preproenkephalin and Hemorrhage

The ventrolateral medulla (VLM) has three functionally defined regions that contain catecholamine-synthesising neurons (rostral C1, caudal C1 and A1 regions). Many neuromessengers can alter cardiovascular functions in the VLM. The aims of this study were, first to validate the utility of real-time RT-PCR SYBR Green assay for quantitation of mRNA expression levels of neuromessengers in small site-specific neuronal populations in the VLM, and second to compare the basal mRNA levels of the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY) and preproenkephalin (ENK) in the three regions and third to examine the effects of haemorrhage on the expression of these three genes. Rats were anaesthetised with sodium pentobarbital and divided into three groups: perfused, sham-operated and haemorrhaged. A 15% haemorrhage was carried out on the haemorrhaged group. It was found that there are regional differences in the level of mRNA expression for all the three genes: with, in general, decreases from the rostral to caudal regions of VLM. A 15% haemorrhage significantly induced expression of PNMT in the rostral C1 region and NPY in the caudal C1 and A1 regions but had no effect on ENK at any sites, suggesting a differential regulation on the expression of these three genes in the VLM. Our results also demonstrate that real-time RT-PCR is a sensitive and accurate method for quantitative studies on neurotransmitter gene expressions in restricted brain regions.

Topics: Animals; Benzothiazoles; Blood Pressure; Diamines; Enkephalins; Fluorescent Dyes; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Hemorrhage; Male; Medulla Oblongata; Neuropeptide Y; Organic Chemicals; Phenylethanolamine N-Methyltransferase; Protein Precursors; Quinolines; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

In the present study, we examined the expression of the gene encoding for the production of the opioid peptide enkephalin in specific brain regions during hemorrhage in the conscious rat. Rats were subjected to fixed-volume (8 ml/300 g) bleeding via jugular vein within 10 min. Total RNA was isolated from rat brain tissue using the hot phenol/chloroform method. Preproenkephalin transcript levels were quantified using Northern blot hybridization of total RNA with alpha-32P-labeled cDNA probe, and autoradiograms were scanned with a densitometer. The ppENK transcript in midbrain was significantly elevated following one and two hours of hemorrhage compared to control group (P < 0.01). The ppENK transcript in spinal cord was also significantly increased after two hours of hemorrhage, when compared with control group (P < 0.01). In the brainstem, ppENK transcript was significantly decreased after both 1 and 2 hr of hemorrhage compared with control values (P < 0.01). These results indicate that hemorrhage acutely changes levels of the transcript of ppENK in specific brain regions. It is possible that the changes in brainstem ppENK transcript may be a mechanism by which certain inhibitory effects of enkephalins on important brain cardiovascular regulatory centers are mediated.

Topics: Animals; Brain Chemistry; Brain Stem; Disease Models, Animal; Enkephalins; Gene Expression Regulation; Hemorrhage; Male; Mesencephalon; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord