Proenkephalin Gene Research Articles

Comparison of synenkephalin and methionine enkephalin immunocytochemistry in rat brain

Immunocytochemistry using an antiserum to the C-terminal octapeptide of synenkephalin, proenkephalin(63–70), was performed throughout the rat brain and revealed numerous immunopositive fibers and some cell bodies. The morphology and distribution of synenkephalin immunoreactivity was extremely similar to that of a commercial methionine enkephalin (Met-ENK) antiserum. Colchicine pretreatment allowed the immunostaining of cell bodies not otherwise possible without pretreatment, but did not affect the distribution of immunoreactive fibers. Using 6 μm serial sections, we were able to colocalize synenkephalin and Met-ENK immunoreactivities in gigantocellular neurons of the medullary reticular formation. Preabsorption of the antiserum with [Tyr 63]proenkephalin(63–70) octapeptide (YEESHLLA) completely eliminated immunoreactivity in the rat brain, while preabsorption with all other peptides used had no detectable effect. We conclude that our antiserum to synenkephalin is specific for enkephalinergic cell bodies, fibers and terminals. The synenkephalin antiserum used in these studies may have advantages over other antisera utilized for immunocytochemical detection of proenkephalin gene expression.

Developmental expression of proenkephalin mRNA and peptides in rat striatum

The development of proenkephalin (PE) gene expression in the rat striatum was examined at the mRNA and peptide levels. Immunocytochemistry was performed with antisera generated to the PE-specific peptide product Met-enkephalin-Arg-Gly-Leu (MERGL). The distribution of immunostaining was compared with the distribution of PE mRNA, determined by in situ hybridization with an oligonucleotide probe. PE mRNA first appeared at E16 in the caudal ventrolateral striatum, followed at E17–18 by the appearance of MERGL immunoreactivity in a similar distribution. The anatomical gradients of PE gene expression were similar to the pattern of histogenesis of striatal neurons, suggesting that the timing of PE gene expression is related to the time of neuronal withdrawal from the mitotic cycle. The relation of the development of PE gene expression to the known patterns of striatal histogenesis, neurochemical compartmentalization and dopaminergic innervation is discussed.

Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium.

Proenkephalin mRNA has previously been shown to be expressed in the rodent uterus with varying levels during the estrous cycle. To examine for the potential regulation of proenkephalin gene expression by steroid hormones in a primate displaying a menstrual cycle and to define the functional tissue within the uterus expressing this transcript, we have used Northern blot analysis of extracted RNA from isolated uterine tissue subtypes from normal adult rhesus macaques obtained during the menstrual cycle and from ovariectomized females under different physiological steroid hormone treatments. A strong band of proenkephalin mRNA of 1.3 kilobases was detected almost exclusively in the proliferative endometrium from monkeys in the follicular phase of the cycle. No proenkephalin mRNA was detected in secretory endometrium obtained from monkeys in the luteal phase. When ovariectomized macaques were implanted with silastic capsules of 17 beta-estradiol, proenkephalin mRNA was detected in the endometrium but not the myometrium of the estradiol-treated animals. No proenkephalin mRNA was detected in ovariectomized control animals. Under these conditions, we were unable to detect proenkephalin mRNA in ovariectomized macaques implanted with separate silastic capsules of 17 beta-estradiol and progesterone or in decidual tissue from early or late pregnancy. These results suggest that in the primate uterus 1) proenkephalin mRNA is expressed primarily in the endometrium of the uterus, 2) expression of the proenkephalin gene is regulated by 17 beta-estradiol in the endometrium, and 3) this effect of estradiol is antagonized by progesterone.

Localization of striatal opioid gene expression, and its modulation by the mesostriatal dopamine pathway: An in situ hybridization study

In situ hybridization was used to study the macroscopic distribution and regulatory control of proenkephalin mRNA and prodynorphin mRNA in rat striatum. While proenkephalin mRNA was widely distributed throughout the striatum, levels of prodynorphin mRNA were highest in the medial and ventral portions of the striatum. Furthermore, in contrast to the results for proenkephalin mRNA, the levels of prodynorphin mRNA appeared higher in the nucleus accumbens than in the striatum. The mesostriatal dopaminergic pathway was destroyed by discrete, unilateral injection of 6-hydroxy-dopamine (6-OHDA) into either the substantia nigra or the neighboring ventral tegmental area (VTA). Lesions of the substantia nigra caused a dramatic ipsilateral increase in the hybridization signal for proenkephalin mRNA, but no change was observed in the hybridization signal for prodynorphin mRNA. Similar effects were seen with VTA lesions. Since destruction of the mesostriatal dopamine system elevates the levels of proenkephalin mRNA, but not of prodynorphin mRNA, in the striatal target neurons, it appears that the mesostriatal pathway exerts a tonic and selective suppression of striatal proenkephalin gene expression at the mRNA level.

Expression and Regulation of the Proenkephalin Gene in Rat Sertoli Cells

Previous studies have shown that multiple forms of proenkephalin RNA are present in the rat and mouse testis that are cell type specific: a 1700-nucleotide transcript, unique to the testis, found in spermatogenic cells, and a 1450-nucleotide form of somatic cell origin. In the present studies, rat Sertoli cells are shown to contain the 1450-nucleotide form of proenkephalin RNA. The concentration of this transcript is quite low immediately after Sertoli cell isolation, but increases after 1 day in culture to a constant level. Proenkephalin RNA levels are stimulated severalfold by FSH and cAMP, but are not significantly affected by LH or testosterone. These studies demonstrate that Sertoli cells are at least one source of the somatic, 1450-nucleotide form of proenkephalin RNA in the rat testis. The expression of opioid peptide genes by Sertoli cells as well as by germ and Leydig cells is further evidence for the postulated autocrine or paracrine functions of these peptides in the testis.

Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: temporal qualities and synergism with progesterone

Estrogen has been shown to increase proenkephalin (PE) mRNA levels in neurons of the ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM). In this series of experiments, we examined the temporal qualities of this induction by determining both the latency of the estrogen-induced elevation in PE mRNA levels and the rate at which the message levels decline following removal of estrogen. In addition we have examined the effects of progesterone on PE gene expression in the VL-VM of estrogen-primed rats. The latency of the estrogen-induced elevation in PE mRNA levels was found to be relatively short: PE mRNA levels were increased 2-fold within 1 h of estrogen replacement. Following estrogen removal the levels of PE mRNA declined rapidly. Progesterone treatment attenuated this decline, prolonging the estrogen-induced elevation of PE mRNA levels. These results suggest that estrogen rapidly increases PE mRNA levels through a mechanism that probably involves alterations in both the rate of appearance and the rate of degradation of the message. Together, the short latency of the estrogen-induced elevation and the rapid rate of decay following estrogen removal indicate that PE gene expression is highly sensitive to fluctuating estrogen levels. The effect of progesterone suggests that this enkephalinergic system may be regulated by both estrogen and progesterone during the estrous cycle.

An AP-2 Element Acts Synergistically with the Cyclic AMP- and Phorbol Ester-Inducible Enhancer of the Human Proenkephalin Gene

An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP- and phorbol ester-inducible transcription of the human proenkephalin gene. We report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

An AP-2 element acts synergistically with the cyclic AMP- and phorbol ester-inducible enhancer of the human proenkephalin gene.

An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP- and phorbol ester-inducible transcription of the human proenkephalin gene. We report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

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.

Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription.

Synthesis of the endogenous opioid precursor, proenkephalin, is regulated by neurotransmitters and membrane depolarization. These events act through second messenger dependent signal transduction pathways via a short inducible DNA enhancer to regulate transcription of the proenkephalin gene. Two DNA elements located within this enhancer are essential for the transcriptional response to cAMP and phorbol ester. Inactivation of either element by mutation or by alteration of their stereospecific alignment eliminates inducible enhancer activity. The promoter distal element, ENKCRE-1, in the absence of a functional adjacent ENKCRE-2 element, has no inherent capacity to activate transcription. However, in the presence of a functional ENKCRE-2 element, this element synergistically augments cAMP and phorbol ester inducible transcription. The promoter proximal element, ENKCRE-2, is essential for both basal and regulated enhancer function. Four different protein factors found in HeLa cell nuclear extracts bind in vitro to the enhancer region. ENKTF-1, a novel enhancer binding protein, binds to the DNA region encompassing ENKCRE-1. The transcription factors AP-1 and AP-4 bind to overlapping sites spanning ENKCRE-2, and a fourth transcription factor, AP-2, binds to a site immediately downstream of ENKCRE-2. The binding of ENKTF-1 to mutant ENKCRE-1 sequences in vitro correlates with the in vivo inducibility of the mutant elements suggesting that ENKTF-1 acts in combination with factors that recognize the ENKCRE-2 domain to regulate cAMP inducible transcription. Together, the two DNA elements, ENKCRE-1 and ENKCRE-2 and the protein factors with which they interact, play a critical role in the transduction and reception of signals transmitted from cell surface receptors to the proenkephalin nuclear transcription complex.

Gonadal Steroids Regulate Proenkephalin Gene Expression in a Tissue-Specific Manner within the Female Reproductive System

Proenkephalin gene expression undergoes marked changes within the female reproductive system of rodents during the estrous cycle and in pregnancy. In order to define the factors responsible for this regulation, the effects of 17-beta-estradiol (E2) and progesterone (P4) have been examined in the ovary and uterus. In the ovary of the rat and hamster, E2 and P4 were without effect on proenkephalin RNA levels when injected individually. However, P4 increased ovarian transcript abundance 2- to 3-fold after pretreatment of animals with E2. In the uterus of either species, E2 had little effect but P4 alone stimulated both proenkephalin RNA abundance and total content severalfold. Glucocorticoids and androgen reproduced this stimulatory effect on proenkephalin transcript levels. The interaction between E2 and P4 on proenkephalin gene expression in the uterus varied with species. In the rat, E2 inhibited stimulation by P4, while in the hamster uterus the two hormones had a synergistic effect, producing a 15-fold elevation of proenkephalin RNA abundance and a 50-fold increase in total uterine content. These distinct steroid responses appear to account for tissue- and species-related differences in the variation of proenkephalin gene expression during the estrous cycle in the rodent ovary and uterus. The stimulatory effect of P4 was shown to involve direct steroid action on the uterus and to be inhibited both by the steroid antagonist RU-486 and the transcriptional inhibitor actinomycin D. These data are consistent with receptor-mediated activation of proenkephalin gene transcription in uterine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of striatal prodynorphin mRNA levels by the raphe-striatal pathway

Peptides derived from proenkephalin and prodynorphin are known to be located in striatal projection neurones. Anatomical studies have shown the presence of a major serotonergic projection from the dorsal raphe nucleus to the striatum, although the function of this pathway is unknown. We have used in situ nucleic acid hybridization to investigate a possible role of this serotonergic innervation in the regulation of striatal opioid gene expression. The levels of proenkephalin mRNA and prodynorphin mRNA were determined in rat striatum and nucleus accumbens. Destruction of the dorsal raphe nucleus by microinjection of 5,7-dihydroxytryptamine failed to affect the levels of proenkephalin mRNA in any region. However, the levels of prodynorphin mRNA were significantly reduced in the medial nucleus accumbens and caudomedial striatum. This implies that activity in the raphe-striatal serotonin pathway mediates a tonic and selective enhancement of prodynorphin gene expression in the striatal target cells. This is in contrast to the tonic suppression of proenkephalin gene expression mediated by the mesostriatal dopamine pathway.

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla.

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.

Expression of the proenkephalin gene in human neuroblastoma cell lines

Several human tumour cell lines were screened for secretion of proenkephalin-derived peptides with an antiserum directed to its N-terminus, Met-enkephalin-Arg 6,Phe 7 and for proopiomelanocortin-derived peptides with an antiserum to β-endorphin. The neuroblastoma SK-N-MC cell line secreted Met-enkephalin-Arg 6,Phe 7-immunoreactive peptides in relatively high amounts into the culture medium, although processing was not complete and there was no evidence for free Met-enkephalin-Arg 6,Phe 7. Gene expression was confirmed by the presence of proenkephalin mRNA and proenkephalin-derived polypeptides in extracts of the SK-N-MC cells and also in the neuroblastoma SH-SY5Y cell line. In the latter cells, however, the expression was approximately 3 times lower, there was less processing of proenkephalin and no evidence for secretion.

Expression of opioid genes in bovine seminal vesicles.

In seminal vesicles, the organ producing most of seminal plasma in the bovine species, the pro-opiomelanocortin and the proenkephalin genes are transcribed and translated, and their translation products processed into opioid peptides, which are secreted into the seminal plasma. By using a micro-organ preparation of seminal vesicles we found that, after 20 h of incubation with labelled methionine, a multiplicity of opioids was produced. Among these, [Met]enkephalin and beta-endorphin were positively identified, whereas in the newly formed secretion only [Met]enkephalin was detected. This may be correlated to the finding that the concentration of beta-endorphin in an extract of seminal plasma was one order of magnitude lower than that of [Leu]enkephalin and [Met]enkephalin. These findings expand the picture of the presence of opioid peptides in the male reproductive tract, indicating that they should have a role(s) in the physiology of reproduction, not only in the hypothalamus-pituitary-gonadal axis, determining the reproductive potential, but also in the so-termed sex accessory glands, determining the actual events leading to reproduction. To our knowledge this is also the first case studied of opioid peptides produced as exocrine hormones.

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.

Proenkephalin gene expression in the PC12 pheochromocytoma cell line: stimulation by sodium butyrate.

The differentiation promoter sodium butyrate increases the content of Met5-enkephalin-Arg6-Gly7-Leu8 (Met5-enk-RGL)-immunoreactive peptides in PC12 pheochromocytoma cells, which, unlike mature adrenomedullary chromaffin cells, contain exceedingly low levels of opioid peptides. These butyrate-induced enkephalin-immunoreactive peptides, which are specific products of the proenkephalin gene, consist principally of two high mol wt forms of amino-terminally extended Met5-enk-RGL. These high mol wt peptides, with apparent mol wt of 20,000 and 10,000, are approximately the same size as the two major immunoreactive peptides found in adult New England Deaconess Hospital rat adrenal. The low mol wt Met5-enk-RGL-immunoreactive peptide found in butyrate-treated cells is similar in size to authentic Met5-enk-RGL, which is not found in the adrenal medulla of the adult rat. When PC12 cells are grown as a tumor in vivo, the amount of Met5-enk-RGL-immunoreactive peptide increased only slightly above the level found in control cells grown in vitro and consisted exclusively of the highest mol wt immunoreactive species. In PC12 cells, the butyrate-stimulated elevation in the content of Met5-enk-RGL-immunoreactive peptides may involve changes in transcription, since the peptide increase is preceded by a 2- to 3-fold increase in the level of proenkephalin mRNA. These results suggest that the PC12 cell line may be useful for investigating those factors that control the initial expression and processing of proenkephalin-derived peptides during embryogenesis.

Differential expression of opioid peptide genes by testicular germ cells and somatic cells.

Spermatogenic cells have been previously shown to be a major site of testicular proenkephalin gene expression. Using RNA gel-blot analysis of purified mouse and hamster germ cells and of testes from prepuberal and germ cell-deficient mutant mice, we now have demonstrated that, in addition to its previously described expression by somatic (Leydig) cells, the gene for a second opioid peptide precursor, pro-opiomelanocortin (POMC), is also expressed by spermatogenic cells. Of particular significance is the finding that the RNAs for proenkephalin and POMC are differentially regulated during spermatogenesis. Two forms of POMC RNA were detected in mouse testis, a larger component 675- to 750-nucleotides (nt) in size common to somatic and spermatogenic cells and a smaller 625-nt RNA found only in pachytene spermatocytes. Two distinct, cell-specific proenkephalin RNAs were also shown to be present in mouse testis: a 1700-nt transcript previously shown to be expressed by spermatogenic cells and a 1450-nt form associated with somatic cells. These data suggest that proenkephalin- and POMC-derived peptides are produced by both somatic cells and germ cells in the testis and in germ cells these two families of opioid peptides may function at different stages of spermatogenesis.

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.

Chapter 2 Use of gene transfer approaches to study regulation of neuropeptide gene expression

This chapter discusses gene transfer techniques to study the regulation and expression of the human proenkephalin gene and several other opioid genes. A mouse anterior pituitary tumor-derived corticotrophic cell line has been transfected with the human proenkephalin gene and the characteristics of several of the isolated clones are discussed in the chapter. It also discusses the expression of proenkephalin in three transient systems: (1) proenkephalin fusion genes, (2) Xenopus oocytes, and (3) vaccinia virus. Gene transfer experiments can be divided into two major categories—namely, stable transfections and transient assay systems. To study stably transfected cell lines, a clone of eukaryotic cells that has integrated the foreign gene into its chromosomal DNA is isolated. The major advantage of this system is the ability to isolate stable cell lines, which can be grown indefinitely in culture and used to study a variety of regulatory phenomena. The gene of interest can be introduced into eukaryotic cells by several different methods, including (a) chemical methods, such as the calcium phosphate precipitation method, (b) physical methods, such as microinjection or electroporation, (c) viral mediated DNA transfer, and (d) the fusion of DNA-containing membranous vesicles, such as liposomes, the envelopes of Sendai virus particles, or protoplasts to cells.