Rat brain preproenkephalin mRNA. cDNA cloning, primary structure, and distribution in the central nervous system

A cDNA-clone library was constructed from poly-adenylated RNA of Fischer rat striatum and screened for inserts coding for the enkephalin precursor preproenkephalin. The insert of one positive clone, pRPE2, was sequenced and found to contain the coding sequence (810 bases), as well as 316 and 155 bases of the 3' and 5' untranslated regions, respectively, of rat preproenkephalin mRNA. The primary structure of rat preproenkephalin (269 amino acids, Mr 30,932) is similar to those of previously sequenced bovine and human preproenkephalins (78 and 82% matched residues, respectively), and contains four copies of Met-enkephalin, one of Leu-enkephalin, one of Met-enkephalin-Arg6-Gly7-Leu8, and one of Met-enkephalin-Arg6-Phe7. Cell-free translation of rat striatal mRNA selected by hybridization with pRPE2 DNA resulted in the synthesis of a 31,000-Da protein. Southern analysis of rat genomic DNA with 32P-labeled pRPE2 fragments is consistent with a single preproenkephalin gene. A 32P-labeled pRPE2 fragment hybridized specifically with preproenkephalin mRNA (approximately 1500 bases) on Northern blots of polyadenylated or total RNA of all brain regions but not liver. Relative abundances of preproenkephalin mRNA in total RNA of specific regions of the rat central nervous system, determined by a sensitive dot-blot hybridization assay, had the following order: striatum much greater than hypothalamus, pons-medulla, spinal cord greater than cerebellum, midbrain, frontal cortex greater than hippocampus, thalamus. The pRPE2 probe is useful for the analysis of the dynamics of preproenkephalin mRNA in rat neurons.     

A new species of enkephalin precursor mRNA with a distinct 5'-untranslated region in haploid germ cells

To elucidate the primary structure of preproenkephalin (A) mRNA expressed by haploid germ cells (round spermatids) in rat testis, we have screened a lambda gt11 cDNA library for preproenkephalin cDNA inserts. The largest cDNA insert contained a protein-coding sequence encoding 269 amino acid residues as well as 327 and 309 bases of the 5'- and 3'-untranslated regions, respectively. The protein-coding region plus 3'-untranslated region of the mRNA was over 99% homologous to that of brain preproenkephalin mRNA, whereas the 5'-untranslated region contained a distinct sequence including a partial sequence of intron A of the preproenkephalin gene [(1984) J. Biol. Chem. 259, 14301-14308; (1984) J. Biol. Chem. 259, 14309-14313]. Northern blot analysis using a 5'-end-specific probe showed that this type of preproenkephalin mRNA exists exclusively in the germ cells.     

Preproenkephalin mRNA and Methionine-Enkephalin Content Are Increased in Mouse Striatum After Treatment with Nicotine

Abstract: A single dose of nicotine increased methionine-enkephalin (Met-Enk) immunoreactivity in the striatum of mice in a time-dependent manner. Met-Enk content reached a maximum by ∼1 h after nicotine and returned to control values by 6 h. The response to nicotine was blocked by pretreating animals with the nicotinic receptor antagonist mecamylamine. In contrast, pretreating mice with the muscarinic receptor antagonist atropine or the dopamine receptor antagonist haloperidol did not block the response. A single dose of nicotine also increased mRNA for the precursor peptide preproenkephalin (PPE). The increase of PPE mRNA preceded that of Met-Enk and reached a maximum by ∼30 min after nicotine. PPE mRNA levels returned to near normal by ∼3 h and increased again by 6 h after nicotine. Daily administration of nicotine for 14 days increased Met-Enk content and PPE mRNA in the striatum of mice as well. Taken together, our results suggest that nicotinic receptors modulate Met-Enk content and PPE mRNA in the mouse striatum.     

Modulation of striatal enkephalinergic neurons by antipsychotic drugs

In this paper we review the detailed mechanisms underlying the modulation of enkephalinergic neurons by dopaminergic neurons in rat striatum. Several lines of evidence, which showed that striatal levels of [Met5]enkephalin (ME) increase after the nigrostriatal dopaminergic pathway was interrupted by hemitransection or direct administration of 6-hydroxydopamine to the substantia nigra, or after repeated injections of either reserpine or haloperidol, suggest that dopamine (DA) plays an important role in regulating the metabolism of ME-containing neurons in the striatum. The increase in ME content after repeated injections of haloperidol was found in areas heavily innervated by DA neurons such as striatum or nucleus accumbens but not in hypothalamus, brain stem, and hippocampus. Further studies suggest that striatal cholinergic interneurons may partially mediate the action of haloperidol on enkephalinergic neurons. Several studies have been carried out to determine whether the elevation of striatal ME content after haloperidol treatment was caused by an increase in the synthesis or by a decrease in the utilization of ME. The rate of decline of striatal ME content in haloperidol-treated rats was steeper than that of controls after intraventricular injection of cycloheximide, which indicated that haloperidol accelerates the turnover of ME. This hypothesis was confirmed by our recent findings that the level of mRNA coding for preproenkephalin A, determined by cell-free translation and blot hybridization with cDNA clones, is increased after repeated injections of haloperidol.     

Enkephalin precursor gene expression in postmeiotic germ cells

Preproenkephalin mRNA was detected in rat testis after postnatal day 30. A high abundance of preproenkephalin mRNA was present in the spermatids (i.e., spermatogenic cells at postmeiotic stage) purified from mature rat testis. The mRNA size of the spermatids was about 1900 bases, which was larger than that of other tissues expressing the preproenkephalin gene. The spermatids contained small amounts of enkephalin-containing proteins, but little or no Met-enkephalin. Preproenkephalin gene expression in the germ cells suggests the connection between gametogenesis and nervous system.     

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.     

Expression of the enkephalin precursor gene in rat Sertoli cells. Regulation by follicle-stimulating hormone

Searching for somatic cells expressing the preproenkephalin (A) gene in the testis, we have isolated Sertoli cells from the testes of 20-day-old rats. Cultured Sertoli cells contained a single species (about 1.5 kb) of preproenkephalin mRNA, and follicle-stimulating hormone (FSH) transiently increased the mRNA abundance to a maximum (about 30 molecules per cell) at 12 h. Various compounds that activate the cyclic AMP system in Sertoli cells similarly increased the abundance of preproenkephalin mRNA. Moreover, FSH increased intracellular Met-enkephalin immunoreactive peptides in Sertoli cells. Thus, the preproenkephalin gene expression in Sertoli cells is positively regulated by FSH through the cyclic AMP system.     

Herpes Simplex Virus 1-Mediated Transfer of Preproenkephalin A in Rat Dorsal Root Ganglia

Abstract: Recombinant herpes simplex virus-1 encoding the rat preproenkephalin A (HSVLatEnk₁) was generated for driving the expression of preproenkephalin A-derived peptides in dorsal root ganglia of rats in vivo. Three weeks after infection via the hind footpads, quantitative RT-PCR and in situ hybridization experiments showed a strong expression of preproenkephalin A mRNA in lumbar dorsal root ganglia. In addition, a 40–160% increase in radioimmunoassayable Met-enkephalin-like material concentrations was found in the dorsal spinal cord and dorsal root ganglia, respectively, at the lumbar level in HSVLatEnk₁-infected rats as compared with animals infected with β-galactosidase-encoding recombinant herpes simplex virus-1 or control rats. These data demonstrate the efficacy of the preproenkephalin A encoding vector and suggest that it should help in elucidating the role of Met-enkephalin-containing primary afferent fibers in pain transmission and/or control.     

Dopamine-Dependent Postnatal Development of Enkephalin and Tachykinin Neurons of Rat Basal Ganglia

The influence of deprivation of the neurotransmitter dopamine (DA) on the development of [Met5]-enkephalin (ME) and substance P (SP) neuropeptide systems of the striatum was investigated in Sprague-Dawley rats. The neurotoxin 6-hydroxydopamine (6-OHDA) was used to induce DA deficiency on postnatal day 3 in rats, and the animals were killed at different postnatal time points until 35 days of age. The levels of ME and SP were determined by radioimmunoassay, and the abundance of preproenkephalin (PPE) and preprotachykinin (PPT) mRNA in the striatum was assessed by Northern blot hybridization analysis. The concentrations of DA, 5-hydroxytryptamine (5-HT), and their acid metabolites were determined by HPLC with electrochemical detection. The postnatal development of the PPE-derived peptide ME and the PPT-derived peptide SP closely paralleled the appearance of the respective mRNAs coding for these peptides. The dopaminergic lesion with 6-OHDA led to a marked depletion of DA and its metabolites but produced an increase in content of 5-HT and its metabolite in the striatum. The lesion did not affect the ME and PPE mRNA levels in the striatum up to 25 days but increased the levels at 35 days. In contrast, a decreased developmental expression in SP and PPT mRNA was observed throughout the observation period. The lesion failed to influence the development of the mRNA coding for the structural protein beta-actin. The results indicate that the normal development of enkephalin, tachykinin, and 5-HT systems of the striatum is dependent on the availability of DA, the integrity of dopaminergic neurons, or both. The studies provide evidence for an interrelationship and interdependence between the development of neurotransmitter and neuropeptide systems. It is suggested that an early developmental abnormality in the DA system could permanently alter the neuropeptide systems, which in turn could influence the progression and expression of the DA-deficiency state parkinsonism, Lesch-Nyhan disease, or both.     

Glucocorticoids and cyclic AMP synergistically regulate the abundance of preproenkephalin messenger RNA in neuroblastoma-glioma hybrid cells

Regulation of preproenkephalin gene expression was studied in NG108-15 neuroblastoma-glioma hybrid cells. Untreated cells contain 20-120 fg preproenkephalin mRNA per microgram cellular RNA. Treatment of cells with a glucocorticoid (e.g. dexamethasone) for 24 hr or 8 days elevated the abundance of this mRNA to 3 or 9 times the control, respectively. Treatment with 8-bromo-cyclic AMP or an adenylate cyclase activator such as prostaglandin E1 or forskolin elevated preproenkephalin mRNA to twice the control or less. Treatment with both glucocorticoid and forskolin for 24 hr or 8 days markedly increased preproenkephalin mRNA to 5-8 and 30 times the control, respectively. Intracellular Met-enkephalin immunoreactivity was increased in parallel with the mRNA abundance. The results demonstrate that preproenkephalin gene expression is synergistically regulated by glucocorticoids and cAMP.     

Differences in ME-LI and VIP-LI in discrete brain regions of seizure-naive and seizure-experienced El mice

In an attempt to elucidate the relationship between endogenous methionine-enkephalin (ME) and vasoactive intestinal polypeptide (VIP) with generalized seizures, we determined regional brain levels of ME-like and VIP-like immunoreactivity (ME-LI and VIP-LI) in El mice during and after seizures induced by repeated tossing stimulation. The levels of ME-LI in the striatum and hippocampus of seizure-naive El mice (El–) were lower than those of the control ddY mice, the mother strain of El mice. Conversely, the level of VIP-LI in the medulla oblongata and pons of El– was higher than that of ddY mice. The level of ME-LI in the striatum of seizure-experienced El mice (El+) killed 96 hours after three consecutive seizures was high, while levels of VIP-LI in the striatum and hypothalamus were low, in comparison to those of El– mice. A detailed time-course study revealed that seizures in El mice caused (1) significant decreases in levels of ME-LI in the striatum and hippocampus during seizures, (2) a significant decrease of VIP-LI content in the striatum 3 hours after seizures, and (3) a significant increase in hypothalamic VIP-LI 9 hours after seizures. These observations suggest that ME and VIP may play some role in El mouse seizures.     

Acute and Persistent Suppression of Preproenkephalin mRNA Expression in the Striatum Following Developmental Hypoxic-Ischemic Injury

Abstract: The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.     

Dopaminergic Regulation of Cannabinoid Receptor mRNA Levels in the Rat Caudate-Plitamen: An In Situ Hybridization Study

Abstract: By quantitative in situ hybridization, we examined in vivo in the rat caudate-putamen the effects on levels of cannabinoid receptor mRNA of an interruption of dopamine neurotransmission for up to 1 month, by either 6-hydroxydopamine lesioning of the medial forebrain bundle or dopamine receptor blockade. We found, in a first set of experiments, that unilateral 6-hydroxydopamine dopa-minergic deafferentation of the striatum (characterized by a contralateral turning behavior in response to apomor-phine, the almost complete disappearance of the tyrosine hydroxylase hybridization signal in the substantia nigra, and an increase of preproenkephalin A mRNA level in the striatum) was associated with significantly increased (45%) cannabinoid receptor mRNA levels in the homolateral caudate-putamen. In a second set of experiments, treatments with the dopamine D₁ receptor antagonist SCH-23390, haloperidol, and the D₂ receptor antagonist sulpiride induced significantly higher cannabinoid receptor mRNA levels (respectively, 67, 34, and 27%) in the caudate-putamen. These observations suggest for the first time that, in vivo, cannabinoid receptor gene expression in the caudate-putamen is under the negative control of dopamine receptor-mediated events.     

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .     

Cocaine and methamphetamine differentially affect opioid peptide mRNA expression in the striatum

In general, administration of methamphetamine and cocaine alters preprodynorphin and preproenkephalin mRNA levels in striatum. However, no study has directly compared the effects of these stimulants on opioid peptides in striatum. This study used in situ hybridization to compare directly the effects of cocaine and methamphetamine on preprodynorphin and preproenkephalin mRNAs in distinct striatal regions. Male Sprague-Dawley rats received a single administration of 15 mg/kg methamphetamine or 30 mg/kg cocaine and were killed 30 min or 3 h later. Methamphetamine and cocaine differentially affected preprodynorphin mRNA in striatum after 3 h. Densitometric analysis of film autoradiograms revealed that cocaine, but not methamphetamine, significantly increased preprodynorphin. This effect was seen throughout rostral striatum and dorsally in caudal striatum. However, specific analysis of "patches" in which preprodynorphin expression is high revealed a significantly greater effect of methamphetamine versus cocaine. In contrast, both cocaine and methamphetamine had similar effects on preproenkephalin mRNA, decreasing levels after 30 min in rostral striatum and in the core of nucleus accumbens. These data suggest that methamphetamine and cocaine have distinct postsynaptic consequences on striatal neurons.     

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.