The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl− absorption linked to Na+ were investigated in isolated perfused tubules using Cl−-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl− (a i Cl ) is 11.3 ± 0.5 mm, the basolateral (V 1 ), apical (V 2 ), and transepithelial (V 3 ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, a i Cl falls by 1.3 ± 0.3 mm and V 2 hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na+ from the lumen causes a i Cl to fall further by 2.3 ± 0.4 mm and V 2 to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a i Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a i Cl . The effects of luminal HCO− 3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO− 3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO3 exchange. Several lines of evidence favor conductive Cl− transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a i Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO− 3-free Ringer, the equilibrium potential for Cl− does not differ from the membrane potential V 2 . Finally, the logarithmic changes in a i Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl− channels at the apical and basolateral cell membranes of the proximal tubule.
The Journal of Membrane Biology – Springer Journals
Published: Feb 2, 2014
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