The mechanism of potentiation of the glutamate-induced neurotoxicity by serum albumin. A possible role of nitric oxide.

Potentiation of the delayed (Glu)-induced neurotoxicity by serum albumin (SA) was studied in experiments with cultured cerebellar granule cells. The delayed neuronal death (DND) was evaluated by counting neurons containing or excluding Trypan Blue 4 h after treatment with Glu. Cytoplasmic Ca2+ ([Ca2+]i) was measured in individual Fura-2-loaded neurons. It was shown that a 15-min application of bovine SA (4 mg/ml) together with Glu (100 microM, 10 microM glycine, Mg2+-free solution) enhanced DND in the culture 1.7 times (43.1+/-3.1%) with respect to the effect induced by Glu alone (24.6+/-0.6%). The bovine SA application did not change the dynamics of [Ca2+]i response during a short-term (1 min) and long-term (15 min) Glu-treatment. DND was prevented by simultaneous application of Glu and inhibitor of NO-synthase N omega-nitro-L-arginine methyl ester (L-NAME), 100 microM) (10.8+/-1.0%) as well as by the application of Glu with SA and L-NAME (9.8+/-1.2%). In order to evaluate the role of nitric oxide (NO) in the SA effect, the cells were incubated for 15 min with the NO-donors sodium nitroprusside (SNP, 10 and 100 microM) and sodium nitrite (NaNO2, 10 and 100 microM) together with SA and in its absence. SA also greatly enhanced the DND induced by SNP and NaNO2. Thus, the DND after simultaneous treatment with SA and SNP was 16.3+/-2.5% (10 microM) or 29.6+/-2.1% (100 microM), and 9.6+/-0.8% (10 microM) and 19.7+/-2.1% after treatment with SNP alone. Exposure to SA together with NaNO2 led to the DND increase up to 26.5+/-1.9% (10 microM) and 37.7+/-3.5% (100 microM) in comparison with 7.4+/-2.0% (10 microM) and 18.9+/-0.8% (100 microM) in experiments with NaNO2 alone. Taking into account the ability of NO and NO2 to oxidize unsaturated fatty acids and the ability of SA to bind them after their hydrolytic removal, we suggested that the SA-induced potentiation of Glu neurotoxicity resulted from exacerbation of the toxic effects of NO and other trace radicals on the neuronal membranes. This hypothesis was supported by the finding that SA also enhanced the neurotoxicity of the lipid prooxidant FeCl2. The simultaneous 15-min application of FeCl2 (10 microM) and SA caused a 51.5+/-4.0% increase in DND, which exceeded 2.4 times the effect produced by FeCl2 alone (21.3+/-2.3%).

Bepridil exacerbates glutamate-induced deterioration of calcium homeostasis and cultured nerve cell injury.

Application of 50 microM bepridil (BPD) to cultured nerve cells did not greatly affect the resting cytoplasmic Ca2+ concentration ([Ca2+]i) but caused its pronounced increase both during prolonged glutamate (GLU, 100 microM) treatment and, especially, in the postglutamate period in case of partial [Ca2+]i recovery. In contrast, in cells exhibiting a high [Ca2+]i plateau in the postglutamate period, BPD application either did not cause any additional elevation of [Ca2+]i or caused a very small increase. Under identical conditions replacement of external Na+ by Li+ or N-methyl-D-glucamine (NMDG) either did not change [Ca2+]i or produced a very small increase, strongly indicating that the BPD-evoked Ca2+ responses could not be explained solely by Na+/Ca2+ exchange inhibition but resulted from some other BPD effects. Indeed, in experiments with Rhodamine 123-loaded neurons it has been shown that 50 microM BPD induced prominent mitochondrial depolarization which is known to abolish the mitochondrial Ca2+ uptake. Finally it was revealed that BPD application to the cell culture either in the period of a prolonged (15 min) GLU action or, especially, in the postglutamate period greatly exacerbated delayed neuronal death, apparently due to a complex inhibitory action of the drug on both Ca2+ buffering and Ca2+ extrusion systems.

Effect of a prolonged glutamate challenge on plasmalemmal calcium permeability in mammalian central neurones. Mn2+ as a tool to study calcium influx pathways.

The rate of Mn(2+)-induced fluorescence quenching (RFQ) was used as a relative measure of plasma membrane Ca2+ permeability (PCa) in fura-2-loaded cultured hippocampal neurons and cerebellar granule cells during and after protracted (15-30 min) glutamate (GLU) treatment. Some limitations of this method were evaluated using a kinetic model of a competitive binding of Mn2+ and Ca2+ to fura-2 in the cell. In parallel experiment a contribution of Ca2+ influx to the cytoplasmic Ca2+ ([Ca2+]i) was repeatedly examined during and following a prolonged GLU challenge by short-duration "low-Ca2+ trials" (50 microM EGTA) and by measurements of 45Ca2+ uptake. Experiments failed to reveal a putative persistent increase in PCa that earlier was thought to underlie Ca2+ overload of the neuron caused by its toxic GLU treatment. By contrast, a sustained increase of [Ca2+]i was found to be associated with a progressive decrease in PCa and Ca2+ influx both in the period of GLU application and after its termination. These findings give new evidence in favour of the hypothesis that the GLU-induced Ca2+ overload of the neuron mainly from an impairment of its Ca2+ extrusion systems.

Changes of cytoplasmic pH in frog nerve fibers during K(+)-induced membrane depolarization.

Frog sciatic nerve and its thin bundles were loaded with fluorescein diacetate in order to monitor changes in cytoplasmic pH (pHi) caused by high K+ depolarization. Isosmotic substitution of external Na+ by K+ at pHo 7.3 led to a steady concentration-dependent (20-120 mM K+) decrease in pHi. Elevation of pHo from 7.3 to 8.5 prevented or even reversed these pHi changes, indicating their strong dependence on transmembrane H+ fluxes. The depolarization-induced intracellular acidification could not be prevented or decreased by any of the following treatments: removal of external Ca2+; application of the Ca2+ antagonists Ni2+ and Co2+; blockade of K+ channels by TEA; addition to the external solution of Zn2+, a blocker of putative voltage-sensitive H+ channels. By contrast, blockade of Na+ channels by 1-3 microM TTX prevented the effect of high K+ concentrations on pHi. It is concluded that the decrease in pHi induced by a prolonged membrane depolarization in frog nerve fibers is mainly due to an enhanced H+ influx through non-inactivating Na+ channels.

Effects of repetitive stimulation, veratridine and ouabain on cytoplasmic pH in frog nerve fibres: role of internal Na+.

Changes of cytoplasmic pH (pHi) in frog nerve fibres during repetitive stimulation have been measured using the fluorescent pH indicator dye fluorescein diacetate (FDA). Under control conditions repetitive (10-50 Hz) stimulation caused only a very small decrease in pHi (by 0.015-0.06 pH units). Modification of Na+ channels by veratridine (VER, 10 microM) greatly increased this stimulus-evoked (SE) internal acidification. Blockade of the Na(+)-K+ pump by ouabain (0.5 mM) enhanced the effects VER and prevented pHi recovery after the termination of repetitive stimulation. A similar inhibition of post-stimulatory recovery of pHi was observed after replacement of external Na+ with Li+, which is not accepted by the Na(+)-K+ pump instead of Na+. These data suggest that SE intracellular acidification in nerves results from or is closely associated with an increase in [Na+]i. Treatments that promote Na+ influx and accumulation of Na+ inside the fibre enhance reduction of pHi. Li+ can be substituted for Na+ in this process.