The inner medullary collecting duct (IMCD) is the final portion of the mammalian renal tubule that is able to significantly regulate systemic acid-base balance. Although the H+ transporters of this segment are relatively well studied, little is known regarding the mechanisms of HCO3- transport. The mechanisms of HCO3- transport in primary cultures of rabbit IMCD were studied using the pH-sensitive
dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, in CO2/HCO3(-)-containing solutions at 37 degrees C. Removal of Cl- from the extracellular
solution caused reversible intracellular alkalinization, demonstrating the presence of Cl-/HCO3- exchange. Alkalinization with Cl- removal was independent of changes in membrane potential, did not require the presence of extracellular Na+, and was inhibited by the disulfonic
stilbene,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (
DIDS, 10(-4) M). Half-maximal intracellular pH (pHi) recovery with readdition of Cl- to the extracellular
solution occurred at a Cl- concentration of 37.4 +/- 5.7 mM. When rabbit IMCD were cultured on permeable support membranes, Cl-/HCO3- exchange activity was found only on the basolateral membrane. However, there was no evidence of
band 3 protein immunoreactivity. In contrast, no evidence for Na(+)-(HCO3-)n > 1 cotransport activity was found. Depolarization of IMCD cells by acute increases in extracellular K+ did not alter pHi, nor was Na(+)-dependent, 5-(N-ethyl-N-isopropyl)amiloride-insensitive pHi recovery from an
acid load inhibited by
DIDS (10(-4) M). Finally, recovery from intracellular
alkalosis induced by incubation in 0 mM Cl-, 50 mM HCO3- extracellular
solution required Cl- and was independent of Na+. These studies indicate that the major mechanism of HCO3- transport in primary cultures of the rabbit IMCD is via a
band 3 protein-negative, Na(+)-independent, basolateral,
Cl-/HCO3- exchanger.