) to 0.4 mM SNC-stimulated cells reduced [Ca2+]i to basal levels, and the addition of DTT to NEM-stimulated cells locked [Ca2+]i at high levels. The stimulatory effects of SNC but not NEM were not abolished by pretreatment with DTT. These findings suggest that modification of the oxidation status of the sulfhydryl groups on the caffeine-sensitive receptors by SNC or NEM regulates Ca2+ channel activity in a reversible manner. SNC did not stimulate NA release by itself but did inhibit ionomycin-stimulated NA release. In contrast, NEM stimulated NA release in the absence of extracellular CaCl2 and further enhanced ionomycin-stimulated NA release. Ca2+ mobilization by SNC from the caffeine-sensitive pool was not a sufficient factor, and other factors stimulating NA release may be negatively regulated by SNC.     

Effect of Potassium on the Release of [3H]Noradrenaline from Rabbit and Human Pulmonary Artery

The release of [3H]noradrenaline ( [3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+-free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic alpha 2-adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+-free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of NA, K-ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+-free solution, the results may indicate involvement of the sodium pump in NA release.     

The Role of GlycineB Binding Site and Glycine Transporter (GlyT1) in the Regulation of [3H]GABA and [3H]Glycine Release in the Rat Brain

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [³H]-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [³H]GABA overflow with an EC₅₀ value of 0.09 mM. The [³H]GABA releasing effect of NMDA was an external Ca²⁺-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [³H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycine_B site, decreased the [³H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [³H]GABA outflow. These data suggest that glycine_B binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [³H]glycine (IC₅₀ 33 and 16 M) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [³H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [³H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [³H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na⁺ for superfusion of hippocampal slices produced an increased [³H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [³H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [³H]glycine efflux even when buffer with low Na⁺ concentration was applied.     

Study of the Mechanism of Release of [3H]GABA from a Teleost Retina In Vitro

Abstract: γ-Aminobutyric acid (GABA) is thought to be a neurotransmitter in the vetebrate retina. We studied the voltage and Ca²⁺ dependency of the process of release of [³H]GABA from the retina of the teleost Eugenes plumieri, using a microsuperfusion technique. Two depolarizing agents, veratridine and high potassium, produced a concentration-dependent release of [³H]GABA. The veratridine effect was inhibited in Na⁺-free solution, but was not affected by 1 μM tetrodotoxin. A substantial inhibition (about 75%) of the veratridine-and potassium-stimulated release of [³H] GABA occurred in Ca²⁺-free medium. Inhibitors of the Ca²⁺ channel, such as Mg²⁺(20 mM), La³⁺ (0.1 mM), and methoxy-verapamil (4 μM-0.4 mM), inhibited the veratridine-and K⁺-stimulated release. However, Co²⁺ and Cd²⁺ caused a potentiation and no change of the K⁺-and veratridine-stimulated release, respectively. This release process is apparently specific, since both depolarizing agents were unable to release [³H]methionine, a nontransmitter amino acid, under the same experimental conditions. Autoradio-graphic studies with [³H]GABA, using the same incubation conditions as for the release experiments, showed a high density of silver grains over the horizontal cells with almost no accumulation by amacrine cells and Muller cells. β-Alanine and nipecotic acid were used as two relative specific inhibitors of the glial and neuronal GABA uptake mechanisms, respectively. Only a small heteroexchange with [³H]GABA was found with β-alanine, and no inhibition of the subsequent veratridine-stimulated release. On the other hand, nipecotic acid produced a strong heteroexchange with [³H]GABA and lacked the capacity to induce the veratridine-stimulated release of [³H]GABA. These results suggest a voltage-and Ca²⁺-dependent neuronal release of [³H]GABA from retina.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

The Na+-Ca2+ Exchange Inhibitor KB-R7943 Inhibits High K+-Induced Increases in Intracellular Ca2+ Concentration and [3H]Noradrenaline Release in the Human Neuroblastoma SH-SY5Y

The effects of the Na⁺-Ca²⁺ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943) on depolarization-induced Ca²⁺ signal and [³H]noradrenaline release were examined in SH-SY5Y cells. KB-R7943 at 10 M significantly inhibited high K⁺-induced increase in intracellular Ca²⁺ concentration. KB-R7943 also inhibited high K⁺-evoked release of [³H]noradrenaline from the cells. These findings suggest that the Na⁺-Ca²⁺ exchanger in the reverse mode is involved at least partly in depolarization-induced transmitter release.     

Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [³H]GABA release induced by ouabain, veratridine and Na⁺ substitution by other monovalent cations (K⁺, Rb⁺, Li⁺, and choline). We observed that ouabain is unable to release [³H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [³H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na⁺ and it is inhibited by extravesicular Ca²⁺ (1 mM) under conditions which appear to permit its entry. However, veratridine-induced [³H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na⁺ by other monovalent cations promotes [³H]GABA release by altering the Na⁺ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li⁺, we observed that the fraction of [³H]GABA released relatively to the total amount of neurotransmitter released by K⁺ or Rb⁺ is about 28% and 68%, respectively. Since the replacement of Na⁺ by K⁺, Rb⁺, and Li⁺ causes different levels of membrane depolarization, and the replacement of Na⁺ by choline causes hyperpolarization of the vesicles, these results suggest that, in parallel to the [³H]GABA release, which is directly proportional to the level of membrane depolarization, this neurotransmitter can be released by decreasing the external Na⁺, which reflects an elevation of the Na⁺ concentration gradient (inout). Like veratridine-induced release, the depolarization-induced release of [³H]GABA by SPM vesicles is inhibited by Ca²⁺, which suggests that this divalent cation interfers with the cytoplasmic GABA release mechanism.Abbreviations used ATPase adenosine triphosphatase - GABA -aminobutyric acid - Mes 2 (N-morpholino)-ethanosulfonic acid - SPM synaptic plasma membranes - membrane potential     

Extracellular Mg regulates the intracellular Na concentration in rat sublingual acini

The intracellular free Na⁺ concentration ([Na⁺]_i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg²⁺ in the regulation of the stimulated [Na⁺]_i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na⁺]_i was approximately 4-fold greater in the absence of extracellular Mg²⁺. When Na⁺ efflux was blocked by the Na⁺,K⁺-ATPase inhibitor ouabain, the stimulated [Na⁺]_i increase was comparable to that seen in an Mg²⁺-free medium. Moreover, ouabain did not add further to the stimulated [Na⁺]_i increase in an Mg²⁺-free medium suggesting that removal of extracellular Mg²⁺ may inhibit the Na⁺ pump. In agreement with this assumption, ouabain-sensitive Na⁺ efflux and rubidium uptake were reduced by extracellular Mg²⁺ depletion. Our results suggest that extracellular Mg²⁺ may regulate [Na⁺]_i in sublingual salivary acinar cells by modulating Na⁺ pump activity.     

Two different modes of inhibition of the rat brain Na+,K+-ATPase by triterpene glycosides,psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na⁺,K⁺-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na⁺,K⁺-ATPase with I₅₀ values of 1×10⁻⁴ M and 3×10⁻⁴ M, respectively. PsA significantly stimulated [³H]ATP binding to Na⁺,K⁺-ATPase, weakly increased [³H]ouabain binding to the enzyme, and inhibited K⁺-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [³H]ATP binding to Na⁺,K⁺-ATPase, and had no effect on [³H]ouabain binding to the enzyme. K⁺-Phosphatase activity was inhibited by PsB in parallel with Na⁺,K⁺-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na⁺,K⁺-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

Uptake and release for glutamine and glutamate in a crude synaptosomal fraction from rat brain

[14C]Glutamine uptake in a crude synaptosomal (P2) fraction, (representing the sum of [¹⁴C]glutamine accumulated and [¹⁴C]glutamate formed by hydrolysis), is distinct from glutamate uptake. Glutamine uptake is Na⁺-independent and unaffected by the Na⁺–K⁺-ATPase inhibitor ouabain, whereas glutamate uptake is Na⁺-dependent and inhibited by ouabain. The uptake of both glutamine and glutamate is unaffected by the gamma-glutamyltransferase inhibitor, Acivicin. This indicates that glutamine uptake is not mediated by a carrier, as distinct from that of glutamate, and also not linked to gamma-glutamyl-transferase. Na⁺ affects the distribution of glutamine-derived glutamate by increasing the synaptosomal content and reducing that of the medium. When glutamate release from synaptosomes preloaded with [¹⁴C]glutamate is measured by superfusion technique in order to prevent reuptake, Na⁺ has been found to inhibit release in a non-depolarizing medium (Ringer buffer with no Ca²⁺) of the [¹⁴C]glutamate as well as of endogenous glutamate. The specific activity of the [¹⁴C]glutamine-derived glutamate in the incubation medium is much higher than that in the synaptosomes, indicating that there exists a readily releasable pool of newly formed glutamate in addition to another pool. The latter glutamate pool is partially reduced by Na⁺.Special Issue Dedicated to Dr. Abel Lajtha.