DIFFERENTIATION OF THE EXPRESSION OF ALDOSTERONE SYNTHASE AND 11-BETA-HYDROXYLASE MESSENGER-RNA IN THE RAT ADRENAL-CORTEX BY REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION

The adrenocortical enzymes of the steroidogenic late pathway in the ra t 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 enz ymes are highly homologous and are encoded by the genes CYP11B2 and CY P11B1, respectively. The purpose of the present study is to describe t he development of two sets of primers and the reverse transcription-po lymerase chain reaction (RT-PCR) conditions that are capable of discri minating between rat P450aldo and P45011 beta mRNAs. The P450aldo prim er set did not amplify full length cDNA P45011 beta plasmid and the P4 5011 beta primer set did not amplify full length cDNA P450aldo plasmid indicating minimal crosstalk. The fidelity of the PCR primers and met hod was further established by sequencing the PCR products and demonst ration of virtual identity with the published sequences of P450aldo an d 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 similar to 8-fold greater expression in P450aldo mRNA on low vs high sodium intake. Similar results were found when single hemicapsules wer e subjected to RT-PCR, demonstrating the sensitivity of the method. We conclude that the two sets of PCR primers and the RT-PCR method descr ibed are capable of evaluating the expression of the highly homologous mRNAs for P450aldo and P45011 beta with great precision and sensitivi ty.