Characterization of [3H]GABA release from striatal slices: evidence for a calcium-independent process via the GABA uptake system

GABA can be released by depolarization even in the absence of external Ca2+. To investigate the underlying mechanism of this phenomenon, GABA release was studied using slices prepared from rat striatum. Slices were preincubated with [3H]GABA in the presence of beta-alanine and superfused with Krebs buffer. Total tritium efflux was measured as an index of GABA release. Electrical stimulation at 2 Hz for 3 min elevated resting tritium efflux approximately two-fold. Decreasing external Ca2+ to 0.1 mM increased basal tritium efflux and reduced electrically evoked overflow, while omitting Ca2+ entirely (and adding 1 mM EGTA) increased both basal efflux and evoked overflow. Tetrodotoxin (5 microM) abolished the evoked release of tritium but did not affect the resting outflow in either normal or Ca2+-deficient conditions. In the presence of normal Ca2+, nipecotic acid (0.1-1 mM), an inhibitor of GABA transport into neurons as well as glia, enhanced both spontaneous efflux and evoked overflow of tritium. Nipecotic acid also increased spontaneous release when external Ca2+ was reduced or removed; however, under these conditions electrically evoked overflow was reduced. These results suggest that the electrically evoked release of [3H]GABA from striatal slices is of neuronal origin, but can occur in part in the absence of external Ca2+. They further suggest that this Ca2+-independent release, which may co-exist with the Ca2+-dependent release, takes place via the same carrier system utilized for high-affinity GABA uptake.     

Tyrosine availability determines stimulus-evoked dopamine release from rat striatal slices

Direct evidence that precursor levels can affect catecholamine release from brain cells has not previously been presented. We observe a dose-dependent relationship between the tyrosine (Tyr) concentration of the superfusing medium and the amount of dopamine (DA) released from rat striatum by trains of electrical pulses. In the absence of exogenous tyrosine, tissue Tyr and DA levels are reduced following stimulation. These findings suggest that DA release from striatal neurons may not be sustained if fluctuations occur in the supply of Tyr to the brain.     

Coactivation of multiple tightly coupled calcium channels triggers spontaneous release of GABA

Voltage-activated Ca(2+) channels (VACCs) mediate Ca(2+) influx to trigger action potential-evoked neurotransmitter release, but the mechanism by which Ca(2+) regulates spontaneous transmission is unclear. We found that VACCs are the major physiological triggers for spontaneous release at mouse neocortical inhibitory synapses. Moreover, despite the absence of a synchronizing action potential, we found that spontaneous fusion of a GABA-containing vesicle required the activation of multiple tightly coupled VACCs of variable type.     

N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain

The role of voltage-dependent calcium channels (VDCCs) in the nicotinic acetylcholine receptor (nAChR)-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) was investigated in chick brain slices. Whole cell recordings of neurons in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv) nuclei showed that cadmium chloride (CdCl2) blocked the carbachol-induced increase of spontaneous GABAergic IPSCs, indicating that VDCCs might be involved. To conclusively show a role for VDCCs, the presynaptic effect of carbachol on SpL and LGNv neurons was examined in the presence of selective blockers of VDCC subtypes. omega-Conotoxin GVIA, a selective antagonist of N-type channels, significantly reduced the nAChR-mediated enhancement of gamma-aminobutyric acid (GABA) release in the SpL by 78% compared with control responses. Nifedipine, an L-type channel blocker, and omega-Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the enhancement of GABAergic IPSCs. In the LGNv, omega-Conotoxin GVIA also significantly reduced the nAChR-mediated enhancement of GABA release by 71% from control values. Although omega-Agatoxin-TK did not block the nicotinic enhancement, L-type channel blockers showed complex effects on the nAChR-mediated enhancement. These results indicate that the nAChR-mediated enhancement of spontaneous GABAergic IPSCs requires activation of N-type channels in both the SpL and LGNv.     

Release of taurine, GABA and dopamine from rat striatal slices: mutual interactions and developmental aspects

The spontaneous and potassium-stimulated release of preloaded taurine and GABA from striatal slices of adult and 7-day-old rats were studied using a superfusion system. Particular attention was paid to mutual interactions of taurine and GABA with dopamine in the release processes. Potassium stimulation (50 mM) enhanced taurine release more in the immature than in the adult striatum, whereas the response was the opposite with GABA release. Spontaneous taurine efflux was increased by dopamine and apomorphine, whereas stimulated release was suppressed by these agents in both age groups. This dopamine effect was partially antagonized by haloperidol, suggesting that dopaminergic systems were able to modify taurine release, possibly via dopaminergic receptors. Dopamine and apomorphine had similar but more inconsistent effects on striatal GABA release, which were not, however, mediated through conventional dopamine receptors. Stimulation with 25 mM K+ caused an 11-fold increase in striatal dopamine release: this effect was potentiated by taurine, while the actions of GABA on dopamine release were variable.     

Voltage-gated, margatoxin-sensitive potassium channels, but not calcium-gated, iberiotoxin-sensitive potassium channels modulate acetylcholine release in rat striatal slices

We evaluated the effects of iberiotoxin, an inhibitor of Slo-type Ca2+-activated potassium channels and two inhibitors of Shaker-type voltage-gated potassium channels margatoxin and dendrotoxin on acetylcholine outflow in rat striatal slices. An in vitro perfusion with 100 nM margatoxin or dendrotoxin induced a concentration-dependent and tetrodotoxin-sensitive enhancement in spontaneous acetylcholine release. In contrast, a perfusion with iberiotoxin did neither modulate basal, nor electrically- or N-methyl-d-aspartate-induced transmitter release. Therefore, Slo-type Ca2+-activated K+-channels do not seem to contribute significantly to cholinergic neurotransmission within rat striatal slices. As the Kv1.2 subtype represents the only common high affinity binding site of margatoxin and dendrotoxin and the effects of these toxins are not additive, this subtype is suggested to be the channel utilized by margatoxin and dendrotoxin to release acetylcholine in this model.     

Met-enkephalin selectively increases the spontaneous release of amino acid neurotransmitters from rat striatal slices

Met-enkephalin (1 microM) increased the spontaneous release of endogenous glutamate (+155%), taurine (+80%) and glycine (+50%) from rat striatal slices, but was without effect in the cerebral cortex. This effect was calcium-dependent and significantly reduced in the presence of naloxone (1 microM). Naloxone alone had no effect on release of any substance. Release of aspartate, gamma-aminobutyric acid and [3H]acetylcholine was not significantly affected. Met-enkephalin did not affect potassium (35 mM)-evoked neurotransmitter release, nor did it affect the uptake of D-[3H]aspartate, [3H]taurine or [14C]glycine. The data indicate that opioid receptor activation selectively increases the spontaneous calcium-dependent release of putative amino acid neurotransmitters in the rat striatum.     

Muscarinic receptors mediate direct inhibition of GABA release from rat striatal nerve terminals

The effects of acetylcholine (ACh) on the depolarization-evoked release of [3H]gamma-aminobutyric acid ([3H]GABA) have been investigated using synaptosomes prepared from rat corpus striatum and depolarized by superfusion with 9 mM KCl. Acetylcholine inhibited the [3H]GABA overflow in a concentration-dependent manner. The maximal effect was about 50%. The IC50 value (concentration producing half-maximal effect) amounted to 1 microM, in the absence of acetylcholinesterase inhibitors. The effect of ACh on the K(+)-evoked [3H]GABA release was counteracted by the muscarinic receptor antagonist atropine, but not by the nicotinic receptor antagonist mecamylamine or by the selective M1 antagonist pirenzepine. The data show that muscarinic receptors with low affinity for pirenzepine are localized on GABAergic nerve endings in rat corpus striatum where they may directly inhibit the release of GABA.     

Acetylcholine release from striatal slices of young adult and aged Fischer 344 rats

The release of endogenous and newly synthesized acetylcholine (ACh) was examined in neostriatal slices prepared from young adult (10-month) and aged (28-month) Fischer 344 rats. Both spontaneous and potassium-stimulated release were tested after various in vitro incubation times (1, 3 or 5 hr). The potassium-stimulated release of ACh from slices of 28-month rats was decreased by 53% when tested after incubating the slices 1 hr. The age-related differences in ACh release lessened if the slices were incubated for longer times (3 or 5 hr) before monitoring release. The spontaneous release of ACh was similar among the slices from both age-groups and at all times points monitored. When the neostriatal slices were incubated in medium supplemented with deuterated choline, the release of both the endogenous and newly synthesized ACh from slices of 28-month rats was decreased by 33% when tested after a 1-hr incubation, but was again similar to that released from slices of 10-month rats when tested after a 3-hr incubation. Choline release from slices of the 28-month rats was similar to that released from the slices of the 10-month rats when acetylcholinesterase (AChE) was inhibited during the release incubation. In slices with intact AChE activity, however, the age-related difference in choline release was similar to that observed for ACh release when AChE was inhibited. That is, when AChE activity was intact, the potassium-stimulated choline release from slices of 28-month rats was less than that released from slices of 10-month rats when release was tested after a 1-hr incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ryanodine receptor calcium release channels

The ryanodine receptors (RyRs) are a family of Ca2+ release channels found on intracellular Ca2+ storage/release organelles. The RyR channels are ubiquitously expressed in many types of cells and participate in a variety of important Ca2+ signaling phenomena (neurotransmission, secretion, etc.). In striated muscle, the RyR channels represent the primary pathway for Ca2+ release during the excitation-contraction coupling process. In general, the signals that activate the RyR channels are known (e.g., sarcolemmal Ca2+ influx or depolarization), but the specific mechanisms involved are still being debated. The signals that modulate and/or turn off the RyR channels remain ambiguous and the mechanisms involved unclear. Over the last decade, studies of RyR-mediated Ca2+ release have taken many forms and have steadily advanced our knowledge. This robust field, however, is not without controversial ideas and contradictory results. Controversies surrounding the complex Ca2+ regulation of single RyR channels receive particular attention here. In addition, a large body of information is synthesized into a focused perspective of single RyR channel function. The present status of the single RyR channel field and its likely future directions are also discussed.     

Investigations of the cholinergic modulation of GABA release in rat thalamus slices

The thalamus receives a dense cholinergic projection from the pedunculopontine tegmentum. A number of physiological studies have demonstrated that this projection causes a dramatic change in thalamic activity during the transition from sleep to wakefulness. Previous anatomical investigations have found that muscarinic type 2 receptors are densely distributed on the dendritic terminals of GABAergic interneurons, as well as the somata and proximal dendrites of GABAergic cells in the thalamic reticular nucleus. Since these structures are the synaptic targets of cholinergic terminals in the thalamus, it appears likely that thalamic pedunculopontine tegmentum terminals can activate muscarinic type 2 receptors on GABAergic cells. To test whether activation of muscarinic type 2 receptors affects the release of GABA in the thalamus, we have begun pharmacological studies using slices prepared from the rat thalamus. We have found that the application of the nonspecific muscarinic agonist, methacholine, and the muscarinic type 2-selective agonist, oxotremorine.sesquifumarate, diminished both the baseline, and K(+) triggered release of [(3)H]GABA from thalamic slices. This effect was calcium dependent, and blocked by the nonselective muscarinic antagonist atropine, the muscarinic type 2-selective antagonist, methoctramine, but not the muscarinic type 1 antagonist, pirenzepine. Thus, it appears that one function of the pedunculopontine tegmentum projection is to decrease the release of GABA through activation of muscarinic type 2 receptors. This decrease in inhibition may play an important role in regulating thalamic activity during changes in states of arousal.     

NMDA- and kainate-evoked GABA release from striatal neurones differentiated in primary culture: differential blocking by phencyclidine

N-Methyl-D-aspartate (NMDA) stimulated (EC50 = 19.4 +/- 1.9 microM) gamma-aminobutyric acid (GABA) release from highly purified striatal neurones differentiated in primary culture. NMDA effect was inhibited (i) in a competitive manner by DL-2-amino-5-phosphonovalerate (APV) and (ii) in a non-competitive manner by phencyclidine (PCP). Kainate (KA) also stimulated GABA release, but this effect was never inhibited by PCP despite the multiple conditions tested (KA stimulation performed, after or not NMDA application, in the presence or not of NMDA). The existence of two distinct receptor-channel complexes on striatal neurones selectively activated by NMDA and KA is discussed.     

Presynaptic effect of L-glutamic acid on the release of dopamine in rat striatal slices

The release of [³H] dopamine ([³H] DA) was estimated in serial superfusate fractions of rat striatal slices continuously superfused with L-[3,5-³H]-tyrosine. L-glutamic acid (5 · 10^?5 M), but not the D-stereoisomer, increased the spontaneous release of [³H] DA (60%). The stimulating effect of L-glutamic acid was still observed in the presence of tetrodotoxin (5 · 10^?7 M), suggesting that the amino-acid acts at a presynaptic site. Moreover, atropine (10^?6 M) or pempidine (10^?5 M) which blocks the acetylcholine (ACh) evoked release of [³H] DA did not reduce the stimulatory effect of L-glutamic acid on [³H] DA release, thus excluding the possible intervention of striatal cholinergic neurons. The data obtained support the hypothesis of a direct control of DA release from nerve terminals by glutamatergic neurons.     

Endothelin-triggered brain damage under hypoglycemia evidenced by real-time monitoring of dopamine release from rat striatal slices

The role of endothelin in the pathogenesis of hypoglycemic brain damage in rats was evaluated using an in vitro model with which we could directly monitor the release of dopamine from striatal slices. There was no evidence of impairment in case of non-exposure of the slices to endothelin during 20-40 min of hypoglycemia. The response all but disappeared in striatal slices stimulated with endothelin 10(-5) M twice during 20 min of hypoglycemia. Endothelin-triggered hypoglycemic damage was not observed in the absence of extracellular Ca2+ or in the presence of nifedipine 10(-6) M. Our findings provide strong evidence that endothelin is one etiological factor in the development of hypoglycemic/ischemic brain injury, as a result of interaction with specific receptors which activate the voltage-sensitive Ca2+ channel.     

Calcium-independent release of gamma-aminobutyrate from nerve processes in the developing rabbit retina

Retinas from 3- and 10-day-old rabbits, and from young (29 days), or adult animals were used to study in parallel the development of synaptic vesicles in amacrine cells and the Ca2+ dependence of the K+-stimulated [3H]gamma-aminobutyrate release from them. Few synaptic vesicles were observed in the amacrine cell processes in retinas from the 3-day-old rabbits. The number of vesicles significantly increased between 3 and 10 days and increased further between day 10 and the adult animal. The Ca2+ dependence of the K+-stimulated release decreased with increasing age. There is thus a poor correlation between the Ca2+ dependent transmitter release and the number of synaptic vesicles in the nerve terminal, favouring the existence of a Ca2+ dependent nonvesicular process for the [3H]gamma-aminobutyrate release in the rabbit retina.     

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.     

DOPA cyclohexyl ester potently inhibits aglycemia-induced release of glutamate in rat striatal slices

Brain ischemic insult causes glutamate release and resultant neuronal cell death. We here show that L-3,4-dihydroxyphenylalanine (DOPA) is a positive regulatory factor for glutamate release elicited by a mild brain insult using in vitro superfused rat striatal slices as a model system. Glucose deprivation for 18 min elicited release of glutamate, DOPA and dopamine (DA). Either tetrodotoxin (TTX) (1 microM) or alpha-methyl-p-tyrosine (alpha-MPT) (1 mM), a tyrosine hydroxylase inhibitor reduced markedly each of these releases. NSD-1015 (20 microM), an aromatic L-amino acid decarboxylase inhibitor restored the inhibition by alpha-MPT of glutamate and DOPA but not DA release. DOPA cyclohexyl ester (DOPA CHE) (0.3-1 microM), a competitive DOPA antagonist, concentration-dependently suppressed aglycemia-induced glutamate release, the effect which was mimicked neither by S-sulpiride nor SCH23390, a DA D(1) or D(2) receptor antagonist, respectively. Zonisamide (1-1000 microM), an anticonvulsant or YM872 (1 microM), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) a receptor antagonist produced no effect on aglycemia-induced glutamate release. DOPA CHE thus showed a relatively potent inhibitory action on aglycemia-induced glutamate release among several neuroprotective agents tested.     

GABA, taurine and learning: release of amino acids from slices of chick brain following filial imprinting.

The intermediate and medial hyperstriatum ventrale (IMHV) is a forebrain region in the domestic chick that is a site of information storage for the learning process of imprinting. We enquired whether imprinting is associated with learning-related increases in calcium-dependent, potassium-stimulated release of neurotransmitter amino acids from the IMHV. Chicks were hatched and reared in darkness until 15-30 h after hatching. They then either remained in darkness or were trained for 2 h by exposure to an imprinting stimulus. One hour later, the chicks were given a preference test and a preference score was calculated from the results of this test, as a measure of imprinting. Chicks were killed 2 h after training. Slices from the left and right IMHV of trained and untrained chicks were superfused with Krebs' solution either with or without calcium and the superfusate assayed for arginine, aspartate, citrulline, GABA, glutamate, glycine and taurine using high-performance liquid chromatography. For calcium-containing superfusates from the left IMHV, preference score was significantly correlated with potassium-stimulated release of (i) GABA (r=0.51, 23 d.f., P=0.008) and (ii) taurine (r=0.77, 23 d.f., P<0.0001). There was no significant difference between the mean values of trained and untrained chicks for either compound. However, examination of the variance of the data indicated that release of both GABA and taurine increased as a result of learning. No significant correlation between preference score and release was found for any of the amino acids from the right IMHV, nor for control tissue from the left IMHV superfused with calcium-free solution. These results demonstrate that the learning process of imprinting is associated with increases in releasable pools of GABA and taurine and/or membrane excitability in the left IMHV.     

Mechanotransduction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels

Changes in strain distribution across the vertebrate skeleton induce modelling and remodelling of bone structure. This relationship, like many in biomedical science, has been recognised since the 1800s, but it is only the recent development of in vivo and in vitro models that is allowing detailed investigation of the cellular mechanisms involved. A number of secondary messenger pathways have been implicated in load transduction by bone cells, and many of these pathways are similar to those proposed for other load-responsive cell types. It appears that load transduction involves interaction between several messenger pathways, rather than one specific switch. Interaction between these pathways may result in a cascade of responses that promote and maintain bone cell activity in remodelling of bone. The paper outlines research on the early rapid signals for load transduction and, in particular, activation of membrane channels in osteoblasts. The involvement of calcium channels in the immediate load response and the modulation of intracellular calcium as an early signal are discussed. These membrane channels present a possible target for manipulation in the engineering of bone tissue repair.     

Mechanotransduction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels

Changes in strain distribution across the vertebrate skeleton induce modelling and remodelling of bone structure. This relationship, like many in biomedical science, has been recognised since the 1800s, but it is only the recent development of in vivo and in vitro models that is allowing detailed investigation of the cellular mechanisms involved. A number of secondary messenger pathways have been implicated in load transduction by bone cells, and many of these pathways are similar to those proposed for other load-responsive cell types. It appears that load transduction involves interaction between several messenger pathways, rather than one specific switch. Interaction between these pathways may result in a cascade of responses that promote and maintain bone cell activity in remodelling of bone. The paper outlines research on the early rapid signals for load transduction and, in particular, activation of membrane channels in osteoblasts. The involvement of calcium channels in the immediate load response and the modulation of intra-cellular calcium as an early signal are discussed. These membrane channels present a possible target for manipulation in the engineering of bone tissue repair.