Proenkephalin Gene Expression Research Articles

Effects of hexarelin and isolation stress on the Met-enkephalin system in young lambs

Koziec K., S canes C.G., Z ubel-Łojek J., Gajewska A. 2023. Effects of hexarelin and isolation stress on the Met-enkephalin system in young lambs. Folia Biologica (Kraków) 71: 195-206.Stress stands out as the primary instigator of numerous diseases – ranging from cardiovascular and gastro- intestinal to diabetes and nervous disturbances – in most developed nations. Endogenous opioid peptides (EOP), particularly Met-enkephalin, play a crucial role in mitigating the up-regulation of the hypothalamo- pituitary-adrenal axis during stress responses, consequently reducing the risk of serious diseases. Hexarelin, a synthetic analog of Met-enkephalin, has been predominantly investigated for its impact on growth hormone (GH) release in both human subjects and rodent models. This study was undertaken to evaluate the influence of isolation stress and/or hexarelin administration on various Met-enkephalin-related parameters in a novel animal model – 3-month-old lambs. Four distinct groups were established: a control group, a group intravenously injected with hexarelin, a group subjected to 60 min of isolation stress from the herd, and a group treated with both hexarelin and stress. Blood and hypothalamus samples were collected to analyze cortisol and Met-enkephalin profiles, proenkephalin (PENK) gene expression, Met-enkephalin concentration, in vitro Met-enkephalin secretion, and opioid receptor binding. The findings revealed a significant impact of stress on all assessed parameters. Hexarelin alone led to a decrease in cortisol levels and Met-enkephalin synthesis, release, and receptor binding in the hypothalamus. When administered prior to stress, hexarelin potentiated the responses of opioid parameters to isolation. These results, for the first time, demonstrate that hexarelin interacts with Met-enkephalin, modulating the stress response at both central and peripheral levels in growing lambs. It is suggested that hexarelin plays a crucial role during stress responses; however, further research on its effects should be conducted concurrently with the examination of opioid profiles.

Increased ethanol intake in prodynorphin knockout mice is associated to changes in opioid receptor function and dopamine transmission

The purpose of this study was to examine the role of the prodynorphin gene in alcohol sensitivity, preference and vulnerability to alcohol consumption. Handling-induced convulsion (HIC) associated to alcohol, alcohol-induced loss of righting reflex (LORR), hypothermic effects in response to acute ethanol challenge, blood ethanol levels (BELs), conditioned place preference, voluntary ethanol consumption and preference, tyrosine hydroxylase (TH), dopamine transporter (DAT) and proenkephalin (PENK) gene expression, and µ-, δ- and κ-opioid agonist-stimulated [S(35) ]- guanosine 5'-triphosphate-binding autoradiography were studied in prodynorphin knockout (PDYN KO) and wild-type (WT) mice. There were no differences in HIC, LORR or the decrease in body temperature in response to acute ethanol challenge between PDYN KO and WT mice. PDYN KO mice presented higher BEL, higher ethanol-conditioned place preference and more ethanol consumption and preference in a two-bottle choice paradigm than WT mice. These findings were associated with lower TH and higher DAT gene expression in the ventral tegmental area and substantia nigra, and with lower PENK gene expression in the caudate-putamen (CPu), accumbens core (AcbC) and accumbens shell (AcbSh) in PDYN KO. The functional activity of the µ-opioid receptor was lower in the CPu, AcbC, AcbSh and cingulate cortex (Cg) of PDYN KO mice. In contrast, δ- and κ-opioid receptor-binding autoradiographies were increased in the CPu and Cg (δ), and in the CPu, AcbC and Cg (κ) of PDYN KO. These results suggest that deletion of the PDYN gene increased vulnerability for ethanol consumption by altering, at least in part, PENK, TH and DAT gene expression, and µ-, δ- and κ-opioid receptor functional activity in brain areas closely related to ethanol reinforcement.

Regulated Proenkephalin Expression in Human Skin and Cultured Skin Cells

Skin responds to environmental stressors via coordinated actions of the local neuroimmunoendocrine system. Although some of these responses involve opioid receptors, little is known about cutaneous proenkephalin expression, its environmental regulation, and alterations in pathology. The objective of this study was to assess regulated expression of proenkephalin in normal and pathological skin and in isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells. The proenkephalin gene and protein were expressed in skin and cultured cells, with significant expression in fibroblasts and keratinocytes. Mass spectroscopy confirmed Leu- and Met-enkephalin in skin. UVR, Toll-like receptor (TLR)4, and TLR2 agonists stimulated proenkephalin gene expression in melanocytes and keratinocytes in a time- and dose-dependent manner. In situ Met/Leu-enkephalin peptides were expressed in differentiating keratinocytes of the epidermis in the outer root sheath of the hair follicle, in myoepithelial cells of the eccrine gland, and in the basement membrane/basal lamina separating epithelial and mesenchymal components. Met/Leu-enkephalin expression was altered in pathological skin, increasing in psoriasis and decreasing in melanocytic tumors. Not only does human skin express proenkephalin, but this expression is upregulated by stressful stimuli and can be altered by pathological conditions.

Forebrain PENK and PDYN gene expression levels in three inbred strains of mice and their relationship to genotype-dependent morphine reward sensitivity

Vulnerability to drug abuse disorders is determined not only by environmental but also by genetic factors. A body of evidence suggests that endogenous opioid peptide systems may influence rewarding effects of addictive substances, and thus, their individual expression levels may contribute to drug abuse liability. The aim of our study was to assess whether basal genotype-dependent brain expression of opioid propeptides genes can influence sensitivity to morphine reward. Experiments were performed on inbred mouse strains C57BL/6J, DBA/2J, and SWR/J, which differ markedly in responses to morphine administration: DBA/2J and SWR/J show low and C57BL/6J high sensitivity to opioid reward. Proenkephalin (PENK) and prodynorphin (PDYN) gene expression was measured by in situ hybridization in brain regions implicated in addiction. The influence of the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI), which attenuates effects of endogenous PDYN-derived peptides, on rewarding actions of morphine was studied using the conditioned place preference (CPP) paradigm. DBA/2J and SWR/J mice showed higher levels of PDYN and lower levels of PENK messenger RNA in the nucleus accumbens than the C57BL/6J strain. Pretreatment with nor-BNI enhanced morphine-induced CPP in the opioid-insensitive DBA/2J and SWR/J strains. Our results demonstrate that inter-strain differences in PENK and PDYN genes expression in the nucleus accumbens parallel sensitivity of the selected mouse strains to rewarding effects of morphine. They suggest that high expression of PDYN may protect against drug abuse by limiting drug-produced reward, which may be due to dynorphin-mediated modulation of dopamine release in the nucleus accumbens.

Increased vulnerability to 6-hydroxydopamine lesion and reduced development of dyskinesias in mice lacking CB1 cannabinoid receptors

Motor impairment, dopamine (DA) neuronal activity and proenkephalin (PENK) gene expression in the caudate-putamen (CPu) were measured in 6-OHDA-lesioned and treated (L-DOPA+benserazide) CB1 KO and WT mice. A lesion induced by 6-OHDA produced more severe motor deterioration in CB1 KO mice accompanied by more loss of DA neurons and increased PENK gene expression in the CPu. Oxidative/nitrosative and neuroinflammatory parameters were estimated in the CPu and cingulate cortex (Cg). CB1 KO mice exhibited higher MDA levels and iNOS protein expression in the CPu and Cg compared to WT mice. Treatment with L-DOPA+benserazide (12 weeks) resulted in less severe dyskinesias in CB1 KO than in WT mice. The results revealed that the lack of cannabinoid CB1 receptors increased the severity of motor impairment and DA lesion, and reduced L-DOPA-induced dyskinesias. These results suggest that activation of CB1 receptors offers neuroprotection against dopaminergic lesion and the development of L-DOPA-induced dyskinesias.

Alterations of prodynorphin gene expression in the rat mesocorticolimbic system during heroin self-administration

Opiate-induced alterations in the gene expression of the opioid propeptides prodynorphin (PDYN) and proenkephalin (PENK) in the brain have previously been described. However, there are no studies examining opioid propeptide system alterations due to long-lasting heroin self-administration. In our study, using in situ hybridization, we measured PDYN and PENK mRNA levels in the dorsal striatum, central nucleus of amygdala (CEA), and nucleus accumbens (NAcc) shell and core in rats after 6 weeks of heroin self-administration (fixed ratio 5, 0.02 mg/kg/infusion of heroin i.v.) and their respective saline or heroin “yoked” control. Our results show an increase in the PDYN mRNA level in the CEA and NAcc shell and no changes of PENK gene expression after heroin self-administration. In addition, to dissociate pharmacological effects of heroin from those produced by motivational processes driving active heroin intake on the PDYN and PENK gene expression, we compared effects of response-dependent (contingent) and response-independent (noncontingent — “yoked” heroin control) heroin administration. We found no differences between contingent and noncontingent groups of rats. In conclusion, our results indicate neuroadaptations in the PDYN but not PENK gene expression in rat limbic forebrain during heroin self-administration. We show that such changes depend on direct pharmacological actions of heroin rather than contingent heroin intake. These neuroalterations probably occur as an adaptation to long-lasting heroin exposure and may underlie changes leading to compulsive drug use and addiction.

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.

Time dependent alterations on tyrosine hydroxylase, opioid and cannabinoid CB 1 receptor gene expressions after acute ethanol administration in the rat brain

The aim of this study was to examine the differential regulation after acute ethanol administration on tyrosine hydroxylase, proenkephalin and cannabinoid CB 1 receptor gene expressions in selected areas of the rat brain. Rats received an intragastric administration of 3 g/kg ethanol and were killed by decapitation at 1, 2, 4, 8 and 24 h. The results showed an activation of tyrosine hydroxylase gene expression in the ventral tegmental area and the substantia nigra, increased proenkephalin gene expression in the caudate–putamen, nucleus accumbens core and shell, central and medial amygdala, ventromedial hypothalamic nucleus and the paraventricular hypothalamic nucleus. In contrast, a significant decrease in the cannabinoid CB1 receptor gene expression was found in caudate–putamen, central amygdala and ventromedial hypothalamic nucleus. In conclusion, the results suggest that an acute dose of ethanol induces neuroplastic alterations in proenkephalin, tyrosine hydroxylase and cannabinoid CB1 receptor gene expressions that may contribute to trigger the rewarding effects of ethanol consumption.

Manipulation of fatty acid amide hydrolase functional activity alters sensitivity and dependence to ethanol

The aim of this study was to examine the role of fatty acid amide hydrolase (FAAH) on ethanol sensitivity, preference, and dependence. The deletion of FAAH gene or the inhibition of FAAH by carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (0.1 mg/kg) markedly increased the preference for ethanol. The study further reveals that URB597 specifically acts through FAAH and that cannabinoid-1 (CB(1)) receptor is critical for N-arachidonoyl ethanolamide (AEA) mediated ethanol-reinforced behavior as revealed by lack of URB597 effect in both FAAH and CB(1)-/- mice compared with vehicle-treated -/- mice. The FAAH -/- mice displayed a lower sensitivity to hypothermic and sedative effects to acute ethanol challenge. The FAAH -/- mice also exhibited a reduction in the severity of handling-induced convulsions following withdrawal from chronic ethanol exposure. The CB(1) receptor and proenkephalin gene expressions, and CB(1) receptor and mu-opioid (MO) receptor-mediated G-protein activation were found to be significantly lower in the caudate-putamen, nucleus accumbens core and shell of FAAH -/- than +/+ mice. Interestingly, the MO receptor-stimulated G-protein signaling was greater in the striatum of FAAH -/- than +/+ mice following voluntary ethanol consumption. These findings suggest that an elevation in the AEA content and its action on the limbic CB(1) receptor and MO receptor might contribute to ethanol-reinforced behavior. Treatment with drugs that decrease AEA tone might prove useful in reducing excessive ethanol consumption.

Increases in preproenkephalin mRNA levels in the Syrian hamster: The influence of glucocorticoids is dependent on age and tissue

In adult hamsters, basal proenkephalin (Penk) gene expression in adrenals is independent of glucocorticoids and glucocorticoid receptor blockade, by RU 486, increases striatal preproenkephalin (PPenk) mRNA levels. However, glucocorticoids maintain both basal and induced Penk gene expression in rat adrenal (medulla) and striatum. This suggests species and tissue-specific differences in Penk gene regulation. Since studies show temporal coordination in Penk gene expression in developing hamster adrenal and striatum, we tested the hypothesis that increasing PPenk mRNA levels are dependent, while basal levels are independent of glucocorticoids in developing hamsters. To facilitate this study, we examined the influence of glucocorticoids on the temporal increases in developing hamster PPenk mRNA observed in adrenals between postnatal days 0 and 4 and in striatum between postnatal days 12 and 48. PPenk mRNA levels were determined in hamster pups after treatment with increasing doses of metyrapone (an 11β hydroxylase inhibitor) or with the glucocorticoid receptor antagonist RU 486 ± metyrapone between postnatal days 2 and 4. Levels were also determined 36 days after hypophysectomy at age 16–17 days. Although plasma glucocorticoid levels and/or the influence from glucocorticoids were reduced, only developmental increases in PPenk mRNA are influenced by glucocorticoids in hamster adrenals, while basal adrenal mRNA levels are unchanged. However, pituitary influence on striatal PPenk mRNA levels appears complex and may involve steroid and/or non-steroid factors. These results suggest that glucocorticoids regulate hamster Penk gene expression via a mechanism that varies with age and tissue and functions during the induction of the Penk gene and not to maintain basal gene expression. Possible mechanisms and species variation are discussed.

Contingency does not contribute to the effects of cocaine self-administration on prodynorphin and proenkephalin gene expression in the rat forebrain

Neuroadaptations in the brain opioid systems produced by chronic exposure to drugs of abuse may contribute to the drug dependence and addiction. Although regulation of the gene expression of the opioid propeptides proenkephalin (PENK) and prodynorphin (PDYN) by psychostimulants has previously been described, little attention has been paid to dissociating effects of pharmacological actions of the drugs from those produced by motivational processes driving active drug intake in self-administration paradigms. In the present study, effects of response-dependent (contingent) and response-independent (noncontingent) cocaine administration on the PENK and PDYN gene expression in the rat forebrain have been directly compared using the “yoked” self-administration procedure. The i.v. cocaine treatment lasted for 5 weeks, and rats were sacrificed 24 h after the last self-administration session. In situ hybridization analysis revealed that levels of the PDYN mRNA were significantly increased in the caudate/putamen, to the same extent in rats self-administering cocaine as in animals receiving noncontingent injections of the drug at the same frequency and dosage. No changes in the expression of the PDYN gene were detected in the nucleus accumbens or in the central nucleus of amygdala. Levels of the PENK mRNA remained unaltered in all the above-mentioned forebrain regions of rats receiving contingent or noncontingent cocaine injections. The obtained data indicate that up-regulation of the PDYN gene expression in the caudate/putamen results from direct pharmacological actions of cocaine rather than from the motivational and cognitive processes underlying active self-administration of the drug.

Ketogenic diet decreases the level of proenkephalin mRNA induced by kainic acid in the mouse hippocampus

The ketogenic diet (KD) has been used to control medically refractory epilepsy in children for more than 80 years. Despite the clinical efficacy of the KD, its underlying bases are still obscure. Previous work from our laboratory has established that the KD has an antiepileptic and neuroprotective effect in the kainic acid (KA)-induced seizure model. The neuronal excitation caused by KA leads to increases in the expression of a variety of genes, including immediate-early genes and opioid peptides derived from proenkephalin (PENK) and prodynorphin (PDYN). In particular, the up-regulated PENK gene that is induced by KA in the hippocampal dentate granule cells has proconvulsant properties. PENK is regulated by the c-jun amino-terminal kinase (JNK) signaling pathway, the crucial role of which is involved in the regulation of transcription factors, such as Jun and Fos. In the present study, we examined the effect of the KD on the increase of PENK, Fos, Jun, AP-1 DNA-binding activity and JNK gene expression induced by KA in the mouse hippocampus. Using in situ hybridization and northern blot analysis, we found that the KD significantly decreases the level of PENK gene expression induced by KA of the granular cells in the hippocampus. In addition, we have also found that KD diminished KA-induced AP-1 DNA-binding activity, Fos and Jun expression, and phoshorylated form of the three types of JNKs. These results suggest that the KD suppresses KA-induced activation of JNK signaling pathways, followed by a decrease of PENK gene expression in the hippocampus, thereby resulting in antiepileptic effects.

Time course of opioid and cannabinoid gene transcription alterations induced by repeated administration with fluoxetine in the rat brain

This study examined the time course effects (8, 16 and 31 days) of fluoxetine administration (1 mg/kg, p.o./day) on serotonin transporter (5-HTT), opioid, tyrosine hydroxylase (TH) and cannabinoid CB 1 receptor gene expressions in selected regions of the rat brain. Treatment with fluoxetine progressively decreased (35–55%) 5-HTT gene expression in dorsal raphe nucleus at 8, 16 and 31 days. The results revealed that fluoxetine administration decreased (30%) proenkephalin gene expression in nucleus accumbens shell (AcbS) and caudate-putamen (CPu) (31 days) but was without effect in nucleus accumbens core AcbC. A pronounced and time related decrease (25–65%) in prodynorphin gene expression was detected in AcbC, AcbS, CPu, hypothalamic supraoptic and paraventricular nuclei at all time points as well as in proopiomelanocortin gene expression (20–30%) in the arcuate nucleus (ARC) of the hypothalamus. On days 16 and 31, tyrosine hydroxylase gene expression in ventral tegmental area and substantia nigra and cannabinoid CB 1 receptor gene expression in the CPu decreased (approximately 45–50% from vehicle). In conclusion, fluoxetine by inhibiting the reuptake of serotonin produced pronounced and time related alterations in genes involved in the regulation of emotional behaviour, suggesting that these neuroplastic changes may be involved, at least in part, in the clinical efficacy of this drug in neuropsychiatric disorders.

Ginsenosides attenuate methamphetamine-induced behavioral side effects in mice via activation of adenosine A 2A receptors: possible involvements of the striatal reduction in AP-1 DNA binding activity and proenkephalin gene expression

Current evidence suggests that ginsenosides inhibit methamphetamine (MA)-induced changes in behavior, but the precise mechanisms that underlie this effect are yet to be determined. We examined the role of adenosine receptors in the ginsenoside-induced changes in hyperlocomotion and conditioned place preference (CPP) in mice that occurred in response to administration of MA (2 mg/kg, i.p. × 1 or 2 mg/kg, i.p. × 6). Changes in circling behavior paralleled changes in CPP in the presence of MA. Pre-treatment with ginsenosides (50 or 150 mg/kg, i.p.) attenuated the MA-induced circling behavior and CPP. This attenuation was reversed by the adenosine A 2A receptor antagonist 1,3,7-trimethyl-8-(3-chrostyryl)xanthine (CSC; 0.5 and 1.0 mg/kg) in a dose-dependent manner, but neither the adenosine A 1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 0.5 and 1.0 mg/kg) nor the A 2B receptor antagonist alloxazine (ALX; 1.5 and 3.0 mg/kg) had any such effect. MA-induced increases in activator protein (AP)-1 DNA binding activity, Fos-related antigen immunoreactivity (FRA-IR), proenkephalin mRNA expression, and proenkephalin-like immunoreactivity were reduced consistently in the striatum of animals that were pretreated with ginsenosides. These reductions were largely prevented by CSC, but not by CPT or ALX. Our results suggest that the stimulation of A 2A receptors by ginsenosides attenuates the changes in behavior and the increases in AP-1 DNA binding activity, FRA-IR, and proenkephalin gene expression in mouse striatum that are induced by MA.

P.1.167 Chronic treatment with fluoxetine produced time realted alterations in opioid, cannabinoid and tirosine hydroxilase gene expression in selected regions of the rat 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.

Behavioural and gene transcription alterations induced by spontaneous cannabinoid withdrawal in mice.

This study examined behavioural signs that occur during tolerance development to cannabinoid treatment and hormonal and gene expression alterations induced by spontaneous cannabinoid withdrawal in mice. Tolerance to CP-55,940 treatment developed for hypothermia, ambulatory and exploratory locomotor activity. Cessation of cannabinoid treatment resulted in a behavioural withdrawal syndrome characterized by a pronounced increase in ambulatory activity and rearings. Corticosterone plasma concentrations dramatically increased 24 and 72 h after cessation of cannabinoid treatment. Similarly, an increase (40%) in cannabinoid [35S]GTPgammaS binding autoradiography was detected on days 1 and 3 of abstinence. Spontaneous cannabinoid withdrawal produced time-related significant alterations in gene transcription: (i) decreased (20%) tyrosine hydroxylase (TH) mRNA levels in the ventral tegmental area and increased (50%) in substantia nigra; (ii) increased proenkephalin (PENK) gene expression more than 100% in caudate-putamen, nucleus accumbens, olfactory tubercle and piriform cortex; (iii) increased (20-40%) pro-opiomelanocortin (POMC) gene expression in the arcuate nucleus of the hypothalamus. These results suggest that spontaneous cannabinoid withdrawal occur after cessation of CP-55,940 treatment. This 'syndrome' includes behavioural, hormonal and gene transcription alterations that seems to be part of the regulation of neuronal plasticity induced by spontaneous cannabinoid withdrawal.

Gender differences in proenkephalin gene expression response to ∆9-tetrahydrocannabinol in the hypothalamus of the rat

Chronic exposure to delta9-tetrahydrocannabinol (delta9-THC) produces an activation of preproenkephalin (PENK) gene expression in the rat hypothalamus. The levels of circulating gonadal steroids concurrently modulate this neuropeptide in male and female rats. However, whether gonadal steroids regulate delta9-THC effects on PENK gene expression in the hypothalamus of male and female rats remains unknown. To test this hypothesis, experiments were carried out on intact, 2-week-gonadectomized, 1-week-gonadectomized, 1-week-dihydrotestosterone (DHT) replaced male rats, and 2-week-gonadectomized, 1-week-gonadectomized, 1-week-oestradiol replaced female rats. One week after hormonal replacement, animals were treated with vehicle or delta9-THC (5 mg/kg/day, i.p. 7 days). In males, delta9-THC administration to intact animals induced PENK mRNA in the paraventricular nucleus (PVN) and ventromedial nucleus (VMN) of the hypothalamus. Orchidectomy did not affect basal PENK mRNA levels in the PVN, but reduced PENK mRNA levels in the VMN. However, delta9-THC treatment induced PENK gene expression to the same extent in both hypothalamic nuclei of intact, castrated and DHT-replaced males. In females, ovariectomy decreased PENK gene expression in PVN and VMN. delta9-THC administration increased PENK gene expression in castrated females, but had no effect in the oestradiol-replaced group. Taken together, these results suggest gender differences in the response of chronic exposure to cannabinoids on PENK gene expression in the hypothalamus. Furthermore, it appears that alterations in opioid gene expression induced by cannabinoids in female rats depend upon the presence or absence of circulating oestradiol.

Intrinsic GABA neurons inhibit proenkephalin gene expression in slice cultures of rat neostriatum

In the neostriatum, the proenkephalin gene is expressed in medium spiny GABA neurons, which project to the globus pallidus. The expression is activated by glutamatergic projections from the neocortex via NMDA receptors. In these experiments we have used slice cultures of rat neostriatum to study the role of GABA in proenkephalin gene expression. Our results show that GABA is released from neostriatal neurons and negatively regulates the proenkephalin gene expression induced by NMDA receptor stimulation. The GABAA receptors involved seem to be colocalized with NMDA receptors on the projection neurons, which express the proenkephalin gene. In further experiments, we have found that the proenkephalin gene expression is not only activated by neocortical projection neurons but also by intrinsic striatal neurons as well as by projections from the thalamus. All these glutamatergic afferents enhance the proenkephalin gene expression via NMDA receptors. Their efficacy is regulated by endogenous GABA.

Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-kappaB.

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the CNS. In this study we have demonstrated that reactive oxygen species (ROS) dramatically induce the expression of two neuropeptide genes, the opioid proenkephalin (pENK) and the opioid-related proorphanin FQ (pOFQ; also known as pronociceptin) in primary astrocytes. Hydrogen peroxide (H2O2) treatment dose-dependently increased pENK and pOFQ mRNA levels with a maximal effect ( approximately 15-fold increase) being detected at 50 microM concentration. Exposing the astrocyte cultures to hypoxia and subsequent re-oxygenation also led to a profound elevation of pOFQ and pENK mRNA levels. Western blot analysis and immunocytochemistry revealed that H2O2 treatment elicited the phosphorylation and nuclear translocation of ERK 1/2 and p38 MAP kinases. Blockade of the p38 or the ERK MAP kinase pathways (by SB202190 and PD98059, respectively) prevented the H2O2-induced increase in pENK and pOFQ mRNA levels indicating a central role for these cascades in the regulation of pOFQ and pENK genes in response to oxidative stress. Regulation of pOFQ and pENK gene expression by ERK and p38 activation may be mediated through the transcription factor cAMP-response element binding protein (CREB). We observed CREB phosphorylation in response to H2O2, which was also prevented by SB202190 and PD98059. The nuclear factor-kappaB (NF-kappaB) pathway appears to be involved exclusively in the induction of pOFQ transcription by H2O2, as NF-kappaB inhibitors antagonized the effect of oxidative stress on pOFQ, but not on pENK expression. The profound induction of these genes by oxidative stress and these other factors may suggest a role for orphanin FQ and enkephalin in injury and stress responses of the CNS and neuropathophysiological conditions involving reactive oxygen species.

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.