Proenkephalin mRNA Levels Research Articles

Loss of mRNA levels, binding and activation of GTP-binding proteins for cannabinoid CB 1 receptors in the basal ganglia of a transgenic model of Huntington’s disease

Data obtained from the basal ganglia of postmortem Huntington’s disease (HD) brains have revealed that the level of cannabinoid CB 1 receptors in striatal efferent neurons decreases in parallel to the dysfunction and subsequent degeneration of these neurons. These findings, and others from rat models of HD generated by lesions with mitochondrial toxins, suggest that the loss of CB 1 receptors may be involved in the pathogenesis of the disease. To explore further the changes in the endocannabinoid system, as well as the potential of endocannabinoid-related compounds, we examined the status of CB 1 receptors in the HD94 transgenic mouse model of HD. These mice express huntingtin exon 1 with a polyglutamine tract of 94 repeats in a tissue-specific and conditional manner using the tet regulatable system. They develop many features of HD, such as striatal atrophy, intraneuronal aggregates and progressive dystonia. In these animals, we analyzed mRNA levels for the CB 1 receptor, in addition to the number of specific binding sites and the activation of GTP-binding proteins by CB 1 receptor agonists. mRNA transcripts of the CB 1 receptor were significantly decreased in the caudate-putamen of HD transgenic mice compared to age-matched littermate controls. The decrease concurred with a marked reduction in receptor density in both the caudate-putamen and its projection areas such as the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. Furthermore, the efficacy of CB 1 receptor activation was reduced in the globus pallidus, as determined by agonist-induced [ 35S]GTPγS binding, and tended towards a decrease in the substantia nigra. None of these changes was seen in the cerebral cortex and hippocampus, despite high levels of expression of the mutant protein in these regions. The decrease in CB 1 receptor levels was accompanied by a decrease in the proenkephalin-mRNA levels but not in substance P-mRNA levels. Taken together, these results suggest that the loss of CB 1 receptor might be preferential to the enkephalinergic CB 1 receptor-containing striatopallidal neurons, and further implicate the CB 1 receptor to the subsequent HD symptomatology and neuropathology.

Changes in prodynorphin and POMC gene expression in several brain regions of rat fetuses prenatally exposed to Delta(9)-tetrahydrocannabinol.

Recently, we demonstrated that prenatal Delta(9)-tetrahydrocannabinol (Delta(9)-THC) exposure alters proenkephalin mRNA levels in several brain regions of rat fetuses. In the present study, we analyzed mRNA levels of the two other opioid peptide precursors, prodynorphin and pro-opiomelanocortin (POMC), in several brain nuclei of rat fetuses which were exposed daily to Delta(9)-THC from day 5 of gestation. Prenatal Delta(9)-THC exposure altered POMC and prodynorphin mRNA levels in most of the brain areas studied at different fetal ages, but the effects were sex-dependent. Thus, POMC mRNA levels increased in Delta(9)-THC-exposed females, but decreased in Delta(9)-THC-exposed males at GD21 in the arcuate nucleus, cerebral cortex and habenular nuclei. POMC mRNA levels also increased in the arcuate nucleus and cerebral cortex of Delta(9)-THC-exposed fetuses at GD18. Prodynorphin mRNA levels were not altered by the prenatal Delta(9)-THC exposure in the striatum, cerebral cortex, hippocampus and hypothalamic structures of fetuses at GD16 and GD18, but a sexually dimorphic response was observed at GD21. Thus, prodynorphin mRNA levels increased in the cerebral cortex, hippocampus and paraventricular hypothalamic nucleus of Delta(9)-THC-exposed females, whereas no changes were observed in Delta(9)-THC-exposed males. In summary, Delta(9)-THC exposure altered the prenatal development of POMC and prodynorphin mRNA levels in several brain structures. Changes in POMC were similar to those reported previously for proenkephalin, increases in females but decreases in males, whereas changes in prodynorphin were only observed in females.

Extinction of Cocaine Self-Administration Produces a Differential Time-Related Regulation of Proenkephalin Gene Expression in Rat Brain

The purpose of this study was to examine the time course effects of extinction of cocaine self-administration behavior on proenkephalin (PENK) gene expression in caudate-putamen nucleus (ST), nucleus accumbens (Acc), olfactory tubercle (Tu), piriform cortex (Pir), ventromedial hypothalamic nucleus (VMN), and central amygdala (Ce) as measured by in situ hybridization histochemistry. Seventy-two littermate male Lewis rats were randomly assigned in triads to one of three conditions: (1) contingent intravenous self-administration of 1 mg/kg/injection of cocaine (CONT); (2) noncontingent injections of either 1 mg/kg/injection of cocaine (NONCONT); or (3) saline yoked (SALINE) to the intake of the self-administering subject. The self-administering rats were trained to self-administer cocaine under a FR5 schedule of reinforcement for a minimum of 3 weeks. After stable baseline levels of drug intake had been reached, saline was substituted for drug. Following this first extinction period, cocaine self-administration was reinstated for an additional period of 2 weeks. Immediately after cessation of the last session of cocaine self-administration (day 0) and 1- , 5- , and 10-day after the second extinction period, animal brains in each triad were removed to be processed for in situ hybridization. PENK mRNA levels were significantly higher in the cocaine groups when compared with SALINE group in the ST, Acc, Pir, and Tu regions on days 0, 1, 5, and 10 of the extinction and lower in the Ce region of CONT group when compared to NONCONT and SALINE groups on days 1, 5, and 10 of the extinction period. In the VMN nucleus, PENK mRNA content in CONT group versus NONCONT and SALINE groups was also lower, but there were statistically significant differences only on day 5. These results suggest that changes in PENK gene expression after contingent cocaine administration might be involved in cocaine withdrawal states.

The comparative analysis of proenkephalin mRNA expression induced by cholera toxin and pertussis toxin in primary cultured rat cortical astrocytes

In rat astrocytes, incubation with cholera toxin (CTX; 0.1 μg/ml) for 8 h increased proenkephalin (proENK) mRNA level (10-fold), which was further increased by dexamethasone (DEX; 1 μM) (2.2-fold as much as CTX alone). Although pertussis toxin (PTX; 0.1 μg/ml) did not affect the basal proENK mRNA level, DEX significantly increased proENK mRNA level in PTX-treated cells (6-fold). The inhibition of protein synthesis by cycloheximide (CHX; 15 μM) also increased proENK mRNA level in PTX-treated cells (5.2-fold), but not in CTX-stimulated cells. The treatment with CTX, but not PTX, increased c-Fos and Fra-2 protein levels as well as AP-1, CRE, or ENKCRE-2 DNA binding activity, but neither toxin affected Fra-1, c-Jun, JunB, and JunD protein levels. CHX significantly attenuated CTX-induced increase of c-Fos or Fra-2 protein level and AP-1, CRE, or ENKCRE-2 DNA binding activity, although CHX alone did not affect the basal AP-1, CRE, and ENKCRE-2 DNA binding activities. Phosphorylated CREB level was increased by both CTX and PTX, although the magnitude of phosphorylation of CREB by PTX was much less than that by CTX. In addition, CHX further or persistently increased PTX- or CTX-induced phosphorylated CREB levels in parallel with increases in proENK mRNA. However, DEX did not alter the basal or stimulated phosphorylated-CREB level. These results suggest that the elevation of phosphorylation of CREB rather than AP-1 level may be involved in CTX-induced and CHX-dependent-PTX-induced increase of proENK mRNA level. In addition, AP-1 expression or CREB phosphorylation appears not to be involved the potentiative action of DEX on proENK mRNA expression in CTX- and PTX-treated astrocytes.

The differential molecular mechanisms underlying proenkephalin mRNA expression induced by forskolin and phorbol-12-myristic-13-acetate in primary cultured astrocytes

In rat astrocytes, forskolin (FSK; 5 μM) and phorbol-12-myristic-13-acetate (PMA; 2.5 μM) increase the proenkephalin (proENK) mRNA level via different pathways. FSK-induced proENK mRNA expression is independent of protein de novo synthesis, and well correlated with CREB phosphorylation. This is in contrast to PMA-induced proENK mRNA expression that is dependent on protein de novo synthesis and is well correlated with the increase of AP-1 DNA binding activity rather than CREB phosphorylation. Differential regulation of AP-1 proteins by PMA and FSK was also observed. While c-Fos, Fra-2 and JunB were increased in response to either stimuli, only Fra-1, c-Jun and JunD were increased by PMA. The combined treatment with FSK and PMA additively increased the proENK mRNA level, which was correlated with AP-1 or ENKCRE-2 DNA binding activity, and CREB phosphorylation. Dexamethasone (DEX; 1 μM) further enhanced FSK- or PMA-induced proENK mRNA expression, which was not correlated with the activation of AP-1 expression and CREB phosphorylation, suggesting that synergistic interaction of glucocorticoid with PKA or PKC pathway for the regulation of proENK mRNA expression appears to be mediated by other pathways rather than CREB and AP-1 families.

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase (‘enkephalinase’) gene

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271–2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of μ receptor density in crude brain homogenates of NEP knockout mice was observed, while δ- and κ-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident–intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.

Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain

Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1–24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.

ARC POMC mRNA and PVN α-MSH are lower in obese relative to lean Zucker rats

Effects of obesity on gene expression for opioid peptides and neuropeptide-Y (NPY) in the arcuate nucleus (ARC), and on opioid peptides and α-melanocyte stimulating hormone (α-MSH) in the paraventricular nucleus (PVN) were examined in obese Zucker rats (18 weeks old). Obese Zucker rats are insulin-resistant, diabetic and hyperleptinemic as indicated by high serum glucose, insulin and leptin levels. ARC proOpiomelanocortin (POMC) mRNA levels were significantly lower in the obese relative to lean Zucker rats and ARC proNeuropeptide Y (proNPY) mRNA levels were higher ( P<0.05). There were no differences in proDynorphin and proEnkephalin mRNA levels in the ARC ( P>0.05). Obese Zucker rats had lower α-MSH and dynorphin A 1–17 peptide levels in the paraventricular nucleus (PVN) ( P<0.05), but did not have lower PVN β-endorphin peptide levels ( P>0.05). The decrease in POMC in the ARC and decrease in α-MSH in the PVN seen in the obese Zucker rat in the present study suggest that reduced activity of the melanocortin system in the ARC to PVN pathway may contribute to the related hyperphagia. Reduced activity of the melanocortin system in the ARC to PVN pathway may be due to a disturbance of leptin signaling coupling to POMC.

Prenatal Δ 9-tetrahydrocannabinol exposure modifies proenkephalin gene expression in the fetal rat brain: sex-dependent differences

Perinatal Δ 9-tetrahydrocannabinol (Δ 9-THC) exposure in rats resulted in enhanced morphine self-administration behavior, naloxone-precipitated withdrawal signs or changes in pain sensitivity, which have been related to changes in μ-opioid receptor binding and/or proenkephalin mRNA levels in several brain regions. However, despite exposure of these animals to Δ 9-THC from fetal ages, the effects were studied only when animals matured, whereas there is no study on possible changes caused by this cannabinoid during the prenatal ontogeny of opioidergic neurons. The purpose of the present study was to examine the changes in proenkephalin mRNA levels, measured by using in situ hybridization, in several brain nuclei of rat fetuses that had been daily exposed to Δ 9-THC from the 5th day of gestation. Results were as follows. Prenatal Δ 9-THC exposure altered proenkephalin mRNA levels in most of the brain areas studied at different fetal ages, but the effects were different between sexes. Thus, proenkephalin mRNA levels increased in females, but decreased in males that had been prenatally exposed to Δ 9-THC. This was observed in the caudate–putamen, hypothalamic paraventricular and ventromedial nuclei and cerebral cortex. No changes were observed, however, in the subventricular zones of the caudate–putamen, neocortex and nucleus accumbens. In summary, prenatal Δ 9-THC exposure produced a sex-dependent effect in proenkephalin mRNA levels in several brain structures of rat fetuses.

Stimulation of astrocyte-enriched culture with arachidonic acid increases proenkephalin mRNA: involvement of proto-oncoprotein and mitogen activated protein kinases

In astrocyte-enriched cultures, arachidonic acid (AA, 100 μM) significantly increased the proenkephalin (proENK) mRNA level (4.9-fold at 8 h). In addition, AA also increased several AP-1 proteins, such as c-Fos, Fra-1, Fra-2, JunB, JunD, and c-Jun, or AP-1 and ENKCRE-2 DNA-binding activity. As well as AP-1 proteins and their DNA-binding activities, proENK mRNA level induced by AA was reduced by the pretreatment with 15 μM of cycloheximide (CHX; 1.6-fold). AA-dependent increase of proENK mRNA is not mediated by cyclooxygenase- or lipoxygenase-dependent metabolites, or free radicals, because the AA-induced increase of proENK mRNA levels was not affected by indomethacin (10 μM), nordihydroguaiaretic acid (10 μM), or N-acetylcysteine. However, as well as proto-oncoprotein levels, such as Fra-1, Fra-2, c-Jun, JunB, but not JunD, AA-induced increase of proENK mRNA was significantly reduced by the pretreatment with 10 μM of PD98059 (1.3-fold) or 10 μM of SB203580 (1.8-fold). These results strongly suggest that AA rather than one of its metabolites is involved in the increase of proENK mRNA. In addition, the activation of both the p38 and ERK pathways appears to be involved in the AA-induced increase of proENK mRNA via activating the expression of proto-oncoprotein, such as Fra-1, Fra-2, c-Jun, and JunB.

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.

Olfactory bulbectomy increases prepro-enkephalin mRNA levels in the ventral striatum in rats

The effects of bilateral olfactory bulbectomy (OBX) on prepro-enkephalin, thyrotropin-releasing hormone, and D-2 receptor mRNA levels in the ventral striatum were examined by in situ hybridization histochemistry. Pre- pro-enkephalin mRNA levels were significantly increased in the olfactory tubercle (OT), but not in the nucleus accumbens, 14 days following bilateral OBX. Levels of D-2 receptor mRNA were also increased in the OT, though to a lesser degree. Prepro-thyrotropin-releasing hormone mRNA was unaffected by OBX. A separate experiment revealed no effect of OBX on enkephalin gene expression 7 days following surgery but a comparable elevation in pre- pro-enkephalin mRNA 14 and 28 days post-surgery. The findings are consistent with previously-reported effects of dopamine lesions on striatal gene expression, suggesting that the observed effects may be mediated by deafferentation-induced alterations in dopaminergic transmission in the OT. Altered dopaminergic function in the OT may be particularly relevant to the ‘anhedonia’ that has been associated with the olfactory bulbectomized rat model of depression.

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.

Time-dependent differences of repeated administration with Δ 9-tetrahydrocannabinol in proenkephalin and cannabinoid receptor gene expression and G-protein activation by μ-opioid and CB 1-cannabinoid receptors in the caudate–putamen

The purpose of the present study was to examine the time-related effects of repeated administration of Δ 9-tetrahydrocannabinol during 1, 3, 7 and 14 days on cannabinoid and μ-opioid receptor agonist-stimulated [ 35 S ]GTPγS binding, and CB 1 cannabinoid receptor and proenkephalin gene expression in the caudate–putamen. Repeated administration with Δ 9-tetrahydrocannabinol produced a time-related reduction in cannabinoid receptor synthesis and activation of signal transduction mechanisms in the caudate–putamen. Indeed, WIN-55,212-2-stimulated [ 35 S ]GTPγS binding decreased 24% on day 1 and then progressively decreased finding a 42% decrease on day 14. Similarly, CB 1 cannabinoid receptor mRNA levels decreased (22%) on day 3, reaching 50% reduction on day 7. In contrast, a pronounced increase is detected in DAMGO-stimulated [ 35 S ]GTPγS binding and proenkephalin mRNA levels in the caudate–putamen. The highest degree of increase was reached on day 7 of the treatment (35% of proenkephalin mRNA levels and 62% of DAMGO-stimulated [ 35 S ]GTPγS binding) and then values slightly decreased on day 14. Taken together, the results of the present study indicate that, in the caudate–putamen, repeated administration with Δ 9-tetrahydrocannabinol produces a time-related increase in proenkephalin gene expression and μ-opioid receptor activation of G-proteins, and a time-related decrease in CB 1 cannabinoid receptor gene expression and reduction in CB 1 cannabinoid receptor activation of G-proteins. These results also suggest a possible interaction between the cannabinoid and opioid systems in the caudate–putamen which may be potentially relevant in the understanding of the alterations of motor behavior that occur after prolonged exposure to cannabinoids.

Differential basal proenkephalin gene expression in dorsal striatum and nucleus accumbens, and vulnerability to morphine self-administration in Fischer 344 and Lewis rats

We have previously shown that the acquisition rate of intravenous morphine self-administration under a fixed ratio one (FR1) schedule of reinforcement was greater in Lewis (LEW) than Fischer 344 (F344) rats. The purpose of the present experiment was to examine the relative motivational properties of morphine (1 mg/kg) or food under progressive ratio (PR) schedules of reinforcement in LEW and F344 rats. In addition, by using in situ hybridization histochemistry we have measured in both strains of rats the basal level of proenkephalin (PENK) gene expression in dorsal striatum and nucleus accumbens (NAcc). The results show that LEW rats responded to significantly higher breaking points (BPs) than F344 rats for intravenous morphine self-administration. In contrast, no differences were found in BPs for food pellets. Basal PENK mRNA levels were significantly higher in the dorsal striatum and nucleus accumbens of F344 than in LEW rats. Taken together, these results reveal a strain difference in the reinforcing efficacy of morphine and in the basal PENK gene expression in brain regions involved in the reinforcing actions of opiates. These data also suggest that the strain differences in opiate self-administration behavior found in this and other studies may be related, at least in part, to differences in basal opioid activity between LEW and F344 rats.

Prodynorphin and proenkephalin mRNAs are increased in rat brain after acute and chronic administration of gamma-hydroxybutyrate

The effects of gamma-hydroxybutyrate (GHB) on prodynorphin (PD) and proenkephalin (PE) mRNA expression were examined using in situ hybridization histochemistry in discrete rat brain structures rich in GHB receptors. A single dose of GHB (500 mg/kg i.p.) increased striatal PE mRNA levels (+60%) between 15 and 90 min after injection. An increase in PD mRNA expression was observed in the frontal cortex (+90%) 6 h after GHB administration. Chronic exposure to GHB (500 mg/kg i.p. twice a day) for 10 days induced significant increases in both PE and PD mRNA levels in different brain regions examined, suggesting that PD and PE mRNA expressions are modulated by the endogenous GHBergic system.

The enkephalinergic osteoblast.

The enkephalinergic osteoblast.

Prostaglandin E 2 increases proenkephalin mRNA level in rat astrocyte-enriched culture

The effect of prostaglandin E 2 (PGE 2) on proenkephalin (proENK) mRNA expression in primary cultured rat astrocytes was studied. The proENK mRNA level was significantly increased about 3.3-fold 4 h after PGE 2 (10 μM) treatment and this increase was potentiated by the pre-treatment with cycloheximide (CHX; 15 μM) about 1.7-fold as much as PGE 2 alone treated cells. The pretreatment with staurosporine (1 μM) completely inhibited the increase of PGE 2-induced proENK mRNA level, although only a partial inhibition of PGE 2-induced proENK mRNA level (≈1.5-fold) by H89 (10 μM) was observed. The increase of PGE 2-induced proENK mRNA level was not affected by the pretreatment with PD98059 (1, 5, and 10 μM), ω-conotoxin GIVA (1 μM), nimodipine (1 μM), calmidazolium (1 μM), or KN-62 (1 μM). In addition to the proENK mRNA level, PGE 2 also increased c-Fos (≈4.3-fold), Fra-1 (≈3.8 fold), and Fra-2 (≈8.2-fold) protein levels at 4 h after drug treatment. However, c-Jun, JunB, and JunD protein levels were not affected by PGE 2. Indeed, PGE 2 failed to up-regulate c- jun mRNA expression as well as its protein product. Surprisingly, although three Jun proteins were not induced by PGE 2, AP-1 and ENKCRE-2 DNA binding activities were increased by PGE 2, (≈5 and ≈2.8-fold, respectively) and which were effectively reduced by CHX (≈2.5 and 2-fold, respectively). In western blot analyses, PGE 2 enhanced the phosphorylation of CREB (≈2.6-fold at 1 h), and CHX showed a potentiative effect on PGE 2-induced CREB phosphorylation (≈1.7 fold at 1 h) which is similar to the action on proENK mRNA regulation. Our results suggest that PGE 2 increases proENK mRNA expression via activating serine/threonine protein kinase such as PKA, but not calcium/calmodulin dependent protein kinase and MAPK. In addition, phosphorylation of CREB rather than the increase of AP-1 may have a possible role at least early stage in PGE 2-induced proENK mRNA level and CHX-evoked potentiation.

Corticosterone can facilitate as well as inhibit corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus.

We have used in situ hybridization to investigate how basal levels of circulating corticosterone modulate CRH gene transcription in the neuroendocrine parvicellular part of the hypothalamic paraventricular nucleus (PVHmpd) during sustained hypovolemia. In the absence of the stressor, the accumulation rate of the CRH primary transcript exhibited a dose dependency on low maintained levels of plasma corticosterone similar to that previously reported for the mature messenger RNA (mRNA); levels declined as plasma corticosterone increased. In response to hypovolemia, the absence of corticosterone compromised CRH gene transcription mechanisms to mount the activated response seen in intact animals. However, adrenalectomized rats with low doses of corticosterone (insufficient to normalize thymus weights) showed an augmented mRNA response compared with that in intact animals. When replaced corticosterone normalized thymus weights, the magnitude of the mRNA response was reduced to that seen in intact animals. In contrast to CRH gene regulation, PVHmpd proenkephalin mRNA levels were unaffected by corticosterone concentrations. These results suggest that corticosterone affects CRH gene transcription in the PVHmpd using two mechanisms: first, inhibition, which probably uses type II glucocorticoid receptor-dependent mechanisms and contributes to classic negative feedback; and second, facilitation, which is seen at low plasma concentrations and maintains gene transcription in the presence of sustained stress, possibly using type I mechanisms. This suggests that one reason why adrenal insufficiency severely compromises survival of sustained stress is that CRH gene transcription cannot be maintained without previous exposure to low levels of plasma corticosterone.

Effects of Chronic Ethanol Consumption and Aging on Proenkephalin and Neurotensin

We examined the combined effects of chronic ethanol consumption and aging on mRNA and peptide for proenkephalin (PE), the precursor of met- and leu-enkephalin. This study also evaluated the effects of aging and alcohol on the level of the mRNA encoding the common precursor of neurotensin (NT) and neuromedin N (NN). PE mRNA and NT/NN mRNA were quantitated in multiple brain areas of 5- and 24-month-old male Fischer 344 rats. Aging, but not chronic ethanol consumption, altered PE mRNA and peptide levels. Aging was accompanied by a loss of PE peptide and PE mRNA in the rostral striatum. In aged rats, PE mRNA was also reduced in the shell region of the nucleus accumbens. The decline in PE mRNA in the rostral striatum and shell region of the nucleus accumbens was caused by a reduction in the number of cells that contain PE mRNA. The percentage of PE mRNA-containing neurons that express a high amount of PE mRNA was also lower in the rostral striatum of 24-month-old rats. The effects of aging may impair motor function and alter the rewarding properties of ethanol consumption. Neither aging nor alcohol changed the PE mRNA levels in the core region of the nucleus accumbens, the frontal cortex, and the piriform cortex. In contrast to PE mRNA, neither aging nor chronic ethanol consumption affected NT/NN mRNA in the regions analyzed. Normal NT/NN mRNA levels were found in the lateral septum and two hippocampal brain areas: the dorsal subiculum and CA1 regions.