Colocalization of H⁺-ATPase and band 3 anion exchanger in rabbit collecting duct intercalated cells

Two major types of interca lated cells (IC) have been previously defined in rabbit collecting duct: α-cells have a basolateral band 3-like anion exchanger and secrete H⁺, whereas β-cells bind peanut agglutinin (PNA) apically and are believed to secrete HCO₃^-. To further define IC types, we double-labeled kidney sections with anti-H⁺-ATPase antibodies and with either an anti-band 3 antibody or PNA. We found four patterns of staining: 1) IC with apical H⁺-ATPase and basal band 3, a configuration consistent with ongoing H⁺ secretion, which prevailed in the inner stripe of outer medulla (OMCD_i); 2) diffuse H⁺-ATPase labeling across the cell and basal band 3, which was most numerous in the outer stripe of outer medulla (OMCD_o); 3) IC with "bright" apical peanut lectin, diffuse H⁺-ATPase, and no band 3, which was abundant in the cortical collecting duct (CCD) and probably represents HCO₃^--secreting cells; and 4) "hybrid" cells with various staining combinations (e.g., apical lectin binding and apical H⁺-ATPase), which although they are uncommon, were seen in the CCD. Consistent with this immunocytochemical finding of hybrid cells, cell-sorting studies on isolated CCD IC showed that 6-18% of PNA-positive cells also stained positively for band 3. We conclude that 1) band 3-positive IC in the OMCD vary axially. Most OMCD_i IC are probably active proton secretors, whereas up to one-half of OMCD_o IC may be latent H⁺ secretors. 2) The diffuse H⁺-ATPase pattern in putative β-cells differs from comparable results in the rat and is not consistent with a "reversed" α-cell. HCO₃^- secretion by β-cells may be driven by an H⁺ extrusion mechanism other than the α-cell pump re-sorted to the basolateral membrane. 3) The possibility of hybrid cells that might combine α- and α-cell transport proteins suggests a mechanism for functional reversal of collecting duct IC polarity.