AimTo 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.MethodsAnimals 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.ResultsIn 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%.ConclusionsThe 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.
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 Ca 2+ -stimulated GABA release. The state of hypoxia was induced by exposure of 10-to 12-day-old rats to a respiratory medium with low O 2 content (4% О 2 and 96% N 2 ) 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 [ 3 Н]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 [ 3 Н]GABA. The means of the initial rate of [ 3 Н]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 [ 3 Н]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 [ 3 Н]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 [ 3 Н]GABA in the cortex and hippocampus was nearly the same and showed no changes under the influence of hypoxia. The non-stimulated release of [ 3 Н]GABA after the influence of hypoxia increased in all structures, while the depolarization-induced Ca 2+ -dependent release of [ 3 Н]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.
The effects of four xanlhine derivatives, caffeine, caffeine benzoate, theophylline, and bromtheophylline, on sodium channels in internally perfused rat dorsal root ganglion neurons were studied under voltage
Local anesthetics and arrhythmic drugs induce both tonic and phasic blockade of voltage-dependent sodium channels in the neuronal membrane. The phasic blockade of molecular receptors depends on their previous activity and, thus, is qualified as use-dependent. Concentrations of the above-mentioned agents necessary for the induction of phasic blockade are one or two orders smaller than those necessary for the induction of tonic blockade. The molecular mechanisms underlying use-dependent blockade remain unstudied. We examined the effect of a novel analgesic, D57, a pyrrole imidazole derivative, on sodium channels in neurons of the rat sensory ganglia (the above blocker demonstrates the effects of both types). Concentrations providing 50% blocking of sodium currents in the resting state (tonic blocking) were 360 ± 18 and 420 ± 27 μM, while the Hill coefficients were 1.87 ± 0.09 and 1.41 ± 0.1 for tetrodotoxin-sensitive (ТТХ-S) and tetrodotoxin-resistant (TTX-R) sodium currents, respectively. Parameters of inactivation of the channels of both types changed significantly upon application of D57. For example, alterations of the voltage of half-inactivation (∆V 0.5 ) in the presence of 250 μM D57 were -11.2 ± 0.4 and -16.7 ± 0.5 mV for ТТХ-S and TTX-R sodium currents, respectively. After approximation of the ∆V 0.5 shifts in solutions containing different concentrations of D57 with the use of the modified Bean equation, we obtained a mean power equal to 2.2 ± 0.3. Analysis of the dependence of macroconstants of the blockade on the membrane potential and the frequency of stimulating impulses showed that, in the case of a shift of the potential from -120 to -80 mV, the constant of dissociation of D57 from channel molecules decreased 10 times, while the efficiency of the blockade of currents demonstrated only a 4.5-fold enhancement. An increase in the stimulation frequency from 0.5 to 3.3 sec -1 led to a 30-fold rise in the rate constant of the direct reaction (K on ), whereas the reverse reaction rate (K off ) increased 2.5 times. Analysis of the kinetics of the blockade using the Chernov model showed that a change in the stimulation frequency from 0.5 to 3.3 sec -1 led to an increase in the rate of association of the analgesic with sodium channels at the membrane potential of -120 mV, while dissociation at the potential of -80 mV was intensified two to three times. At the potentials of -80 mV and -120 mV, association and dissociation were accelerated 15-20 and 5-6 times, respectively. A probable molecular mechanism underlying phasic blockade is discussed. We hypothesize that phasic blockade is based on the formation of a planar structure under conditions of interaction of the corresponding aromatic groupings in the molecules of pharmacological agents with a certain phenyl residue of polypeptide chains of sodium channel α subunits (aromatic-aromatic interaction).
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