DISTRIBUTION OF ADRENOCORTICOID RECEPTORS IN THE RAT CNS MEASURED BY COMPETITIVE PCR AND CYTOSOLIC BINDING

Combined quantitative polymerase chain reaction (PCR) and cytosolic bi nding assay techniques are used to measure mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA, K-d, and B-max in various rat central nervous system (CNS) regions, namely amygdala, hypothalamu s, hippocampus, cortex, pituitary, and cervical, thoracic, and lumbar spinal cord. Two internal standards (i.s.) cDNA were cloned for quanti tative PCR purposes. The i.s. templates differed from the respective w ild-type (wt) templates for a single base-pair mutation introduced by PCR that generated a unique restriction site, thus allowing amplificat ion products arising from coamplification of sol and i.s. to be distin guished. Results show that cerebellum, which displayed average B-max v alues for both receptors, contained the highest level of MR and GR mRN A. Hippocampus also had a high level of MR mRNA. Low mRNA content was found in the hypothalamus for MR and GR as well as in the cortex for G R. High B-max values for both MR and GR were found in the lumbar spina l cord, despite a modest mRNA content. The lowest B-max values were fo und in the cortex for both receptors. It is, therefore, concluded that mRNA content and B-max are not closely correlated in the rat CNS. The se data suggest a differential regulation of various adrenocorticoid r eceptor isoforms. Moreover, this quantitative PCR method is very sensi tive and can be used to assay small amounts of material in order to ob tain absolute measurements of mRNA expression.