Role of h+, k+-atpase in phi regulation in inner medullary
collecting duct cells in culture.
Ono, Shuichi, Jayarama Guntupalli, and Thomas D. Dubose, Jr.
Division of Renal Diseases and Hypertension, Department of Internal
Medicine
APStracts 3:0001F, 1996.
Studies in inner medullary collecting duct (IMCD) cells in primary
culture have proposed two mechanisms for Na+-independent hydrogen ion
transport in the IMCD: an H+- ATPase, and an H+,K+-ATPase. In the
present study, we have employed two sources of IMCD cells: cells in
primary culture derived from the terminal papilla of the Munich-
Wistar rat (IMCDp), and an established murine cell line (mIMCD-3), in
order to define the predominant mechanism(s) of Na+-independent pHi
recovery in the IMCD. In confluent monolayers of IMCDp and mIMCD-3
cells, intracellular pH was measured using the pH sensitive dye BCECF
following addition and withdrawal of NH4Cl. Removal of K+ completely
abolished Na+-independent pHi recovery in both IMCDp (DpHi[EPSILON]
min-1 = 0.039 +/- 0.006 to 0.005 +/- 0.003, P<0.001) and in
mIMCD-3 (DpHi [EPSILON] min-1 = 0.055 +/- 0.009 to -0.003 +/- 0.002,
P<0.001) cells, respectively. In mIMCD-3 cells, K+-dependent pHi
recovery was abolished by either of two specific inhibitors of the
H+,K+-ATPase: Sch 28080 (5 or 10 [mu]M) or A80915A (10[mu]M). In
contrast, bafilomycin A1 (2.5 and 10 nM), an inhibitor of the H+
-ATPase, failed to attenuate K+-dependent pHi recovery. Moreover,
sequence verified mouse gastric and colonic a H+,K+- ATPase probes
hybridized to total RNA from mIMCD-3 cells. Based on these findings,
we conclude that Na+-independent pHi recovery from an acid load in
both IMCDp and mIMCD-3 cells is critically dependent on extracellular
K+. That K+-dependent pHi recovery was inhibited by both Sch 28080
and A80915A, but not by bafilomycin A1, suggests that the predominant
mechanism by which Na+-independent pHi recovery is accomplished in
IMCD is through the H+,K+-ATPase. Expression of both gastric and
colonic a H+,K+-ATPase mRNA in mIMCD-3 cells, suggests that one or
both of these H+,K+-ATPases may be responsible for proton secretion
in the IMCD.
Received 21 March 1995; accepted in final form 5 December 1995.
APS Manuscript Number F96-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 22 January 96