Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells.
MDCK cells were grown to confluent monolayers on permeant filter supports; pH was analysed by using the pH-sensitive fluorescent probe 2'7'-biscarboxyethyl-5,6-carboxyfluorescein and a routine spectrofluorometer equipped with a perfusion cuvette [Krayer-Pawlowska et al. (1990) J Membr Biol 120:173-183]. Superfusion of the basolateral (but not apical) cell surface with Na(+)-containing solutions led to immediate recovery of pHi from an acid load (NH4 prepulse). This pHi recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent Km for Na+ of about 25 nM Na+ and an apparent Ki for inhibition by dimethylamiloride of around 0.2 microM; inhibition by dimethylamiloride was competitive with Na+ interaction. Lowering pHi prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent Vmax for Na/H exchange is increased more than tenfold by lowering the pHi from 7.0 to 6.7 without an effect on apparent Km values for Na+ interaction. It is concluded that MDCK cells (strain I) grown on a permeant support contain only basolateral Na/H exchange activity, most likely Na/H-1 [for nomenclature see Igarashi et al. (1991) Kidney Int 40:S84-S89].
Vilella
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S
Department of Physiology, University of Zürich, Switzerland.
Guerra
L
L
Helmle-Kolb
C
C
Murer
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H
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002138:Calibration; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010477:Perfusion; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; C039614:ethylisopropylamiloride