Proenkephalin mRNA Levels Research Articles

Perinatal Δ9-tetrahydrocannabinol exposure reduces proenkephalin gene expression in the caudate-putamen of adult female rats

Perinatal Δ 9-tetrahydrocannabinol ( Δ 9-THC) exposure in rats affects several behavioral responses, such as opiate self-administration behavior or pain sensitivity, that can be directly related to changes in opioidergic neurotransmission. In addition, we have recently reported that the administration of naloxone to animals perinatally exposed to Δ 9-THC produced withdrawal responses, that resemble those observed in opiate-dependent rats. The purpose of the present study was to examine the basal opioid activity in the brain of adult male and female rats that had been perinatally exposed to Δ 9-THC. To this aim, proenkephalin mRNA levels were measured, by using in situ hybridization histochemistry, in the caudate-putamen, nucleus accumbens, central amygdala and prefrontal cingulate cortex. The results showed a marked reduction in proenkephalin mRNA levels in the caudate-putamen of Δ 9-THC-exposed females as compared to oil-exposed females, whereas no changes were observed between Δ 9-THC- and oil-exposed males. There were no differences in proenkephalin mRNA levels in the nucleus accumbens, central amygdala and prefrontal cingulate cortex between males and females perinatally exposed to Δ 9-THC and their respective controls, although a certain trend to decrease was observed in Δ 9-THC-exposed females. In summary, perinatal exposure to Δ 9-THC exposure decreased proenkephalin gene expression in the caudate-putamen of adult rats, although this effect exhibited a marked sexual dimorphism since it was only seen in females. This result is in agreement with a previous observation from our laboratory that females, but not males, that had been perinatally exposed to Δ 9-THC, self-administered more morphine in adulthood. This suggests that low levels of proenkephalin mRNA may be used as a predictor of greater vulnerability to opiates.

Chronic administration of cannabinoids regulates proenkephalin mRNA levels in selected regions of the rat brain

This study was designed to examine the interactions between the cannabinoid and enkephalinergic systems in the rat brain. To this aim, we have examined the effects of subchronic (5 days) administration (10 mg·kg −1·day −1; i.p.) of Δ 9-tetrahydrocannabinol (THC) or R-methanandamide (AM356) and chronic (18 days) administration with the synthetic cannabinoid receptor agonist CP-55,940 (1 mg·kg −1·day −1; i.p.) on proenkephalin (PENK) mRNA levels in several brain regions of the rat. Twenty micrometer brain sections from striatum, nucleus accumbens, paraventricular nucleus, ventromedial nucleus, periaqueductal grey matter and mammillary nucleus were hybridized with an oligonucleotide probe complementary to PENK using in situ hybridization technique. Subchronic administration of THC or AM356 increased PENK mRNA levels in the ventromedial nucleus of the hypothalamus, (82%) and (39%), in the periaqueductal grey matter, (97%) and (49%), and mammillary nucleus, (43%) and (9%), respectively. In contrast, both drugs were without effect in the striatum and nucleus accumbens. On the other hand, chronic administration of CP-55,940 increased PENK mRNA levels in the striatum (44%), nucleus accumbens (25%), paraventricular (31%) and ventromedial nuclei of the hypothalamus (41%). These results revealed that chronic cannabinoid administration increases opioid gene expression in the rat central nervous system and suggest an interaction between the cannabinoid and enkephalinergic systems that may be part of a molecular integrative response to behavioral and neurochemical alterations that occur in cannabinoid drug abuse.

RU-486 blocks stress-induced enhancement of proenkephalin gene expression in the paraventricular nucleus of rat hypothalamus

The purpose of this study was to investigate the glucocorticoid (GC) mediated regulation of corticotropin-releasing hormone (CRH) and proenkephalin (PE) gene expressions in the paraventricular nucleus (PVN) of the hypothalamus during physical stress induced by a single intraperitoneal (i.p.) injection of hypertonic saline (9% NaCl). Previous intracerebroventricular (i.c.v.) administration of the type II glucocorticoid receptor (GR) antagonist RU-486 (20 ng/rat), increased the basal CRH mRNA levels in the PVN but had no effect on PE gene expression. Stress induced by injection of hypertonic saline increased both CRH and PE mRNA levels in PVN. Administration of RU-486 completely blocked the stress-induced increase of PE mRNA levels, but failed to alter the CRH mRNA levels in the PNV. These data suggests that, under these experimental conditions, endogenous GC are necessary for a normal PE response to hypertonic saline stress.

The stimulation of rat astrocytes with phorbol-12-myristate-13-acetate increases the proenkephalin mRNA: involvement of proto-oncogenes

The effect of phorbol-12-myristate-13-acetate (PMA) on the regulation of proenkephalin (proENK) mRNA level, ENKCRE-2 or AP-1 DNA binding activity, and the mRNA and protein levels of proto-oncogenes (c- fos, fra-1, and c- jun) in primary cultured rat astrocytes were studied. The proENK mRNA level was elevated at 4 h after the treatment of PMA (2.5 μM) without altering the intracellular proENK protein level, and this increase was attenuated by pre-treatment with cycloheximide (CHX; 15 μM), a protein synthesis inhibitor. Both AP-1 and ENKCRE-2 DNA binding activities were markedly increased at 1–4 h by PMA treatment and these PMA-induced responses were inhibited by pre-treatment with CHX, showing that the increase of proENK mRNA level was well correlated with the AP-1 and ENKCRE-2 DNA binding activities. In contrast, although the phospho-CREBP level was also increased by PMA at 0.5–1 h, the pre-treatment with CHX further increased the PMA-induced phospho-CREBP level. In addition, PMA caused the induction of c- fos, c- jun and fra-1 mRNA level and, especially, PMA-induced increase of fra-1 mRNA level was further enhanced by CHX treatment at 4 h. Furthermore, western immunoblot assay showed that PMA caused induction of c-Fos, Fra-1, and c-Jun protein levels. PMA-induced increases of proto-oncoproteins levels were also inhibited by CHX treatment. The results suggest that newly synthesized AP-1 proteins, such as c-Fos, Fra-1, and c-Jun may play important roles in the regulation of PMA-induced proENK gene expression in cultured rat astrocytes. Phospho-CREB protein appears not to be involved in the regulation of PMA-induced proENK gene expression.

Ethanol withdrawal enhances the prodynorphin system activity in the rat nucleus accumbens

The present study investigated the effects of ethanol withdrawal after its chronic administration on endogenous opioid systems in the nucleus accumbens of rats. An in situ hybridization study showed an increase in the prodynorphin mRNA level at 24 and 48 h (by 189 and 146%, respectively) after ethanol withdrawal, whereas the proenkephalin mRNA level remained unchanged. Furthermore, after a 48 h withdrawal period, the level of α-neoendorphin ( αNEO), a prodynorphin-derived peptide, was significantly decreased (by 48%), that effect being associated with the enhancement of the K +-stimulated release of that peptide from nucleus accumbens slices. At 96 h after ethanol withdrawal, only the basal release of αNEO was elevated, while other parameters returned to the control level. Our data indicate that after 48 h of ethanol withdrawal, prodynorphin neurons are highly activated. The increased supply of endogenous κ opioid receptor agonists in the nucleus accumbens at that time may promote aversive states during ethanol withdrawal.

Glutamate metabotropic receptor agonist 1 S,3 R-ACPD induces internucleosomal DNA fragmentation and cell death in rat striatum

Glutamate metabotropic receptor mediated mechanisms have been implicated in both neuroprotection and neurotoxicity. To characterize these mechanisms further in vivo, the effects of an intrastriatally injected metabotropic receptor agonist, trans-(1 S,3 R)-1-amino-1,3-cyclopentanedicarboxylic acid (1 S,3 R-ACPD), were studied alone and together with N-methyl- d-aspartate (NMDA) or kainic acid (KA) receptor agonists on DNA fragmentation and nerve cell death. 1 S,3 R-ACPD induced internucleosomal DNA fragmentation of striatal cells in a dose-dependent manner. TUNEL and propidium iodide staining showed DNA fragmentation and profound nuclear condensation around the injection site. Fragmented nuclei were occasionally seen under light microscopy. Internucleosomal DNA fragmentation induced by 1 S,3 R-ACPD was attenuated by the protein synthesis inhibitor cycloheximide as well as by the non-selective and selective metabotropic receptor antagonists l-(+)-2-amino-3-phosphonopionic acid ( l-AP3), ( R S)-aminoindan-1,5-dicarboxylic acid and ( R S)-α-methylserine- o-phosphate monophenyl ester, respectively. The 1 S,3 R-ACPD (100–900 nmol) induced death of striatal neurons was suggested by the reduction in NMDA and D 1 dopamine receptors by up to 13% ( P<0.05) and 20% ( P<0.05) as well as by the decline in GAD 67 mRNA (25%, P<0.01) and proenkephalin mRNA levels (35%, P<0.01). Interestingly, 1 S,3 R-ACPD attenuated internucleosomal DNA fragmentation induced by NMDA, but potentiated that induced by KA. These results suggest that metabotropic receptor stimulation leads to the death of striatal neurons by a mechanism having the biochemical stigmata of apoptosis. Moreover, metabotropic receptor stimulation evidently exerts opposite effects on pre- or postsynaptic mechanisms contributing to the NMDA and KA-induced apoptotic-like death of these neurons.

Lactation decreases mRNA levels of opioid peptides in the arcuate nucleus of the rat

The state of lactation results in increased food intake to compensate for the increased energy expenditure to produce nutrients supplied to the offspring. In this study, Sprague-Dawley female rats lactating for 10–16 days, and rats 7 days post-lactation were implanted with osmotic minipumps infusing either naltrexone (NTX) (70 μg/h) or saline (0.9%) over a 48 h period. mRNA levels of pro-dynorphin (proDYN), pro-opiomelanocortin (POMC) and pro-enkephalin (proENK) were measured in the arcuate nucleus (ARC) and whole pituitary of both groups. In both saline- and NTX-treated lactating subjects, food intake was higher than in post-lactating subjects ( P<0.01). In post-lactating subjects, NTX decreased food intake by 27% during the infusion period ( P<0.05). There were no significant differences in body weight between the treatment groups; however, naltrexone decreased body weight gain in both lactating and post-lactating subjects. In both saline and NTX-treated lactating subjects, ARC mRNA levels of proDYN, POMC and proENK were significantly decreased compared with the saline or NTX-treated post-lactating subjects ( P<0.01). NTX did not significantly influence gene expression of opioid peptides in the ARC in either the lactating or the post-lactating subjects. Neither the lactation condition nor NTX administration significantly changed mRNA levels of proDYN, POMC or proENK in whole pituitary. Thus, as has been noted in energy-deprived rats, opioid peptide gene expression is decreased in the ARC of lactating rats, a period during which rats have increased energy requirements.

Alterations in the estrogen sensitivity of hypothalamic proenkephalin mRNA expression with age and prenatal exposure to alcohol

Studies suggest that exposure to alcohol in utero causes reproductive and neuroendocrine deficits in adult female rats. The ventromedial nucleus of the hypothalamus (VMN) is an estrogen-sensitive brain region which is regarded as a primary locus for modulating female reproduction. Proenkephalin (PE) mRNA expression in the VMN is dramatically increased by estrogen and this elevation is thought to be involved in modulating female reproductive behavior and neuroendocrine function. To examine whether prenatal alcohol exposure has long-term effects on the ability of estrogen to influence hypothalamic PE mRNA levels, female rats at 2–3, 6–7 or 15–18 months of age, derived from alcohol- or control-fed dams, were studied. 7 days following ovariectomy, animals received either estrogen or sham treatment for 2 days prior to sacrifice. PE mRNA levels in the VMN and striatum were determined by in situ hybridization histochemistry. Film autoradiogram density, numbers of PE mRNA-expressing cells and exposed silver grains/cell were analyzed. Estrogen treatment increased hybridization density, the number of PE mRNA-expressing cells and PE mRNA (grains) level/cell in the VMN of normal adult female rats. In old rats, estrogen increased the number of PE mRNA-expressing cells without up-regulating PE mRNA grain density/cell. In fetal alcohol-exposed (FAE) female rats, the number of cells that expressed PE mRNA did not increase following estrogen treatment at any age. Elevation of grain density/cell following estrogen was observed in FAE animals but only at 7–8 months of age. Overall, these data indicate that the estrogen responsiveness of PE mRNA expression in the VMN declines with age and, furthermore, prenatal exposure to alcohol blunts estrogen's effects on PE mRNA expression in the adult VMN. These finding may help to explain the mechanisms underlying the loss of reproductive function observed in FAE females.

Long-term potentiation in perforant path/granule cell synapses is associated with a post-synaptic induction of proenkephalin gene expression

Enkephalin peptides released from hippocampal mossy fibres lower the threshold for the generation of long-term potentiation (LTP) at the mossy fibre synapses. High frequency stimulation of the hippocampal dentate gyrus, sufficient to induce mossy fibre LTP, is associated with increased expression of the proenkephalin gene in the granule cells. We show here that a similar elevation in proenkephalin mRNA levels is observed, in anaesthetised rats, following stimulation of the perforant path sufficient to induce LTP in the perforant path/granule cell synapses. This strengthens the evidence implicating granule cell enkephalins as mediators of functional plasticity in the hippocampus. Furthermore, the results hint at a form of `domino plasticity', where potentiation of transmission at the perforant path/granule cell synapses is subsequently followed by an enkephalin-mediated potentiation of transmission at the mossy fibre synapses.

Dextromethorphan blocks opioid peptide gene expression in the rat hippocampus induced by kainic acid

We have previously shown that dextromethorphan (DM) antagonizes kainic acid (KA)-induced neurotoxicity. Accumulating evidence indicates that the induction of seizure activity causes profound alterations in the levels of hippocampal opioid peptide mRNA. The present study was performed to further explore the effect of DM on KA-induced seizures as measured by hippocampal opioid peptide mRNA levels. Both Northern blot and in situ hybridization methods were used to examine the proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels in the rat hippocampus. The robust seizure activity induced by KA correlated with a significant increase in hippocampal opioid peptide mRNA levels. Pretreatment of rats with DM decreased hippocampal PENK and PDYN mRNA levels and seizure activity induced by KA. Hippocampal PDYN mRNA levels fell quickly but PENK mRNA levels fell rather slowly, indicating that the PENK and PDYN mRNAs are differentially regulated. Our results demonstrate that DM modulates opioid peptide gene expression induced by KA, and that DM protects against KA-induced seizures.

Effects of single and repeated morphine administration on the prodynorphin, proenkephalin and dopamine D2 receptor gene expression in the mouse brain.

Effects of single (20 mg/kg i.p.) and repeated morphine administration (increasing doses: from 10 to 50 mg/kg i.p. twice daily for 7 days) on the proenkephalin (PENK), prodynorphin (PDYN) and dopamine D2 receptor (D2) mRNA levels in the nucleus accumbens and striatum of the mouse were investigated. As shown by an in situ hybridization, a single dose of morphine had no significant effect on the PDYN, PENK and D2 mRNA levels in the nucleus accumbens and striatum. Repeated treatment with morphine increased the PDYN mRNA level in both those structures after 2 and 72 h. In contrast to PDYN, the PENK mRNA level was reduced in the nucleus accumbens and remained unchanged in the striatum following repeated morphine administration. Repeated morphine had no effect on the D2 mRNA level in the nucleus accumbens and striatum after 2 h, and decreased it in the nucleus accumbens after 72 h only. The above results indicate that repeated morphine leads to long-lasting upregulation of the PDYN gene expression in the mouse nucleus accumbens and striatum; on the other hand, the PENK and D2 mRNA gene expressions are either inhibited or remain unchanged, significant changes being observed in the nucleus accumbens only.

Proenkephalin gene expression in the brainstem regulates post-exercise hypotension.

The opioid receptor antagonist naloxone reverses the reduction in blood pressure following exercise. We have previously demonstrated that compared to genetically matched controls, spontaneously hypertensive rats (SHR) have decreased proenkephalin mRNA levels in the nucleus tractus solitarius (NTS), the caudal (CVLM) and rostral ventrolateral medulla (RVLM) of the brainstem. We hypothesized that in SHR an acute bout of exercise would increase proenkephalin mRNA in the NTS and RVLM. Female 12-week-old SHR (n = 4/group) were randomly assigned to exercise and control groups. Mean arterial pressure (MAP) and heart rate were recorded at rest and every 5 min for 30 min following: (1) 40 min of treadmill running at 30 m/min, 10% grade; or (2) 40 min of rest on the treadmill. Rats were sacrificed 30 min post-exercise or post-rest. Exercise induced increases in MAP and heart rate, approximately 18 mmHg and approximately 140 beats/min, respectively, P < 0.001. There were no differences in pre-exercise/rest MAP between groups, or in control rats following rest on the treadmill, 162.5 +/- 3 vs. 163.1 +/- 4 mmHg, control and control after treadmill rest, respectively; NS P > 0.05. The pre- to post-exercise reduction in MAP after 40 min of treadmill running was from 164 +/- 5.1 to 146 +/- 2.0 mmHg (P < 0.001) as recorded 30 min post-exercise. At 30 min post-exercise proenkephalin mRNA levels in the NTS, CVLM and RVLM were increased: 97, 198 and 227%, respectively, P < 0.01. These data reconfirm the existence of post-exercise hypotension in SHR and suggest that increases in enkephalin synthesis and release in the NTS, CVLM, and RVLM may be involved in regulating post-exercise hypotension.

Regulation of the expression of the proenkephalin gene in cultured meningeal fibroblasts: opposite effects of alpha 1- and beta 2-adrenoceptors.

Meningeal fibroblasts express the proenkephalin gene during embryonal development but terminate the expression shortly before birth. When brought into primary culture at postnatal day 1, the fibroblasts again express the gene. Activation of protein kinase A reduces this expression and thus may contribute to its prenatal termination. Since the noradrenergic innervation of the meninges begins around the time of birth, it was investigated in the present study, how adrenergic agonists affected the levels of proenkephalin mRNA in cultured fibroblasts. The beta 2-adrenoceptor agonists salbutamol and procaterol increased the levels of endogenous cAMP and diminished the concentration of proenkephalin mRNA indicating that the cultured fibroblasts possessed this beta-subtype. In contrast, noradrenaline increased the level of proenkephalin mRNA in a concentration-dependent manner. This effect was independent of endogenous cAMP and was mediated by alpha 1-adrenoceptors. The data indicate that the noradrenergic innervation of the meninges at the time of birth is not responsible for the termination of the proenkephalin gene expression.

Molecular mechanisms underlying the regulation of proenkephalin gene expression in cultured spinal cord cells.

The regulation of proenkephalin (proENK) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells in the present study. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12 or 24 h and total RNA and proteins were isolated for Northern and Western blot analyses. The proENK mRNA level began to increase within an hour, then reached and remained at a peak 3-12 h after stimulation by both forskolin and PMA. The increased proENK mRNA level in forskolin-treated cells was slightly decreased 24 h after the stimulation, whereas the level of proENK mRNA returned to basal levels in PMA-treated cells. A Western blot assay revealed that the intracellular level of proENK protein was not changed by treatment with either forskolin or PMA. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase of proENK mRNA. However, pretreatment with nimodipine (an L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (an N-type Ca2+ channel blocker; 1 microM), calmidazolium (a calmodulin antagonist; 1 microM) or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) attenuated the forskolin- or PMA-induced increase of proENK mRNA levels. Dexamethasone (1 microM) did not affect the forskolin-induced increase of proENK mRNA levels. Our results suggest that the elevation of proENK mRNA levels in the spinal cord is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proENK mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced alterations in proENK mRNA.

Co-expression of steroid hormone receptors in opioid peptide-containing neurons correlates with patterns of gene expression during the estrous cycle.

The anteroventral periventricular nucleus (AVPV) of the preoptic region represents an essential component of neural pathways regulating gonadotropin secretion, and contains sexually dimorphic populations of neurons that express dynorphin or enkephalin. In the present study we used in situ hybridization to measure prodynorphin (PDYN) and proenkephalin (PENK) mRNA in the AVPV of intact animals killed on each day of the cycle. Levels of PDYN mRNA were lowest in animals killed on the afternoon of proestrus and then increased by over 60% by the morning of the following day. Expression of PENK mRNA was generally stable during the cycle, but a small yet significant reduction was detected on proestrus relative to levels of PENK mRNA in animals killed on the day of diestrus. In addition, we used double in situ hybridization to demonstrate that the majority of PDYN mRNA-containing neurons express both estrogen (50%) and progesterone receptor (85%) mRNAs. Only one quarter of the PENK-containing neurons also co-express estrogen receptor mRNA, and fewer than 10% of the PENK mRNA neurons express PR mRNA. Thus, the differential expression of PDYN and PENK during the cycle generally correlates with distinct differences in the degree of colocalization of ER and PR mRNA in PDYN and PENK mRNA-containing neurons in the AVPV.

Molecular characterization of immune derived proenkephalin mRNA and the involvement of the adrenergic system in its expression in rat lymphoid cells

Proenkephalin (PENK), a classically defined opioid gene, was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine systems. In the last few years, it was demonstrated, however, that significant levels of PENK mRNA and PENK-derived peptides are transiently expressed in cells of the immune system. Very little is known about the molecular mechanisms regulating this transient expression. In order to investigate those mechanisms, we examined the in vivo expression of PENK mRNA in mesenteric lymph nodes after exposing rats to lipopolysaccharide. In the present study we demonstrate that: (1) promoter usage and splicing of PENK mRNA function similarly in mesenteric lymph nodes as in neural cells; (2) PENK expression in mesenteric lymph nodes is modulated by adrenaline via adrenergic receptors; and (3) the adrenergic system participates in the modulation of the LPS induced PENK mRNA expression. These results provide more evidence for the involvement of opioids in neuro-immune interactions.

Palatability-induced hyperphagia increases hypothalamic Dynorphin peptide and mRNA levels

Opioid involvement in regulating the intake of highly palatable diets was studied by examining the effect of feeding either a cornstarch-based diet (CHO) or a high fat diet containing sucrose (Fat/Sucrose) on hypothalamic opioid levels. Rats received either CHO ad libitum, Fat/Sucrose ad libitum, Fat/Sucrose pair-fed to the caloric intake of CHO, or Fat/Sucrose at 60% of ad libitum Fat/Sucrose intake. Animals receiving Fat/Sucrose ad libitum consumed more calories and gained more weight than animals receiving CHO ( P < 0.001). Relative to CHO, ad libitum intake of Fat/Sucrose elevated proDynorphin mRNA levels in the arcuate and Dynorphin A 1–17 levels in the paraventricular nucleus (PVN) ( P < 0.05), but did not affect arcuate mRNA levels of proEnkephalin or proOpiomelanocortin (POMC), or PVN levels of Met-Enkephalin or β-Endorphin. Pair-feeding the Fat/Sucrose diet to the level of intake of the CHO diet resulted in levels of proDynorphin and Dynorphin A 1–17 that were similar in the two diet groups. Pair-feeding Fat/Sucrose reduced mRNA levels of proDynorpin, proEnkephalin and POMC, and Dynorphin A 1–17 levels, relative to ad libitum feeding of Fat/Sucrose. Met-Enkephalin and β-Endorphin were not affected by dietary treatment. Feeding Fat/Sucrose at 60% of ad libitum intake resulted in mRNA levels of proDynorphin, proEnkephalin and POMC, and Dynorphin A 1–17 levels that were similar to those observed in CHO group. Hypothalamic Dynorphin A 1–17 and proDynorphin mRNA levels are stimulated by feeding a highly palatable diet rich in fat and sucrose. The increased synthesis may be due in part to a palatability-induced overconsumption of calories. Caloric restriction of the same diet decreases mRNA levels of proDynorphin, proEnkephalin and POMC, as well as levels of Dynorphin A 1–17.

Effects of morphine on forskolin-stimulated pro-enkephalin mRNA levels in rat striatum: a model for acute and chronic opioid actions in brain.

Opioid agonists inhibit adenylyl cyclase in brain through Gi-coupled receptors. One potential biological role for this reaction would be to decrease pro-enkephalin mRNA synthesis by decreasing intracellular cAMP levels and preventing stimulation of gene expression via the cAMP regulatory element (CRE). To determine whether such effects occur in vivo, rats were injected i.c.v. with a water-soluble analog of forskolin, 7-beta-deacetyl-7 beta[gamma-(morpholino-butyryl] butyryl] forskolin (DMB-forskolin), to stimulate the CRE. Pro-enkephalin mRNA levels were assayed in striatum by Northern blot analysis. The treatment with the forskolin analog increased striatal pro-enkephalin mRNA levels approximately 2-fold in 4 h. When rats were injected with morphine (20 mg/kg i.p.) 1 h before DMB-forskolin administration, the stimulation of pro-enkephalin mRNA levels was eliminated. This acute effect of morphine was blocked by co-administration with 10 mg/kg naloxone. When rats were chronically treated with morphine for 8 days, then injected with morphine (20 mg/kg i.p.) 1 h before DMB-forskolin administration, the inhibitory effect of morphine was lost (i.e. DMB-forskolin increased pro-enkephalin mRNA levels by 2 fold in either control and morphine-treated rats). These data not only demonstrate the in vivo relevance of opioid-inhibited adenylyl cyclase in the control of pro-enkephalin mRNA levels, but also show that this model is useful for studying how this signal transduction system is attenuated during the development of tolerance.

Rat meningeal fibroblasts in primary culture express the proenkephalin gene

When brought into primary culture, rat meningeal fibroblasts contained proenkephalin-mRNA detected with Northern blot hybridization. In contrast, splenic fibroblasts did not express the gene under the same culture conditions. In situ hybridization showed that the meningeal fibroblasts did not uniformly express the gene: groups of positive cells were surrounded by cells with low or no proenkephalin-mRNA. Some fibroblasts which contained the mRNA species took up bromo-deoxyuridine indicating that the expression of the gene also occurred in proliferating cells, but was not restricted to this group. In chromaffin and astroglial cells, activation of protein kinase A or C with 8 Br.cAMP or O-tetradecanoyl 13-phorbolacetate, respectively, increases the expression of the gene. In meningeal fibroblasts, however, both agents reduced the levels of proenkephalin-mRNA.In the case of 8Br.cAMP, this effect was blocked by the protein synthesis inhibitor cycloheximide indicating that a newly-synthesized protein was involved. Cultured meningeal fibroblasts appear to be useful for studies on the cell specificity of the expression of this peptide gene as well on its regulation.

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.