Comparative study of catecholamine synthesizing enzymes in adrenal medulla of CRH knock-out mice, their CRH (+/+) mates and Sprague-Dawley rats.

OBJECTIVE

Corticotropin-releasing hormone deficient mice (CRH-KO) serve as an interesting model to understand the role of CRH in the regulation of adrenomedullary system. The aim of this study was to compare tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) on the levels of gene expression and protein in adrenal medulla of CRH-KO mice, their CRH (+/+) mates and Sprague-Dawley (SD) rats.

METHODS

Levels of TH and PNMT mRNA were determined by reverse transcription with subsequent polymerase chain reaction (RT-PCR) and quantified relatively to the housekeeper glyceraldehyde-3-phosphate dehydrogenase. The amount of TH and PNMT protein was determined by Western blot analysis and visualized by enhanced chemiluminiscence.

RESULTS

We detected a clear signal of 645 bp for TH mRNA and of 260 bp for PNMT mRNA in adrenal medulla of rats and CRH (+/+) mice, with higher concentration of TH and PNMT mRNA in rat adrenal medulla. Subsequently, TH and PNMT immunoprotein was measured and we found significantly higher amount of TH and also PNMT protein in the rat compared to CRH (+/+) mice. On the other hand, the amount of TH and PNMT immunoprotein in adrenal medulla of CRH-KO mice was significantly lower compared to CRH (+/+) mice.

CONCLUSIONS

Our results indicate the lower production of adrenomedullary TH and PNMT protein in CRH (+/+) mice compared to rats, which reflects the lower gene expression of these enzymes in adrenal medulla of mice. We also demonstrated the differences in TH and PNMT protein levels between CRH (+/+) and CRH-KO (-/-) mice.