Subcellular Distribution of Opioid Peptides in Rat Hypothalamus and Pituitary

Homogenates of rat anterior lobe (AL) and neurointermediate lobe (NIL) pituitary and rat hypothalamus were subjected to subcellular fractionation and density gradient centrifugation. The subcellular distribution of immunoreactive dynorophin A (ir-Dyn A) in NIL was found to be similar to that of ir-arginine vasopressin (ir-AVP). ir-Dyn A migrated as a discrete band on sucrose density gradients, which corresponded in sedimentation rate to that of ir-AVP, suggesting that these two peptides are stored within organelles of similar size and density. Two other products of prodynorphin, ir-alpha-neoendorphin (ir-alpha-nEND) and ir-Dyn A-(1-8) also comigrated with ir-AVP. ir-[Leu5]-enkephalin (ir-LE), which may be a product of prodynorphin or proenkephalin, was also found to migrate in this region of the gradient. When a homogenate of rat hypothalamus was prepared using a method that has been developed for synaptosome isolation, ir-Dyn A was found to comigrate with Na+/K+-activated adenosine triphosphatase (Na/K-ATPase), a synaptosomal marker enzyme. Using a more concentrated homogenate ir-Dyn A was found to migrate to a less dense region where peptide-containing synaptic vesicles have previously been localized. When a synaptosomal preparation was lysed in hypotonic solution a shift was seen in the migration rate of ir-Dyn A to this region of the gradient (containing putative synaptic vesicles). Thus the bulk of hypothalamic dynorphin appears to be present within synaptosome-like structures which, upon lysis, release a less dense, smaller subcellular organelle corresponding in sedimentation characteristics to other types of peptide-containing synaptic vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postmortem Changes in Rat Brain Extracellular Opioid Peptides Revealed by Microdialysis

Microdialysis combined with a solid-phase radioimmunoassay was used to monitor changes in extracellular opioid peptide levels in the rat globus pallidus/ventral pallidum as a result of terminal brain ischemia. Ischemia was induced by anesthetic overdose or by severance of blood vessels supplying the brain. In control animals the recovered immunoreactivity increased an average of 13-fold in the 30-min sample following anesthetic overdose. Perfusion of a calcium-free, 10 mM EGTA-containing medium through the dialysis probe significantly attenuated the amplitude of this response, with the average increase being only threefold. Shorter sampling intervals (5 min) indicated that release of opioid peptide material into the extracellular environment occurs within the first 5 min of ischemia resulting from severance of the blood supply to the brain. HPLC analysis identified the majority of the postmortem-induced immunoreactive material as Met- and Leu-enkephalin.     

Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α-N-Acetyl Derivative

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH₂ terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ?15 fmol of β-endorphin per incubation tube, i.e., ? 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH₂ terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.     

Stress Impacts the Regulation Neuropeptides in the Rat Hippocampus and Prefrontal Cortex

Adverse life experiences increase the lifetime risk to several stress‐related psychopathologies, such as anxiety or depressive‐like symptoms following stress in adulthood. However, the neurochemical modulations triggered by stress have not been fully characterized. Neuropeptides play an important role as signaling molecules that contribute to physiological regulation and have been linked to neurological and psychiatric diseases. However, little is known about the influence of stress on neuropeptide regulation in the brain. Here, we have performed an exploratory study of how neuropeptide expression at adulthood is modulated by experiencing a period of multiple stressful experiences. We have targeted hippocampus and prefrontal cortex (PFC) brain areas, which have previously been shown to be modulated by stressors, employing a targeted liquid chromatography‐mass spectrometry (LC‐MS) based approach that permits broad peptide coverage with high sensitivity. We found that in the hippocampus, Met‐enkephalin, Met‐enkephalin‐Arg‐Phe, and Met‐enkephalin‐Arg‐Gly‐Leu were upregulated, while Leu‐enkephalin and Little SAAS were downregulated after stress. In the PFC area, Met‐enkephalin‐Arg‐Phe, Met‐enkephalin‐Arg‐Gly‐Leu, peptide PHI‐27, somatostatin‐28 (AA1‐12), and Little SAAS were all downregulated. This systematic evaluation of neuropeptide alterations in the hippocampus and PFC suggests that stressors impact neuropeptides and that neuropeptide regulation is brain‐area specific. These findings suggest several potential peptide candidates, which warrant further investigations in terms of correlation with depression‐associated behaviors.     

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

Abstract: The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague-Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c, twice daily for 10 days), and total cytochrome P450 content, the activity of N ADPH-cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine-injected rats: the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH-cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213-227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH-cytochrome c reductase, BROD, and PROD, and levels of P4502B1 /2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.     

Down-Regulation of Decapping Protein 2 Mediates Chronic Nicotine Exposure-Induced Locomotor Hyperactivity in Drosophila

Long-term tobacco use causes nicotine dependence via the regulation of a wide range of genes and is accompanied by various health problems. Studies in mammalian systems have revealed some key factors involved in the effects of nicotine, including nicotinic acetylcholine receptors (nAChRs), dopamine and other neurotransmitters. Nevertheless, the signaling pathways that link nicotine-induced molecular and behavioral modifications remain elusive. Utilizing a chronic nicotine administration paradigm, we found that adult male fruit flies exhibited locomotor hyperactivity after three consecutive days of nicotine exposure, while nicotine-naive flies did not. Strikingly, this chronic nicotine-induced locomotor hyperactivity (cNILH) was abolished in Decapping Protein 2 or 1 (Dcp2 or Dcp1) -deficient flies, while only Dcp2-deficient flies exhibited higher basal levels of locomotor activity than controls. These results indicate that Dcp2 plays a critical role in the response to chronic nicotine exposure. Moreover, the messenger RNA (mRNA) level of Dcp2 in the fly head was suppressed by chronic nicotine treatment, and up-regulation of Dcp2 expression in the nervous system blocked cNILH. These results indicate that down-regulation of Dcp2 mediates chronic nicotine-exposure-induced locomotor hyperactivity in Drosophila. The decapping proteins play a major role in mRNA degradation; however, their function in the nervous system has rarely been investigated. Our findings reveal a significant role for the mRNA decapping pathway in developing locomotor hyperactivity in response to chronic nicotine exposure and identify Dcp2 as a potential candidate for future research on nicotine dependence.     

Role of Acetylcholine Receptors and Dopamine Transporter in Regulation of Extracellular Dopamine in Rat Carotid Body Cultures Grown in Chronic Hypoxia or Nicotine

Abstract: Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for ～12 days in normoxia (Nox; 20% O₂), chronic hypoxia (CHox; 6% O₂), or chronic nicotine (CNic; 10 µM nicotine, 20% O₂), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (～15-min) exposure to hypoxia (5% O₂; ～6× basal), high extracellular K⁺ (30 mM; ～10× basal), nomifensine (1 µM; a selective DA uptake inhibitor; ～3× basal), and nicotine (100 µM; ～5× basal), but not methylcholine (300 µM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (～9× control), the stimulatory effect of high K⁺ (3–4× basal) and acute hypoxia (～2× basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (～9× control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (～2× basal), nicotine, nomifensine, and high K⁺ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 µM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 µM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Differential Regulation of Neuronal Nicotinic Receptor Binding Sites Following Chronic Nicotine Administration

Abstract: The deleterious effect of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic neurons of the substantia nigra is well established. In addition, increased glutamatergic drive to basal ganglia output nuclei is considered a likely contributor to the pathogenesis of Parkinson's disease. One possibility for the increased excitatory tone may be related to an impairment in glutamate uptake. As astrocytes possess efficient transport mechanisms for both MPTP and glutamate, we have examined the effect of this agent on d -aspartate uptake into these cells. Treatment of cultures with 50 µ M MPTP for 24 h decreased uptake by 39%. Kinetic analysis revealed that this effect was due to a 35% decrease in V _max with no change in the K _m. Treatment with deprenyl, a monoamine oxidase B inhibitor, produced a complete reversal of MPTP-induced uptake inhibition, but was ineffective following exposure of cells to the MPTP metabolite, 1-methyl-4-phenylpyridinium (MPP⁺). Removal of MPTP from cultures resulted in a complete restoration of glutamate uptake after 24 h. These results show that MPTP reversibly compromises glutamate uptake in cultured astrocytes, which is dependent on the conversion of MPTP to MPP⁺. Such findings suggest that the glutamate transporter in astrocytes plays an important role in MPTP-induced neurotoxicity and possibly in parkinsonism.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Summary Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Effects of Chronic Morphine Treatment on β-Endorphin-Related Peptides in the Caudal Medulla and Spinal Cord

Abstract: The effects of chronic morphine treatment on β-endorphin (βE)-immunoreactive (βE-ir) peptide levels were determined in the rat caudal medulla and different areas of the spinal cord. Seven days of morphine pelleting had no effect on total βE-ir peptides in the caudal medulla. In contrast, it significantly increased βE-ir peptide concentrations in the cervical and thoracic regions of the spinal cord compared with placebo-pelleted controls, whereas in the lumbosacral region this trend did not reach statistical significance. Injections of the opiate receptor antagonist naloxone 1 h before the rats were killed had no effect on the morphine-induced increases in the cord. Chromatographic analyses revealed that enzymatic processing of βE-related peptides in the spinal cord seemed unaffected by the morphine and/or naloxone treatments. In light of previous data showing that morphine down-regulates βE biosynthesis in the hypothalamus, the present results suggest that the regulation of βE-ir peptides in the spinal cord is distinct from that found in other CNS areas. These data provide support for previous results suggesting that βE-expressing neurons may be intrinsic to the spinal cord.     

Developmental Regulation of Neuregulin1 Isoforms and erbB Receptor Expression in Intact Rat Dorsal Root Ganglia

Neuregulins (NRGs) are a family of growth factors which bind to the erbB family of tyrosine kinase receptors. The exact nature and interaction of specific NRG isoforms and erbB receptors that occur during the development of the nervous system have not been reported. In order to better understand the role that different NRG isoforms and erbB receptors play in the differentiation, proliferation, and survival of neurons and glial cells, we isolated protein and mRNA from dorsal root ganglia of rat pups between embryonic day (E) 13 and postnatal day (P) 15. The relative expression levels of the NRGs and erbB receptors for the different time points were compared using both Western and RT-PCR analyses. NRG1-type1α protein levels were highest at E-13 and then decreased by approximately 40% and remained constant through P-15. In contrast, mRNA levels for NRG1-type1α remained constant from E-15 to P-15. The protein levels for NRG1-type 1β were similar to NRG1-type1α at E-13 with an approximate 40% increase in the levels at E-15 and E-17 followed by a decrease to E-13 levels for the remainder of the developmental time periods. The mRNA levels for NRG1-type1β remained constant from E-15 to P-15. The protein and mRNA expression patterns for each erbB receptor were distinctive. The protein levels for erbB-2 were highest at E-19 while erbB-3 levels were highest at E-17 and E-18. ErbB-4 protein levels were highest at E-13 and decreased through P-15. The developmental pattern for erbB-2 and erbB-4 mRNA levels had no relation to that of the corresponding protein levels while the mRNA levels for erbB-3 were highest at E-17 and E-18 similar to the pattern observed for the erbB-3 protein levels. We concluded that both NRG and erbB expression in dorsal root ganglia are mostly translationally controlled and that NRG1 isoforms and their erbB receptors are not coordinately regulated. Special issue article in honor of Dr. George DeVries.     

Effects of Chronic and Subchronic Nicotine on Tyrosine Hydroxylase Activity in Noradrenergic and Dopaminergic Neurones in the Rat Brain

Chronic nicotine (0.8 mg/kg by daily subcutaneous injection) over a 7 to 28-day period was found to increase the activity of tyrosine hydroxylase in predominantly noradrenergically innervated regions but not in dopaminergic projection areas. Increases in tyrosine hydroxylase activity were observed in dopaminergic cell body regions only after nicotine treatment for 3 to 5 days. The increase in tyrosine hydroxylase activity in noradrenergic neurones was evident first in the cell bodies in the locus coeruleus from 3 to 7 days, reaching 223% of control activities, and was followed by increases of up to 205% in the terminals up to 3 weeks later. It was then established that nicotine for 7 days was sufficient to increase the activity of the enzyme to the same extent in the terminals at 21 days even without further nicotine administration. This is consistent with axonal transport preceded by induction of the enzyme in noradrenergic cell bodies, whereas "delayed activation" might account for the transient effect seen in dopaminergic cell body regions. The response in the locus coeruleus to nicotine for 7 days was completely blocked by daily preinjection with mecamylamine but not with hexamethonium, which is consistent with the effect of nicotine on tyrosine hydroxylase being mediated by central nicotinic receptors.     

Regulation of Endogenous ADP-Ribosylation by Acute and Chronic Lithium in Rat Brain

Abstract: In the present study, we investigated the effects of lithium on endogenous ADP-ribosylation in rat brain. It was found that addition of lithium in vitro inhibits endogenous ADP-ribosylation activity in extracts of frontal cortex at therapeutically relevant concentrations. Inhibition is observed at concentrations as low as 0.3 m M and is maximal at 1 m M when 50% inhibition is obtained. A similar degree of inhibition of endogenous ADP-ribosylation was observed for all substrate proteins identified, including G_sα, suggesting that lithium's effect may be achieved at the level of ADP-ribosyltransferases and not specific substrate proteins. In contrast to lithium, chloride salts of sodium and potassium do not alter endogenous ADP-ribosylation activity in frontal cortex. To assess the possible in vivo relevance of this in vitro action of lithium, we studied the effect of chronic lithium administration on levels of endogenous ADP-ribosylation in frontal cortex. It was found that chronic lithium treatment, in contrast to the inhibitory effect of the drug in vitro, produced a >35% increase in endogenous ADP-ribosylation activity. A similar degree of increase was observed for all of the substrate proteins identified. These novel findings raise the possibility that certain endogenous ADP-ribosyltransferases are among the acute targets of lithium in the brain and that adaptations in these enzymes may be part of the mechanisms underlying lithium's long-term effects on brain function.     

Dynorphin-Related Opioid Peptides in the Neurointermediate Pituitary of Rats Are Not α-N-Acetylated

Immunoreactive dynorphin in the neurointermediate pituitary of rats was found to consist of four different molecular weight forms. The three larger molecular weight forms, with apparent molecular weights of 4800, 3200, and 1700, constituted more than 80% of the total dynorphin immunoreactivity, and each liberated leucine-enkephalin but not alpha-N-acetyl-leucine-enkephalin upon enzymatic treatment with trypsin followed by carboxypeptidase B. Only a minor portion of the smallest dynorphin-related molecular weight form, dynorphin-(1-8), released alpha-N-acetyl-leucine-enkephalin upon enzymatic cleavage. This suggests that the vast majority of dynorphin-related peptides in the rat neurointermediate pituitary is not alpha-N-acetylated. The exceptionally high opiate-like activity of the molecular weight 1700 dynorphin suggests that this dynorphin-related opioid peptide may constitute the major part of opioid activity in the neurointermediate pituitary of rats.     

反义c－fos和c－jun抑制IL－1诱导阿片肽分泌

白细胞介素-1(IL-1)及阿片肽作为神经调质参与了神经细胞兴奋性毒性作用.以大鼠大脑皮层神经细胞为研究对象,探讨了IL-1、阿片肽和c-fos、c-jun表达产物之间的关系.结果表明,IL-1β能诱导大脑皮层神经细胞c-fos、c-jun mRNA瞬时短暂表达,15min增高,30min达高峰,c-fos mKNA 2h回至基线水平,c-jun mRNA 8h回至基线水平;联合应用c-fos、c-jun反义寡核苷酸能部分抑制IL-1诱导的大脑皮层神经细胞脑啡肽及β-内啡肽分泌增加,呈一定量效关系,相应意义寡核苷酸无抑制作用.提示IL-1促进大脑皮层神经细胞脑啡肽及β-内啡肽分泌作用部分受Fos和Jun蛋白调控.