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 mem brane of epithelial cells. We evaluated the use of a multiwell-multilabel r eader to investigate basolateral NHE-1 in confluent MDCK cells and compared the results with data obtained using an imaging system equipped with a fil ter perfusion system. Using the multiwell-multilabel reader we obtained vir tually 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 ad dition 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 stim ulation of protein kinase C using phorbol myristate acetate. In summary, ou r data indicate that multiwell-multilabel readers are suitable to investiga te physiology and regulation of basolateral NHE. Thus, multiwell-multilabel readers offer a valid and convenient alternative to investigate basolatera l transporters. (C) 2001 Academic Press.