Fluorescent characterization of collecting duct cells: a second H+-secreting type

We have used three fluorescent probes to label acid-base transporting cells with specific physiological properties in the rabbit collecting duct. Rhodamine albumin identified cells active in luminal endocytosis; rhodamine peanut agglutinin (PNA) identified cells with apical surface PNA ligands; and 6-carboxyfluorescein (6-CF) diacetate identified cells with alkaline pH or acetazolamide-sensitive esterase activity. More than 90% of all cells identified by PNA or rhodamine albumin selectively concentrated 6-CF. Axial heterogeneity of the identified cells was clearly evident along the collecting duct. In the midcortical collecting duct the predominant labeled cell (108 +/- 15/mm) was positive for PNA and 6-CF. These cells were less prevalent (69 +/- 10/mm) in inner cortical collecting ducts and absent from the outer medullary collecting duct. Cells that labeled only with 6-CF (with no detectable luminal endocytosis or PNA binding) showed the opposite distribution. They were the predominant identified cell in the inner stripe of the outer medulla (126 +/- 20/mm), and were less common in the cortical collecting duct. Because the former segment secretes H+, it was likely that these cells were H+-secreting cells. We used excitation ratio microspectrofluorometry of 6-CF to measure cytosolic pH (pHi approximately 7.2) and found evidence for a basolateral DIDS-sensitive Cl- -HCO3- exchanger and a Na+-independent luminal H+ pump. The previously described endocytic H+-secreting cell was seen at its highest concentration in the outer stripe (39 +/- 6/mm). Finally, 5-10% of identified cells did not stain selectively with 6-CF in cortical collecting ducts (solely endocytic or PNA binding). The function of these latter types could not be established. These studies suggest that the distribution and number of these populations of cells may determine the direction and magnitude of H+ transport along the collecting duct.