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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 (H[sub 2]O[sub 2]) treatment dose-dependently increased pENK and pOFQ mRNA levels with a maximal effect (∼15-fold increase) being detected at 50 µm 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 H[sub 2]O[sub 2] 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 H[sub 2]O[sub 2]-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 H[sub 2]O[sub 2], which was also prevented by SB202190 and PD98059. The nuclear factor-κB (NF-κB) pathway appears to be involved exclusively in the induction of pOFQ transcription by H[sub 2]O[sub 2], as NF-κB 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. [ABSTRACT FROM AUTHOR]

The article presents an abstract of the paper "Induction of Proenkephalin and Proorphanin FQ mRNA Expression by Oxidative Stress in Astrocytes: Role of ERKs, p38 Map Kinases and NFκB," which will be presented at the 30th Annual Meeting of the American Society for Neurochemistry to be held in New Orleans, Louisiana from March 14-17, 1999. It shows that the induction of neuropeptide genes may suggest a role for orphanin FQ in the stress response of neuropathophysiological conditions.

Purpose: The study's purpose was to measure the impact of anti-reflux needleless connector usage in prevention of intraluminal thrombotic occlusions among central venous catheters, as represented by alteplase usage, in a home infusion patient population., Methods: An18-month before-and-after cohort study of a single home infusion intervention was conducted to compare occlusion outcomes with use of two types of needleless connectors-neutral and anti-reflux-in preventing catheter occlusions, which have been reported to occur in 28% of home infusion patients, resulting in treatment delays, increased nursing encounters and emergency room visits, and higher overall pharmacy costs for supplies and alteplase., Results: A total of 552,707 patient therapy days were studied: 42.5% in the neutral needleless connector group (n = 235,004 therapy days) and 57.5% in the anti-reflux needleless connector group (n = 317,703 therapy days). The rate of alteplase usage with neutral versus anti-reflux needleless connectors was 4.4% versus 2.2% per 1,000 therapy days, with median alteplase use of 112 (95% CI, 89-169) units versus 82 (95% CI, 68-109) units (P < 0.001). Implementation of anti-reflux connectors reduced occlusions and alteplase usage by 48%., Conclusion: Statistical evidence demonstrated that use of anti-reflux needleless connectors with central venous access devices reduced the need for alteplase in the study population. Since 10% of patient occlusions were within 7 days after home infusion admission, future research may indicate that placement of anti-reflux needleless connectors at the time of in-hospital insertion can improve patient outcomes. This quality improvement measure reduced central catheter occlusions, alteplase costs, and the number of required nursing and emergency room visits., (© American Society of Health-System Pharmacists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study basis: As a byproduct of protein metabolism, serum uric acid is a controversial risk factor and is the focus of several recent studies in the field of cardiovascular disease. Whether serum uric acid is involved in the development of these pathologies alone or in conjunction with other factors is a matter of debate. Objective: The objective of this study is to assess the direct relationship between serum uric acid and the ejection fraction. Methods: A retrospective study of 303 patients with heart failure, classified according to the ESC guidelines, was conducted, and several parameters, along with the relationship between serum uric acid and ejection fraction, were characterized. Results: A direct relationship between the level of serum uric acid and the ejection fraction was established ( p = 0.03); patients with higher uric acid had an increased risk of having a lower ejection fraction. Conclusions : Serum uric acid, even when asymptomatic, is linked with the level of the ejection fraction of the left ventricle.

A polymorphism of the human Brain Derived Neurotrophic Factor (BDNF) gene that produces a valine-to-methionine substitution at codon 66 (Val66Met) is linked to adult anxiety and mood disorders, possibly through effects on brain circuitry function. Associations between BDNF gene variants and brain activity have not been explored in anxious and depressed adolescents. The current study investigated the association between BDNF genotype and amygdala-hippocampal responses to emotional stimuli in adolescents with anxiety disorders and/or major depressive disorder (MDD) and in healthy adolescents. Twenty-seven unmedicated patients with acutely-impairing current anxiety disorders and/or MDD and 31 healthy adolescents, matched on age, gender and IQ, rated their fear of fearful, angry, neutral and happy facial expressions during collection of fMRI data on the amygdala and hippocampus. Left and right amygdala and hippocampal responses were analyzed using repeated-measures analyses of variance models, with diagnosis (patients, healthy) and genotype (Met-carriers, Val/Val homozygotes) as between-group factors and facial expression (fearful, angry, neutral, happy) as a within-subject factor. Significant effects of diagnosis and diagnosis-by-genotype interactions (F's>4, p's<0.05) characterized activations in amygdala and anterior hippocampal regions. Greater activations in patients than healthy adolescents were found. Critically, these hyperactivations were modulated by BDNF genotype: Met-carriers showed greater neural responses of emotional faces than Val/Val homozygotes in patients only. These data are first to demonstrate the contribution of BDNF gene variants to the neural correlates of adolescent anxiety and depression. Early "gene-brain" linkages may lay the foundation for longer-term patterns of neural dysfunction in affective disorders., (Copyright 2009 Elsevier Inc. All rights reserved.)

Chronic alcoholism leads to gray matter shrinkage and induces the formation of superoxide anions (O(2)(-)) that can cause neuronal cell death. The mitochondrial superoxide dismutase 2 (SOD2) enzyme is critical in the metabolism of superoxide. An Ala16Val polymorphism putatively affects SOD2 enzyme activity in vivo. Brain volumes of 76 treatment-seeking alcohol-dependent individuals were measured with a 1.5T MRI. Intracranial tissue margins were manually outlined on coronal sections. Gray matter, white matter, sulcal, and ventricular CSF volumes were estimated using intensity-based K-means clustering. Ala16Val (rs4880) and a second haplotype tagging SNP, rs10370, were genotyped. The q-value package was used to correct for multiple comparisons. In the alcoholics, cerebrospinal fluid and intra-cranial volumes showed significant differences across the six diplotype categories. The homozygous Ala16-containing diplotype rs10370TT-rs4880GG was associated with lowest gray matter ratio (greater shrinkage; p=0.005). Presence of one or two copies of the low activity Ala16 allele was a risk factor for lower gray matter volume in alcoholics below the median alcohol consumption (p=0.03) but not in alcoholics above this level. White matter ratio was associated with sex (p=0.002) and lifetime total alcohol consumption (p=0.01) but not with diplotypes. In this exploratory analysis, a putative functional missense variant of SOD2 appears to influence gray matter loss in alcoholics. This may be due to impaired clearance of reactive oxygen species formed as a result of alcohol exposure. The risk/protective effect was observed in alcoholics with lower levels of lifetime alcohol consumption. Highest levels of exposure may overwhelm the protective action of the SOD2 enzyme.

Background: Associations between a functional polymorphism in the serotonin transporter gene and amygdala activation have been found in healthy, depressed, and anxious adults. This study explored these gene-brain associations in adolescents by examining predictive effects of serotonin transporter gene variants (S and L(G) allele carriers vs. L(A) allele homozygotes) and their interaction with diagnosis (healthy vs. patients) on amygdala responses to emotional faces., Methods: Functional magnetic resonance data were collected from 33 healthy adolescents (mean age: 13.71, 55% female) and 31 medication-free adolescents with current anxiety or depressive disorders (or both; mean age: 13.58, 56% female) while viewing fearful, angry, happy, and neutral facial expressions under varying attention states., Results: A significant three-way genotype-by-diagnosis-by-face-emotion interaction characterized right amygdala activity while subjects monitored internal fear levels. This interaction was decomposed to map differential gene-brain associations in healthy and affected adolescents. First, consistent with healthy adult data, healthy adolescents with at least one copy of the S or L(G) allele showed stronger amygdala responses to fearful faces than healthy adolescents without these alleles. Second, patients with two copies of the L(A) allele exhibited greater amygdala responses to fearful faces relative to patients with S or L(G) alleles. Third, although weaker, genotype differences on amygdala responses in patients extended to happy faces. All effects were restricted to the fear-monitoring attention state., Conclusions: S/L(G) alleles in healthy adolescents, as in healthy adults, predict enhanced amygdala activation to fearful faces. Contrary findings of increased activation in patients with L(A)L(A) relative to the S or L(G) alleles require further exploration.

Background: Genetic polymorphisms that influence serotonin (5-hydroxytryptamine, 5HT) neurotransmission are candidates for contributing to susceptibility to posttraumatic stress disorder (PTSD). The objective of our study was to determine if a variable length polymorphism for the promoter regions of the 5HT transporter (5HTTLPR), and/or a substitution polymorphism in the promoter region for the 5HT2A receptor, would be associated with PTSD in a trauma exposed population of adult African-Americans., Methods: Using a case control design, 118 participants recruited from the primary care clinics and the campus of a historically black university who met inclusion criteria including trauma exposure provided blood samples for genomic DNA. PTSD criteria were determined by the Clinician Assessment of PTSD Scale (CAPS) and criteria for other psychiatric disorders by the Structured Clinical Interview for DSM-IV (SCID). 5HTTLPR and 5HT2A-1438A/G were genotyped using established methods. Associations of genotypes with lifetime PTSD, and models testing associations of allele "dose", were analyzed., Results: Fifty-five (47%) participants met lifetime criteria for PTSD and 26 (22%) met criteria for (mostly comorbid) major depression. The 5HT2A (lower expressing) G allele was significantly associated with PTSD. We did not find significant associations with 5HTTLPR., Conclusions: Our findings suggest a relationship between genetic variation in the 5HT2A promoter region and PTSD.

Understanding inter-individual differences in stress response requires the explanation of genetic influences at multiple phenotypic levels, including complex behaviours and the metabolic responses of brain regions to emotional stimuli. Neuropeptide Y (NPY) is anxiolytic and its release is induced by stress. NPY is abundantly expressed in regions of the limbic system that are implicated in arousal and in the assignment of emotional valences to stimuli and memories. Here we show that haplotype-driven NPY expression predicts brain responses to emotional and stress challenges and also inversely correlates with trait anxiety. NPY haplotypes predicted levels of NPY messenger RNA in post-mortem brain and lymphoblasts, and levels of plasma NPY. Lower haplotype-driven NPY expression predicted higher emotion-induced activation of the amygdala, as well as diminished resiliency as assessed by pain/stress-induced activations of endogenous opioid neurotransmission in various brain regions. A single nucleotide polymorphism (SNP rs16147) located in the promoter region alters NPY expression in vitro and seems to account for more than half of the variation in expression in vivo. These convergent findings are consistent with the function of NPY as an anxiolytic peptide and help to explain inter-individual variation in resiliency to stress, a risk factor for many diseases.

Transient reductions in serotonin levels during tryptophan depletion (TD) are thought to impair reward processing in healthy volunteers, while another facet of the serotonergic system, the serotonin transporter (5-HTTLPR) short allele polymorphism, is implicated in augmented processing of aversive stimuli. We examined the impact and interactions of TD and the serotonin promoter polymorphism genotype on reward and punishment via two forms of instrumental learning: passive avoidance and response reversal. In this study, healthy volunteers (n=35) underwent rapid TD or control procedures and genotyping (n=26) of the 5-HTTLPR for long and short allele variants. In the passive avoidance task, tryptophan-depleted volunteers failed to respond sufficiently to rewarded stimuli compared to the control group. Additionally, long allele homozygous individuals (n=11) were slower to learn to avoid punished stimuli compared to short allele carriers (n=15). TD alone did not produce measurable deficits in probabilistic response reversal errors. However, a significant drug group by genotype interaction was found indicating that in comparison to short allele carriers, tryptophan-depleted individuals homozygous for the long allele failed to appropriately use punishment information to guide responding. These findings extend prior reports of impaired reward processing in TD to include instrumental learning. Furthermore, they demonstrate behavioral differences in responses to punishing stimuli between long allele homozygotes and short allele carriers when serotonin levels are acutely reduced.

Rationale: Genotype at the 5' promoter region (5-HTTLPR) of the serotonin transporter has been implicated in moderating the effects of acute tryptophan depletion on neurocognitive functioning. Acute tryptophan depletion has been associated with the processing of fear-relevant cues, such as emotional expressions, but the effect of genotype at the 5-HTTLPR has not been assessed., Objective: The present study investigated the effects of acute tryptophan depletion on the recognition of standardized facial expressions of emotions in healthy volunteers classified as ll homozygotes or s carriers., Materials and Methods: A double-blind between-groups design was used with volunteers randomly selected to ingest capsules containing an amino acid mixture specifically lacking tryptophan, or placebo capsules containing lactose. 5 h after capsule ingestion, subjects were required to identify anger, disgust, fear, happiness, sadness, and surprise expressions that progressed from neutral to each full emotional expression in 5% steps., Results: Tryptophan depletion significantly impaired the recognition of fearful facial expressions in s carriers but not ll homozygotes. This impairment was specific to fear expressions. No significant differences in the recognition of other expressions were found. Free tryptophan levels were correlated with fear recognition in s carriers but not ll homozygotes., Conclusions: The effects of acute tryptophan depletion on the processing of emotional expressions varies as a function of genotype at the 5-HTTLPR. Depletion impairs the recognition of fear in s carriers but not ll homozygotes. This finding reinforces the importance of considering genotype when assessing the behavioral effects of pharmacologic modulation.

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are expressed in the substantia nigra (SN), a brain area containing dopamine neurons that degenerate in Parkinson's disease. Endogenous N/OFQ facilitates nigral glutamate release and inhibits nigrostriatal dopamine transmission and motor behavior. Here, we present evidence suggesting that endogenous N/OFQ may contribute to Parkinson's disease. Pharmacological blockade of the SN N/OFQ-NOP receptor system attenuated parkinsonian-like akinesia/hypokinesia in 6-hydroxydopamine hemilesioned or haloperidol-treated rats, whereas deletion of the NOP receptor gene conferred mice partial protection from haloperidol-induced motor depression. The antiparkinsonian action of NOP receptor antagonists was associated with reduction of glutamate release in the SN. In 6-hydroxydopamine hemilesioned rats, enhancement of N/OFQ expression and release was detected in the lesioned compared with the unlesioned SN, indicating that parkinsonism may be associated with overactivation of the N/OFQ-NOP receptor system in the SN. Finally, deletion of the N/OFQ gene conferred mice partial protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of SN dopamine neurons. Based on these data, we propose that NOP receptor antagonists may represent a novel approach for combined (symptomatic and neuroprotective) therapy of Parkinson's disease.

The beta-adrenergic receptors (beta-AR) are G protein-coupled receptors activated by epinephrine and norepinephrine and are involved in a variety of their physiological functions. Previously, three beta-AR genes (ADRB1, ADRB2 and ADRB3) were resequenced, identifying polymorphisms that were used in genetic association studies of cardiovascular and metabolic disorders. These studies have produced intriguing but inconsistent results, potentially because the known functional variants: ADRB1 Arg389Gly and Gly49Ser, ADRB2 Arg16Gly and Gln27Glu, and ADRB3 Arg64Trp provided an incomplete picture of the total functional diversity at these genes. Therefore, we created marker panels for each beta-AR gene that included the known functional markers and also other markers evenly spaced and with sufficient density to identify haplotype block structure and to maximize haplotype diversity. A total of 27 markers were genotyped in 96 US Caucasians and 96 African Americans. In both populations and for each gene, a single block with little evidence of historical recombination was observed. For each gene, haplotype captured most of the information content of each functional locus, even if that locus was not genotyped, and presumably haplotype would capture the signal from unknown functional loci whose alleles are of moderate abundance. This study demonstrates the utility of using beta-AR gene haplotype maps and marker panels as tools for linkage studies on beta-AR function.

The alpha 2-adrenergic receptors (alpha2-AR) mediate physiological effects of epinephrine and norepinephrine. Three genes encode alpha2-AR subtypes carrying common functional polymorphisms (ADRA2A Asn251Lys, ADRA2B Ins/Del301-303 and ADRA2C Ins/Del322-325). We genotyped these functional markers plus a panel of single nucleotide polymorphisms evenly spaced over the gene regions to identify gene haplotype block structure. A total of 24 markers were genotyped in 96 Caucasians and 96 African Americans. ADRA2A and ADRA2B each had a single haplotype block at least 11 and 16 kb in size, respectively, in both populations. ADRA2C had one haplotype block of 10 kb in Caucasians only. For the three genes, haplotype diversity and the number of common haplotypes were highest in African Americans, but a similar number of markers (3-6) per block was sufficient to capture maximum diversity in either population. For each of the three genes, the haplotype was capable of capturing the information content of the known functional locus even when that locus was not genotyped. The alpha2-AR haplotype maps and marker panels are useful tools for genetic linkage studies to detect effects of known and unknown alpha2-AR functional loci.

Nociceptin/orphanin FQ (N/OFQ) is a recently identified opioid-related neuropeptide. Earlier in vitro studies revealed regulation of N/OFQ expression by injury-induced factors, such as ciliary neurotrophic factor, inflammatory cytokines, and reactive oxygen species. We have extended our studies to in vivo experiments investigating the effect of traumatic brain injury on N/OFQ gene expression and peptide levels in the rat brain. Stab wound injury to the rat cerebral cortex led to a significant increase in N/OFQ mRNA levels in the vicinity of the injury, with the largest induction being seen at 24 h post-injury. Quantitative in situ hybridization revealed an almost twofold increase in the number of cells expressing N/OFQ, and the signal intensities within cells were also elevated. Stab wound injury leads to proliferation and hypertrophy of astrocytes, which respond to injury-related factors in vitro by up-regulating N/OFQ expression. However, in vivo N/OFQ co-localized exclusively with the neuronal marker, NeuN, following injury. N/OFQ expression was not detected in caspase-3-positive neurons undergoing apoptosis following injury, and increased N/OFQ expression was spatially more extended than the secondary injury-induced responses, such as astrogliosis and neuronal degeneration. Elevation of N/OFQ immunoreactivity closely followed the increase in N/OFQ gene expression as determined by immunohistochemistry. N/OFQ selectively activates the NOP receptor (ORL-1), but we did not detect parallel changes in levels of NOP receptor mRNA following injury, indicating regulation of the nociceptin system at the peptide and not the receptor level. In summary, a profound and prolonged up-regulation of N/OFQ expression in neurons surrounding a stab wound lesion to cerebral cortex was detected. The function of N/OFQ up-regulation in injury-induced responses in the brain is currently under investigation.

Ceramide is generated in response to inflammatory mediators and oxidative stress. Since these stimuli dramatically increase nociceptin/orphanin FQ (N/OFQ) gene expression in astrocytes we evaluated the regulation of N/OFQ by ceramide and the signaling mechanisms involved. We found that ceramide induced N/OFQ mRNA levels 22-fold after 24 h. In astrocytes ceramide stimulated the JNK, p38 and ERK MAP kinase pathways and also led to the activation of the transcription factors CREB and NFkappaB. Using specific inhibitors of signaling pathways we determined that N/OFQ gene induction is mediated by ERK and p38 MAP kinases. ERK activation may be induced by protein kinase C and it leads to CREB phosphorylation. The NFkappaB pathway also appears to be crucial for ceramide-induced N/OFQ gene expression., (Copyright 2002 Lippincott Williams & Wilkins)

In the central nervous system, glial cells play an important role in inflammatory and immune responses, and opioid peptides have been identified as essential mediators between the nervous and the immune systems. We report the profound upregulation of the opioid-related nociceptin/orphanin FQ (N/OFQ) by inflammatory mediators in astrocytes. The bacterial endotoxin, lipopolysaccharide (LPS), and the proinflammatory cytokines, interleukin-beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), induced levels of N/OFQ mRNA and immunoreactivity. HPLC analysis of the immunoreactivity in astrocyte extracts revealed that a large molecular weight precursor for N/OFQ is being synthesized and released in response to LPS and astrocytes appear to lack the enzymes required to process the precursor protein. Western blot analysis showed that LPS treatment elicited the activation of ERK 1/2 and p38 MAP kinases. Blockade of the p38 or the ERK MAP kinase pathways prevented the LPS-induced increase in N/OFQ mRNA levels indicating a role for these cascades in the regulation of N/OFQ genes in response to LPS. Regulation of N/OFQ gene expression by ERK and p38 activation may be mediated through the transcription factor CREB. We observed CREB phosphorylation in response to LPS, which was also prevented by SB202190 and PD98059. The NFkappaB pathway also appears to be involved in the induction of N/OFQ transcription by LPS, since NFkappaB inhibitors antagonized the effect of LPS on N/OFQ expression. Regulation of N/OFQ by inflammatory mediators in astrocytes may suggest a role for N/OFQ in neural-glial communication and in inflammatory responses in certain neuropathophysiological conditions.

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.

We have identified the gene encoding nociceptin/orphanin FQ (N/OFQ), the novel opioid-like neuropeptide, as responsive to ciliary neurotrophic factor (CNTF). N/OFQ mRNA levels were induced five- and ninefold by CNTF in striatal and cortical neurons. In primary astrocytes CNTF also increased N/OFQ mRNA levels. CNTF is a multifunctional cytokine that mediates the development and differentiation of both neurons and astrocytes and supports the survival of various neurons. CNTF is also an injury-induced factor in the brain playing a crucial role in astrogliosis. The mechanism by which CNTF elicits these effects is not well understood, but it is likely to involve regulation of specific genes. CNTF regulation of N/OFQ expression was sensitive to the kinase inhibitors H-7 and genistein but not to inhibition of protein synthesis. This pharmacological profile is consistent with CNTF activating the Janus protein tyrosine kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway to induce N/OFQ transcription. In nuclear extracts of CNTF-treated striatal neurons DNA binding of STAT proteins was increased. Radioimmunoassays revealed elevated N/OFQ immunoreactivity in striatal neurons after CNTF treatment. Expression of the related proenkephalin gene was not affected by CNTF in either neuronal or glial cultures. Regulation of N/OFQ expression by CNTF might point to a possible function of N/OFQ during development and after neural injury.

The regulation of nociceptin/orphanin FQ (N/OFQ) gene expression by neuronal activity and by activation of the cyclic AMP signaling pathway in primary neuronal and astroglial cultures is described. Neuronal activity mimicked by veratridine-mediated depolarization profoundly increased N/OFQ gene expression in primary striatal neurons. Calcium entry through L-type, but not N-type, voltage-sensitive calcium channels activated by depolarization appears to be involved, because nitrendipine and nifedipine, but not omega-conotoxin, reduced the induction of N/OFQ expression by veratridine. A selective inhibitor of calcium/calmodulin kinases (KN-62) also antagonized the depolarization-induced increase in N/OFQ mRNA levels, suggesting a role for these enzymes in the activity-dependent induction of N/OFQ gene expression. Constitutively expressed transcription factors may mediate N/OFQ gene expression levels, because veratridine induction of N/OFQ transcription was insensitive to the protein synthesis inhibitor cycloheximide. Regulation of N/OFQ gene expression by depolarization and cyclic AMP is not restricted to striatal neurons, because similar regulation was also observed in neuronal cultures derived from the cerebral cortex. Veratridine did not increase N/OFQ mRNA levels in primary astrocyte cultures; however, elevated intracellular cyclic AMP levels lead to a dramatic, 30-fold induction of N/OFQ mRNA levels in these cells.

Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long-term changes in neuronal phenotype and function. Although activity-dependent induction of immediate-early genes has been extensively studied, much less is known about the late-response genes. We have investigated the activity-dependent regulation of delta-opioid receptor (DOR) mRNA levels in NG108-15 cells. Transsynaptic activation was mimicked by depolarization with 55 mM KCl or veratridine. Both treatments lead to a time-dependent increase of DOR mRNA levels. Ca2+ entry through L-type voltage-dependent Ca2+ channels activated by depolarization appears to be involved, because L-type channel blockers reduced the induction of DOR expression. Ca2+ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN-62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization-induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin-dependent enzymes are involved in the activity-dependent induction of DOR gene expression. Induction of DOR gene expression by an activity-dependent increase in intracellular Ca2+ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.

The effects of activation of the adenylyl cyclase-protein kinase A pathway on the expression of delta-opioid receptor mRNA in the NG108-15 neuroblastoma x glioma cell line has been investigated. Activation of prostaglandin E1 (PGE1) receptors, which are positively coupled to adenylyl cyclase, resulted in a reduction in delta-receptor messenger RNA levels. Direct stimulation of adenylyl cyclase by forskolin or treatment of cells with the cyclic AMP analogue dibutyryl cyclic AMP (db-cAMP) mimicked the effect of PGE1. Down-regulation in receptor protein levels, as measured by loss of radioligand binding sites, was also observed and its extent correlated well with the decrease in the amount of delta-opioid receptor transcripts. D-Ser2-Leu-enkephalin-Thr6 (DSLET) inhibition of adenylyl cyclase activity was also diminished after db-cAMP treatment. Inhibitors of protein kinase A (PKA) partially reversed the PGE1- and db-cAMP-mediated repression of the delta-opioid receptor mRNA levels. The rate of degradation of delta-opioid receptor mRNA in the presence of actinomycin D was not altered in response to db-cAMP, suggesting that mRNA stability is not reduced by PKA action. The regulation of delta-opioid receptor mRNA levels by db-cAMP was not sensitive to the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis is not required in this process.

Levels of mRNA coding for mu and delta opioid receptors were evaluated by competitive PCR in regions of rat brain where adaptive changes in opioid regulation of adenylyl cyclase occur following chronic morphine treatment. Correlation between receptor protein and mRNA levels after exposure to full and partial agonists were also investigated in NG108-15 cells. Different basal gene expression levels of mu and delta opioid receptors were found in the caudate-putamen, nucleus accumbens, thalamus and periaquaductal gray. After continuous morphine treatment for 7 days both mu and delta opioid receptor mRNA levels were unchanged in all four regions compared with saline-treated controls. In NG108-15 cells marked down-regulation of delta opioid receptors as measured by radioligand binding was observed after 5 or 24 h full agonist (D-Ser3-Leuenkephalin; DSLET) treatment. Morphine, a partial agonist at delta receptors, did not reduce receptor number at either time point. The reduction in delta receptor binding after DSLET treatment was not accompanied by significantly diminished levels of delta receptor mRNA. Unaltered mu and delta receptor mRNA levels in brain after chronic morphine exposure make it unlikely that adaptive changes in the transcription of these genes play a role in the observed tolerance in opioid regulation of adenylyl cyclase.