Detection of plasma membrane Ca2+-ATPase activity in mouse T lymphocytes by flow cytometry using fluo-3-loaded vesicles
✍ Scribed by William G. Telford; Richard A. Miller
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 726 KB
- Volume
- 24
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
The plasma membrane Ca*+-ATPase (PMCA) is the primary means by which many cell types pump calcium out of the cytosol following release of calcium from internal stores, returning intracellular calcium concentrations to normal levels. Traditional methods for measuring PMCA activity utilizing isotopic calcium uptake into inside-out (10) membrane vesicles have poor specificity for PMCA activity and require large numbers of cells. A flow cytometric method has been devised that allows the measurement of calcium uptake in I 0 vesicles using the fluorescent calcium chelator fluo-3. I 0 vesicles from mouse lymphocytes were loaded with flu03 pentapotassium salt and analyzed by flow cytometry following treatment with buffered calcium and/or ATP. I 0 vesicles appeared as a subpopulation of low forward-scatterlow side-scatter events, which were distinguishable from higher side-scatter debris. Treatment of vesicles with calcium and ATP resulted in a 5-fold to 3O-fold increase in I 0 vesicle fluo-3 fluorescence. Measurement of uptake kinetics gave K,,5 values of approximately 0.2-0.8 pM and 2 m M for calcium-and ATP-stimulated PMCA activity, respectively, which were consistent with published values obtained by other methods. Broad specificity P-type ATPase inhibitors and more narrowly specific PMCA and calmodulin inhibitors all blocked calcium uptake, whereas thapsigargin (an endoplasmidsarcoplasmic reticulum (EWSR-AT- Pase) inhibitor) had no effect, indicating that the assay provides a specific measure of vesicular PMCA activity. Flow cytometric analysis, therefore, may represent a useful approach for quantifying PMCA activityinmammahncells. o 19% WiIey-Lii, ~n c .