Sodium uptake assay

Pyrethroid insecticides (deltamethrin, NRDC 157, cismethrin), DDT analogs ( p,j> -DDT, (>,j> -DDT, methoxychlor, EDO), and a DDT-pyrethroid hybrid compound (GH401) enhanced veratridine-dependent sodium uptake by mouse brain synaptosomes The effectiveness of these compounds in the sodium uptake assay was in good agreement with their acute mammalian toxicities. , -DDT also enhanced veratridine-dependent sodium uptake by fish brain synaptosomes These findings demonstrate the utility of ion flux assays to study interactions of insecticides with sodium channels in the central nervous system and to explore species differences in insecticide target site sensitivity ... 

Sodium uptake assay. Assays using mouse brain synaptosomes were performed as described previously (6., ), except that insecticides were introduced to resuspended synaptosomes in 0.2-0.4 yl of ethanol rather than as a residue in the incubation tube. This amount of ethanol improved the delivery of insecticides, thereby increasing the reproducibility of the assay, and had no measurable effect on veratridine-dependent sodium channel activation. These methods were also used for assays with fish brain membranes, except that all buffers were augmented with sucrose to give osmolarities equivalent to the 0 7 M sucrose used for membrane isolation. 

Waltz F, Pillette L, Ambroise Y (2010) Anon-radioactive iodide uptake assay for sodium iodide symporter function. Anal Biochem 396(1/91-95... 

Our preliminary results with fish brain preparations suggest that ion flux techniques may be valuable in studies of target site differences between species. We have demonstrated veratridine-stimulated, tetrodotoxin-sensitive sodium uptake in a vesicular preparation from fish brain, thus confirming the presence of functional sodium channels in this preparation. Our results with , -DDT in this system also agree well with the action of DDT analogs and pyrethroids in mouse brain assays. Further studies wih both preparations should allow the exploration of target site differences between mammals and fish that have been inferred from whole animal toxicity studies. 

Two-Site Assay Protocol for the Measurement of Human a-Fetopro-tein. Purified antibodies to human a-fetoprotein (AFP) are prepared by allowing 1 ml of high titer antiserum (binding capacity approximately 125 p,g of AFP per milliliter) to react with 1 mg of AFP immunoadsorbent (protein uptake 120 p,g per milligram of cellulose) for 48 hr at 4°. Elution of high affinity antibody is carried out as described above for the preparation of purified insulin antibody. The antibody solution is diluted to 100 ml in 50 vaM sodium hydrogen carbonate buffer (pH 9.6) and used to coat 500 plastic tubes as described above. 

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]. 

Greenamyre JT, Higgins DS, Young AB (1990) Sodium-dependent D-aspartate binding is not a measure of presynaptic neuronal uptake sites in an autoradiographic assay. Brain Res., 5II, 310-318. 

Another example is the indole alkaloid ibogaine. Radioligand binding assays revealed that ibogaine interacted with a wide variety of receptors at concns. of 1-100 p.M. These included the opiate, 5-HT2, 5HT3, and muscarinic [, 2 receptors, and the dopamine, norepinephrine, and serotonin uptake sites. Furthermore, ibogaine interacted with N-methyl-D-aspartic acid (NMDA) associated ion and sodium ion channels. This broad spectrum of activity may in part be responsible for ibogaine s putative anti-addictive activity [80]. 

In vivo electron transport assays demonstrate that the site of Ca /Na action is Photosystem II. Ascorbate/DAD to methyl viologen rates (measured as O2 uptake) in the presence of DCMU are not affected by depletion of Ca " and Na" " while water to benzoquinone rates (measured as 2 evolution) in the presence of DBMIB show a decline concommitent with the loss of complete photosynthetic electron transport. Jji vitro examination of partial reactions within PS II reveals that the site of the cation effect excludes the water-splitting enzyme and occurs prior to the site of DCMU inhibition diphenylcarbazide-mediated silicomolybdate photoreduction after heat inactivation of the water-splitting enzyme shows diminished rates in membranes prepared from cells partially depleted of calcium and sodium. 

---