The effect of anaesthetics on the uptake and release of gamma-aminobutyrate and D-aspartate in rat brain slices.

1 The effect of various concentrations of thiopentone, pentobarbitone, methohexitone, hydroxydione, alphaxalone/alphadolone, ketamine, alpha-chloralose, and urethane on the transport of radiolabelled gamma-aminobutyric acid (GABA) and D-aspartate was investigated. 2 Uptake of the amino acids was weakly inhibited, if at all, by the anaesthetics and it is unlikely that such effects contribute significantly to their physiological function. 3 The spontaneous efflux of GABA and D-aspartate was not detectably altered by any of the drugs tested. 4 Thiopentone, pentobarbitone, methohexitone and hydroxydione inhibited K+-stimulated GABA and D-aspartate release. The other anaesthetics had no effect on K+-stimulated amino acid release. 5 The rank order of potency of the inhibitors of K+-stimulated amino acid release did not correlate with their anaesthetic potency. Furthermore not all inhibitors appeared to be very effective at anaesthetic concentrations. 6 It is concluded that although it is possible that inhibition of excitatory transmitter release may be involved in the anaesthetic action of some anaesthetics, for many of the substances tested in this study such as mechanism does not appear to be implicated.     

Regional differences in the effects of isoflurane on neurotransmitter release

Stimulus evoked neurotransmitter release requires that Na⁺ channel-dependent nerve terminal depolarization be transduced into synaptic vesicle exocytosis. Inhaled anesthetics block presynaptic Na⁺ channels and selectively inhibit glutamate over GABA release from isolated nerve terminals, indicating mechanistic differences between excitatory and inhibitory transmitter release. We compared the effects of isoflurane on depolarization-evoked [³H]glutamate and [¹⁴C]GABA release from isolated nerve terminals prepared from four regions of rat CNS evoked by 4-aminopyridine (4AP), veratridine (VTD), or elevated K⁺. These mechanistically distinct secretegogues distinguished between Na⁺ channel- and/or Ca²⁺ channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC₅₀ = 0.42 ± 0.03 mM) more potently than GABA release (IC₅₀ = 0.56 ± 0.02 mM) from cerebral cortex (1.3-fold greater potency), hippocampus and striatum, but inhibited glutamate and GABA release from spinal cord terminals equipotently. Na⁺ channel-specific VTD-evoked glutamate release from cortex was also significantly more sensitive to inhibition by isoflurane than was GABA release. Na⁺ channel-independent K⁺-evoked release was insensitive to isoflurane at clinical concentrations in all four regions, consistent with a target upstream of Ca²⁺ entry. Isoflurane inhibited Na⁺ channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC₅₀ = 0.30 ± 0.03 mM) more potently than GABA release (IC₅₀ = 0.67 ± 0.04 mM) from cortex (2.2-fold greater potency). The magnitude of inhibition of Na⁺ channel-mediated 4AP-evoked release by a single clinical concentration of isoflurane (0.35 mM) varied by region and transmitter: Inhibition of glutamate release from spinal cord was greater than from the three brain regions and greater than GABA release for each CNS region. These findings indicate that isoflurane selectively inhibits glutamate release compared to GABA release via Na⁺ channel-mediated transduction in the four CNS regions tested, and that differences in presynaptic Na⁺ channel involvement determine differences in anesthetic pharmacology.     

Anaesthetic drugs: linking molecular actions to clinical effects

The use of general anaesthetics has facilitated great advantages in surgery within the last 150 years. General anaesthesia is composed of several components including analgesia, amnesia, hypnosis and immobility. To achieve these components, general anaesthetics have to act via multiple molecular targets at different anatomical sites in the central nervous system. Much of our current understanding of how anaesthetics work has been obtained within the last few years on the basis of genetic approaches, in particular knock-out or knock-in mice. Anaesthetic drugs can be grouped into volatile and intravenous anaesthetics according to their route of administration. Common volatile anaesthetics induce immobility via molecular targets in the spinal cord, including glycine receptors, GABA(A) receptors, glutamate receptors, and TREK-1 potassium channels. In contrast, intravenous anaesthetics cause immobility almost exclusively via GABA(A) receptors harbouring beta3 subunits. Hypnosis is predominantly mediated by beta3-subunit containing GABA(A) receptors in the brain, whereas beta2 subunit containing receptors, which make up more than 50% of all GABA(A) receptors in the central nervous system, mediate sedation. At clinically relevant concentrations, ketamine and nitrous oxide block NMDA receptors. Unlike all other anaesthetics in clinical use they produce analgesia. Not only desired actions of anaesthetics, but also undesired side effects are linked to certain receptors. Respiratory depression involves beta3 containing GABA(A) receptors whereas hypothermia is largely mediated by GABA(A) receptors containing beta2 subunits. These recent insights into the clinically desired and undesired actions of anaesthetic agents provide new avenues for the design of drugs with an improved side-effect profile. Such agents would be especially beneficial for the treatment of newborn children, elderly patients and patients undergoing ambulatory surgery.     

The TREK K2P channels and their role in general anaesthesia and neuroprotection

Two-pore-domain K+ (K(2P)) channels are a diverse and highly regulated superfamily of channels that are thought to provide baseline regulation of membrane excitability. Of these, the TREK channels are expressed highly in the human CNS, and can be activated by temperature, membrane stretch and internal acidosis. In addition, TREK channels are sensitively activated by certain polyunsaturated fatty acids that have been shown to have neuroprotective activity and by volatile and gaseous general anaesthetics. New data derived from studies of knockout animals suggest that TREK-1 might have an important role in the general anaesthetic properties of volatile agents, such as halothane, and provide an explanation for the neuroprotective properties of polyunsaturated fatty acids.     

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca(2+) channels and adenylyl cyclase. Through Ca(2+) imaging and immunocytochemistry, we have recently shown that adenosine A(1), GABA(B) and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca(2+) channels by analysing this process in nerve terminals from mice lacking the alpha(1B) subunit (Ca(v) 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca(2+) channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABA(B) and adenosine A(1). Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A(1), GABA(B) and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca(2+) channel type but rather, it is associated to functional mGlu7 receptors.     

Inhibitory effects of intravenous anaesthetic agents on K(+)-evoked glutamate release from rat cerebrocortical slices. Involvement of voltage-sensitive Ca(2+) channels and GABA(A) receptors

It is widely accepted that most general anaesthetic agents depress the central nervous system (CNS) by potentiation or activation of the GABA(A) receptor-mediated Cl(-) conductance. These agents also reportedly inhibit voltage-sensitive Ca(2+) channels (VSCCs), thus depressing excitatory transmission in the CNS. However, in this regard there are few functional data at the level of neurotransmitter release. In this study we examined the effects of VSCC antagonists and a range of intravenous anaesthetic agents on K(+)(40 mM)-evoked glutamate release from rat cerebrocortical slices in the absence and presence of the GABA(A) receptor antagonist bicuculline (100 microM). We employed both selective and non-selective VSCC antagonists, the anaesthetic barbiturates thiopental, pentobarbital and phenobarbital, the non-anaesthetic barbiturate barbituric acid, the non-barbiturate anaesthetics alphaxalone, propofol and ketamine and the GABA(A) receptor agonist, muscimol. Glutamate released into the incubation medium was determined by a glutamate dehydrogenase-coupled assay. Omega-agatoxin IV(A) (P-type VSCC), omega-conotoxin MVII(C) (P/Q-type VSCC) and Cd(2+) (non-selective) essentially abolished glutamate release whilst nifedipine (L-type VSCC) and omega-conotoxin GVI(A) (N-type VSCC) reduced release by less than 30%. The concentrations producing 50% of the maximum inhibition (IC(50)) for thiopental, pentobarbital, phenobarbital, alphaxalone, propofol and ketamine were (in microM) 8.3, 22, 112, 6.3, 83 and 120, respectively. Barbituric acid produced a small (about 20%) inhibition. With the exception of ketamine, the IC(50) values for these anaesthetic agents were increased threefold by bicuculline (100 microM). In addition, muscimol significantly inhibited release by 26% with an IC(50) of 1.1 microM. In summary, a range of anaesthetic agents at clinically achievable concentrations inhibit glutamate release and this inhibition of release appears to be due mainly to direct inhibition of P/Q-type VSCCs, although activation of the GABA(A) receptor plays a role in this response.     

GABA, glutamate and substance P-like immunoreactivity release: effects of novel GABAB antagonists.

1. The effects of various GABA receptor ligands on the electrically-evoked release of endogenous GABA, glutamate and substance P-like immunoreactivity from the dorsal horn of rat isolated spinal cord were examined. 2. Exogenous GABA (10-300 microM) significantly decreased the evoked, but not basal, release of endogenous glutamate in a concentration-dependent manner. The GABAA agonist, isoguvacine (1-100 microM), failed to decrease the release of glutamate although it did reduce the release of GABA. Baclofen (0.1-1000 microM), the GABAB agonist, reduced the release of GABA and glutamate in a stereospecific and concentration-dependent manner. 3. The actions of five GABAB antagonists on these release systems were compared. CGP36742, CGP52432, CGP55845A and CGP57250A significantly increased the evoked release of GABA and glutamate. They also reversed the effects of (-)-baclofen in a concentration-dependent manner. On the other hand, while CGP56999A had no effect on glutamate release, it was an effective antagonist of the baclofen-induced inhibition of GABA and substance P release. 4. These results suggest that GABAB receptors on nerve terminals within the dorsal horn spinal cord may be heterogeneous. However, this is based solely on the data obtained with CGP56999A which affected only GABA and substance P, but not glutamate, release.     

The metabotropic glutamate receptor 1 is not involved in the facilitation of glutamate release in cerebrocortical nerve terminals

In this study we have addressed the identification of the metabotropic glutamate receptor (mGluR) involved in the facilitation of glutamate release in nerve terminals from the cerebral cortex. mGluR1 and 5 are coupled to phosphoinositide hydrolysis and the activation of these receptors with the specific agonist 3,5-dihydroxyphenylglycine (DHPG) enhances the release of glutamate. We have examined whether mGluR1 is responsible for this modulatory effect by preparing nerve terminals from mGluR1 deficient mice. The Ca²⁺-dependent glutamate release evoked by a submaximal depolarization is enhanced by the agonist DHPG in nerve terminals from both wild and mutant mice. This result is consistent with the finding that the mGluR agonist also induces a similar increase in the levels of diacylglycerol (DAG) in the nerve terminals from wild and mutant mice. Moreover, the activity-dependent switch from facilitation to inhibition of release, observed when a second stimulation of the receptor is applied shortly after (5 min) the first pulse, was also observed in the mutant mice. These results indicate therefore, that the facilitation of glutamate release is unlikely to be due to the activation of mGluR1 but related to another phosphoinositide coupled mGluR.     

A novel GABA receptor modulates stimulus-induced glutamate release from cortico-striatal terminals

The possibility of there being a population of GABA receptors located on cortico-striatal terminals is suggested by [3H]GABA binding studies. Experiments carried out to investigate the stimulus-induced release of glutamate from cortico-striatal terminals revealed an active modulation by a presynaptic GABA receptor. The differential responses of this receptor to a range of GABA agonists suggest that it may represent a novel type of GABA receptor.     

Cellular and synaptic actions of general anaesthetics.

1. This paper briefly reviews mechanisms by which such widely-used volatile anaesthetics as halothane and isoflurane suppress neural function in the brain. 2. In general, anaesthetics tend to depress neuronal firing and excitatory synaptic transmission, and potentiate synaptic inhibition. 3. According to recent evidence, a particular important action of anaesthetics is to inactivate a variety of both voltage-dependent and agonist-triggered Ca-currents. 4. Activation of K outward currents and Na inward currents probably occurs only with higher doses of anaesthetics. 5. How anaesthetics interfere with Ca-channels remains largely a matter of speculation--though some evidence favours a Ca-mediated action, following Ca2+ release from internal stores, that may account also for potentiation of IPSPs by prolonging the opening of GABA-activated Cl- channels. 6. Whatever its precise underlying mechanism, a suppression of Ca-influx into pre-synaptic terminals could well account for the depression of excitatory synaptic transmission.     

Pre- and postsynaptic volatile anaesthetic actions on glycinergic transmission to spinal cord motor neurons

1. A common anaesthetic endpoint, prevention of withdrawal from a noxious stimulus, is determined primarily in spinal cord, where glycine is an important inhibitory transmitter. To define pre- and postsynaptic anaesthetic actions at glycinergic synapses, the effects of volatile anaesthetic agents on spontaneous and evoked glycinergic currents in spinal cord motor neurons from 6 - 14-day old rats was investigated. 2. The volatile anaesthetic agents enflurane, isoflurane and halothane significantly increased the frequency of glycinergic mIPSCs, enflurane to 190.4% of control+/-22.0 (mean+/-s.e.m., n=7, P<0.01), isoflurane to 199.0%+/-28.8 (n=7, P<0.05) and halothane to 198.2%+/-19.5 (n=7, P<0.01). However without TTX, isoflurane and halothane had no significant effect and enflurane decreased sIPSC frequency to 42.5% of control+/-12.4 (n=6, P<0.01). All the anaesthetics prolonged the decay time constant (tau) of both spontaneous and glycine-evoked currents without increasing amplitude. With TTX total charge transfer was increased; without TTX charge transfer was unchanged (isoflurane and halothane) or decreased (enflurane). 3. Enflurane-induced mIPSC frequency increases were not significantly affected by Cd(2+) (50 microM), thapsigargin (1 - 5 microM), or KB-R7943 (5 microM). KB-R7943 and thapsigargin together abolished the enflurane-induced increase in mIPSC frequency. 4. There are opposing facilitatory and inhibitory actions of volatile anaesthetics on glycine release dependent on calcium homeostatic mechanisms and sodium channels respectively. Under normal conditions (no TTX) the absolute amount of glycinergic inhibition does not increase. The contribution of glycinergic inhibition to anaesthesia may depend on its duration rather than its absolute magnitude.     

Concentration-dependent isoflurane effects on depolarization-evoked glutamate and GABA outflows from mouse brain slices.

The synaptic concentrations of glutamate and gamma-aminobutyric acid (GABA) are modulated by their release and re-uptake. The effects of general anaesthetics on these two processes remain unclear. This study evaluates the effects of isoflurane, a clinically important anaesthetic, on glutamate and GABA release and re-uptake in superfused mouse cerebrocortical slices. Experiments consisted of two 1.5-min exposures to 40 mM KCl in 30 min intervals. During the second exposure, different concentrations of isoflurane with and without 0.3 mM L-transpyrrolidine-2,4-dicarboxylic acid (PDC, a competitive inhibitor of glutamate uptake transporter) or 1 mM nipecotic acid (a competitive inhibitor of GABA uptake transporter) were introduced. The ratios of the second to first KCl-evoked increases in glutamate and GABA were used to determine the isoflurane concentration-response curves. The results can be described as a sum of two independent processes, corresponding to the inhibitions of release and re-uptake, respectively. The EC50 values for the inhibitions of release and re-uptake were 295+/-16 and 805+/-43 microM for glutamate, and 229+/-13 and 520+/-25 microM for GABA, respectively. Addition of PDC did not significantly affect glutamate release but shifted the re-uptake curve to the left (EC50= 315+/-20 microM). Nipecotic acid completely blocked GABA uptake, rendering isoflurane inhibition of GABA re-uptake undetectable. Our data suggest that isoflurane inhibits both the release and re-uptake of neurotransmitters and that the inhibitions occur at different EC50's. For GABA, both EC50's are within the clinical concentration range. The net anaesthetic effect on extracellular concentrations of neurotransmitters, particularly GABA, depends on the competition between inhibition of release and that of re-uptake.     

General anaesthetics: effects on transmitter release

In this report, the physiological effects, observations and events leading to transmitter release in both central nervous system and peripheral synapses are discussed. The presynaptic modulation of transmission at the central nervous system and at the neuromuscular junction was investigated using electrophysiological and neurochemical techniques. It was concluded that various general anaesthetics may affect the presynaptic mechanism of transmission, but these effects were controversial. For example, terminal excitability, which is taken as an index for presynaptic activity, could either be reduced or increased by general anaesthetics. Similar conflicting effects have been reported for the action of general anaesthetics on spontaneous and evoked release of ACh, uptake and release of intracellular Ca2+, and choline transport into the presynaptic nerve terminals.     

Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes

Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.     

Anaesthetics depress the sensitivity of cortical neurones to L-glutamate.

1 The effects of general anaesthetics on the responses of neurones to iontophoretically applied L-glutamate have been examined in slices of the guinea-pig olfactory cortex in vitro. 2 Concentrations of pentobarbitone, ether, methoxyflurance, trichloroethylene and alphaxalone that are known to depress synaptic transmission in the prepiriform cortex also depressed the sensitivity of prepiriform neurones to L-glutamate. 3 Halothane, in concentrations that depress synaptic transmission (less than 1%) did not alter sensitivity of neurones to glutamate. Higher concentrations (greater than 1% produced a dose-related depression of the glutamate sensitivity of neurones. 4 All four volatile anaesthetics tested caused some cells to alter their glutamate-evoked firing pattern to one in which the spike discharges were more closely grouped. Pentobarbitone and alphaxalone had no such effect. 5 If the sensitivity of the neurones to the endogenous excitatory transmitter is affected by anaesthetics in the same way as the glutamate-sensitivity, these results suggest that halothane depresses synaptic transmission by decreasing the amount of transmitter released from the nerve terminals, whereas the other anaesthetics depress the sensitivity of the post-synaptic membrane to the released transmitter.     

Anaesthetic suppression of transmitter actions in neocortex.

1. The effects of general anaesthetics were investigated on neuronal sensitivities to transmitter substances, which were determined by iontophoretic applications of acetylcholine, glutamate, N-methyl-D-aspartate (NMDA) and gamma-aminobutyrate (GABA) during intracellular recording in in vitro slice preparations of neocortex (guinea-pig). 2. In most of the 65 neurones studied, perfusion of isoflurane (0.5-2.5 minimum alveolar concentration (MAC)) or Althesin (25-200 microM) and, in some cases, halothane (0.5-2 MAC), markedly reduced the depolarizing responses and associated membrane conductance changes evoked by dendritic applications of acetylcholine, glutamate, NMDA and GABA. 3. The order of depression was acetylcholine greater than glutamate or NMDA much greater than GABA. This selectivity could also be assessed from the EC50 for the isoflurane-induced depression of the just-maximal responses to acetylcholine, which was 0.9 MAC compared with an EC50 = 1.9 MAC for the suppression of glutamate responses. The selectivity was less pronounced in the case of the actions of Althesin, where the EC50s were 75 microM for the depression of acetylcholine responses and 90 microM for the depression of glutamate responses. 4. The hyperpolarizing responses observed when GABA was applied near the perikaryon in 7 neurones, were slightly reduced (approximately 15%) in 4, and unchanged in 3 neurones during anaesthetic application. 5. The pronounced depression of the responsiveness to the putative arousal transmitters and an observed blockade of acetylcholine-induced potentiation of glutamate actions suggest that anaesthetics produce unconsciousness, at least in part, by interfering with subsynaptic mechanisms of neocortical activation.     

Presynaptic receptors for dopamine, histamine, and serotonin

Presynaptic receptors for dopamine, histamine and serotonin that are located on dopaminergic, histaminergic and sertonergic axon terminals, respectively, function as autoreceptors. Presynaptic receptors also occur as heteroreceptors on other axon terminals. Auto- and heteroreceptors mainly affect Ca(2+) -dependent exocytosis from the receptor-bearing nerve ending. Some additionally subserve other presynaptic functions.Presynaptic dopamine, histamine and serotonin receptors are involved in various (patho)physiological conditions. Examples are the following:Dopamine autoreceptors play a role in Parkinson's disease, schizophrenia and drug addiction. Dopamine heteroreceptors affecting the release of acetylcholine and of amino acid neurotransmitters in the basal ganglia are also relevant for Parkinson's disease. Peripheral dopamine heteroreceptors on postganglionic sympathetic terminals influence heart rate and vascular resistance through modulation of noradrenaline release. Blockade of histamine autoreceptors increases histamine synthesis and release and may support higher CNS functions such as arousal, cognition and learning. Peripheral histamine heteroreceptors on C fiber and on postganglionic sympathetic fiber terminals diminish neuropeptide and noradrenaline release, respectively. Both inhibititory effects are beneficial in myocardial ischemia. The inhibition of neuropeptide release also explains the antimigraine effects of some agonists of presynaptic histamine receptors. Upregulation of presynaptic serotonin autoreceptors is probably involved in the pathogenesis of major depression. Correspondingly, antidepressant treatments can be linked with a reduced density of 5-HT autoreceptors. 5-HT Heteroreceptor activation diminishes acetylcholine and GABA release and may therefore increase anxiety. In the periphery, presynaptic 5-HT heteroreceptor agonists shorten migraine attacks by inhibition of the release of neuropeptides from trigeminal afferents, apart from their constrictive action on meningeal vessels.     

General anaesthetics and field currents in unclamped, unmyelinated axons of rat olfactory cortex.

1. The effects of seven general anaesthetics and one local anaesthetic having a wide range of physical and chemical properties were studied on nerve terminal Na- and K-mediated currents in slices of olfactory cortex. These currents were measured from the groups of fine unmyelinated axons traversing the surface of the olfactory cortex and which give off synapses en passant. The amplitude of the K-current was visualized by depolarizing the axons via an electrode polarization. 2. The anaesthetics tested were ketamine (0.1-2 mM), pentobarbitone (0.1-5 mM), urethane (5-200 mM), halothane (0.5-5 mM), ether (10-200 mM), alphaxalone (0.001-0.05 mM), diisopropylphenol (0.05-0.5 mM) and lignocaine (0.01-0.5 mM). All had depressant effects on the axonal Na-current (at the higher concentrations tested) and on the K-current (at slightly lower concentrations). The apparent lower potency on the Na-current was considered to be due to a masking of effect as a consequence of the reduction in the K-mediated membrane rectification rather than any real difference in the susceptibilities of the Na and K-currents. 3. Some of the depressant effect of pentobarbitone and alphaxalone was gamma-aminobutyric acid (GABA)-mediated as indicated by the reduced potency in the presence of bicuculline. The actions of ketamine and halothane were unaffected by bicuculline. 4. For some anaesthetics these axonal depressant effects might contribute to general anaesthesia, while for other substances the relatively high concentrations necessary would suggest that this mode of action does not produce effective anaesthesia in vivo.     

Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca^2 + ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca^2 + concentration ([Ca^2 +]_C), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na⁺/Ca^2 + exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca^2 + entry and ERK/synapsin I signaling pathway.     

The action of volatile anaesthetics on stimulus-secretion coupling in bovine adrenal chromaffin cells.

1. The action of four volatile anaesthetics, ethrane, halothane, isoflurane and methoxyflurane on stimulus-secretion coupling has been studied in isolated bovine adrenal medullary cells. All four agents inhibited the secretion of adrenaline and noradrenaline evoked by 500 microM carbachol at concentrations within the anaesthetic range. Total catecholamine secretion induced by stimulation with 77 mM potassium was also inhibited but at higher concentrations. All four agents inhibited the 45Ca influx evoked by stimulation with 500 microM carbachol and the 45Ca influx in response to K+-depolarization. 2. When total catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding halothane or methoxyflurane to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. 3. The action of methoxyflurane on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells, which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. 4. Halothane and methoxyflurane inhibited the carbachol-induced secretion of catecholamines in a non-competitive manner. 5. Halothane and methoxyflurane inhibited the increase in 22Na influx evoked by carbachol. For halothane and methoxyflurane this inhibition of Na influx appears to be sufficient to account for the inhibition of the evoked catecholamine secretion. 6. We conclude that the volatile anaesthetics ethrane, halothane, isoflurane and methoxyflurane inhibit the secretion of adrenaline and noradrenaline induced by carbachol at concentrations that lie within the range encountered during general anaesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)