Mechanisms of bicarbonate transport by cultured rabbit inner medullary collecting duct cells

Abstract

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.