Abstract

The effect of the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD) on exocytotic, transporter-mediated, tonic release, the ambient level and uptake of L-[(14)C]glutamate was assessed in rat brain synaptosomes using different methodological approaches of MβCD application. The addition of 15 mM MβCD to synaptosomes (the acute treatment, AT) immediately resulted in the extraction of cholesterol and in a two times increase in the extracellular L-[(14)C]glutamate level. When 15 mM MβCD was applied to synaptosomes for 35 min followed by washing of the acceptor (the long-term pretreatment, LP), this level was only one-third higher than in the control. The opposite effects of MβCD on tonic L-[(14)C]glutamate release and glutamate transporter reversal were found in AT and LP. Tonic release was dramatically enlarged in AT, but decreased after LP. Transporter-mediated release was increased several times in AT, but attenuated in LP. Depolarization-evoked exocytotic release of L-[(14)C]glutamate was completely lost in AT, whereas after LP, it was decreased by half in comparison with the control. Na(+)-dependent L-[(14)C]glutamate uptake was decreased by ~60% in AT, whereas in LP, it was lowered by ~40% only. The presence of MβCD in the incubation media during AT caused dramatic dissipation of the proton gradient of synaptic vesicles that was shown with the pH-sensitive dye acridine orange, whereas after LP, no statistically significant changes were registered in synaptic vesicle acidification. It was concluded that the diverse changes in glutamate transport in AT and LP were associated with the difference in the functional state of synaptic vesicles.

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