[Release of glutamate from cytosol of synaptosomes under conditions of experimental hypergravity].

The release of L-[14C]glutamate via Na+-dependent glutamate transporters functioned in the reverse mode was investigated in cortical synaptosomes under centrifuge-induced hypergravity. The protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) induced increase in [Na+]i, depolarized the plasma membrane, dissipated the proton gradient across synaptic vesicles and mitochondrial membrane, caused a fall in both the ATP level and the ATP/ADP ratio. 35 mM KCl-stimulated L-[14C]glutamate release from synaptosomes preliminary treated with 1 microM FCCP considerably increased from 27.0+/-2.2 % to 35.0+/-2.3 % of total accumulated synaptosomal label after centrifuge-induced hypergravity as compared to control animals (P< or =0.05). We found the competitive nontransportable glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate to inhibit FCCP and high KCL-stimulated release of L-[14C]glutamate. The release would be expected to occur via plasma membrane glutamate transporters. Transportable inhibitor of glutamate transporters-DL-threo-beta-hydroxyaspartate (DL-THA) induced heteroexchange of L-[14C]glutamate from enlarged by FCCP cytosolic pool of the neurotransmitter. DL-THA-evoked release of L-[14C]glutamate was also increased significantly after hypergravity. Combined application of KCl, DL-THA and FCCP unmasked dramatic changes in the activity of the glutamate transporters functioning in the reverse mode after centrifuge-induced G-loading.

[Methyl-beta-cyclodextrin influences glutamate transport in the rat brain nerve terminals by depletion of membrane cholesterol].

Role of membrane cholesterol in direct and reversed function of Na+ -dependent glutamate transporters and exocytosis was investigated. The depletion of membrane cholesterol by methyl-beta-cyclodextrin (MebetaCD) resulted in a dose-dependent significant reduction of the L-[14C]glutamate uptake by synaptosomes. Treatment of synaptosomes with 15 mM MebetaCD caused a decrease in the velocity of L-[14C]glutamate uptake by 49 +/- 4% (P < or = 0.05). The depolarization stimulated Ca2+ -dependent glutamate release that occurred via reverse functioning of glutamate transporters decreased insignificantly for 1 min from 8.0 +/- 0.4% to 6.7 +/- 0.4% of total accumulated synaptosomal label after MebetaCD treatment. The depletion of membrane cholesterol resulted in a reduction of the depolarization evoked exocytotic release from 8.0 +/- 1.0% to 4.2 +/- 1.0% of total synaptosomal label. Thus, cholesterol depletion was found to decrease significantly the Na+ -dependent uptake and exocytotic release of glutamate.

[Modulating effect of glutamate transporter inhibitors on accumulation and release of the neuromediator by the brain nerve terminals in rats].

L-[14C]glutamate uptake and release processes in nerve terminals has been investigated using the nontransportable and transportable competitive inhibitors of glutamate transport as tools. The effects of DL-threo-beta-benzyloxyaspartate (DL-TBOA) and DL-threo-beta-hydroxyaspartate (DL-THA) on the accumulation of L-[14C]glutamate have been evaluated after the exposure of rats to centrifuge-induced hypergravity. Both analogs potently inhibited the L-[14C]glutamate uptake in a dose-dependent manner (100 microM glutamate, 30 s incubation period). The IC50 values for DL-TBOA calculated on the basis of curves of non-linear regression kinetic analysis was 18 +/- 2 microM and 11 +/- 2 microM (p < or = 0.05) before and after the exposure to artificial gravity, respectively. L-THA, transportable analog, exhibited similar inhibitory characteristics (18 +/- 2 and 12 +/- 2 microM, respectively). We have also demonstrated that DL-TBOA exerted slighter effect on depolarization-evoked carrier-mediated L-[14C]glutamate release in control rats in comparison with gravity-loaded ones. Thus, DL-TBOA had complex effect on glutamatergic transmission, inhibited uptake and release of L-glutamate, and perhaps, became more potent under centrifuge-induced hypergravity.

[Study on the effect of hypergravity stress on L-glutamate uptake by rat brain nerve endings]. / Issledovanie vliianiia gipergravitatsionnogo stressa na aktivnoe pogloshchenie L-glutamata nervnymi okonchaniiami golovnogo mozga krys.

Using rat brain synaptosomes, we have investigated the effect of hypergravity on the kinetic parameters of Na(+)-dependent, high-affinity L-glutamate transport activity. The time-course of L-[14C]-glutamate uptake and dependence of L-[14C]-glutamate uptake velocity on glutamate concentrations were analyzed. K(m) and Vmax of this process have been determined. The hypergravity stress was created by centrifugation of rats for 1 hour at 10 g. We observed no differences in K(m) values between the control rats (10.7 +/- 2.5 microM) and animals exposed to hypergravity (6.7 +/- 1.5 microM). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate was not sensitive to hypergravity stress. In contrast, the maximal velocity of glutamate uptake changed in hypergravity conditions. Vmax reduced from 12.5 +/- +/- 3.2 nmol/min per 1 mg of protein (control group) to 5.6 +/- 0.9 nmol/min per 1 mg of protein (animals, exposed to hypergravity stress). The possible mechanisms of attenuation of the glutamate transporter activity without modifying K(m) of glutamate uptake were discussed.