Quantitative evaluation of catecholamine enzymes gene expression in adrenal medulla and sympathetic Ganglia of stressed rats.
Stress-induced changes in mRNA levels of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) have been expressed as relative arbitrary units compared with a control group. The aim of this study was to quantify basal and stress-induced levels of TH, DBH, and PNMT mRNAs in rat adrenal medulla (AM) and stellate ganglia (SG) by the RT-competitive PCR method using corresponding competitors of known concentration. In rats stressed by immobilization (IMO) once for 2 h, the concentration of mRNAs was determined in various intervals after the end of stress stimulus. In SG, the basal concentration of TH mRNA was 0.017 amol/ng of total RNA, which is approximately 30 times lower than in the AM (0.460 amol/ng RNA). The basal concentration of DBH mRNA in SG was 2.60 amol/ng of total RNA, which is about 150 times more than TH mRNA in SG but only two times less than DBH mRNA in the AM in which PNMT mRNA is present in the highest concentration. After a single 2-h IMO, the peak elevation of TH and DBH mRNA concentration in SG occurred 24 h after the termination of stress stimulus, when their AM mRNA concentrations were already at control values. Presence of PNMT mRNA levels in the SG, of control and stressed rats has been demonstrated for the first time. Repeated IMO (7 days, 2 h daily) did not produce further increase in the mRNA concentrations compared with the elevated values found in adapted control groups. Levels of TH protein were significantly increased only after repeated IMO in SG and AM. Thus, our data show for the first time the exact concentrations of TH, DBH, and PNMT mRNA in SG and AM of rats under control and stress conditions. The lowest concentration of TH mRNA in the AM and SG supports the hypothesis that tyrosine hydroxylation is the rate-limiting step in catecholamine biosynthesis.
Kvetnansky
R
R
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovak Republic. ueenkvet@savba.sk
Micutkova
L
L
Rychkova
N
N
Kubovcakova
L
L
Mravec
B
B
Filipenko
M
M
Sabban
E L
EL
Krizanova
O
O
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004299:Dopamine beta-Hydroxylase / Q000235:genetics*; D005728:Ganglia, Sympathetic / Q000201:enzymology*; D015870:Gene Expression; D007103:Immobilization; D008297:Male; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013312:Stress, Physiological / Q000201:enzymology / Q000235:genetics*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; D014446:Tyrosine 3-Monooxygenase; D004299:Dopamine beta-Hydroxylase; D010625:Phenylethanolamine N-Methyltransferase