H,k-atpase in the rcct-28a rabbit cortical collecting duct cell
line.
Campbell, W. Grady, I. David Weiner, Charles S. Wingo and Brian D.
Cain.
Department of Biochemistry and Molecular Biology1, and Division of
Nephrology, Hypertension, and Transplantation, University of Florida
College of Medicine, Gainesville, Florida 32610, and Gainesville
Veterans Affairs Medical Center2, Gainesville, Florida 32608 USA
APStracts 5:0182F, 1998.
In the present work, we demonstrate that the rabbit cortical
collecting duct cell line RCCT-28A possesses three distinct H,K
-ATPase catalytic subunits (HKa). Intracellular measurements of RCCT
-28A cells using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)
-carboxyfluorescein (BCECF) indicated that the mechanism accounting
for recovery from an acid load exhibited both K+-dependence and
sensitivity to Sch- 28080 characteristic of H,K-ATPases. Recovery
rates were 0.022_0.005 pH units/min in the presence of K+,
0.004_0.002 in the absence of K+, and 0.002_0.002 in the presence of
Sch-28080. The mRNAs encoding the HKa1 subunit and the H,K-ATPase b
(HKb) subunit were detected by reverse transcriptase polymerase chain
reaction (RT-PCR). In addition, two HKa2 species were found by RT-PCR
and 5ae rapid amplification of cDNA ends (RACE) in the rabbit renal
cortex. One was homologous to HKa2 cDNAs generated from other species
and the second was novel. The latter, referred to as HKa2c, encoded
an apparent 61 residue amino-terminal extension that bore no homology
to reported sequences. Antipeptide antibodies were designed based on
this extension, and these antibodies recognized a protein of the
appropriate mass in both rabbit renal tissue samples and RCCT-28A
cells. Such findings constitute very strong evidence for expression
of the HKa2c subunit in vivo. The results suggest that the rabbit
kidney and RCCT-28A cells express at least three distinct H,K
-ATPases.
Received 1 May 1998; accepted in final form 8 October 1998.
APS Manuscript Number F100-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 10 November 1998