Increase In Proenkephalin mRNA Levels Research Articles

Unilateral 6-hydroxydopamine lesions of nigrostriatal dopaminergic neurons increased CB 1 receptor mRNA levels in the caudate-putamen

It has been recently suggested that the effects of cannabinoids on motor behavior might be different in rats with lesions of nigrostriatal dopaminergic neurons than in controls. In the present study, we examined the possible alteration in the status of cannabinoid CB1 receptors in the basal ganglia of rats with unilateral lesions of those neurons caused by 6-hydroxydopamine. We used two different experimental groups depending on the duration of the period of recovery after the lesion, and comparisons were done between the lesioned and nonlesioned sides at the level of the basal ganglia. Both groups of lesioned rats exhibited a similar marked reduction in tyrosine hydroxylase (TH)-mRNA levels, measured by in situ hybridization, in the substantia nigra of the lesioned side. In the same way, lesioned rats exhibited the characteristic rotational behavior after a single injection of apomorphine and the intensity of this rotation was stable at the two times analyzed after the lesion. Also as expected, lesioned rats exhibited an increase in proenkephalin mRNA levels in the caudate-putamen, whereas mRNA levels of substance P decreased, although differences between the two times of recovery analyzed were observed in this case. We did not find any significant changes in CB1 receptor binding, measured by [3H]WIN-55,212,2 autoradiography, or in the activation of signal transduction mechanisms, measured by WIN-55,212,2-stimulated [35S]GTPgammaS binding autoradiography, between the lesioned and nonlesioned sides at the level of the lateral caudate-putamen, globus pallidus and substantia nigra in both groups of lesioned rats. However, we found a significant increase in levels of CB1 receptor-mRNA transcripts, measured by in situ hybridization, in the lesioned side in both the lateral and medial caudate-putamen. This occurred 7-10 weeks after the lesion, but the increase was markedly waned after 17-18 weeks. In summary, the unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons originated a marked increase in CB1 receptor-mRNA levels in cell bodies of striatal efferent neurons, although accompanied by no changes in CB1 receptor binding and activation of signal transduction mechanisms. This supports a critical role for dopamine in the control of CB1 receptor gene expression. However, the magnitude of the effect significantly waned as a function of the duration of the period after lesion.

Long-term increase of Sp-1 transcription factors in the hippocampus after kainic acid treatment

Systemic administration of kainic acid (KA), a glutamate receptor agonist, causes robust seizures and has been used as an excellent rodent model for human temporal lobe epilepsy. Recently, we have demonstrated that a single injection of KA increases the steady-state levels of proenkephalin (PENK) mRNA in the rat hippocampus for at least one year. However, the molecular mechanisms underlying this long-term increase in PENK mRNA levels have not been clearly defined. To determine the possible involvement of the Sp-1 transcription factors in this regulation, electrophoresis mobility-shift assays were used to study the expression of Sp-1 factors in the hippocampus after KA treatment. The results showed that there are long-lasting increases in Sp-1 DNA-binding activity. The Sp-1 DNA-binding complexes were only competed by the non-radioactive Sp-1 element and not by ENKCRE2, AP-1 or CRE elements, indicating the specificity of Sp-1 DNA-binding activity. Since the expression of Sp-1 parallels the time course of long-lasting increase in the expression of PENK mRNA and mossy fiber sprouting after KA treatment, we hypothesize that the increase in Sp-1 activity may be associated with the long-term changes in the plasticity of hippocampal function after KA-induced seizures.

N-methyl-D-aspartate and nitric oxide regulate the expression of calcium/calmodulin-dependent kinase II in the hippocampal dentate gyrus.

Injection of small volumes of N-methyl-D-aspartate (NMDA) or Sin-1 molsidomine (a nitric oxide releasing agent) onto the dendrites of granule cells in the hippocampal dentate gyrus leads to changes in the level of expression of a number of genes. There is a fall in prodynorphin mRNA levels with a corresponding increase in proenkephalin mRNA levels. Similar changes in opioid gene expression occur following the induction of long-term potentiation (LTP). We report here that at short time periods (1-6 h) after injections of NMDA or sin-1 molsidomine, there is an increase in the levels of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha), consistent with a report of elevated CaMKII alpha mRNA in postsynaptic neurons in the CA1 region of the hippocampus following LTP induction [54]. However, we also report that 24 h after injection of NMDA or sin-1, there is a dramatic decrease in CaMKII alpha mRNA levels in the vicinity of the injection. This effect is specific for CaMKII alpha mRNA, in that many other mRNA species are not affected, and occurs in the dendritic population of CaMKII alpha mRNA as well as in the pool of mRNA in the granule cell bodies. The effect is blocked by an inhibitor of cGMP-dependent protein kinase. The biphasic regulation of CaMKII alpha mRNA may be of considerable functional importance for the long-term response of granule cells to local stimulation of NMDA receptors or NO release.

Induction of c- fos gene expression is not responsible for increased proenkephalin mRNA levels in the hippocampal dentate gyrus following NMDA stimulation

The increased levels of proenkephalin mRNA in the dentate gyrus following hippocampal stimulation have been assumed to be a consequence of the transient induction of c- fos. Injection of 50 μM NMDA in vivo onto the dendrites of a small number of granule cells causes a pronounced but highly localised elevation in proenkephalin mRNA levels 24 h later, whereas vehicle has no effect. In contrast, there is a widespread induction of c- fos mRNA throughout the dentate gyrus, 45 min after injection of either vehicle or NMDA, suggesting that induction of c- fos expression is unrelated to the subsequent, anatomically discrete, increase in proenkephalin mRNA levels.

Adrenal secretion of BAM-22P, a potent opioid peptide, is enhanced in rats with acute cholestasis.

The adrenal gland is known to produce and release endogenous opioids into the circulation. Bovine adrenal medulla docosapeptide (BAM-22P) is a potent opioid agonist, derived from the proenkephalin A gene, which is present in the adrenal medulla. This study was undertaken to determine whether BAM-22P is released into plasma during acute cholestatic liver injury, which increases plasma total opioid activity. Acute cholestasis was induced by bile duct ligation or administration of the hepatotoxin alpha-naphthylisothiocyanate. Plasma levels of BAM-22P were determined by a sensitive radioimmunoassay, and the specificity of the assay was confirmed using high-performance liquid chromatography. Plasma BAM-22P levels was cholestatic rats were significantly higher than those in control rats. This increase in plasma BAM-22P levels was completely prevented by adrenalectomy. Adrenal steady-state levels of proenkephalin mRNA, as determined by Northern blot hybridization analyses, were also increased significantly in cholestatic rats. These increases in proenkephalin mRNA levels were not paralleled by changes in adrenal BAM-22P peptide levels, which were similar in cholestatic rats and their respective controls. Similar levels of proenkephalin mRNA expression were observed in innervated and denervated adrenal glands from cholestatic rats, suggesting that the increase in adrenal proenkephalin mRNA levels in acute cholestasis is not due to splanchnic nerve activation. Thus acute cholestasis in the rat is associated with adrenal secretion and accumulation in plasma of the highly potent opioid peptide BAM-22P and an augmentation of adrenal proenkephalin mRNA expression. The increase in plasma BAM-22P levels may contribute substantially to the increase in total circulating opioid activity documented in cholestatic rats.

Differential dopaminergic regulation of proenkephalin and prodynorphin mRNAs in the basal ganglia of rats

Proenkephalin and prodynorphin mRNA levels in the caudate-putam en and in the nucleus accumbensof rats were measured by in situ hybridization 2,4 and 8 weeks following unilateral lesion of the medial forebrain bundle by 6-hydroxydopamine (6-OHDA). After 2 weeks a 60% increase of the levels of proenkephalin mRNA in the ipsilateral caudate-putamen was observed which declined to 20% above control after 8 weeks. A smaller increase in the levels of proenkephalin mRNA of about 20% was observed in the nucleus accumbens after 2 weeks and no significant alteeration could be observed 4 and 8 weeks after lesioning. The levels of prodynorphin mRNA in the ipsilateral caudate putamen decreased 20% below control and returned to control levels 4 and 8 weeks post-lesion. In contrast, in the nucleus accumbens a persistent ipsilateral decrease of prodynorphin (20–30%) was found 2, 4 and 8 weeks post-lesion. These findings indicate, that lesions of the mesostriatal dopamine (DA) system differentially influences opioidergic gene expression in distinct areas of the caudate-putamen. The lesions cause an increase in proenkephalin mRNA levels which was higher in the caudate-putamen than in the nucleus accumbens and tend to be reversible. Conversely, the lesion caused a persistent decrease in the levels of prodynorphin mRNA in the nucleus accumbens and a small and transient decrease in the caudate-putamen.

Proenkephalin Gene Expression in C6 Rat Glioma Cells: Potentiation of Cyclic Adenosine 3′,5′- Monophosphate-Dependent Transcription by Glucocorticoids

Glucocorticoids enhance proenkephalin gene expression in several cell types. To elucidate the mechanism(s) involved, we analyzed the potentiation by dexamethasone of the cAMP-dependent increase in proenkephalin mRNA levels elicited by forskolin in C6 rat glioma cells. This potentiation did not require ongoing protein synthesis. In nuclear run-on transcription assays, dexamethasone alone did not alter proenkephalin transcription, but strongly increased the magnitude and duration of transcriptional elevation by forskolin through a direct action not requiring ongoing protein synthesis. Dexamethasone did not alter basal or stimulated cAMP levels. To search for functionally cooperative glucocorticoid and cAMP regulatory elements, we transfected C6 cells with plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of rat proenkephalin sequences from bases -5800 to +703. Maximum stimulation of transiently expressed CAT activity by forskolin required more than 145 and 190 or fewer base pairs of 5'-flanking sequence, implicating sequences up-stream from the previously described cAMP-inducible enhancer. Dexamethasone reduced forskolin-stimulated CAT expression from plasmids with 190 or more base-pairs of 5'-flanking sequence, an effect apparently involving multiple up-stream regions. Dexamethasone also reduced forskolin-stimulated CAT mRNA levels in C6 cells stably transfected with proenkephalin/CAT chimeric genes in the presence or absence of proteins synthesis. In summary, we demonstrate that glucocorticoids and cAMP synergize positively in regulating transcription of the endogenous gene, but interact negatively in regulating the chimeric constructs, which may lack the context or distal element(s) required for positive synergism.

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.

Human Chorionic Gonadotropin Regulates Expression of the Proenkephalin Gene in Adult Rat Leydig Cells

The gene encoding the proenkephalin precursor is expressed in both progenitor spermatogenic cells and somatic cells in the rat testis. The 1750-nucleotide (nt) germ cell-specific proenkephalin mRNA is the predominant form detected in the adult rat. The 1400-nt somatic cell form has previously been shown to be expressed in cultured Sertoli cells derived from sexually immature rats, and FSH treatment increases expression of the proenkephalin gene in that experimental system. In the present report we demonstrate that the 1400-nt proenkephalin transcript is present in a freshly isolated, enriched preparation of adult rat Leydig cells. This gene continues to be expressed when the Leydig cells are maintained in primary culture. Treatment of cultured Leydig cells with hCG or a cAMP analog leads to a rapid increase in proenkephalin-mRNA levels. The expression of the proenkephalin gene throughout rat development in the two major somatic cell types of the testis further suggests that proenkephalin-derived peptides play an important role in modulating testicular function. The gene encoding another of the opioid peptide precursors, POMC, has previously been reported to be expressed in rat Leydig cells. We were unable to detect POMC transcripts in cultured Leydig cells. POMC mRNA is abundant in a germ cell-enriched cell fraction.

Estrogen Increases Proenkephalin Messenger Ribonucleic Acid Levels in the Ventromedial Hypothalamus of the Rat

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.

Multiple regulation of proenkephalin gene expression by protein kinase C.

In the present study we investigated the role of protein kinase C (Ca2+/phospholipid-dependent enzyme)-mediated processes in the regulation of proenkephalin gene expression in primary cultures of bovine adrenal chromaffin cells. Activators of protein kinase C such as 1-oleoyl-2-acetylglycerol, mezerein, and the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate induced a time-dependent increase in proenkephalin mRNA levels, whereas the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. The increase in phorbol ester-induced proenkephalin mRNA was potentiated by low concentrations of the Ca2+ ionophore A23187, suggesting an interaction between protein kinase- and Ca2+-mediated processes in the regulation of proenkephalin mRNA. The phorbol ester-induced stimulation does not appear to be mediated by an interaction with the cAMP-generating system or increases in Ca2+ uptake. However, when proenkephalin mRNA levels were stimulated by KCl (10 mM) and the dihydropyridine BayK8644, PMA exhibited an inhibitory effect on proenkephalin mRNA, which was detectable at a 10-fold lower concentration of PMA than the stimulatory effect. This inhibitory effect appears to be mediated by an inhibition of Ca2+ entry through voltage-dependent Ca2+ channels, as suggested by 45Ca2+ uptake experiments. Thus, the net effect of PMA depends on and varies with the state of voltage-dependent Ca2+ channel activity. A third mode of action by protein kinase C to modulate proenkephalin gene expression is by interaction with the phosphatidylinositol second messenger system. Stimulation of phosphoinositide hydrolysis and proenkephalin mRNA by histaminic H1-receptor activation was inhibited by low concentrations of PMA. We suggest that protein kinase C may act as a positive and negative regulator of proenkephalin gene expression by interacting with at least three receptor-coupled second messenger systems.

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.