Radioligands from brain membrane

Guinea pig brain membranes, 0.5 mg of protein/sample, were incubated with 12 different concentrations of the compounds in the presence of receptor-specific radioligands at 25°C, in a final volume of 1 ml of 50 mM Tris-HCl, pH 7.5. Nonspecific binding was determined using 1 M naloxone. Data are the mean values S.E.M. from three experiments, performed in triplicate. 

Fig. 1. Saturation binding of a cannabinoid receptor ligand to brain membranes. Shown are the results of a typical saturation assay of [ H]SR141716A (a CBi-selective antagonist) in rat cortex membranes. Various concentrations (0.05-5.5 nAf) of radioligand were incubated in assay buffer with 0.1% BSA and rat cortex membrane homogenates (20 jig/tube) for 1 h at 30°C in the absence (total DPM) and presence (nonspecific DPM) of 5 pM unlabeled SR141716A. Specific DPM and nonspecific DPM shown are the mean of three values measured in consecutive assay tubes in the same rack. Specific DPM were calculated by subtracting mean nonspecific DPM from mean total DPM at each concentration of [ H]SR141716A.

A variety of in vitro assays have been developed that minimize or avoid live animal experimentation. Several capitalize on the sodium channel s affinity for these toxins. Neuronal cell lines lyse in the presence of veratridine, a sodium channel activator, and ouabain, which prevents removal of the excessive sodium ions allowed in by veratridine. In the presence of both these drugs, a sodium channel blocker such as saxitoxin rescues the cells. Cellular viability can then be measured by adding tetrazolium salts that are metabolized by living cells to a colored product. Alternatively, isolated cellular membranes, typically from brain tissue, are used to bind radiolabeled saxitoxin. After incubating receptor and radioligand in the presence of a test sample, any radiolabeled saxitoxin bound to the cell membranes are deposited onto filters by vacuum pressure. Radioactivity from the labeled saxitoxin is then measured with a signal reduction indicating the presence of saxitoxin. 

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