Synaptosomes Permeabilization

Acidification of Small Synaptic Vesicles in SLO-Permeabilized Synaptosomes... 

Uptake of glutamate results in an acidification of the intravesicular lumen. This acidification can be indicated by the fluorescent dye acridine orange, which accumulates in acid compartments. Synapto-somes were prepared (McMahon etal., 1992) and stored as pellets of about 1 mg overlaid with 200 p.1 Krebs-Ringer-HEPES buffer on ice. Acidification was performed with either glutamate or chloride (Hell et al., 1992 Hartinger and Jahn, 1993). To get rid of the non-vesicular glutamate extensive washing of the permeabilized synaptosomes is required. 

Resuspend permeabilized synaptosomes in 1 ml acidification buffer containing 200 [xM ouabain, 100 pM vanadate, 100 pM oligomycin B pre-warmed to 32 °C,... 

Figure 2 illustrates representative experiments using rat brain synaptosomes. Acidification can be measured only in permeabilized synaptosomes. 

Regulation of Vesicular Transmitter Transporters in Permeabilized Cells and Synaptosomes... 

Transmitter uptake into secretory vesicles is an ATP-dependent process. So far these studies have been restricted to isolated secretory vesicles, where intracellular substances necessary for regulation may be lost during purification. Neuroendocrine cells or synaptosomes permeabilized with both pore-forming toxins can be used to study the regulation of transmitter storage without the necessity of purifying secretory vesicles. 

Fig. 2. Acidification of small synaptic vesicles by glutamate and chloride in synap-tosomes. The acidification assay was performed as described in section 3.2 of this chapter. Two representative experiments with intact (upper trace) or SLO-permeabilized (lower trace) synaptosomes are shown. The ordinate gives the changes of absorbance obtained (A 492-530). Final concentrations of potassium glutamate (Glut), KCl, and ammonium sulfate (NH/) were 10 mM, 45 mM and 30 mM, respectively. The uptake of glutamate and chloride result in an acidification of the lumen of small synaptic vesicles, which increases the vesicular uptake of acridine orange, resulting in a decrease in the amount of extravesicular dye. This acidification can be only observed when the plasma membrane is permeabilized...

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