Chloride uptake assay

Chloride flux assay. Synaptic vesicles were prepared from brains of male ICR gice (20-30 g Blue Spruce Farms, Altamont, NY) (15) Assays of °C1 uptake involved preincubation (10-20 min) of vesicles with carrier solvent (ethanol or acetone, 0.5-1 yl) or insecticide, followed by incubation (4 sec) with bCl with or without GABA or added insecticides and isolation of labelled vesicles by rapid vacuum filtration. Detailed descriptions of the assay are published elsewhere ( 15,1 7) Abamectin stock solutions were prepared in absolute ethanol in silanized glass vials Abamectin in ethanol (1 yl) was added to give concentrations of 3 nM-3 yM in a final volume of 200 yl of vesicle suspension during preincubation or 3 yM in a final volume of 200 yl of bCl uptake medium. 

Recent studies have grouped lindane with the cyclodienes on the basisLjaf its potent and stereospecific inhibition of [3H]DHPTX (.2,3) and [3DS]TBPS (4 J>) binding. However, the quantitative correlations between potency in the chloride flux assay, potency in the [3 >S]TBPS binding assay, and acute toxicity that were observed for the cyclodienes do not extend to lindane. Lindane was a very weak inhibitor of chloride uptake (I 1 mM J7). Inclusion of the data... 

In a study of six mercury compounds, mercury chloride, mercury nitrate, sodium ethylmercurithi-osalicylate, methyl mercury chloride, mercury acetate and phenylmercury acetate in MDCK cells, LLC-PKl cells and human primary proximal tubular cells (hPTC) and non-renal cell lines (SAOS and Hep G2) it was found that all mercury compounds were toxic to all cell types as evidenced by neutral red uptake, thymidine incorporation and the MTT assay [189]. However, sodium ethylmercurithiosalicylate, methyl mercury chloride and phenylmercury acetate were one order of magnitude more toxic than the other compounds. In addition the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) potentiated the toxicity of all mercury compounds [189]. In a study using primary rabbit proximal tubular cells it was also shown that methyl mercury chloride is more toxic than mercury chloride [190]. Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg " occurred within 6-24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CHgHg" occurred within 3 hr of exposure and was concentration- dependent [190]. 

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...

Count duplicate 10 jjL aliquots of the stopped suspensions (specific and nonspecific uptake and background) and calculate the total amount of chloride in each incubation, assuming that 10 pL represents 10/1080, or 0,93% of the total dpm. The amount of accumulated in the synaptoneurosomes is expressed in nmol Ch by comparison with the total amount of Cl" m the assay, and normalized to the amount of synaptoneurosomal protein (in mg)... 

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