Expression of preproenkephalin A gene and presence of Met-enkephalin in dorsal root ganglia of the adult rat.
The expression of the preproenkephalin A gene was investigated in adult rat dorsal root ganglia (DRG). A radioimmunoassayable Met-enkephalin (ME)-like material was detected in 0.1 M HCl extracts of rat DRG, representing approximately 60 pg of ME equivalents/mg of protein. Chromatographic analyses indicated that the major component of the ME-like material coeluted with authentic ME. In northern blot experiments on total RNA extracted from DRG, a cDNA probe corresponding to the entire coding region of rat preproenkephalin A mRNA yielded a single band of the expected size for this mRNA, i.e., 1.5 kb. Polymerase chain reaction (PCR) experiments were carried out with DRG, striatum, and liver cDNAs using two primers flanking the 1,371-1,771 base region of the preproenkephalin A gene. Thirty PCR cycles performed on both striatum and DRG cDNAs generated a single band of 400 bp, as expected, whereas only trace amounts of this product were detectable using liver cDNAs. Nucleotide sequencing of the PCR product obtained with DRG cDNAs revealed a 100% homology with the 1,371-1,771 sequence of the preproenkephalin A gene. In situ hybridization with a cRNA probe showed that about 3.5% of DRG cells expressed the preproenkephalin A transcript. However, most of these cells probably did not process proenkephalin to enkephalins, as thorough immunohistochemical investigations with anti-ME antibodies allowed the detection of only one in approximately 6,000 cells (in 30 sections of DRG) that exhibited ME-like immunoreactivity. Cells expressing preproenkephalin A mRNA were intermediate-sized neurons, suggesting that primary afferent ME-containing fibers belong to the A category and may participate in a local (spinal) inhibitory control of nociception.
Pohl
M
M
INSERM U 288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Collin
E
E
Bourgoin
S
S
Conrath
M
M
Benoliel
J J
JJ
Nevo
I
I
Hamon
M
M
Giraud
P
P
Cesselin
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015740:Calcitonin Gene-Related Peptide / Q000032:analysis / Q000096:biosynthesis; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000032:analysis / Q000096:biosynthesis*; D004745:Enkephalins / Q000032:analysis / Q000096:biosynthesis*; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011498:Protein Precursors / Q000032:analysis / Q000096:biosynthesis*; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004745:Enkephalins; D011498:Protein Precursors; D004744:Enkephalin, Methionine; C033660:preproenkephalin; D015740:Calcitonin Gene-Related Peptide