Exocytotic Release of [3H]-Acetylcholine by Ouabain Involves Intracellular Ca2+ Stores in Rat Brain Cortical Slices

1. The effect of ouabain on the release of [3H]acetylcholine ([3H]ACh) in rat brain cortical slices was investigated. 2. The ouabain-induced release of [3H]ACh was calcium-independent and not blocked by EGTA. 3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect suggesting the involvement of intracellular calcium stores. 4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhibited by 73% the ouabain-induced release of [3H] ACh, suggesting exocytotic release of the neurotransmitter. 5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by 57 and 66% respectively, the ouabain-elicited release of [3H]ACh in brain cortical slices. 6. Confocal microscopy and calcium imaging showed that ouabain increased the levels of [Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma. 7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellular stores that can increase [3H] ACh exocytosis from rat brain cortical slices.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Effects of short-term hypoxia on [3H]glutamate release from preloaded hippocampal and cortical synaptosomes

The effect of short-term hypoxia on the release of [³H]glutamate from preloaded hippocampal and cortical synaptosomes was studied in a rapid superfusion system. The technique minimised the loss of released glutamate by reuptake. The results indicated that the effects of short term hypoxia were qualitatively similar to those reported in previous studies using more long-term hypoxia, but were significantly smaller. The non-Ca²⁺-dependent efflux of glutamate from cortical synaptosomes was increased by hypoxia as was the Ca²⁺-dependent release from hippocampal tissue. Possible mechanisms for these findings were discussed. The small amplitude of these changes in comparison to the effects seen in slowly perfused tissue in vitro and in vivo indicated that the contribution made by changes in neuronal efflux to the overall increase in extracellular glutamate seen in hypoxia is relatively minor.     

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

[³H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca²⁺-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [³H]purine release. Nimodipine only at the concentration of 10 M modified [³H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [³H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of⁴⁵Ca²⁺ by the cultures. The treatment of cells with the Ca²⁺ agonist Bay K 8644 did not affect [³H]purine release or the⁴⁵Ca²⁺ uptake. The drug did not either alter [Ca²⁺]_i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca²⁺ discharge, significantly reduced the evoked [³H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca²⁺ ATPase, was able to increase either the culture [³H]purine release or the [Ca²⁺]_i. Together, the findings indicate that voltage-sensitive calcium channels (VSCC_s) of the neuronal N and L-types are not involved in the modulation of [³H]purine release from rat cultured astrocytes whereas Ca²⁺ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC_s seem to be able to affect [³H]purine outflow with mechanisms other than VSCC gating.     

Photoaffinity labelling of Ca2+ channels with [3H]azidopine

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited steroselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr approximately 145 000. The covalent labelling of the Mr approximately 145 000 band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Presynaptic Modulation of K+-Evoked [3H]Dopamine Release in Striatal and Frontal Cortical Synaptosomes of Normotensive and Spontaneous-Hypertensive Rats

Regional differences in presynaptic [³H]dopamine ([³H]DA) release and its modulation by D₂ DA-receptors between the frontal cortex and striatum obtained from Wystar-Kyoto (WKY) and spontaneous-hypertensive rats (SHR) have been evaluated using superfused synaptosomes. Synaptosomal tritium content was significantly lower in the frontal cortex than in the striatum in both SHR and WKY (45% and 48%, respectively), but no differences in tritium content were obtained between strains. However, the 15 mM K⁺-evoked [³H]DA overflow was lower in the SHR as compared to WKY rats in both brain regions (striatum 23%, frontal cortex 21). Concentration-response curves for quinpirole (1nM-10 M)-mediated inhibition of 15mM K⁺-evoked [³H]DA release showed no differences between SHR and WKY. These results suggest that SHR has less ability to release [³H]DA as compared to WKY rats, but SHR did not show differences in the autoregulation of such release in both the frontal cortex and striatum.     

Ca2+-Dependent and Ca2+-Independent [3H]GABA Release from Rat Brain Synaptosomes: the Effect of Temperature

The main inhibitory neurotransmitter GABA in the mammalian brain is distributed in the nerve terminals between two pools, vesicular (synaptic vesicles) and cytosolic. GABA is released from these pools by different mechanisms; there are calcium-activated exocytotic release and calcium-independent sodium-dependent release from the cytosolic pool (resulting from the membrane GABA transporter reversal). We investigated the influence of temperature on [³H]GABA release from rat brain synaptosomes, which was induced by stimulation of both these processes. In addition, we used -latrotoxin as a stimulant of [³H]GABA release. Synaptosomes from the rat brain were used in the experiments. 4-Aminopyridine (4-AP) and high [KCl] were applied to stimulate calcium-activated and calcium-independent [³H]GABA release, respectively. 4-AP-evoked [³H]GABA release was of the same intensity at 37 and 25°C (10.1 ± 1.2 and 10.1 ± 0.8% of total [³H]GABA incorporated into the synaptosomes, respectively). The effect of 4-AP on the ⁴⁵Ca²⁺ influx into synaptosomes was also temperature-independent: 0.775 ± 0.075 and 0.725 ± 0.100 nmol/min/mg of protein at 37 and 25°C, respectively. A drop in the effect of 4-AP was observed only at 15°C. When synaptosomes were depolarized with 50 mM KCl, a temperature decrease from 37°C to 25°C resulted in a twofold drop in the [³H]GABA release, from 20.5 ± 1.4 to 10.3 ± 0.7%; at 15°C [³H]GABA release dropped to less than one-third of the norm (6.0 ± 0.5%). -Latrotoxin-stimulated [³H]GABA release was diminished from 32.5 ± 2.5 at 37°C to 17.2 ± 1.3 at 25°C and 5.9 ± 0.4% at 15°C and was not affected by the presence or absence of calcium in the medium. It seems likely that the observed effect of temperature can be interpreted as based on the temperature dependence of the -latrotoxin insertion into the membrane. It is suggested that the pattern of the temperature sensitivity of GABA release from the synaptosomes can be used as a criterion for identification of the mode of neurotransmitter release.     

Characterization of divalent cation-induced [3H]Acetylcholine release from EGTA-treated rat hippocampal synaptosomes

Calcium-naive synaptosomes were used to assess the effects of divalent cations on [³H]acetylcholine release from rat hippocampal homogenates. Following equilibration with calcium-free buffer (containing 10M EGTA), calcium reversibly increased [³H]acetylcholine efflux (up to five-fold) while causing no measurable efflux of lactate dehydrogenase. When substituted for calcium, strongtium and barium behaved similarly although barium exhibited three-fold greater efficacy. In the presence of elevated potassium, 4-aminopyridine or tetraethylammonium, the secretagogue efficacy of calcium (but not barium) was markedly increased. The release-promoting effects of both cations were inhibited by lanthanum, magnesium, cadmium, and -conotoxin but were insensitive to nifedipine and cobalt (both 10 M). In addition, stimulation of muscarnic cholinergic autoreceptors substantially inhibited both calcium and barium-evoked [³H]acetylcholine release. Taken together, these results indicate that cation-evoked transmitter release from calcium-naive synaptosomes is subject to normal neuroregulatory mechanisms and therefore should be useful for investigating presynaptic modulation of neuronal exocytosis.     

Effect of Subtype-Specific Ca2+-Antagonists and Ca2+-Free Media on the Field Stimulation-Evoked Release of ATP and [3H]Acetylcholine from Rat Habenula Slices

The involvement of different subtypes of voltage-sensitive Ca²⁺ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [³H]acetylcholine ([³H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [³H]ACh were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca²⁺ channel blocker -conotoxin GVIA (-CgTX, 0.01–1 M) reduced the stimulation-evoked release of ATP and [³H]ACh in a dose-dependent manner. Similarly, the P-type Ca²⁺ channel antagonist -agatoxin IVA (-Aga IVA) (0.05 M) and the inorganic Ca²⁺ channel blocker Cd²⁺ (0.2 mM) inhibited the outflow of both transmitters, while Ni²⁺ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca²⁺ antagonists. Long-term perfusion (i.e., 90 min) with Ca²⁺ free solution inhibited the evoked-release of ATP and [³H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [³H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca²⁺]₀ condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca²⁺ chelators, and dipyridamole (2 M), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca²⁺-free media. Tetrodotoxin (TTX, 1 M), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca²⁺]₀ conditions. In summary, we showed that both N- and P-type Ca²⁺ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [³H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [Ca²⁺]₀ conditions an additional release of ATP occurs, which is not associated with action potential propagation.     

Voltage-Sensitive Ca2+ Channels Involved in Nicotinic Receptor-Mediated [3H]Dopamine Release from Rat Striatal Synaptosomes

Abstract: The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [³H]dopamine release from striatal synaptosomes, and this action is both Na⁺ and Ca²⁺ dependent and is blocked by Cd²⁺. This suggests that stimulation of presynaptic nicotinic receptors results in Na⁺ influx and local depolarisation that activates voltage-sensitive Ca²⁺ channels, which in turn provide the Ca²⁺ for exocytosis. Here we have investigated the subtypes of Ca²⁺ channels implicated in this mechanism. [³H]Dopamine release evoked by anatoxin-a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a ⁸⁶Rb⁺ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 µM) blocked anatoxin-a-evoked [³H]dopamine release by 60% but had no significant effect on ⁸⁶Rb⁺ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker ω-agatoxin IVA (90 nM) also inhibited KCl-evoked release by ～30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker ω-conotoxin MVIIC (1 µM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [³H]dopamine release by activation of N-type Ca²⁺ channels. In contrast, KCl-evoked [³H]dopamine release appears to involve both N-type and P-type channels.     

Effect of ethanol on [3H]Dopamine release in rat nucleus accumbens and striatal slices

Ethanol (10–200 mM) transiently increased tritium overflow from superfused rat nucleus accumbens slices previously incubated with [³H]dopamine (DA) and [¹⁴C]choline. The effect was greater in striatal tissue and did not appear to be a non-specific membrane effect since [¹⁴C]acetylcholine (ACh) release was not affected. Lack of antagonism by picrotoxin suggested that -aminobutyric acid (GABA) receptors were not involved. Calcium was not a requirement and the DA uptake blocker, nomifensine, was without effect. Ethanol appeared to be causing [³H]DA release into the cytoplasm. K⁺-stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens and striatal slices was not affected. Clonidine-mediated inhibition of the K⁺-evoked release of [³H]DA remained unaltered. Ethanol attenuated the isoproterenol-induced enhancement of [³H]DA release. Ethanol therefore appeared to interact with components of the DA terminal causing a transient increase in the release of neurotransmitter without impairing K⁺-evoked release but apparently interfering with the isoproterenol-induced effect.     

L型钙流和反向钠-钙交换在豚鼠心室肌细胞兴奋-收缩偶联中的作用

目的 :比较和探讨L型钙流 [ICa(L) ]和反向钠—钙交换 (NCX)在触发豚鼠心室肌细胞兴奋—收缩偶联中的作用。方法 :以分离的豚鼠单个心室肌细胞为对象 ,采用膜片钳和单细胞收缩测量技术 ,给予 35℃的各种含药物细胞外液快速灌流 ,同时记录ICa(L) 和细胞收缩。结果 :①在 +10mV的钳制电压 ,使用硝苯地平 (Nif) 10～ 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L ,阻滞ICa(L) 越多 ,细胞收缩被阻滞得越多 ,呈线性相关。②在 +5 0mV的钳制电压 ,Nif 10 0 μmol/L以及Nif 30 μmol/L +Cd2 +3 0 μmol/L仅能抑制部分细胞收缩 ,但剩余的细胞收缩起始时间明显延迟 ,且能被 5mmol/LNi2 +所阻滞。③在 +10 0mV的钳制电压 ,细胞收缩起始时间较 +5 0mV明显延迟 ,且不能被Nif 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L所阻滞。结论 :在生理条件下 ,ICa(L) 是触发心室肌细胞兴奋—收缩偶联的主要途径 ,但在膜电位 >+5 0mV时 ,反向NCX也参与兴奋—收缩偶联。     

Haloperidol reduces K+-evoked Ca2+-dependentd-[3H]aspartate release from rat hippocampal slices

Rat hippocampal slices preloaded withd-[³H]aspartate, a non metabolizable analogue ofl-glutamate, were superfused with artifical CSF. Depolarization was induced by 53.5 mM K⁺, in the presence of Ca²⁺ (1.3 mM) or Mg²⁺ (5 mM) to determine the Ca²⁺ dependent release. Haloperidol added in the superfusion medium at 100 M reduced by about 60% the Ca²⁺ dependent release ofd-[³H]aspartate. This drug at 20 M or 100 M inhibited the non-activated glutamate dehydrogenase (GDH) but had no effect on GDH activated by ADP (2 mM) or leucine (5 mM). In addition no effect was observed on phosphate activated glutaminase (PAG) in the presence either of 20 mM or 5 mM phosphate. These results indicate that the effect of haloperidol is exerted on presynaptic mechanisms regulating neurotransmitter release.     

Effects of membrane peroxidation on [3H]acetylcholine release in rat cerebral cortical synaptosomes

[³H]Acetylcholine (ACh) release, malonaldehyde formation and⁴⁵calcium-uptake were measured in rat cerebral cortical nerve terminal that were exposed to various concentrations of ferrous and ascorbate ions. At a constant molar ratio of 25:1, ferrous:ascorbate, these ions increased malonaldehyde (MA) synthesis in a concentration-dependent manner. Treatment with these ions in the same ratio also induced a dose-related inhibition of the K⁺-depolarization-induced release of newly synthesized [³H]ACh. Combined exposure to Fe²⁺/ascorbate also reduced calcium ionophore A23187-induced [³H]ACh release. Neither ferrous nor ascorbate ions alone altered depolarization-or ionophore-induced [³H]ACh release over this concentration range. Depolarization- and A23187-induced⁴⁵calcium uptake were not affected by peroxidation, suggesting that membrane peroxidation influenced some process in the release-process subsequent to calcium influx in a manner similar to what is observed during aging.     

Inhibition of the Na+/Ca2+ antiport of heart mitochondria by diethylpyrocarbonate

Diethylpyrocarbonate inhibits Na⁺/Ca²⁺ antiport activity in isolated heart mitochondria. The inhibition is time-dependent with maximum activity developed after 5 min at 25°C. The reaction of diethylpyrocarbonate with the mitochondrial membrane is biphasic with 25–30 nmol mg^–1 reacting rapidly and an additional 30 nmol mg^–1 taken up slowly over a 30-min incubation. Inhibition of mitochondrial Na⁺/Ca²⁺ antiport by diethylpyrocarbonate decreases theV _max of the reaction, and the inhibition cannot be reversed by washing the mitochondria or addition of excess histidine. The inhibition occurs at levels of inhibitor that have little or no effect on Ca²⁺ uptake, Na⁺/H⁺ antiport, or succinate respiration. A portion of the Na⁺-dependent efflux of Ca²⁺ is insensitive to diethylpyrocarbonate and this component is abolished by diltiazem. The mechanism by which diethylpyrocarbonate inactivates Na⁺/Ca²⁺ antiport is still uncertain, but may involve the modification of an unprotonated histidine residue in the transporter.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Mechanisms of [(3)H]glycine release from mouse spinal cord synaptosomes selectively labeled through GLYT2 transporters

Glycine release has been rarely studied. The aim of this work was to characterize the release of the amino acid from spinal cord glycinergic nerve endings selectively pre-labeled through glycine transporters of the GLYT2 type. Purified mouse spinal cord synaptosomes were incubated with [(3)H]glycine in the presence of the GLYT1 blocker N-[(3R)-3-([1,1'-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine hydrochloride and exposed in superfusion to varying concentrations of KCl, 4-aminopyridine (4-AP), or veratridine. KCl (< or = 15 micromol/L), 4-AP (up to 1 mmol/L), and veratridine (< or = 0.3 micromol/L)-provoked [(3)H]glycine release by external Ca2+-dependent, botulinum toxin C(1)-sensitive, exocytosis. The overflows evoked by higher concentrations of K+ or veratridine involved external Ca2+-independent mechanisms of different nature. Only the overflow evoked by 3 or 10 micromol/L veratridine occurred totally (3 micromol/L) or in part (10 micromol/L) by transporter reversal, being sensitive to the GLYT2 blockers 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminociclopentyl)-methyl] benzamide or O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-l-serine; in contrast, the external Ca2+-independent [(3)H]glycine overflow provoked by 50 mmol/L K+ was transporter-independent. This component of K+-evoked overflow and the GLYT2-independent portion of the 10 micromol/L veratridine-evoked overflow, were largely sensitive to the vesicle depletor bafilomycin or BAPTA-AM and were prevented by blocking the mitochondrial Na+/Ca2+ exchanger with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one, indicating the involvement of exocytosis triggered by intraterminal mitochondrial Ca2+ ions.     

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca²⁺ and Na⁺. These results led us to believe that the increase in Na⁺ by taurine could be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through Na⁺-Ca²⁺ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na⁺ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na⁺-Ca²⁺ exchanger blocker on taurine-induced [Ca]_i increase in embryonic chick heart cells. Using Fura-2 Ca²⁺ imaging and Fluo-3 Ca²⁺ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]_i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na⁺ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]_i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]_i. However, low concentrations of the Na⁺-Ca²⁺ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]_i in heart cells appears to be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger.