Characterization of a distinct Na(+)-H+ exchanger in basolateral membranes of human jejunum.
Kinetically distinct Na(+)-H+ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na(+)-H+ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na(+)-H+ exchanger may function in homeostatic intravesicular pH (pHi) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na(+)-H+ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H+ gradient [pHi/extravesicular pH (pHo) = 5.2/7.5] resulted in a Na+ uptake overshoot (1.45 +/- 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 +/- 0.13 nmol/mg protein). Na+ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na+ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K+ and the ionophore valinomycin. Na+ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r2 = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID50 value for amiloride of 29 microM (fourfold lower than ID50 for brush-border Na(+)-H+ exchanger). Kinetic studies of amiloride-sensitive Na+ uptake revealed a maximal velocity = 1.53 +/- 0.19 nmol.mg protein-1.5 s-1 and Michaelis constant = 9.83 +/- 3.5 mM. By varying pHi a sigmoidal effect of internal H+ on Na+ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pHi on Na+ efflux and Na(+)-Na+ exchange was studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
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Ghishan
D000584:Amiloride / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007583:Jejunum / Q000378:metabolism* / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D008566:Membranes / Q000378:metabolism; D008875:Middle Aged; D012964:Sodium / Q000493:pharmacokinetics; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium