Determination of basolateral Na(+)/H(+) exchange activity in MDCK cells using a multiwell-multilabel reader.
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 multiwell-multilabel 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.
Gekle
M
M
Physiologisches Institut, University of Würzburg, Würzburg, 97070, Germany.
Freudinger
R
R
Mildenberger
S
S
Sauvant
C
C
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D004285:Dogs; D001330:Electronic Data Processing / Q000379:methods*; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis*
D017923:Sodium-Hydrogen Exchangers