Studies on the kinetics of Na+/H+ exchange in OK cells: introduction of a new device for the analysis of polarized transport in cultured epithelia.
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 (pHi) 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 pHi recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pHi = 7.68; pHo = 7.4) where Na+/H+ exchange is not detectable, transport rate increased as pHi 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 19 mM at an intracellular pH of 7.1 and 60 mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and the Ki was 3.4 microM. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pHi and processes associated with their polarized expression and regulation.
Krayer-Pawlowska
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D
Department of Physiology, University of Zürich, Switzerland.
Helmle-Kolb
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Montrose
M H
MH
Krapf
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R
Murer
H
H
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007474:Ion Exchange; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium