Studies on the uptake, binding and metabolism of leukotriene B4 by human neutrophils.

Human polymorphonuclear granulocytes (PMN) generate the inflammatory mediator leukotriene B4 (LTB4) as a response to cell activation. In addition, PMN inactivate LTB4 by omega-oxidation resulting in the formation of 20-OH- and 20-COOH-LTB4. The transport of exogenous LTB4 to the metabolizing enzymes is mediated via high- and low-affinity receptor subsets. Uptake of [3H]LTB4 by the cells was carried out in a time-dependent fashion, reaching maximal values after 5 min of incubation. No additional uptake of [3H]LTB4 then occurred. Prestimulation of PMN with phorbol myristate acetate or sodium fluoride resulted in the loss of high- and low-affinity receptors. Deactivating concentrations of LTB4 specifically reduced the high-affinity receptor subset. Prestimulation of PMN with cytochalasin B or with the membrane fluidizer butanol shifted the low-affinity receptors to the high-affinity state. The polyene antibiotic amphotericin B shifted high-affinity receptors to the low-affinity subset. The changes in the receptor expression pattern correlated with the respective conversion rate of exogenously added LTB4. Our results suggest that the distribution of high- and low-affinity receptors is regulated by GTP-binding proteins, the activation of protein kinase C and the organization of the membrane bilayer. In this way, human neutrophils control the respective level of the lipid mediator LTB4.     

Effects of the GABA uptake inhibitor tiagabine on inhibitory synaptic potentials in rat hippocampal slice cultures.

1. The effects of the gamma-aminobutyric acid (GABA) uptake blocker tiagabine on inhibitory synaptic potentials (IPSPs) were examined with microelectrode and whole-cell recording from CA3 pyramidal cells in rat hippocampal slice cultures. 2. Tiagabine (10-25 microM) greatly prolonged the duration of monosynaptic IPSPs elicited in the presence of excitatory amino acid antagonists but had no effect on their amplitude. Part of the prolonged time course resulted from a GABAB receptor-mediated component that was not detectable under control conditions. 3. The mean decay time constant of the underlying GABAA receptor-mediated synaptic current was increased from 16 to 250 ms. Spontaneous miniature IPSPs recorded with whole-cell clamp were unaffected by tiagabine. Pentobarbital sodium, in contrast, increased the decay time constant of both evoked and spontaneous GABAA-mediated currents. 4. Tiagabine (25 microM) inhibited spontaneous and evoked epileptiform bursting induced by increasing the extracellular potassium concentration to 8 mM. 5. We conclude that GABA uptake plays a significant role in determining the time course of evoked IPSPs and also limits the likelihood that GABAB receptors are activated.     

Effects of etifoxine on ligand binding to GABA(A) receptors in rodents

The GABA(A) receptor/chloride ionophore is allosterically modulated by several classes of anxiolytic and anticonvulsant agents, including benzodiazepines, barbiturates and neurosteroids. Etifoxine, an anxiolytic and anticonvulsant compound competitively inhibited the binding of [(35)S]t-butylbicyclophosphoro-thionate (TBPS), a specific ligand of the GABA(A) receptor chloride channel site. To investigate the etifoxine modulatory effects on the different binding sites of the GABA(A) receptor complex, we have examined the effects of etifoxine on binding of the receptor agonist [(3)H]muscimol and the benzodiazepine modulator [(3)H]flunitrazepam in rat brain membrane preparations. The anticonvulsant properties of etifoxine combined with muscimol and flunitrazepam were performed in mice with picrotoxin-induced clonic seizures. Etifoxine modestly enhanced binding of [(3)H]muscimol and of [(3)H]flunitrazepam by increasing the number of binding sites without changing the binding affinity of [(3)H]flunitrazepam. In contrast, the compound decreased the affinity of muscimol for its binding site. In vivo, the combination of subactive doses of etifoxine with muscimol or flunitrazepam produced an anticonvulsant additive effect against the picrotoxin-induced clonic seizures in mice. These results suggest that the interaction of etifoxine on the GABA(A) receptor complex would allosterically modify different binding sites due to conformational changes. Functionally, the resulting facilitation of GABA transmission underlies the pharmacological properties of etifoxine.     

Decrease in GABA immunoreactivity and alteration of GABA metabolism after kindling in the rat hippocampus

Summary The kindling model of epilepsy, induced by tetanic stimulation of Schaffer collateral/commissural fibers, was studied in the rat hippocampus. Gamma-aminobutyric acid immunoreactivity was used to quantify the number of GABA-immunoreactive somata per mm² in CA1 region, 28 days after the last generalized seizure. Comparison of the numbers obtained from kindled animals with those from controls, showed a significant decrease (18%) on the ipsilateral stimulated side but none on the contralateral side. In control rats injection of the GABA-transaminase inhibitor, amino oxyacetic acid (AOAA), led to a 46% increase in the number of cell somata immunoreactive for GABA. This probably results from an accumulation of GABA, reflecting GABA synthesis by glutamate decarboxylase (GAD) activity, in somata of interneurons that had initially a GABA content below the immunocytochemical detection threshold. In kindled rats, 31 days after the last seizure, the number of GABA-immunoreactive cells that could be observed after AOAA-treatment was significantly lower (35% ipsilateral and 25% contralateral) when compared to AOAA-treated controls. This suggests that in kindled animals a GAD dependent increase in GABA content did not take place in a subpopulation of interneurons. The observations for kindled rats are interpreted as a long-term decrease in GABA content and as an alteration in GABA turnover in a subpopulation of interneuron somata, the latter possibly due to a decrease in GAD activity. The long-term enhanced seizure sensitivity, characteristic for kindled animals, may be due to a decreased GABAergic inhibitory control of the neuronal circuitry in the CA1 region of the hippocampus.     

The time course of GABA action on the crayfish stretch receptor: evidence for a saturable GABA uptake

The conductance increase induced by bath application of GABA has been measured in voltage-clamped stretch-receptor neurones of crayfish. A rapid conductance increase was obtained only at GABA concentrations above 3.3 X 10(-4) M. The response to lower GABA concentrations (between 10(-4) and 10(-6) M) developed slowly over 30-60 min. Repetitive application of intermediate GABA concentrations induced postsynaptic conductance changes which were progressively enhanced in their onset and magnitude. In the presence of nipecotic acid or in Na+-free Ringer solutions, the response to all GABA concentrations was rapid and constant for each concentration. The time course of inhibitory postsynaptic currents was unaffected by nipecotic acid. These results suggest the presence of a saturable GABA uptake system which limits the access of bath-applied GABA to postsynaptic receptors. This system has little if any effect on the termination of response to synaptically released GABA.     

Kinetic differences in GABA receptor binding following GABA and bicuculline methiodide pretreatment

The kinetics of [3H]GABA binding were studied in cortical membranes following GABA and bicuculline methiodide (BCM) pretreatment at 37 degrees C. The slower parts of the biphasic ON and OFF rates can be attributed to the high affinity sites. BCM pretreatment decreased the ON and OFF rates of [3H]GABA binding, including the rapid initial ON- and OFF-phases. Decreased association rates of GABA were independent of GABA and concentration and suggested a rate determining dissociation of bound BCM (t1/2 = 35 min).     

Reduced GABA uptake sites in the temporal lobe in schizophrenia

The binding of [3H]nipecotic acid, a ligand for labelling GABA uptake sites in brain, was measured in left and right frontal cortex, polar temporal cortex, hippocampus and amygdala from control and schizophrenic postmortem brains. In schizophrenic brains, single concentration [3H]nipecotic acid binding was reduced bilaterally in amygdala and hippocampus and on the left side only in polar temporal cortex. These data suggest that GABA neurones are involved in the cerebral atrophy of schizophrenia and, in agreement with other studies, that this process is most pronounced in left temporal cortex.     

On the inhibition of GABA release from a preparation of rat brain nerve-endings by SKF 89976-A, a GABA uptake inhibitor.

Fractions enriched in nerve-terminals were made in order to investigate the release of gamma-aminobutyric acid (GABA). A P2-preparation made from whole rat brain was pre-labeled with [3H]GABA and thereafter incubated in high concentrations (1 mM) of the GABA uptake inhibitor SKF 89976-A. The nerve-terminals were thereafter incubated in depolarizing concentrations of KCl and finally the medium and nerve-terminals separated by a filtration procedure. The amount of [3H]GABA present on the filter and in the supernatant was measured by liquid scintillation counting and used to assess the release rate. SKF 89976-A reduced the KCl-induced efflux of [3H]GABA from the nerve-terminals. The inhibition was as effective against KCl-induced [3H]GABA efflux in the presence as well as in the absence of Ca2+. Our finding may demonstrate an inhibition of the inside-out activity of the GABA transporter.     

Inhibition of GABA and benzodiazepine receptor binding by penicillins

Penicillins are thought to be GABA receptor antagonists. In order to determine the affinities of various penicillin derivatives for the GABA receptor, their potencies as inhibitors of specific [3H]GABA binding to rat brain membranes were investigated. All investigated penicillins inhibit specific [3H]GABA binding, with IC50 values ranging from 2 to 60 mM. The results are consistent with the assumption that penicillins are weak GABA receptor antagonists.     

Some functional consequences of GABA uptake by brain cells

Recent electrophysiological studies on the rat hippocampus (in vivo and in vitro) provide further evidence that neuronal and glial uptake of the inhibitory transmitter gamma-aminobutyric acid (GABA) limits the intensity and the duration of effects not only of locally applied exogenous GABA but also of GABAergic inhibitory synaptic potentials (IPSPs). There is good reason to believe that such uptake is at least partly responsible for the 'fading' of GABA action. Moreover, because it is probably driven by the transmembrane Na+ electrochemical gradient and is accompanied by Na+ influx, GABA uptake is potentially electrogenic and therefore may have a depolarizing effect on both neurons and glia.     

Effect of Plasmodium yoelii infection on GABA metabolism of mouse brain

Plasmodium yoelii infection in albino mice decreased the activity of brain glutamic acid decarboxylase (GAD) by about 30 and 48% in crude homogenate and its synaptosomal fraction, respectively. The decrease was evident from 20% parasitemia and remained more or less constant up to 80% parasitemia. The Km values of GAD in normal and infected animals were 1.2 x 10(-2) and 3.3 x 10(-2) mM, respectively, indicating a decrease in enzyme substrate affinity due to infection. The lowered GAD activity rose to slightly above normal by treatment of infected animals with chloroquine. Decrease in GAD activity reflected lower gamma-aminobutyric acid (GABA) levels in the infected brain; however, GABA-transaminase activity was not significantly influenced by infection. It has been proposed that impaired GABA synthesis may be due to hypoxia induced by malarial infection.     

Age-related changes of benzodiazepine and GABA binding sites in the rat retina

The changes in the number and sensitivity of benzodiazepine and GABA binding sites in the rat retina during postnatal development, adulthood and ageing and their functional relationship at different ages have been studied. Data indicate an increase in the total number of both GABA and benzodiazepine binding sites with age. In contrast, the activation of retinal benzodiazepine receptor binding by GABA is significantly reduced in aged rats with respect to young adult and newborn rats. Moreover, the activation of retinal benzodiazepine receptor binding induced by dark exposure of the animals is present in young adult rats but is lost in aged rats. These results suggest that in the retina of aged rats there is an increase of GABA and benzodiazepine receptors which have lost their functional connection.     

Comparison of the effect of the GABA uptake blockers, tiagabine and nipecotic acid, on inhibitory synaptic efficacy in hippocampal CA1 neurones.

The action of the novel gamma-aminobutyric acid (GABA) uptake blocker, tiagabine, has been studied on isolated GABAergic fast inhibitory postsynaptic potentials (IPSP) and currents (IPSC) in rat hippocampal CA1 pyramidal cells in the slice preparation. Tiagabine (20-50 microM) had little effect on the peak amplitude of the IPSC, but caused a robust increase in the half-width (by 109 +/- 15%). These results contrasted with those obtained using the established uptake blocker, nipecotic acid (100 microM to 1 mM), which reduced the amplitude of the IPSC by 35 +/- 6% and caused only a modest prolongation of the recovery phase. These effects, which were poorly reversible, are probably explained by the fact that nipecotic acid is a substrate for the GABA-uptake carrier and can act as a false transmitter. Tiagabine is not transported by the GABA carrier and results with this substance demonstrate the role of uptake in determining the kinetics of activation of GABAA receptors. Tiagabine is proposed as the blocker of choice for the GABA uptake system.     

Benzodiazepine binding after in vivo elevation of GABA

[3H]Diazepam binding was measured in rat cortical membranes. Acute and chronic amino-oxyacetic acid (AOAA) pretreatment greatly increased GABA levels, but did not alter diazepam binding. The GABA normally present was sufficient to maximally stimulate diazepam binding. In in vivo binding studies, acute and chronic AOAA treatment increased the amount of [3H]diazepam in the brain at the time of sacrifice, thus increasing both specific and nonspecific binding and leaving unchanged the fraction of drug specifically bound. Fluctuation of brain GABA may not affect benzodiazepine binding unless the normal concentration is greatly depressed.     

Perinatal hypoxia: different effects of the inhibitors of GABA transporters GAT1 and GAT3 on the initial velocity of [3H]GABA uptake by cortical,hippocampal, and thalamic nerve terminals

Aim

To analyze the effects of highly selective blocker GAT1, NO-711, and substrate inhibitor GAT3, β-alanine, on the initial velocity of [³H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals (synaptosomes) after perinatal hypoxia.

Methods

Animals were divided into two groups: control (n = 17) and hypoxia (n = 12). Rats in the hypoxia group underwent hypoxia and seizures (airtight chamber, 4% O₂ and 96% N₂) at the age of 10-12 postnatal days and were used in the experiments 8-9 weeks after hypoxia.

Results

In cortical synaptosomes, the effects of NO-711 (30 μΜ) and β-alanine (100 μΜ) on [³H]GABA uptake were similar in control and hypoxia groups. In hippocampal synaptosomes, NO-711 inhibited 84.3% of the initial velocity of [³H]GABA uptake in normal conditions and 80.1% after hypoxia, whereas the effect of β-alanine was increased after hypoxia from 14.4% to 22.1%. In thalamic synaptosomes, the effect of NO-711 was decreased by 79.6% in controls and by 70.9% in hypoxia group, whereas the effect of β-alanine was increased after hypoxia from 20.2% to 30.2%.

Conclusions

The effectiveness of β-alanine to influence GABA uptake was increased in hippocampal and thalamic nerve terminals as a result of perinatal hypoxia and the effectiveness of NO-711 in thalamic nerve terminals was decreased. These results may indicate changes in the ratio of active GAT1/GAT3 expressed in the plasma membrane of nerve terminals after perinatal hypoxia. We showed a possibility to modulate non-GAT1 GABA transporter activity in different brain regions by exogenous and endogenous β-alanine.Perinatal hypoxia leads to multiple chronic neurological deficits including mental retardation, learning and memory disability, behavioral abnormalities, and even epilepsy (1). Pathological consequences of early life hypoxia may be a result of disturbance of the highly regulated maturation process. γ-Aminobutyric acid (GABA) as the first functional neurotransmitter in the developing brain fulfils an important signaling function in synapse formation and network construction (2). Hypoxic injury of the developing brain is mainly studied using the hypoxia model, in which rat pups undergo a brief exposure to graded global hypoxia in an airtight chamber (3). This showed a particular vulnerability of GABAergic neurons (4-6) and a long-lasting decrease in thresholds to convulsant action in adult rats that underwent hypoxia at an early age (3,6,7). In rats 10-12 postnatal days old, a single brief episode of moderately graded global hypoxia led to the development of tonic-clonic activity and caused a long-lasting (70-80 days after hypoxia) selective increase in seizure susceptibility in hippocampal slices (3).Sodium- and chloride-dependent GABA transporters (GATs), which belong to the SLC6 superfamily of Na⁺ -dependent transporters, terminate inhibitory synaptic transmission, ie, after release from presynaptic nerve terminals GABA is rapidly removed from the extracellular space by GATs, thereby maintaining optimal ambient level of the neurotransmitter. Chronic neurological abnormalities, which develop after hypoxia at an early age, may be associated with changes in the functioning of GATs (8-10). Our previous experiments on rats that underwent perinatal hypoxia demonstrated a long-lasting increase in the ambient GABA level in cortical and hippocampal nerve terminals, whereas the thalamus was less sensitive to perinatal hypoxia, and thalamic GATs, in contrast to cortical and hippocampal ones, had a lower affinity to GABA (11).Four types of GABA transporters are expressed in the plasma membrane of presynaptic nerve terminals and glial cells, that is, GAT1, GAT2, GAT3, and GAT4. GATs serve as one of the main targets for drugs in the treatment of neurological disorders, and so GABA uptake inhibitors are very promising agents with potential application in epilepsy, anxiety, pain, drug abuse, sleep disorders, and other disorders (8,9). The two most likely candidates for the maintenance of optimal ambient level of GABA in the brain are GAT1 and GAT3 (8). 1,2,5,6-Tetrahydro-1-(2-(((diphenylmethylene)amino)oxy)ethyl)-3-pyridinecarboxylic acid hydrochloride (NO-711) is a potent and selective GAT1 inhibitor with an IC₅₀ of 0.38 µM; IC₅₀ for GAT2 and GAT3 are 729 and 349 µM, respectively (12,13). Noteworthy, mixed GAT inhibitors are shown to have much broader spectrum of anticonvulsant activity than compounds with affinity only for GAT1. There are reports suggesting that non-GAT1 inhibitors are very interesting as potential candidates for future epilepsy treatment (8,14). Since the physiological role of GATs other than GAT1 is not fully determined, these inhibitors can also be pharmacological tools for the research on the biological role of non-GAT1 GABA transporters (15). In this context, there is a great need for the analysis of GABA uptake inhibitors, which can be used as research tools or potential drugs (16). β-alanine, a structural intermediate between α-amino acid (glycine, glutamate) and γ-amino acid (GABA) neurotransmitters, is a substrate inhibitor of GAT3. It should be underlined that exogenous β-alanine is carried across the blood brain barrier (BBB) into the central nervous system (CNS) via a taurine-sensitive β-amino acid transporter (17).The aim of the research was to analyze the effects of highly-selective blocker of GAT1, NO-711, and substrate inhibitor of GAT3, β-alanine, on the initial velocity of [³H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals isolated from control rats (8-9 weeks old) and rats (8-9 weeks old) that preliminary underwent hypoxia and seizures at the age of 10-12 postnatal days (perinatal hypoxia). The research question was how to modulate long-lasting changes in GABA uptake (and ambient level of GABA) in different brain regions after perinatal hypoxia/seizures taking into account that these changes might result from altered functioning of different types of GATs.     

Calcium uptake in mast cells,energy metabolism and histamine secretion

The inhibition of energy metabolism of mast cells causes an inhibition of histamine secretion. As the secretion is generally initiated by the influx of calcium into the cell, we have made correlative studies of the effect of blocking the energy metabolism on calcium uptake and histamine secretion.When the influx of calcium is increased by exposing the cells to low concentrations of saponin or ionophore A23187, histamine release occurs, having the character of a secretory response. Brief incubation of the cells with antimycin A, 10^–9 M–10^–7 M, prior to exposure to saponin or the calcium ionophore gave similar dose-response curves for the inhibitory effect of antimycin A on calcium uptake and histamine release.The inhibition of calcium uptake in untreated mast cells by antimycin A, 10^–9 M–10^–7 M, showed good correlation to the inhibition of anaphylactic histamine release and the release induced by compound 48/80. The antigen-induced histamine release is dependent on extracellular calcium and an inhibition of its uptake by antimycin A could by itself inhibit the release. Compound 48/80 on the other hand induces histamine release both in the presence and absence of calcium, and both are similarly inhibited by 10^–9 M–10^–7 M antimycin A. This indicates that antimycin A has other sites of action apart from the inhibition of the influx of extracellular calcium. The inhibitory effect of antimycin A on compound 48/80-induced histamine secretion in the absence of extracellular calcium may be due to an inhibitio of energy requiring steps in the final phase of the secretory process. These steps could be phosphorylation and activation of enzymes involved in the secretion. Since calmodulin has been demonstrated in a high concentration in the cytosol of the mast cell, and histamine release induced by antigen, compound 48/80, saponin and the calcium ionophore, is inhibited by calmodulin-antagonists, it seems likely that calmodulin-dependent protein kinase plays an important role in the release process.It may then be tentatively concluded that the activation of calcium channels in the plasma membrane as well as the activation of the enzymes involved in the final phase of secretion are both are energyrequiring steps, dependent on ATP.