[Extracellular acidification leads to reactive oxygene species formation in rat brain synaptosomes].

Formation of reactive oxygen species in rat brain synaptosomes was studied using DCFDA fluorescent dye at lowered extracellular pH. It has been shown that decrease in pH value from 7.4 to 7.0 and up to 6.0 leads to increase of fluorescence that is indicative of oxidative stress. The effect is observed regardless of whether Ca ions are present in incubation medium or no. Acidification of the incubation medium induces quenching of fluorescence of previously oxidized form of the dye in experiments without synaptosomes This evidences that increase of dye fluorescence is really associated with reactive oxygen species accumulation. Thus, it has been demonstrated that pH declined up to 7.0 in the incubation medium is sufficient to induce the formation of reactive oxygen species in synaptosomes.

[Y-27632 induces calcium-independent glutamate release in rat brain synaptosomes by the mechanism which is distinct from exocytosis].

The inhibitor of Rho-kinase Y-27632 induces non-secretory exocytosis in PC12 cells. The influence of this compound on central synapses remains uninvestigated. We showed that Y-27632 at the concentration 100 jtM led to spontaneous [14C]glutamate release in synaptosomes, which was not accompanied by plasma membrane depolarization. Membrane potential was registered by fluorescent dye DiSC3(5). Y27632 induced an increase of acridine orange fluorescence, exercising no influence over fluorescence of FM2-10 dye. These results suggest that Rho-kinase inhibition decreases pH gradient of synaptic vesicles not inducing exocytosis. Dissipation of the gradient leads to leakage of neurotransmitters to cytosol pumping them out by plasma membrane transporters. Our results show the involvement of Rho-dependent branch of intracellular signaling in regulation of pH gradient in synaptic vesicles.

[Glutamate induces the formation of free radicals in rat brain synaptosomes].

The influence of glutamate and the agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes has been investigated using the fluorescent dye DCFDA. It was shown that glutamate at concentrations of 100 microM and 1 mM increases the synthesis of reactive oxygen species. This phenomenon was eliminated by removing calcium from incubation medium. The addition of NMDA (100 microM) and kainate (100 microM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, the activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.

[Hypotonic swelling activates the Na*/H* exchange in rat brain synaptosomes].

The influence of hypotonic swelling and hypertonic shrinking on cytosolic pH in synaptosomes was investigated. It was shown that decreasing the osmolarity of incubation medium to 230 mOsm leads to alkalization and increasing the osmolarity of incubation medium to 810 mOsm leads to acidification. Alkalization was inhibited by amiloride, indicating the involvement of the Na+/H+ exchanger. The acidification of cytosol upon hypertonic shrinking was insensitive, to amiloride and the inhibitor of Na+, K+, Cl- cotransport bumetanide. Thus, the Na+/H+ exchange in synaptosomes is activated by hypotonic swelling but not hypertonic shrinking, in contrast with erythrocytes and lymphocytes, which have been investigated earlier.

[Lanthanides induce neurotransmitter release from the vesicular pool in rat brain synaptosomes].

The influence of lanthanoids on exocytosis was investigated. It was shown that gadolinium increases the spontaneous release of the glutamate nonmetabolizing analogue [3H]D-aspartate. It was established using the fluorescent dye acridine orange that gadolinium and lanthanum induce exocytosis. The effect was dose-dependent and was maximum at 300 microM Gd3+. The exocytosis induced by gadolinium was calcium-independent. It is suggested that lanthanides induce a vesicular release of neurotransmitters by the mechanisms common for all polyvalent cations.

[Effect of osmolar concentration of culture media on exocytosis in isolated presynaptic nerve endings of rat brain ]. / Vliianie osmotichnosti inkubatsionnoi sredy na ékzotsitoz v izolirovannykh presinapticheskikh okonchaniiakh neironov mozga krys.

The effect of hypotonic and hypertonic shock on exocytosis in rat brain synaptosomes was studied using the fluorescent dye acridine orange. It was shown that an increase in medium osmolarity leads to calcium-independent exocytosis. The response of the probe was directly proportional to the amount of osmolithes added. A decrease in medium osmolarity to 230 mOsm led to an increase of acridine orange fluorescence, which is comparable with exocytosis occurring by the action of 15 mM KCl. This effect was independent of calcium concentration. It is assumed that, under hypotonic shock, part of neurotransmitters are released from the vesicular pool.

[Influence of calcium ionophore A23187 on neurotransmitter release in rat brain synaptosomes]. / Vliianie kal'tsevogo ionofora A23187 na osvobozhdenie neiromediatorov iz sinaptosom mozga krys.

The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters.