Phosphorimaging detection and quantitation for isotopic ion flux assays.
A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated (22)Na influx in four cultured cell lines provided satisfactory concentration-response data for epibatidine and several other nicotinic agonists. The data were consistent with data obtained using standard 6-well assays. Assays for nicotinic-receptor-mediated (86)Rb efflux produced data similar to data obtained with the (22)Na influx assay. However, assays for (45)Ca influx were not successful, although (45)Ca was readily detected and quantified. Voltage-gated sodium channel-mediated (22)Na influx in a neuroblastoma cell line allowed assay of the effects of such sodium channel activators as batrachotoxin and a pumiliotoxin B/scorpion venom combination. Phosphorimaging detection allows for reliable beta counting of up to 1,200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening.
Fitch
Richard W
RW
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA. rfitch@indstate.edu
Daly
John W
JW
D000470:Alkaloids / Q000494:pharmacology; D001345:Autoradiography / Q000295:instrumentation* / Q000379:methods*; D001496:Batrachotoxins / Q000494:pharmacology; D002132:Calcium Radioisotopes; D002460:Cell Line; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000502:physiology*; D012414:Rubidium Radioisotopes; D012604:Scorpion Venoms / Q000494:pharmacology; D012680:Sensitivity and Specificity; D015222:Sodium Channels / Q000502:physiology*; D012979:Sodium Radioisotopes
D000470:Alkaloids; D001496:Batrachotoxins; D002132:Calcium Radioisotopes; D007212:Indolizines; D010880:Piperidines; D011978:Receptors, Nicotinic; D012414:Rubidium Radioisotopes; D012604:Scorpion Venoms; D015222:Sodium Channels; D012979:Sodium Radioisotopes; C031598:pumiliotoxin B