Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Neuropsychopharmacology and Neurogenetic Aspects of Executive Functioning: Should Reward Gene Polymorphisms Constitute a Diagnostic Tool to Identify Individuals at Risk for Impaired Judgment?

Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest that examination of multifactorial interactions of an individual's genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies, we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission.     

Transmembrane Transport and Release of GABA in the Brain of Rats Subjected to Postnatal Hypoxia

We studied the effects of early postnatal hypoxia on the efficiency of active GABA transport through the plasma membrane of synaptic terminals (synaptosomes) isolated from the cerebral cortex, hippocampus, and thalamus of rats and on non-stimulated and Ca²⁺-stimulated GABA release. The state of hypoxia was induced by exposure of 10- to 12-day-old rats to a respiratory medium with low O₂ content (4% О₂ and 96% N₂) for 12 min (up to the initiation of clonico-tonic seizures). Animals were taken in the experiment 8 to 9 weeks after an episode of hypoxic stress. The intensity of transmembrane transport of GABA was estimated according to accumulation of [³Н]GABA in a coarse synaptosomal fraction. The process was characterized by calculation of the Michaelis constant K _m and also of the initial (within the 1st min) and maximum rates of accumulation of [³Н]GABA. The means of the initial rate of [³Н]GABA accumulation in preparations from the thalamus, cortex, and hippocampus were 205.5 ± 8.8, 266.2 ± 29.6, and 302.3 ± 31.2 pmol/min⋅mg protein, respectively. Hypoxic stress influenced the rates of accumulation of [³Н]GABA in synaptic terminals from the cortex and hippocampus but not in those from the thalamus. According to the characteristics of the response to hypoxic stress, all experimental animals could be classified into two groups. In some rats, accumulation of [³Н]GABA in both cortical and hippocampal synaptosomes decreased insignificantly (by about 15%), while in other animals this parameter increased significantly (by nearly 50%) for the cortex and decreased by 21.5%, on average, for the hippocampus. The affinity of the transporter with respect to [³Н]GABA in the cortex and hippocampus was nearly the same and showed no changes under the influence of hypoxia. The non-stimulated release of [³Н]GABA after the influence of hypoxia increased in all structures, while the depolarization-induced Ca²⁺-dependent release of [³Н]GABA was intensified only in synaptosomes from the cerebral cortex. The mechanisms of development of modifications of GABA-ergic processes under the influence of hypoxic stress in the course of the perinatal period are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 293–302, July–August, 2008.     

Effectiveness of extracellular lactate/pyruvate for sustaining synaptic vesicle proton gradient generation and vesicular accumulation of GABA

The effects of extracellular monocarboxylates pyruvate and lactate on membrane potentials, acidification and neurotransmitter filling of synaptic vesicles were investigated in experiments with rat brain synaptosomes using [(3)H]GABA and fluorescent dyes, potential-sensitive rhodamine 6G and pH-sensitive acridine orange. In experiments investigating accumulation of acridine orange in synaptic vesicles within the synaptosomes, monocarboxylates, similarly to glucose, ensured generation of the vesicle proton gradient by available and recycled vesicles, and pyruvate demonstrated the highest efficacy. An increase in the level of proton gradient correlated with enhanced accumulation of [(3)H]GABA in synaptic vesicles and resulted in enlarged exocytosis and attenuated the transporter-mediated [(3)H]GABA release. Pyruvate added to glucose-contained medium caused more active binding of rhodamine 6G by synaptosomes that reflected mitochondrial membrane hyperpolarization, and this intensification of nerve terminal energy metabolism resulted in an increase in total ATP content by approximately 25%. Pyruvate also prolonged the state of metabolic competence of nerve terminal preparations, keeping the mitochondrial potential and synaptic vesicle proton gradient at steady levels over a long period of time. Thus, besides glucose, the extracellular monocarboxylates pyruvate and lactate can provide sufficient support of energy-dependent processes in isolated nerve terminals, allowing effective functioning of neurotransmitter release and reuptake systems.     

Effects of staurosporine on Ca2+-dependent and Ca2+-independent [14C]GABA release from rat brain synaptosomes

The effect of a protein kinase inhibitor, staurosporine, on Ca²⁺-dependent and Ca²⁺-independent release of [¹⁴C]GABA in isolated rat brain synaptosomes was studied. Calcium-dependent [¹⁴C]GABA release was stimulated by depolarization with a K⁺ channel blocker, 4-aminopyridine (4-AP), or high K⁺ concentration. It has been shown that the effect of 4-AP is Ca²⁺-dependent, while high K⁺ is able to evoke [¹⁴C]GABA release in both Ca²⁺-dependent and Ca²⁺-independent manners. In addition, Ca²⁺-independent [¹⁴C]GABA release was studied using α-latrotoxin (LTX) as a tool. Pretreatment of synaptosomes with staurosporine resulted in pronounced inhibition of 4-AP-stimulated Ca²⁺-dependent [¹⁴C]GABA release. The inhibitory effect of staurosporine on [¹⁴C]GABA release was not due to modulation of 4-AP-promoted⁴⁵Ca²⁺ influx into synaptosomes. If the process of [¹⁴C]GABA release occurred in the Ca²⁺-independent manner irrespectively of what, LTX or high K⁺, stimulated this process, it was not inhibited by staurosporine. Considering the above findings, it is reasonable to assume that the absence of Ca²⁺ in the extracellular medium created conditions for activation of the process of neurotransmitter release without Ca²⁺-dependent dephosphorylation of neuronal phosphoproteins; as a consequence, regulation of exocytotic process was modulated in such a manner that inhibition of protein kinases did not disturb exocytosis.     

Regulation of TRH release by the cultured neonate rat pancreas

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic β-cells.     

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.     

Improvement of metabolic competence of isolated nerve terminals by extracellular pyruvate

Neuronal activity is tightly coupled with brain energy metabolism. Numerous studies have proved that glucose is not a sole energy substrate for neurons; metabolic monocarboxylate intermediates derived from glucose (pyruvate and lactate) released by astrocytes are shown to be taken up and oxidized by neurons, and, moreover, could serve as neuroprotective agents. Herein, we presented the data that extracellular pyruvate (4 mM) in the presence of glucose caused the increase in synaptosomal ATP content from 3.48+/-0.30 to 4.38+/-0.23 nmol/mg of protein. This correlates with the enhanced accumulation of fluorescent dye acridine orange in the available and the recycling synaptic vesicles within the synaptosomes reflecting the improved generation of proton gradient through the synaptic vesicle membrane. We have also demonstrated the effect of extracellular pyruvate on distribution of [3H]GABA between synaptic vesicles and cytoplasm in loaded synaptosomes. To estimate [3H]GABA accumulation into the synaptic vesicles, Ca 2+-dependent 4-aminopyridine-triggered exocytotic neurotransmitter release was studied. Evaluation of cytosolic 1H]GABA pool was performed by measuring the Ca2+-independent transporter-mediated neurotransmitter release evoked by nipecotic acid or high K+. The presence of pyruvate resulted in doubled exocytotic release of [3H]GABA, and significantly attenuated Ca2+-independent release of cytosolic [3H]GABA. Together, these observations provide insight into the important role of glucose metabolic intermediate, pyruvate, in sustaining activity of vesicular inhibitory amino acid transporter and so normal inhibitory transmission. We propose to use pyruvate for keeping up synaptosomal preparations in state of metabolic stability.