Determination of Basolateral Na+/H+ Exchange Activity in MDCK Cells Using a Multiwell–Multilabel Reader
✍ Scribed by Michael Gekle; Ruth Freudinger; Sigrid Mildenberger; Christoph Sauvant
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 90 KB
- Volume
- 296
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Na ؉ /H ؉ exchangers (NHE) are important membrane transport proteins involved in transepithelial transport and cellular pH homeostasis. NHE-1, important for cellular pH and volume homeostasis, is expressed in the basolateral membrane of epithelial cells. We evaluated the use of a multiwell-multilabel reader to investigate basolateral NHE-1 in confluent MDCK cells and compared the results with data obtained using an imaging system equipped with a filter perfusion system. Using the multiwell-multilabel reader we obtained virtually the same values for steady-state pH and NHE-1 activity under control conditions compared to the imaging system. With both setups Na ؉dependent pH recovery after an acid load occurred virtually only after basolateral addition of Na ؉ . Furthermore, Na ؉ -dependent pH recovery was reduced by >80% in the presence of the NHE-1 inhibitor HOE642. In addition, we detected an almost identical increase of NHE-1 activity with the two methods after stimulation of protein kinase C using phorbol myristate acetate. In summary, our data indicate that multiwellmultilabel readers are suitable to investigate physiology and regulation of basolateral NHE. Thus, multiwell-multilabel readers offer a valid and convenient alternative to investigate basolateral transporters.