Effects of new fluorinated analogues of GABA, pregabalin bioisosters, on the ambient level and exocytotic release of [3H]GABA from rat brain nerve terminals.

Recently, we have shown that new fluorinated analogues of γ-aminobutyric acid (GABA), bioisosters of pregabalin (ß-i-Bu-GABA), i.e. ß-polyfluoroalkyl-GABAs (FGABAs), with substituents: ß-CF3-ß-OH (1), ß-CF3 (2); ß-CF2CF2H (3), are able to increase the initial rate of [3H]GABA uptake by isolated rat brain nerve terminals (synaptosomes), and this effect is higher than that of pregabalin. So, synthesized FGABAs are structural but not functional analogues of GABA. Herein, we assessed the effects of synthesized FGABAs (100µM) on the ambient level and exocytotic release of [3H]GABA in nerve terminals and compared with those of pregabalin (100µM). It was shown that FGABAs 1-3 did not influence the ambient level of [3H]GABA in the synaptosomal preparations, and this parameter was also not altered by pregabalin. During blockage of GABA transporters GAT1 by specific inhibitor NO-711, FGABAs and pregabalin also did not change ambient [3H]GABA in synaptosomal preparations. Exocytotic release of [3H]GABA from synaptosomes decreased in the presence of FGABAs 1-3 and pregabalin, and the effects of FGABAs 1 &3 were more significant than those of FGABAs 2 and pregabalin. FGABAs 1-3/pregabalin-induced decrease in exocytotic release of [3H]GABA from synaptosomes was not a result of changes in the potential of the plasma membrane. Therefore, new synthesized FGABAs 1 &3 were able to decrease exocytotic release of [3H]GABA from nerve terminals more effectively in comparison to pregabalin. Absence of unspecific side effects of FGABAs 1 &3 on the membrane potential makes these compounds perspective for medical application.

Synthesis of new fluorinated analogs of GABA, Pregabalin bioisosteres, and their effects on [(3)H]GABA uptake by rat brain nerve terminals.

Fluorinated analogs of natural substances take an essential place in the design of new biologically active compounds. New fluorinated analogs of γ-aminobutyric acid, that is, ß-polyfluoroalkyl-GABAs (FGABAs), were synthesized with substituents: ß-CF3-ß-OH (1), ß-CF3 (2); ß-CF2CF2H (3). FGABAs are bioisosteres of Pregabalin (Lyrica®, Pfizer's blockbuster drug, ß-i-Bu-GABA), and have lipophilicity close to this medicine. The effects of synthesized FGABAs on [(3)H]GABA uptake by isolated rat brain nerve terminals (synaptosomes) were assessed and compared with those of Pregabalin. FGABAs 1-3 (100µM) did not influence the initial velocity of [(3)H]GABA uptake when applied acutely, whereas an increase in this parameter was found after preliminary incubation of FGABAs with synaptosomes. Pregabalin after preliminary incubation with synaptosomes caused unidirectional changes in the initial velocity of [(3)H]GABA uptake. Using specific inhibitors of GAT1 and GAT3, NO-711 and SNAP5114, respectively, the ability of FGABAs 1-3 to influence non-GAT1 and non-GAT3 uptake activity of nerve terminals was analyzed, but no specificity was found. Therefore, new synthesized FGABAs are structural but not functional analogs of GABA (because they did not inhibit synaptosomal [(3)H]GABA uptake). Moreover, FGABAs are able to increase the initial velocity of [(3)H]GABA uptake by synaptosomes, and this effect is higher than that of Pregabalin.

New effects of GABAB receptor allosteric modulator rac-BHFF on ambient GABA, uptake/release, Em and synaptic vesicle acidification in nerve terminals.

Positive allosteric modulators of GABAB receptors have great therapeutic potential for medications of anxiety, depression, etc. The effects of recently discovered modulator rac-BHFF on the key characteristics of GABAergic neurotransmission were investigated in cortical and hippocampal presynaptic nerve terminals of rats (synaptosomes). The ambient level of [(3)H]GABA that is a balance between release and uptake of the neurotransmitter increased significantly in the presence of rac-BHFF (at concentrations 10-30µM). The initial velocity of synaptosomal [(3)H]GABA uptake was suppressed by the modulator. In the presence of GABA transporter blocker NO-711, it was shown that rac-BHFF increased tonic release of [(3)H]GABA from synaptosomes (at concentrations 3-30µM). Rac-BHFF within the concentration range of 0.3-30µM did not enhance inhibiting effect of (±)-baclofen on depolarization-induced exocytotic release of [(3)H]GABA. Rac-BHFF (0.3-30µM) caused dose-dependent depolarization of the plasma membrane and dissipation of the proton gradient of synaptic vesicles in synaptosomes that was shown in the absence/presence of GABAB receptor antagonist saclofen using fluorescent dyes rhodamine 6G and acridine orange, respectively, and so, the above effects of rac-BHFF were not associated with the modulation of presynaptic GABAB receptors. Therefore, drug development strategy of positive allosteric modulation of GABAB receptors is to eliminate the above side effects of rac-BHFF in presynapse, and vice versa, these new properties of rac-BHFF may be exploited appropriately.

The dynamics of changes in hippocampal GABAergic system in rats exposed to early-life hypoxia-induced seizures.

Hypoxia-evoked seizures (H/S) early in life lead to multiple chronic neurological deficits. Here, we present the results of studying GABA release and uptake in hippocampal axon terminals of rats exposed to H/S at 10-12 days of age. We characterized (i) exocytotic release of GABA; (ii) the initial rate of GABA uptake; (iii) the regulation of GABA release by presynaptic GABA(B) receptors. Rats were used for experiments 2, 4 and 8 weeks after H/S. We found that exocytotic [(3)H]GABA release was higher in rats exposed to H/S, and a maximal difference in the release was observed between the control and experimental rats tested 2 weeks after H/S. In contrast, the initial rate of GABA uptake decreased with age, and this tendency was more pronounced in rats exposed to H/S. Using (±)-baclofen and SKF 97541 as agonists of GABA(B) receptor, we revealed that a significant difference in the auto-inhibition of exocytotic [(3)H]GABA release was detected only between the control and experimental adult rats (8 weeks after hypoxia). The inhibitory effect dropped dramatically in the control adults, but only slightly decreased in adult rats exposed to H/S, thus becoming threefold more potent after hypoxic injury. Together, the results show that H/S affects the dynamics of age-dependent changes in the GABAergic system, and that the enhanced GABA(B) receptor-mediated auto-inhibition can be an important factor in weakening the postsynaptic inhibition and in the development of hyperexcitability in rats exposed to H/S.