Regulation of striatonigral prodynorphin peptides by dopaminergic agents

The primary purpose of this study was to examine the regulation of prodynorphin peptides by dopaminergic agents in the central nervous system. The indirectly acting catecholamine agonist D-amphetamine sulfate (AMPH) and the dopamine receptor antagonist haloperidol (HAL) were administered to rats across a variety of treatment schedules and drug doses. The striatum, substantia nigra and hippocampus were dissected and examined by radioimmunoassay for 5 different prodynorphin peptides, covering all 3 opioid domains in the prodynorphin precursor: dynorphin A(1-8) and dynorphin A(1-17) of the dynorphin A domain, dynorphin B(1-13) of the dynorphin B domain, and alpha-neo-endorphin and beta-neo-endorphin of the neo-endorphin domain. In addition, the proenkephalin peptide Met-enkephalin-arg6-gly7-leu8 (MERGL) was examined in the striatum. AMPH administered one hour prior to sacrifice caused a dose-dependent depletion of prodynorphin peptides in both the striatum and substantia nigra. In animals treated with AMPH once each day for 7 days and sacrificed 24 h later, a dramatic dose-dependent increase in prodynorphin peptides was observed in these brain regions. Animals treated with AMPH once each day for 7 days and sacrificed one hour after the final injection showed no changes in prodynorphin peptides. In addition to changes in individual prodynorphin peptides, AMPH treatment caused alterations in the relationships between intermediate peptides (dynorphin A(1-17) and alpha-neo-endorphin) and their immediate products (dynorphin A(1-8) and beta-neo-endorphin). AMPH caused no consistent changes in prodynorphin peptides in the hippocampus, or in MERGL in the striatum. Taken together these data suggest that acute dopaminergic activation causes depletion of dynorphins from striatonigral prodynorphin neurons, presumably due to dopamine-dependent release of these peptides; repeated activation causes repeated release, with a rebound increase in biosynthesis. HAL, in contrast to AMPH caused relatively subtle changes in striatonigral prodynorphin peptides. Although no significant changes in individual prodynorphin peptides were observed, HAL treatment caused a change in the relationship between dynorphin A(1-17) and dynorphin A(1-8), a change opposite in direction to that observed with AMPH treatment. As has been previously reported, repeated HAL administration caused a dose-dependent increase in the proenkephalin peptide MERGL. The relatively subtle effects of HAL on prodynorphin peptides suggests that tonic dopamine activity is not important in the regulation of striatonigral prodynorphin neurons. The potential functional and behavioral significance of the present results are discussed.     

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nogra, striatum (not Leu-enkephalinArg⁶) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg⁶ levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were effected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.     

Localization of striatal opioid gene expression,and its modulation by the mesostriatal dopamine pathway: An in situ hybridization study

In situ hybridization was used to study the macroscopic distribution and regulatory control of proenkephalin mRNA and prodynorphin mRNA in rat striatum. While proenkephalin mRNA was widely distributed throughout the striatum, levels of prodynorphin mRNA were highest in the medial and ventral portions of the striatum. Furthermore, in contrast to the results for proenkephalin mRNA, the levels of prodynorphin mRNA appeared higher in the nucleus accumbens than in the striatum. The mesostriatal dopaminergic pathway was destroyed by discrete, unilateral injection of 6-hydroxydopamine (6-OHDA) into either the substantia nigra or the neighboring ventral tegmental area (VTA). Lesions of the substantia nigra caused a dramatic ipsilateral increase in the hybridization signal for proenkephalin mRNA, but no change was observed in the hybridization signal for prodynorphin mRNA. Similar effects were seen with VTA lesions. Since destruction of the mesostriatal dopamine system elevates the levels of proenkephalin mRNA, but not of prodynorphin mRNA, in the striatal target neurons, it appears that the mesostriatal pathway exerts a tonic and selective suppression of striatal proenkephalin gene expression at the mRNA level.     

Dynorphin levels in parkinsonian patients: Leu-enkephalin production from either proenkephalin A or prodynorphin in human brain

When measured in postmortem parkinsonian brains, dynorphin levels were unchanged, as compared to control brains, in mesencephalic, striatal and corticolimbic areas. A significant reduction in Leu⁵-enkephalin and Met⁵-enkephalin levels had been previously observed in the pallidum and putamen whereas only Met⁵-enkephalin concentrations were decreased in the substantia nigra of parkinsonian brains. These data suggest that L-Enk could be generated either from proenkephalin A in the striatal areas or from prodynorphin in the nigral areas.     

Analysis of opioid and non-opioid end products of pro-dynorphin in the substantia nigra of the rat

The substantia nigra is among the richest pro-dynorphin terminal field regions in the rat brain. We therefore contrasted processing in this area to the known processing in the posterior pituitary. Fractionation of acid extracts of the posterior pituitary by gel filtration followed by analysis by radioimmunoassay indicated that the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) averaged 1:2. The levels of dynorphin A-related end products to alpha-neo-endorphin and bridge peptide (a 2K nonopioid end product of pro-dynorphin) were approximately equimolar; however, the levels of dynorphin B-sized material were 50% lower than dynorphin A levels. Similar analyses of acid extracts of the substantia nigra also indicated that the levels of dynorphin A, alpha-neo-endorphin, and bridge peptide were approximately equimolar. In this terminal field the levels of dynorphin B-sized material were approximately 60% lower than dynorphin A. A striking feature of the nigral system was that the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) averaged 1:16. Thus, in the nigra, dynorphin A(1-17) is primarily a biosynthetic intermediate rather than as an end product.     

Prodynorphin peptide distribution in the forebrain of the Syrian hamster and rat: a comparative study with antisera against dynorphin A, dynorphin B, and the C-terminus of the prodynorphin precursor molecule

The neuroanatomical distribution of the prodynorphin precursor molecule in the forebrain of the male Syrian hamster (Mesocricetus auratus) has been studied with a novel antiserum directed against the C-terminus of the leumorphin [dynorphin B (1-29)] peptide product. C-peptide staining in sections from colchicine-treated hamsters is compared to staining in sections from untreated animals. In addition, the pattern of C-peptide immunostaining in hamster brain is compared to that in the rat brain. Finally, the C-peptide immunolabeling patterns in hamsters and rats are compared to those obtained with antisera to dynorphin A (1-17) and dynorphin B (1-13). Areas of heaviest prodynorphin immunoreactivity in the hamster include the hippocampal formation, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, medial and central amygdaloid nuclei, ventral pallidum, substantia nigra, and numerous hypothalamic nuclei. Although this C-peptide staining pattern is similar to dynorphin staining reported previously in the rat, several species differences are apparent. Whereas moderate dentate gyrus granule cell staining and no CA4 cell staining have been reported in the rat hippocampal formation, intense immunostaining in the dentate gyrus and CA4 cell labeling are observed in the hamster. In addition, the medial preoptic area, bed nucleus of the stria terminalis, and medial nucleus of the amygdala stain lightly for prodynorphin-containing fibers and cells in the rat, compared to heavy cell and fiber staining in the hamster in all three of these regions. In the rat there is no differential staining between tissues processed with the C-peptide, dynorphin A, and dynorphin B antisera, but numerous areas of the hamster brain show striking differences. In most hamster brain areas containing prodynorphin peptides, the C-peptide antiserum immunolabels more cells and fibers than the dynorphin B antiserum, which in turn labels more cells and fibers than dynorphin A antiserum. However, exceptions to this hierarchy of staining intensity are found in the lateral hypothalamus, substantia nigra, arcuate nucleus, and habenula. The differences in staining patterns between rat and hamster are greatest when C-peptide antiserum is used; apparent species differences are present, though less pronounced, in dynorphin B- and dynorphin A-immunostained material.     

Primate model of Parkinson''s disease: alterations in multiple opioid systems in the basal ganglia

A motor disorder similar to idiopathic Parkinson's Disease develops in rhesus monkeys after several daily repeated doses of N-methyl-4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP). The concentrations of peptides derived from proenkephalin A, proenkephalin B, substance P and somatostatin were measured by specific radioimmunoassays in the basal ganglia of MPTP-treated monkeys. In MPTP-treated monkeys, dynorphin B concentration was reduced in the caudate. In the putamen, the concentrations of peptides derived from both proenkephalin A and proenkephalin B were decreased. In the globus pallidus, the concentrations of all opioid peptides tend to be increased, reaching significance only for alpha-neo-endorphin. In the substantia nigra, only Met-enkephalin concentration was reduced, while other peptides derived from either proenkephalin A or proenkephalin B were not changed. Substance P and somatostatin were not changed in any brain area examined. Some of the symptoms associated with Parkinson's Disease may be related to altered activity of endogenous opiates in basal ganglia.     

Co-localization and characterization of immunoreactive peptides derived from two opioid precursors in guinea pig adrenal glands

Peptides derived from both proenkephalin and prodynorphin have been identified in guinea pig adrenal medulla. In extracts of whole adrenal glands radioimmunoassays directed to the prodynorphin-derived peptides alpha-neoendorphin, dynorphin A, and dynorphin B detected high concentrations of immunoreactive material ranging from 113 to 216 pmol/gm. The concentrations measured by radioimmunoassays directed to the proenkephalin products met-enkephalin-Arg-Gly-Leu and met-enkephalin-Arg-Phe were 878 and 484 pmol/gm, respectively. No metorphamide or dynorphin(1-8) could be detected in the adrenals. Leucine-enkephalin immunoreactivity which can be generated from either prodynorphin or proenkephalin could also be measured in the extracts. Gel filtration showed the immunoreactive material, with the exception of that measured by the alpha-neoendorphin radioimmunoassay, to be predominantly of high molecular weight ranging from Mr = 3,000 to 12,000. Immunocytochemistry, using well characterized antisera to alpha-neoendorphin and met-enkephalin-Arg-Gly-Leu, demonstrated that the prodynorphin and proenkephalin products were present in the same cells in the medulla region of the gland. The results show that two opioid peptide precursors can be localized in the same cells and exhibit some common features in their processing. As a relatively homogeneous, localized system, the guinea pig adrenal gland should prove a valuable, in vivo model for the study of co-localized opioid precursors.     

Distribution and characterization of opioid peptides derived from proenkephalin A in human and rat central nervous system

In various areas of rat and human brain and spinal cord the distributions of opioid peptides derived from the proenkephalin A precursor, the heptapeptide [Met]enkephalin-Arg⁶-Phe⁷ (MERF), the octapeptide [Met]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MERGL), and bovine adrenal medulla dodecapeptide (BAM-12P), were determined by a combination of radioimmunoassay, gel filtration, and high-performance liquid chromatography.In the human central nervous system the highest concentrations were seen in the striatum (pallidum > caudate nucleus > putamen) and in substantia nigra, hypothalamus, and periaqueductal gray. Similarly, in rat brain high levels were found in striatum and hypothalamus.Bovine adrenal medulladocosa peptide (BAM-22P) only occurred in the rat brain, but could not be detected in human brain. No MERF, MERGL, BAM-12P, or BAM-22P could be found in either rat or human pituitary.In contrast to MERF, MERGL and BAM-12P, peptides derived from the proenkephalin B precursor, dynorphin₁₋₈ and dynorphin B, showed high concentrations only in substantia nigra and pallidum, but quite low levels in the other regions of human brain and spinal cord.The present study provides evidence that the proenkephalin A precursor known from adrenal medulla also exists in the rat and human central nervous system. Moreover, the identification of BAM-12P in these tissues indicates that cleavage of the precursor molecule must also involve sites different from those with paired basic amino acids.     

Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat

The effect of chronic neuroleptic treatment, using haloperidol or clozapine, on immunoreactive dynorphin peptide and substance P levels in basal ganglia of rats was examined. The drugs were administered i.p. in daily doses for 10 days (haloperidol 1 mg/kg and clozapine 10 mg/kg). Dynorphin A, dynorphin B and substance P were measured in substantia nigra, striatum, globus pallidus and hypothalamus using specific radioimmunoassays. The most prominent effects were observed with with clozapine which increased levels of all measured peptides in substantia nigra. Haloperidol only affected nigral substance P levels which declined, while nigral dynorphin peptide levels remained unchanged. In striatum, haloperidol slightly reduced dynorphin peptides while substance P was unaffected. Clozapine increased striatal substance P but the dynorphin peptides were not affected. Minor changes in dynorphin peptides found in globus pallidus and hypothalamus were not statistically reliable. Substance P was not changed in these structures after either of the two drugs. High molecular weight fragments (greater than or equal to 5,000) from the dynorphin precursor, proenkephalin B, were measured in substantia nigra and striatum using trypsin digestion and subsequent analysis of generated Leu-enkephalin-Arg6. These high molecular weight fragments were found to be affected in the same manner as the dynorphin peptides. This study indicates that the two types of neuroleptic drugs have different modes of interaction on peptide systems in basal ganglia of rats. Dynorphin peptides and substance P were also differentially affected.     

Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats

In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), γ-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 μM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 μM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2–4 mg/kg s.c.) or intracerebrally (1–100 μM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the μ-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-μ subtype of opioid receptors located on nigro-striatal DA neurones.     

The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord

Recurrent exposure to intermittent electrical foot-shock (30 min, twice daily) for 7 days caused an increase in immunoreactive (ir) dynorphin and ir-alpha-neo-endorphin in lumbar and cervical (but not thoracic) spinal cord as measured 16 h following the final session. At this time the level of ir-Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was also increased at the lumbar level. An acute foot-shock depleted spinal cord dynorphin in chronically stressed but not in naive rats. No alterations in levels of ir-dynorphin or ir-MEAGL were seen in discrete brain tissues. In contrast to the brain, where no effects were seen, the levels of beta-endorphin increased in both lobes of the pituitary. This change, however, was not accompanied by an alteration in levels of beta-endorphin in plasma. These data show that chronic foot-shock stress selectively influences particular pools of opioid peptides, predominantly those derived from proenkephalin B in the spinal cord and from proopiomelanocortin in the anterior pituitary. It is suggested that alterations observed in the spinal cord reflect enhanced activity of the proenkephalin B system in response to chronic nociceptive stimulation.     

GABAergic regulation of striatal opioid gene expression.

Peptides derived from prodynorphin and preproenkephalin are located in GABAergic striatal projection neurons. We have used nucleic acid hybridization techniques to investigate the role of GABA in the regulation of striatal opioid peptide gene expression. Rats were treated with the GABA-transaminase inhibitors aminooxy acetic acid, ethanolamine O-sulphate and gamma-vinyl-GABA for one week. The GABA levels in the striatum were significantly elevated after each treatment. The GABA-transaminase-inhibitors decreased the striatal levels of the opioid peptides met-enkephalin and dynorphin(1-8) and concomitantly decreased the concentrations of the mRNAs coding for proenkephalin and prodynorphin. These findings indicate that GABA exerts an inhibitory influence on prodynorphin and proenkephalin gene expression in the striatum. The mechanisms underlying these inhibitions are discussed.     

Systemic administration of kainic acid differentially regulates the levels of prodynorphin and proenkephalin mRNA and peptides in the rat hippocampus.

The effects of systemic kainic acid (KA) administration on hippocampal levels of prodynorphin and proenkephalin mRNA, as well as opioid peptides derived from these precursors, were evaluated. A single subcutaneous injection of KA induced a range of seizure states, from mild wet dog shakes to generalized motor seizures. Northern blot analysis of hippocampal mRNA revealed an increase in both prodynorphin and proenkephalin mRNA levels which corresponded to the intensity of the convulsions. Conversely, hippocampal levels of immunoreactive dynorphin A (1-8) and [Met]5-enkephalin were decreased as a function of seizure frequency and intensity. The time course of KA-induced alterations in prodynorphin and proenkephalin mRNA and peptide levels was also investigated. Hippocampal prodynorphin mRNA levels rose at a dramatic rate. At 3 h following KA administration, mRNA levels were maximally elevated approximately 13-fold. The levels decreased over a 48 h period, eventually reaching control values. In contrast, proenkephalin mRNA levels increased more slowly. At 24 h, a maximal 24-fold increase was observed. At 72 h after injection, proenkephalin mRNA levels were still slightly elevated. In the same experiment, immunoreactive enkephalin peptide levels, although somewhat decreased at 3-12 h, began to increase between 12 and 24 h after injection, and were still rising at 72 h. In marked contrast, immunoreactive dynorphin peptide levels ranged from 40% to 80% of control values at all times tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynorphin peptides in human substantia nigra

Stepwise processing of the prohormone for dynorphin-related peptides, proenkephalin B, may generate a large number of opioid peptides. It is therefore important to perform a chemical characterization of the immunoreactive dynorphin (ir-dyn) found in different tissues. In this study dynorphin peptides in human substantia nigra were characterized. Highly sensitive radioimmunoassays (RIA) directed against the C-terminals of dynorphin A (dyn A) and dynorphin B (dyn B) respectively, were used to measure the levels of dynorphin peptides. Tissue extraction was performed either with MeOH/0.1 M HCl (1/1) or 1 M HAc. Gel filtration on a Sephadex G-50 column revealed three peaks of ir-dyn B, the predominating one coeluting with synthetic dyn B. Ir-dyn A also appeared in three peaks, one of them (20-30%) coeluting with dyn A. The HAc extract contained much higher levels of ir-dyn B as compared to the MeOH/HCl extract, but approximately the same levels of ir-dyn A. Ion exchange separation of the HAc extract somewhat changed the size distribution pattern, with more ir coeluting with the synthetic peptides compared to other molecular weight forms.     

Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.     

Changes of proenkephalin and prodynorphin mRNAs and related peptides in rat brain during the development of deep prepyriform cortex kindling

The effects of deep prepyriform cortex (DPC) kindling on the amount of proenkephalin and prodynorphin mRNAs, Met5-enkephalin (ME) and dynorphin (DYN) in rat brain were examined. Animals received electrical stimulation of the DPC until two consecutive stage 2 seizures (S2) or stage 5 seizures (S5) were attained. The proenkephalin mRNA and ME contents in the entorhinal cortex were increased 24 h after S2 and also 5 min and 24 h post S5. In the hippocampus, the proenkephalin mRNA level was reduced 24 h after S2 but increased 5 min and 24 h after S5. Elevated hippocampal ME concentration was observed 24 h after S2 and S5. Similarly, the ME level in the frontal cortex was increased 24 h after S2 and S5 but the proenkephalin mRNA content was only elevated at S5. In the striatum, the proenkephalin mRNA level was slightly increased 24 h after S2 and S5, but no change in ME content was found. The amount of prodynorphin mRNA in the hippocampus was attenuated only at 24 h after S5, whereas DYN concentration was reduced 5 min after S5. No change in striatal DYN concentration was observed despite a slight elevation of prodynorphin mRNA 24 h post S2 and S5. Six weeks after the last seizure, no difference in ME and DYN was found between kindled and control animals. These findings indicate that the enkephalin-containing perforant pathway in the entorhinal cortex-hippocampal region is particularly sensitive to electrical stimulations applied to the DPC. Its role and importance in the development of kindling are discussed.     

Enkephalin-containing polypeptides in human cerebrospinal fluid

The chemical nature of peptides in human CSF with the enkephalin core sequence from proenkephalin A and proenkephalin B, was investigated. Direct measurements with radioimmunoassay (RIA) were run on enkephalin, enkephalin hexapeptides, dynorphin A, dynorphin A1-8 and dynorphin B. The hexapeptides occurred in about 3 times higher concentration than the corresponding enkephalins. The only dynorphin RIA which gave positive results was the one for dynorphin A. However, most dynorphin A immunoactive material showed higher apparent molecular weight (MW; 3 and 5 kdalton) than the standard (2 kdalton). To identify and quantitate every possible proenkephalin derived peptide with the enkephalin sequence, chromatographic fractions were treated with trypsin. The products, Leu-enkephalin-Arg6 (from proenkephalin B) and Met-enkephalin-Lys6/Arg6 (from proenkephalin A) were measured by specific RIAs and identified by HPLC. In the higher (greater than 5 kdalton) MW interval, there was about 10-fold higher yield of Met-enkephalyl than Leu-enkephalyl hexapeptides. In the intermediate 1-3 kdalton MW interval, most activity derived from proenkephalin B. Finally, from the low MW region, there was about 5 times more proenkephalin A peptides. The main dynorphin A peak at 5 kdalton was transferred to a major Leu-enkephalin-Arg6 peak by trypsin degradation. The data indicate the presence of a whole family of peptides from the two proenkephalin genes in human CSF. Precursors to the peptides supposed to be the active members in the proenkephalin families occur in relatively high concentrations and may provide good markers for activity in these peptide systems.     

Possible interactions between dynorphin and dopaminergic systems in rat basal ganglia and substantia nigra

Chronic haloperidol treatment markedly increases dynorphin-related peptide contents in caudate-putamen, globus pallidus and substantia nigra. Leu-enkephalin levels follow dynorphin-related peptide concentrations in these areas while Met-enkephalin-related peptide contents are unchanged in the substantia nigra following a similar treatment. An acute haloperidol injection had no effect on any opioid peptide levels in the basal ganglia. This suggests that Leu-enkephalin is likely to be derived from prodynorphin in the rat striatonigral pathway. Moreover, the Leu-enkephalin/dynorphin projection appears to be under striatal dopaminergic control.     

Expression of vasopressin and opiates but not of oxytocin genes studied by in situ hybridization in embryonic rat brain primary cultures

Expression of arginine-vasopressin (AVP), oxytocin (OT), dynorphin and enkephalin genes was studied with the in situ hybridization technique in embryonic rat brain serum-free cultures. Neurones were prepared from hypothalamus and extrahypothalamic structures of 16-day-old rat embryos. After 7 days in culture, AVP gene expression occurred in hypothalamic cultures only, whereas ProOT mRNAs were undetectable. By contrast, prodynorphin and proenkephalin mRNAs could be detected in both hypothalamic and extrahypothalamic cultures, however, with a higher number of cells containing proenkephalin mRNAs. These observations demonstrated that AVP, dynorphin and enkephalin, but not OT genes, can be expressed in cultures prepared from embryonic rat brain as young as 16 days old. This is the first report of an early expression of opioid peptide genes within the central nervous system suggesting that opioids could be involved in the early phases of nervous system development.