Propylene glycol releases calcium from mitochondrial stores in rat cerebrocortical synaptosomes

In Ca2+-free medium, propylene glycol (PG) increases the cytosolic-free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes. In the current studies, the authors investigated the intracellular source of this Ca2+. PG (0.5-5% v/v) dose-dependently increased [Ca2+]i in Ca2+-free medium. The increase in [Ca2+]i was completely inhibited by pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP, 10 microM). The inhibitory effect of CCCP was dependent on the concentration and the pretreatment time. These results suggest that, in Ca2+-free medium, PG increases [Ca2+]i in rat cerebrocortical synaptosomes by releasing Ca2+ from mitochondrial stores.     

Propylene glycol increases cytosolic free calcium in rat cerebrocortical synaptosomes

In these studies, the authors investigated the effect of propylene glycol (PG) on the cytosolic free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes using the fluorescent Ca2+ indicator fura-2. PG (0.5-5% v/v) increased [Ca2+]i in a concentration-dependent manner. The PG-induced increase in [Ca2+]i was inhibited approximately 50% by the omission of extracellular Ca2+ or the addition of Ni2+ (100 microM). Decrease of extracellular Na+ (6.2 mM) or addition of tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), or omega-conotoxin MVIIC (1 microM) had no effect on the increase in [Ca2+]i. Also, addition of TMB-8 (100 microM), ryanodine (50 microM) or thapsigargin (1 microM) did not modify the increase in [Ca2+]i in the absence of extracellular Ca2+. These results suggest that PG increases [Ca2+]i in rat cerebrocortical synaptosomes by both stimulating Ca2+ entry through a Ni2+-sensitive pathway and releasing Ca2+ from TMB-8-, ryanodine- and thapsigargin-insensitive Ca2+ stores.     

Measured increase in intracellular Ca during stimulated release of endogenous glutamate from human cerebrocortical synaptosomes

Presynaptic terminals (synaptosomes) prepared from guinea pig and rat cerebral cortex release endogenous glutamate in a Ca²⁺-dependent manner in response to membrane depolarisation. In the present study, synaptosomes were prepared from human cerebral cortex removed in association with temporal lobe resections in epileptic patients. The cytosolic free Ca²⁺ concentration increased from 474±66 before to 649±89 nM after 2 min depolarisation. The basal level of free cytosolic Ca²⁺ is higher and the increase in response to depolarisation is more pronounced in human synaptosomes than observed in animal experiments. The Ca²⁺-dependent glutamate release, estimated as the difference between total — and the Ca²⁺-independent glutamate release, increased from 0 to 5.4±1.9 nmol/mg protein. The released amount of glutamate is larger than reported in animal models. These results demonstrate that membrane depolarisation of synaptosomes from human brain evokes a rapid rise in cytosolic free Ca²⁺ and a more prolonged rise in synaptic, Ca²⁺-dependent glutamate release.     

Lamotrigine inhibition of glutamate release from isolated cerebrocortical nerve terminals (synaptosomes) by suppression of voltage-activated calcium channel activity.

Lamotrigine (LAG) is an antiepileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters. The present study was aimed at investigating the effect of LAG on the 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). LAG inhibited the release of glutamate evoked by 4AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C). In addition, LAG did not alter the resting synaptosomal membrane potential or 4AP-evoked depolarization. Furthermore, ionomycin-evoked glutamate release was not affected by LAG. Based on these results, we suggest that presynaptic calcium influx blockade and inhibition of glutamate release may underlie the mechanism of action of LAG. These action may also contribute to their neuroprotective properties in excitotoxic injury.     

Inhibition of glutamate release by fluspirilene in cerebrocortical nerve terminals (synaptosomes)

Fluspirilene, a neuroleptic drug which is used clinically to treat schizophrenic patients, is a dopamine D2 receptor antagonist. Besides its well-known actions on the dopamine receptors, fluspirilene also displays calcium channel-blocking activity. The aim of this study was to investigate the effect of fluspirilene on the 4-aminopyridine (4AP)-evoked glutamate release in the cerebrocortical nerve terminals (synaptosomes). Fluspirilene reduced 4AP-evoked glutamate release in a concentration-dependent manner. This inhibitory effect was associated with a decrease in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C), which could be completely abolished by the Ca2+ channel blocker omega-CgTX GVIA. Furthermore, fluspirilene did not produce any effect on ionomycin-evoked glutamate release. These results suggest that fluspirilene inhibits glutamate release primarily by reducing presynaptic Ca2+ influx via N-type Ca2+ channels in rat cerebrocortical nerve terminals. This finding implies that presynaptic Ca2+ channel blockade concomitant with inhibition of glutamate release and possibly other neurotransmitters release may contribute to the antischizophrenic action of fluspirilene.     

Fluoxetine depresses glutamate exocytosis in the rat cerebrocortical nerve terminals (synaptosomes) via inhibition of P/Q-type Ca2+ channels

Fluoxetine, an antidepressant that is used clinically in the treatment of mood disorders, is a selective serotonin reuptake inhibitor. In the present study we investigated the effects of fluoxetine on 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). Fluoxetine suppressed the release of glutamate evoked by 4AP in a concentration-dependent manner. This effect was associated with a reduction in the depolarization-evoked increase in cytosolic free calcium levels in the absence of significant effect on the synaptosomal membrane potential. In addition, both ionomycin- and sucrose-evoked glutamate releases were not affected by fluoxetine, indicating that fluoxetine-mediated inhibition of glutamate release is not a direct effect on the exocytotic machinery. Furthermore, the inhibitory action of fluoxetine was completely abolished in synaptosomes pretreated with P/Q type Ca(2+) channel blocker omega-agatoxin IVA (omega-AgTX IVA) or protein kinase C (PKC) stimulator 4beta-phorbol 12, 13-dibutyrate (PDBu). These results suggest that, in cerebrocortical nerve terminals, fluoxetine inhibits glutamate release through the suppression of P/Q type Ca(2+) channel activity. The presynaptic action of fluoxetine is mediated by a PKC-sensitive signaling pathway. Synapse 48:170-177, 2003.     

Facilitation of glutamate release from rat cerebrocortical glutamatergic nerve terminals (synaptosomes) by phosphatidylserine and phosphatidylcholine

Phosphatidylserine (PS) and phosphatidylcholine (PC) have been shown to enhance cognitive function. Considering that brain glutamatergic system is thought to participate in cognitive processing, our objective was to determine the effect of PS and PC on glutamate release from the nerve terminal (synaptosome) freshly isolated from rat cerebral cortex. Data showed that both PS and PC potently facilitate 4‐aminopyridine (4‐AP)‐evoked Ca²⁺‐dependent and Ca²⁺‐independent glutamate release. Facilitation of glutamate release by PS or PC was associated with an increase of 4‐AP‐evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca²⁺]_c). In addition, glutamate release elicited by direct Ca²⁺‐entry with Ca²⁺‐ionophore (ionomycin) was also facilitated by PS or PC. Furthermore, PS‐ or PC‐mediated facilitation of 4‐AP‐evoked glutamate release was superseded or suppressed by protein kinase C (PKC) activator and inhibitor, respectively. Together, these results suggest that PS or PC effects a facilitation of glutamate exocytosis by increasing nerve terminal excitability and Ca²⁺ influx into cerebrocortical nerve terminals through a signaling cascade involving PKC. Synapse 63:215–223, 2009. © 2008 Wiley‐Liss, Inc.     

Chronic stress enhances methamphetamine-induced extracellular glutamate and excitotoxicity in the rat striatum

Striking parallels exist between the neurochemical and toxic effects of stress and methamphetamine. Despite these similarities, no studies have examined how stress may promote the toxic effects of methamphetamine (METH). The current study tested the hypothesis that chronic stress enhances METH toxicity by augmenting glutamate (GLU) release and excitotoxicity in response to METH administration. Adult male Sprague-Dawley rats were exposed to 10 days of unpredictable stress and then received either saline or METH (7.5 mg/kg, i.p., once every 2 h x four injections). Prior exposure to unpredictable stress acutely enhanced the striatal extracellular GLU concentrations in response to METH, and eventually caused proteolysis of the cytoskeleton protein spectrin. Administration of the corticosterone synthesis inhibitor, metyrapone (25 mg/kg, i.p., prior to each stressor), during unpredictable stress attenuated the enhanced striatal GLU release in response to METH, blocked spectrin proteolysis, and attenuated METH-associated toxicity measured by long-term depletions in the dopamine and serotonin tissue content as well as depletions in dopamine and serotonin transporter immunoreactivity of the striatum. In summary, prior exposure to unpredictable stress enhances METH-induced elevations of GLU in the striatum, resulting in long-term excitotoxic damage and an augmentation of damage to dopamine and serotonin terminals. These studies provide a neurochemical basis for how stress contributes to the deleterious effects of METH abuse.     

Measured increase in intracellular Ca(2+) during stimulated release of endogenous glutamate from human cerebrocortical synaptosomes.

Presynaptic terminals (synaptosomes) prepared from guinea pig and rat cerebral cortex release endogenous glutamate in a Ca(2+)-dependent manner in response to membrane depolarisation. In the present study, synaptosomes were prepared from human cerebral cortex removed in association with temporal lobe resections in epileptic patients. The cytosolic free Ca(2+) concentration increased from 474+/-66 before to 649+/-89 nM after 2 min depolarisation. The basal level of free cytosolic Ca(2+) is higher and the increase in response to depolarisation is more pronounced in human synaptosomes than observed in animal experiments. The Ca(2+)-dependent glutamate release, estimated as the difference between total - and the Ca(2+)-independent glutamate release, increased from 0 to 5.4+/-1.9 nmol/mg protein. The released amount of glutamate is larger than reported in animal models. These results demonstrate that membrane depolarisation of synaptosomes from human brain evokes a rapid rise in cytosolic free Ca(2+) and a more prolonged rise in synaptic, Ca(2+)-dependent glutamate release.     

A 23187-stimulated calcium uptake and GABA release by cerebrocortical synaptosomes: effects of high pressure

Summary Guinea pig cerebrocortical synaptosome preparations were used to study the effect of compression to 62 ATA on⁴⁵Ca²⁺ uptake and [³H]GABA release using a calcium ionophore A23187, which bypasses the voltage-sensitive calcium channel. Pressure was found to exert a suppressive effect on the A 23187-induced release of [³H]GABA, while having no significant effect on A 23187-stimulated⁴⁵Ca²⁺ uptake. On the other hand, both depolarization-induced⁴⁵Ca²⁺ uptake and [³H]GABA release were inhibited by pressure exposure. These results suggest that pressure may suppress GABA release by affecting pre-synaptic events subsequent to calcium influx.     

Morphine inhibits glutamate exocytosis from rat cerebral cortex nerve terminals (synaptosomes) by reducing Ca2+ influx

Morphine, a mu-opioid agonist, suppressed the Ca(2+)-dependent release of glutamate that was evoked by exposing cerebrocortical synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by morphine was concentration-dependent and blocked by the nonselective opioid receptor antagonist naloxone. As determined by examining the mechanism of mu-opioid receptor-mediated inhibition of glutamate release, morphine caused a significant reduction in 4-aminopyridine-evoked increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](c)), but failed to alter both 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential and Ca(2+) ionophore (ionomycin)-induced glutamate release. In addition, morphine was not capable of producing further inhibition on 4AP-evoked glutamate release in synaptosomes pretreated with the cannabinoid CB(1) receptor agonist WIN 55212-2, which has been shown to depress glutamate release through a suppression of presynaptic voltage-dependent Ca(2+) channel activity. These data suggest that morphine exerts its inhibitory effect presynaptically, likely through the reduction of Ca(2+) influx into nerve terminals, and thereby inhibits the release of glutamate in the cerebral cortex. This may therefore indicate that mu-opioid receptor agonists have neuroprotective properties, especially in the excessive glutamate release that occurs under certain pathological conditions.     

Chronic stress enhances apomorphine-induced stereotyped behavior in mice: Involvement of endogenous opioids

Mice subjected to repeated stressful experiences showed an increase in stereotypic climbing behavior induced by apomorphine thus suggesting a modified sensitivity of dopaminergic receptors. Naltrexone, injected before each stressful experience, reversed this effect of chronic stress indicating an involvement of endogenous opioids.     

Gyroxin,a toxin from Crotalus durissus terrificus snake venom,induces a calcium dependent increase in glutamate release in mice brain cortical synaptosomes

Gyroxin is a thrombin-like toxin obtained from the venom of the South American rattlesnake, Crotalus durissus terrificus. Literature has reported “gyroxin syndrome” characterized, in mice, as series of aberrant motor behavior, known as barrel rotation, mainly after intraperitoneal administration. Despites several studies, a physiological mechanism of “gyroxin syndrome” are still not completely understood. In this context, alterations on the central nervous system (CNS), especially causing neurotoxic events, are pointed out as likely candidates. Then, we decided to investigate whether gyroxin induces alterations in glutamate release, one of the most important neurotransmitter involved in neurotoxicity. For that, we performed all experiments, in vitro, using a model of mice brain cortical synaptosomes. Notably, our results indicate that the administration of gyroxin on purified presynaptic brain cortical terminals resulted in an extracellular Ca²⁺- dependent raise in glutamate release. Indeed, our results also showed that gyroxin increases intrasynaptosomal calcium (Ca²⁺) levels through acting on voltage gated calcium channels (VGCC), specifically N and P/Q subtypes. Moreover, our data show that gyroxin increases exocytosis rate. Interestingly, these data suggest that gyroxin might induce neurotoxicity by increasing glutamate levels. However, future investigations are needed in order to elucidate the nature of the following events.     

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA_A receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABA_A receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.     

Possible regulation of high-affinity glutamate uptake in synaptosomes of normal and epileptic mice

Glutamate (Glu) uptake is the primary mechanism for its removal from the synapse. In genetic audiogenic seizures (AGS), Glu uptake is elevated prior to the appearance of seizures. Increased Glu uptake is also observed in synaptosomes from normal mice preincubated with lithium or nitroarginine, an NO synthase inhibitor. Pertussis and cholera toxins cause a marked reduction in Glu uptake. In contrast, neither lithium nor nitroarginine affected Glu uptake by synaptosomes from genetic epileptic mice. Arachidonic acid inhibits Glu uptake, whereas synaptosomes from epileptic mouse brain appear to be more sensitive to arachidonic acid as indicated by a shift of the inhibition curve to the left. These observations are indicative of the possible regulation of Glu uptake by second messengers and its alteration in genetic epilepsy.     

Possible regulation of high-affinity glutamate uptake in synaptosomes of normal and epileptic mice

Glutamate (Glu) uptake is the primary mechanism for its removal from the synapse. In genetic audiogenic seizures (AGS), Glu uptake is elevated prior to the appearance of seizures. Increased Glu uptake is also observed in synaptosomes from normal mice preincubated with lithium or nitroarginine, an NO synthase inhibitor. Pertussis and cholera toxins cause a marked reduction in Glu uptake. In contrast, neither lithium nor nitroarginine affected Glu uptake by synaptosomes from genetic epileptic mice. Arachidonic acid inhibits Glu uptake, whereas synaptosomes from epileptic mouse brain appear to be more sensitive to arachidonic acid as indicated by a shift of the inhibition curve to the left. These observations are indicative of the possible regulation of Glu uptake by second messengers and its alteration in genetic epilepsy.     

Deficient NMDA-mediated glutamate release from synaptosomes of schizophrenics

Previous studies from our laboratory indicated that the veratridine-induced release of glutamate and GABA from synaptosomes derived from brains of schizophrenics was decreased. In the present study, synaptosomes were prepared from frozen brain samples from schizophrenics and from controls. Stimulation by 10 μmol/L 2-amino-3-hydroxy-5-methoxylisoxazole-4-propionic acid (AMPA) produced equal glutamate release from both groups. Release induced by either 10 μmol/L kainic acid (KA) or n-methyl-d-asparate (NMDA) was reduced significantly in the preparations derived from schizophrenics. Similarly, the amount of GABA released by 50 μmol/L glutamate was also reduced in the schizophrenic-derived synaptosomes. However, in membranes derived from the crude synaptosomal pellet, no differences between the controls and schizophrenics were observed in measures of total glutamate binding or its displacement by NMDA. The data demonstrate a deficiency in NMDA (and possibly KA) receptor functioning in schizophrenics and support the “second-generation” theories of schizophrenia as a glutamatergic deficiency disorder.     

Ethacrynic acid-induced glutamate release from mouse brain synaptosomes

A loop diuretic, ethacrynic acid (0.3 mM), released glutamate from mouse brain synaptosomes as potently as 40 mM K+, and was more potent than furosemide and bumetanide. Ethacrynic acid-induced glutamate release was suppressed by depletion of Ca2+ or Cl- from the incubation medium. The findings suggest that ethacrynic acid enhances glutamate release through Cl(-)-related depolarization of nerve endings.     

Vitamin B2 inhibits glutamate release from rat cerebrocortical nerve terminals

We examined the effect of riboflavin, vitamin B2, on the release of endogenous glutamate from nerve terminals purified from rat cerebral cortex. The release of glutamate evoked by 4-aminopyridine was inhibited by riboflavin. Further experiments indicated that riboflavin-mediated inhibition of glutamate release (i) results from a reduction of vesicular exocytosis, not from an inhibition of nonvesicular release; (ii) is associated with a decrease in presynaptic N-type and P/Q-type voltage-dependent Ca channel activity. These findings are the first to suggest that, in rat cerebrocortical nerve terminals, riboflavin suppresses voltage-dependent Ca channel activity and in so doing inhibits evoked glutamate release. This finding may explain the neuroprotective effects of vitamin B2 against neurotoxicity.     

Glutaminase inhibition and the release of neurotransmitter glutamate from synaptosomes

Cerebrocortical synaptosomes were incubated with glutamine together with 6-diazo-5-oxo-L-norleucine (DON) and NH4Cl (which are known to inhibit phosphate-stimulated glutaminase) in order to assess the effect of such inhibition on the pool sizes and extent of evoked release of endogenous amino acids, particularly glutamate. DON (5 mM) inhibited glutaminase by 73-89% and NH4Cl (1-4 mM) inhibited the enzyme by 45-53% under the conditions employed. NH4Cl (4 mM) incubated with synaptosomes for 30 min reduced pool sizes of aspartate and glutamate by 28% and inhibited release of glutamate by 55% compared to control release. DON caused a decrease in both the pool size of glutamate (22%) and the extent of veratrine-evoked release of amino acids (21%).