OS 21-07 EXPRESSION OF CATECHOLAMINERGIC SYSTEM GENES IN ADRENAL MEDULLA OR SYMPATHETIC GANGLIA OF SPONTANEOUSLY HYPERTENSIVE RAT.

OBJECTIVE: Catecholaminergic system plays an important role in blood pressure regulation and hypertension development. The available information concerning mRNA expression of catecholaminergic system genes in spontaneously hypertensive rats (SHR) are often contradictory. This might be due to various reference genes used as internal controls. We therefore searched for suitable reference genes for gene expression profiling in adrenal medulla and sympathetic ganglia of SHR and Wistar-Kyoto (WKY) rats, which would enable reliable comparison of mRNA expression of genes of catecholaminergic system between these strains. Moreover, we performed cluster analysis to achieve complex understanding of the role of age and genotype for the expression of catecholaminergic system genes.

DESIGN AND METHOD: Expression of mRNA was measured by quantitative real-time PCR in adrenal medulla and superior cervical ganglia of 4-week-old or 24-week-old SHR and WKY rats. We focused on the expression of genes involved in catecholamine biosynthesis, formation of catecholaminergic vesicles and catecholamine removal from the synaptic cleft. We evaluated 12 reference genes by Normfinder software and compared them as internal controls.

RESULTS: The combination of reference genes Hprt1 and Ywhaz in adrenal medulla and Gapdh and 18S in sympathetic ganglia were chosen as the best ones. In both tissues most differences in mRNA expression were associated with catecholamine biosynthetic pathway which was downregulated in 4-week-old SHR. This attenuation partially persisted during aging in sympathetic ganglia and became even more pronounced in adrenal medulla. The expression of genes related to catecholaminergic vesicles was also attenuated during aging in adrenal medulla of SHR.

CONCLUSIONS: The use of various reference genes for standardization influenced seriously obtained results and their interpretation, indicating the importance of appropriate internal control. Decreased expression of genes of catecholamine biosynthetic and secretory pathway in SHR could be the consequence of feedback inhibition by increased circulating norepinephrine rather than the cause of hypertension in this strain. (GACR16-10349Y, GAUK9662/2014).