ALDOSTERONE-DEPENDENT REGULATION OF NA-K-ATPASE SUBUNIT MESSENGER-RNAIN THE RAT CCD - COMPETITIVE PCR ANALYSIS

In the cortical collecting duet (CCD), aldosterone increases the numbe r of functionally active Na-K-adenosinetriphosphatase (Na-K-ATPase) mo lecules by a mechanism involving an isoform-specific increase in the a bundance of the Na-K-ATPase (alpha(1)- and beta(1)-subunit protein. Ho wever, the molecular basis for the response, particularly in the mamma lian CCD in vivo, has remained unclear. To resolve this issue, reverse transcription (RT) and a competitive polymerase chain reaction (PCR) were employed to study mineralocorticold-dependent regulation of alpha (1)- and beta(1)-subunit mRNA in the rat CCD. Na-K-ATPase subunit-spec ific oligonucleotides primers were used in the PCR to amplify reverse- transcribed subunit mRNA (RT-mRNA) from single microdissected CCD. Con trol 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)-subu nit cDNA), serially diluted, and coamplified with the wild-type Na-K-A TPase subunit RT-mRNA from single CCD. PCR products of predicted size were observed by ethidium bromide staining. Southern blots with an int ernal subunit-specific oligonucleotide confirmed Na-K-ATPase aland bet a(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 cD NA tested so that the amount of subunit mRNA could be determined. A ch ronic reduction in corticosteroid levels by bilateral adrenalectomy (7 days) reduced the apparent level of beta(1)-subunit transcript by 54. 0 +/- 6.3% but not the beta(1)-subunit. Administering aldosterone to p hysiological 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 le ast in part, to mineralocorticoid-dependent control of Na-K-ATPase alp ha(1)-subunit mRNA abundance; and 2) competitive PCR may provide a sen sitive and quantitative tool for determining hormone-dependent regulat ion of mRNA abundance in nephron segments.