Differentiation of the expression of aldosterone synthase and 11 beta-hydroxylase mRNA in the rat adrenal cortex by reverse transcriptase-polymerase chain reaction.
The adrenocortical enzymes of the steroidogenic late pathway in the rat are aldosterone synthase (P450aldo), which catalyzes the production of aldosterone, and 11 beta-hydroxylase (P45011 beta), which catalyzes the production of corticosterone throughout the cortex. These two enzymes are highly homologous and are encoded by the genes CYP11B2 and CYP11B1, respectively. The purpose of the present study is to describe the development of two sets of primers and the reverse transcription-polymerase chain reaction (RT-PCR) conditions that are capable of discriminating between rat P450aldo and P45011 beta mRNAs. The P450aldo primer set did not amplify full length cDNA P45011 beta plasmid and the P45011 beta primer set did not amplify full length cDNA P450aldo plasmid indicating minimal crosstalk. The fidelity of the PCR primers and method was further established by sequencing the PCR products and demonstration of virtual identity with the published sequences of P450aldo and P45011 beta. RT-PCR of mRNA from adrenal capsules (zona glomerulosa) and subcapsules (zona reticularis/fasciculata) from rats demonstrated no effect of sodium diet on the expression of P45011 beta mRNA but an approximately 8-fold greater expresison in P450aldo mRNA on low vs high sodium intake. Similar results were found when single hemicapsules were subjected to RT-PCR, demonstrating the sensitivity of the method. We conclude that the two sets of PCR primers and the RT-PCR method described are capable of evaluating the expression of the highly homologous mRNAs for P450aldo and P45011 beta with great precision and sensitivity.
Oaks
M K
MK
Immunology and Endocrine Research Laboratories, St Luke's Medical Center, Milwaukee, WI 53215, USA.
Raff
H
H
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D019405:Cytochrome P-450 CYP11B2; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012982:Sodium, Dietary / Q000008:administration & dosage; D013252:Steroid 11-beta-Hydroxylase / Q000096:biosynthesis*
D012333:RNA, Messenger; D012982:Sodium, Dietary; D003577:Cytochrome P-450 Enzyme System; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase