Aldosterone-dependent regulation of na/k atpase subunit mrna in the
rat cortical collecting duct: competitive pcr analysis.
Tsuchiya, Ken, Gerhard Giebisch, and Paul A. Welling.
Department of Medicine Kidney Center, Tokyo Women's Medical
College, Tokyo, Japan, Department of Cellular and Molecular
Physiology, Yale University School of Medicine, New Haven, CT 06511,
Department of Physiology University of Maryland School of Medicine,
Baltimore MD
APStracts 3:0009F, 1996.
In the cortical collecting duct (CCD), aldosterone increases the
number of functionally active Na/K ATPase molecules by a mechanism
involving an isoform-specific increase in the abundance of the Na/K
ATPase alpha 1 and beta 1 subunit protein. However, the molecular
basis for the response, particularly in the mammalian CCD in vivo,
has remained unclear. To resolve this issue, reverse transcription
and a competitive polymerase chain reaction (PCR) were employed to
study mineralocorticoid-dependent regulation of alpha 1 and beta 1
subunit mRNA in the rat CCD. Na/K ATPase subunit-specific
oligonucleotides primers were used in the PCR to amplify reverse
transcribed subunit mRNA from single microdissected CCD. Control
templates were constructed (84 bp deletion mutation of the rat Na/K
alpha 1 subunit cDNA and 70bp deletion of the beta 1 subunit cDNA),
serially diluted and co-amplified with the wild-type Na/K ATPase
subunit RT- mRNA from single CCD. PCR products of predicted size were
observed by ethidium bromide staining. Southern blots with an
internal subunit specific oligonucleotide confirmed Na/K ATPase alpha
1 and beta 1 identity. The ratio of the amplified wild type to mutant
PCR products was found to be linear over the range of input control
cDNA tested so that the amount of subunit mRNA could be determined. A
chronic reduction in corticosteroid levels by bilateral adrenalectomy
(7 days) reduced the apparent level of alpha 1 subunit transcript by
54.0+6.3%, but not the beta 1 subunit. Administering aldosterone to
physiological levels is sufficient to restore CCD alpha 1 subunit
mRNA abundance toward control levels within 6 hours. In conclusion:
1) regulation of Na/K ATPase of CCD in vivo can be attributed, at
least in part, to mineralocorticoid-dependent control of Na/K ATPase
alpha 1 subunit mRNA abundance, 2) competitive PCR may provide a
sensitive and quantitative tool for determining hormone-dependent
regulation of mRNA abundance in nephron segments.
Received 26 August 1994; accepted in final form 16 November 1995.
APS Manuscript Number F307-4.
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