Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence.
The importance of chloride channels for the cell is demonstrated by a number of serious human diseases that are due to mutations in chloride channels. The most well-known of these diseases is cystic fibrosis. Investigations into the mechanisms of the disease and possible treatments require the study of chloride fluxes at the level of individual cells. The present study compares two methods for studies of chloride transport: X-ray microanalysis and MQAE fluorescence with image analysis. As an experimental system, the cAMP-activated chloride channel in cultured respiratory epithelial cells was chosen. Both methods showed that stimulation with the cAMP-elevating agents forskolin and IBMX decreased the chloride content of the cells by about 20-27%. Inducing a driving force for chloride by replacing extracellular chloride by nitrate resulted in a chloride efflux that was significantly increased in the presence of forskolin and IBMX. This study shows that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride transport in individual cells.
Andersson
Charlotte
C
Department of Medical Cell Biology, Uppsala University, SE-75123 Uppsala, Sweden. charlotte.andersson@medellbiol.uu.se
Roomans
Godfried M
GM
D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D019411:Clinical Laboratory Techniques; D000242:Cyclic AMP / Q000502:physiology; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D004577:Electron Probe Microanalysis / Q000379:methods*; D004848:Epithelium / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans
D018118:Chloride Channels; D002712:Chlorides; D000242:Cyclic AMP