ISSN:
1432-1424
Keywords:
brush border membrane
;
amiloride
;
tissue culture
;
intracellular pH
;
fluorescence
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na+/H+ exchange at the level of an intact monolayer. Na+/ H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+/H+ exchange activity was measured by monitoring changes in intracellular pH (pH i ) after an acid load, using the pH-sensitive dye 2′7′-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH i , recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH i =7.68; pH o =7.4) where Na+/H+ exchange is not detectable, transport rate increased as pH i decreased. A positive cooperativity characterized the interaction of internal H+ with the exchanger, and suggests multiple H+ binding sites. In contrast, extracellular [Na+] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na+ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and theK i was 3.4 μM. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH i and processes associated with their polarized expression and regulation.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01872400
