Aldosterone-dependent regulation of Na-K-ATPase subunit mRNA in the rat CCD: competitive PCR analysis.

Abstract

In the cortical collecting duct (CCD), aldosterone increases the number of functionally active Na-K-adenosin-etriphosphatase (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 (RT) 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 (RT-mRNA) from single microdissected CCD. Control templates were constructed (84-bp deletion mutation of the rat Na-K-ATPase alpha 1-subunit cDNA and 70-bp deletion of the beta 1-subunit cDNA), serially diluted, and coamplified 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-subunit 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 h. We conclude the following: 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; and 2) competitive PCR may provide a sensitive and quantitative tool for determining hormone-dependent regulation of mRNA abundance in nephron segments.