TY - JOUR
T1 - Aldosterone-dependent regulation of Na-K-ATPase subunit mRNA in the rat CCD
T2 - Competitive PCR analysis
AU - Tsuchiya, Ken
AU - Giebisch, Gerhaed
AU - Welling, Paul A.
PY - 1996
Y1 - 1996
N2 - 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 α1- and β1-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 α1- and β1-subunit mRNA in the rat CCD. Na-K-ATPase subunit-speciflc 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 cq-subunit cDNA and 70-bp deletion of the i-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 α1-and β1-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 α1-subunit transcript by 54.0 ± 6.3% but not the β1-subunit. Administering aldosterone to physiological levels is sufficient to restore CCD ai-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 α1-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.
AB - 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 α1- and β1-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 α1- and β1-subunit mRNA in the rat CCD. Na-K-ATPase subunit-speciflc 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 cq-subunit cDNA and 70-bp deletion of the i-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 α1-and β1-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 α1-subunit transcript by 54.0 ± 6.3% but not the β1-subunit. Administering aldosterone to physiological levels is sufficient to restore CCD ai-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 α1-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.
KW - Cortical collecting duct
KW - Kidney
KW - Polymerase chain reaction
KW - Sodium and potassium homeostasis
KW - Steroid hormone
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U2 - 10.1152/ajprenal.1996.271.1.f7
DO - 10.1152/ajprenal.1996.271.1.f7
M3 - Article
C2 - 8760237
AN - SCOPUS:0029775025
SN - 0002-9513
VL - 271
SP - F7-F15
JO - American Journal of Physiology
JF - American Journal of Physiology
IS - 1 PART 2
ER -