Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Adenosine A1 receptors inhibit Ca channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A₁ receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca²⁺]_i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [³H]ACh, but not the release of [¹⁴C]GABA, was potentiated when adenosine A₁ receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca²⁺]_i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A₁ receptors. Our results show that adenosine A₁ receptors inhibit Ca²⁺ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A₁ receptors.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons

To determine whether the sensitizing actions of prostaglandins on sensory neurons are due to modulation of voltage-sensitive calcium channels (VSCC) we examined the effects of inhibiting these channels on PGE,-induced enhancement of evoked peptide release from isolated dorsal root ganglion neurons. The inhibitory effects of the VSCC blockers on stimulated release were dependent upon the type of chemical agent used to evoke the release. Bradykinin-stimulated release of immunoreactive substance P(iSP) and calcitonin gene-related peptide (iCGRP) was attenuated by the N-type VSCC blocker, ω-conotoxin GVIA (100 nM), but was unaffected by blockade of L-type (1 μM nifedipine) or P-type (200 nM ω-agatoxin IVA) VSCC. In contrast, potassium-stimulated release of peptides was inhibited by nifedipine, but not by co-conotoxin GVIA or ω-agatoxin IVA. None of the VSCC blockers tested attenuated capsaicin-stimulated release of iSP and iCGRP. The combination of 1 μM nifedipine and 100 nM ω-conotoxin GVIA reduced the whole cell calcium current 89% ± 1.7%. Administration of 100 nM PGE₂ potentiated bradykinin- and capsaicin-evoked peptide release by 2–3-fold. Neither nifedipine nor ω-conotoxin GVIA attenuated the PGE₂-mediated potentiation of bradykinin-evoked release, and neither ω-conotoxin GVIA nor w-agatoxin IVA blocked the potentiation of capsaicin-evoked release induced by PGE₂. These results indicate that the sensitizing actions of PGE₂ as measured by enhanced peptide release, are not mediated by L-, N-, or P-type VSCC.     

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.     

Multiple Types of Ca2+ Channels in Mouse Motor Nerve Terminals

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca²⁺-channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω-Aga-IVA (0.3 μM), a blocker of P-type Ca²⁺channels] or ω-conotoxin MVIIC [ω-CTx-MVIIC (2 μM), a P-and Q-type Ca²⁺-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L-type Ca²⁺ channels) plus ω-conotoxin GVIA [ω-CTx-GVIA (2 μM), a blocker of N-type Ca²⁺ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca²⁺ signals, a small decrease in presynaptic Ca²⁺ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4-diaminopyridine (3, 4-DAP, a k⁺-channel blocker) or high Ca²⁺(10 mM) to accelerate and amplify the endplate potentials and Ca²⁺ currents. The endplate potentials amplified by 3, 4-DAP or by high Ca²⁺ correspondingly proved to be quite resistant to both ω-Aga-IVA and ω-CTx-MVIIC; ωAga-IVA exerted only a partial inhibitory effect on endplate potentials, and the ω-Aga-IVA-resistant component was further inhibited by ω-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca²⁺ channel blocker Cd²⁺ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na⁺ and K⁺ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca²⁺ currents in the triangularis sterni also revealed partial inhibition by ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA. Cd²⁺ (300 μM) abolished the toxin-resistant component of the Ca²⁺ current. In contrast, a combination of nifedipine (50 μM) with ω-CTx-GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca²⁺channels, i.e. ω-Aga-IVA-sensitive, ω-CTx-MVIIC-sensitive and toxin-resistant Ca²⁺ channels, coexist in mouse motor nerve terminals.     

Inhibition of N-type Calcium Channels: The Only Mechanism by which Presynaptic α2-Autoreceptors Control Sympathetic Transmitter Release

α₂-Adrenoceptors are known to inhibit voltage-dependent Ca²⁺ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca²⁺ entry, underlie the presynaptic α₂-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [³H]noradrenaline was elicited in the presence of extracellular Ca²⁺ by electrical pulses, 25 mM K⁺ or 10μM nicotine, or by adding Ca²⁺ to otherwise Ca²⁺-free medium when cells had been made permeable by the calcium ionophore A23187 or by α-latrotoxin. Pretreatment of neurons with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA and application of the α₂-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K⁺ or nicotine, but not the overflow caused by Ca²⁺ after permeabilization with α-latrotoxin or A23187. In contrast, the L-type Ca²⁺ channel blocker nitrendipine reduced the overflow due to K⁺ and nicotine, but not the overflow following electrical stimulation or α-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca²⁺ channel agonist BayK 8644, which per se enhanced overflow. In ω-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca²⁺ currents corroborated these results: UK 14304 inhibited Ca²⁺ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α₂-adrenergic effects. Our results indicate that the α₂-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω-conotoxin GVIA-sensitive N-type Ca²⁺ channels. Mechanisms downstream of these channels and voltage-sensitive Ca²⁺ channels other than N-type appear not to be important.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Na influx through Ca channels can promote striatal GABA efflux in Ca-deficient conditions in response to electrical field depolarization

Electrical field depolarization releases γ-aminobutyric acid (GABA) in rat striatal slices in the absence of external Ca²⁺. ω-Conotoxin GVIA (ω-CgTx; 1–50 nM), a neuronal Ca²⁺ channel blocker, inhibits electrically evoked efflux of newly taken up [³H]GABA in a concentration-dependent manner in either normal or Ca²⁺-free medium. This suggests that ion influx occurs through Ca²⁺ channels in the absence of external Ca²⁺ and contributes to the efflux of GABA. Reducing external Na⁺ concentration to 27.25 mM (low [Na⁺]₀ medium) by equimolarly substituting choline chloride for sodium chloride has differential effects on electrically evoked GABA efflux depending on the external Ca²⁺ concentrations. In normal Ca²⁺ medium, electrically evoked GABA efflux increases whereas, in Ca²⁺-free medium, it is greatly inhibited when [Na⁺]₀ is reduced to 27.25 mM. In low [Na⁺]₀ medium, GABA efflux is largely tetrodotoxin (TTX)-sensitive, however, spike firing evoked by antidromic stimulation of striatal cells is inhibited. In Na⁺-free medium, resting GABA efflux increases 17-fold whereas evoked GABA efflux diminishes. In Ca²⁺-free medium, 70 min of incubation with 1–2-bis-(2-aminophenoxy)ethane-N,N,N′,N′ tetraacetoxy methyl ester (BAPTA-AM, 1 μM), an intracellular calcium chelator, increases both resting GABA efflux and electrically evoked GABA overflow by 100%. These results suggest that: (1) in Ca²⁺-free conditions, Na⁺ permeability of cells increases via Ca²⁺ channels and this profoundly affects GABA efflux. (2) Electrical field depolarization is likely to release GABA by directly depolarizing axon terminals. (3) Ca²⁺-independent GABA efflux is not promoted by an increase in intracellular free Ca²⁺ concentration via Na⁺/Ca²⁺ exchange processes from internal pools.     

Stimulation of L-type Ca2+ channel in growth cones activates two independent signaling pathways

Although growth cones respond to various modulators of neurite outgrowth, such as neurotrophins, neurotransmitters, and cell adhesion molecules, the signal-transducing mechanisms for these modulators in growth cones are unclear. Since recent studies have suggested that the signals of these modulators are mediated by Ca²⁺ influx through L-type voltage-sensitive Ca²⁺ channels (VSCCs) in the growth cone, we examined L-type VSCC-dependent signaling pathways, using isolated growth cones (IGCs) from developing rat forebrains. Binding assays revealed that L-type VSCC is enriched in growth cone membrane and gradually decreased in amount developmentally, while N-type VSCC has the opposite tendency. In intact IGCs, Bay K 8644 (BK, an L-type agonist) induced much more rapid elevation of [Ca²⁺]_i than that in adult synaptosomes. Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by protein kinase C (PKC) was enhanced in the IGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca²⁺-dependent degradation of brain spectrin (fodrin) by calpain was also enhanced by BK or GABA, consequently inducing the release of α-actinin from the membrane skeleton of the growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other, indicating that these reactions occur independently. Our results suggest that Ca²⁺ influx through L-type VSCCs activates two distinct signaling branches, probably in the different domains of the growth cone, i.e., Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein, and Ca²⁺-dependent degradation of brain spectrin and the release of α-actinin by calpain. J. Neurosci. Res. 51:682–696, 1998. © 1998 Wiley-Liss, Inc.     

Calcium influx through N- and P/Q-type channels activate apamin-sensitive calcium-dependent potassium channels generating the late afterhyperpolarization in lamprey spinal neurons

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca²⁺-dependent K⁺ channels (K_Ca) activated by calcium influx through voltage-dependent channels during the action potential ( 1 , Neuroreport, 3, 943–945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the K_Ca channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by ω-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2 ± 14.9% (mean ± SD). Application of the P/Q-type calcium channel blocker ω-agatoxin IVA reduced the amplitude of the AHP by 20.3 ± 10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (±)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100–200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive K_Ca channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.     

Properties of the voltage-gated calcium channels mediating dopamine and acetylcholine release from the isolated rat retina

We examined the properties of voltage-gated calcium channels mediating endogenous dopamine (DA) and acetylcholine (ACh) release in the isolated rat retina. Application of 30 mM KCl elicited the release of DA and ACh, and these releases were abolished in Ca²⁺-free medium. The high K⁺-evoked DA release was largely blocked by both of ω-agatoxin IVA and ω-conotoxin MVIIC, P- and Q-type calcium channel antagonists, and partly blocked by isradipine, an L-type calcium channel antagonist, and ω-conotoxin GVIA, an N-type calcium channel antagonist, ω-Agatoxin IVA at a small dose, sufficient to block P-type channels alone, was however without effect. On the other hand, the high K⁺-evoked ACh release was partly blocked by ω-agatoxin IVA and ω-conotoxin MVIIC, but was resistant to isradipine and ω-conotoxin GVIA. Flunarizine, a non-selective T-type calcium channel antagonist, did not inhibit the release of DA and ACh. Cd²⁺ markedly blocked the release of both DA and ACh, Co²⁺ and Ni²⁺ slightly blocked the release of DA, and the release of ACh was not blocked by these two divalent cations. These results suggest that the high K⁺-evoked release of retinal DA is largely mediated by ω-agatoxin IVA and ω-conotoxin MVIIC sensitive calcium channels (probably Q-type channels), while the release of retinal ACh is largely mediated by as yet uncharacterized Cd²⁺ sensitive calcium channels. The properties of voltage-gated calcium channels involved in the release of ACh in the rat retina differ from those of DA.     

Adenosine A1 receptors inhibit Ca2+ channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A1 receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca2+]i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [3H]ACh, but not the release of [14C]GABA, was potentiated when adenosine A1 receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca2+]i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A1 receptors. Our results show that adenosine A1 receptors inhibit Ca2+ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A1 receptors.     

Release of γ-[H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain

In the present study we have investigated the effect of cations and ouabain on Ca²⁺-independent and Ca²⁺-dependent release of γ-[³H]aminobutyric acid ([³H]GABA) from sheep brain synaptosomes. The presence of Na⁺ in the external medium is essential for the Ca²⁺-independent release induced by K⁺ or ouabain. Thus, in the absence of Ca²⁺, ouabain of K⁺ causes the release of [³H]GABA provided that Na⁺ is present in the external medium. Under K⁺-depolarizing conditions, in a Na⁺ medium, either ouabain or Ca²⁺ further increases the [³H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na⁺ is not required for the Ca²⁺-dependent release of [³H]GABA due to K⁺ depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K⁺-depolarization dependent release is absolutely dependent on external Ca²⁺, which suggests that this release of [³H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K⁺-depolarization in a Na⁺ medium. It is likely that the release induced by ouabain or K⁺ involves the membrane carrier which responds to changes in membrane potential.     

Endogenous GABA release from slices of rat cerebral cortex and hippocampus in vitro

Using a rapid, simple and sensitive radioreceptor assay, a Ca²⁺-dependent K⁺-evoked release of endogenous GABA was demonstrated from rat cortical and hippocampal slices in vitro. This evoked-release of endogenous GABA was similar to tha of [³H]GABA release (in its Ca²⁺ dependency) but differed from the latter in having a higher signal to noise level. Neither 5-HT nor a stable enkephalin analogue had any effect on endogenous GABA release from hippocampus slices.     

Excitatory sulphur amino acid-evoked neurotransmitter release from rat brain synaptosome fractions

Summary The neuroactive sulphur-containing amino acids L-cysteate (CA), L-cysteine sulphinate (CSA), L-homocysteine sulphinate (HSA), S-sulpho-L-cysteine (SC) and L-homocysteate (HCA) evoked the release of previously accumulated D-[³H]aspartate from rat brain cerebrocortical and cerebellar synaptosome fractions in a manner that was wholly Ca²⁺-independent. However, analysis of endogenous release by hplc revealed the presence of both Ca²⁺-dependent and -independent components of L-glutamate release but only a Ca²⁺-independent component of L-aspartate release. CA, CSA, HSA and SC but not HCA evoked the release of previously accumulated [³H]GABA from synaptosome fractions by a mechanism shown to comprise both a Ca²⁺-dependent and -independent component. The specific antagonists of the N-methyl-D-aspartate (NMDA) receptor, 3-[(±)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP) and the relatively selective competitive quisqualate (QUIS)/kainate (KA) receptor antagonist, 6-cyano-7-dinitroquinoxalinedione (CNQX), were ineffective in blocking the excitatory sulphur amino acid-evoked release of either D-[³H]aspartate, [³H]GABA or of endogenous established transmitter amino acids.     

Effects of ω-conotoxin MVIIC on veratridine-induced cytotoxicity and cytosolic Ca oscillations

External Ca²⁺ entry through various Ca t+-channel subtypes is responsible for the large oscillations of the cytosolic Ca²⁺ concentrations, [Ca²⁺]_i, and cell death induced by veratridine in primary cultures of bovine chromaffin cells. Blockade by ω-conotoxin GVIA (GVIA) of N-type Ca²⁺ channels, by ω-agatoxin GIVA (IVA) of P-type Ca²⁺ channels, or by furnidipine of L-type Ca²⁺ channels did not afford cytoprotection. However, ω-conotoxin MVIIC (MVIIC), a wide-spectrum blocker of N-, P- and Q-type Ca²⁺ channels greatly protected the cells against the cytotoxic effects of veratridine. Furnidipine further enhanced the cytoprotecting effects of MVIIC. MVIIC but not fumidipine, markedly reduced the oscillations of [Ca²⁺]_i induced by veratridine in single fura-2-loaded chromaffin cells. The results suggest that Ca²⁺ entry through any of the different Ca²⁺ channel subtypes present in bovine chromaffin cells might be cytotoxic. They also support two ideas: (i) that wide-spectrum neuronal Ca²⁺ channel blockers (i.e. MVIIC) might be better cytoprotecting agents than more specific neuronal Ca²⁺ channel blockers (i.e., GVIA, IVA, furnidipine); and (ii) that combined Ca²⁺ channel blockers may provide greater cytoprotection than single compounds.     

A new function for glycine GlyT2 transporters: Stimulation of γ‐aminobutyric acid release from cerebellar nerve terminals through GAT1 transporter reversal and Ca2+‐dependent anion channels

Glycine GlyT2 transporters are localized on glycine‐storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ‐aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule‐associated protein‐2‐positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [³H]GABA and exposed to glycine. Glycine stimulated [³H]GABA release in a concentration‐dependent manner. The glycine effect was insensitive to strychnine or to 5,7‐dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca²⁺ entered by reversal of plasmalemmal Na⁺/Ca²⁺exchangers. A significant portion of the GlyT2‐mediated release of [³H]GABA (about 50% of the external Ca²⁺‐independent release) occurred by reversal of GABA GAT1 transporters. Na⁺ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na⁺/Ca²⁺ exchangers, causing an increase in cytosolic Ca²⁺, which in turn triggered a Ca²⁺‐induced Ca²⁺ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca²⁺ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels. © 2013 Wiley Periodicals, Inc.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

Effect of L-glutamic acid on [C]GABA release from isolated rat retina

The effect of various putative neurotransmitters on the release of [¹⁴C]GABA taken up by isolated rat retina was investigated by a perfusion technique.l-Glutamic acid initially enhanced the GABA release and subsequently reduced it to a level below the baseline rate. This enhancing effect ofl-glutamic acid was Ca²⁺-dependent and tetrodotoxin-sensitive.l-Aspartic acid only reduced the GABA release, whereas kainic acid exhibited only a marked enhancement of its release. Acetylcholine, norepinephrine, dopamine, glycine and taurine did not have any significant effects on GABA release.