Determination of basolateral Na+/H+ exchange activity in MDCK cells using a multiwell-multilabel reader

Citation
M. Gekle et al., Determination of basolateral Na+/H+ exchange activity in MDCK cells using a multiwell-multilabel reader, ANALYT BIOC, 296(2), 2001, pp. 174-178
Citations number
14
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
0003-2697 → ACNP
Volume
296
Issue
2
Year of publication
2001
Pages
174 - 178
Database
ISI
SICI code
0003-2697(20010915)296:2<174:DOBNEA>2.0.ZU;2-E
Abstract
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.