Nh3 permeability of principal cells and intercalated cells measured
by confocal fluorescence imaging.
Yip, Kay-Pong, and I. Kurtz.
Department of Physiology, Brown University, Providence, RI 02912,
UCLA School of Medicine, Division of Nephrology, Los Angeles, CA
90024
APStracts 2:0080F, 1995.
The cortical collecting duct (CCD) is an important site for NH3
secretion in mammalian nephron. However, given the cellular
heterogeneity of this epithelium the transcellular sites for NH3
secretion are unknown . In the present study, a dual-excitation
confocal microscope was designed and optimized to have sufficient
temporal resolution to measure the permeability of ammonia (PNH3)
across the basolateral and apical membrane of principal cells (PC's)
and intercalated cells (IC's) in perfused rabbit cortical collecting
ducts (CCD's). The rate of cellular NH3 influx was calculated from
the time course of increase in intracellular pH (pHi), measured with
2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) after 20 mM
NH4Cl was added to the bath or luminal perfusate. The time course of
increase in pHi was calculated from 488/442 image-pairs stored at a
rate of 4Hz. The apparent basolateral and apical PNH3 of PC's were
36+/-5 and 113+/-11 Nm/s, respectively. The values were 5.0+/-0.7 and
34+/-3 Nm/s after membrane folding correction. The apparent
basolateral and apical PNH3 of IC's were 38+/-6 and 132+/-15 Nm/s.
Correcting for membrane folding, the values were 9.0+/-1.0 and 47+/-5
Nm/s, respectively. The results demonstrate that the apical surface
was more permeable than the basolateral surface in both cell types.
In addition, IC's were more permeable to NH3 than PC's across both
membranes. The transcellular PNH3 of PC's and IC's were 27.3 Nm/s and
29.5 Nm/s respectively. Assuming that the ratio of PC's to IC's is
6:4 in the outer CCD, the predicted transtubular PNH3 is 28.2 Nm/s,
which is consistent with previous studies where apparent whole tubule
PNH3 was estimated using the fluid collection method (20-60 Nm/s).
The data suggest that the contribution of PC's and IC's to
transtubular NH3 secretion will not be dependent on the difference in
transtubular PNH3 values but on the net proton secretory rate of each
cell type.
Received 17 November 1994; accepted in final form 27 April 1995.
APS Manuscript Number F409-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 26 May 1995.