A novel soluble protein factor with non-opioid dynorphin A-binding activity

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.     

Evidence for a Selective Processing of Proenkephalin B into Different Opioid Peptide Forms in Particular Regions of Rat Brain and Pituitary

The distribution of five major products of proenkephalin B [dynorphin1-17, dynorphin B, dynorphin1-8, alpha-neo-endorphin and beta-neo-endorphin] was studied in regions of rat brain and pituitary. The distribution pattern of immunoreactive (ir) dynorphin B (= rimorphin) was found to be similar to that of ir-dynorphin1-17, with the highest concentrations being present in the posterior pituitary and the hypothalamus. HPLC and gel filtration showed the tridecapeptide dynorphin B to be the predominant immunoreactive species recognized by dynorphin B antibodies in all brain areas and in the posterior pituitary. In addition, two putative common precursor forms of dynorphin B and dynorphin1-17 with apparent molecular weights of 3,200 and 6,000 were detected in brain and the posterior pituitary. The 3,200 dalton species coeluted with dynorphin1-32 on HPLC. In contrast with all other tissues, anterior pituitary ir-dynorphin B and ir-dynorphin1-17 consisted exclusively of the 6,000 dalton species. Concentrations of dynorphin1-8 were several times higher than those of dynorphin1-17 in striatum, thalamus, and midbrain while posterior pituitary, hypothalamus, pons/medulla, and cortex contained roughly equal concentrations of these two opioid peptides. No dynorphin1-8 was detected in the anterior pituitary. Concentrations of beta-neo-endorphin were similar to those of alpha-neo-endorphin in the posterior pituitary. In contrast, in all brain tissues alpha-neo-endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of beta-neo-endorphin. These findings indicate that differential proteolytic processing of proenkephalin B occurs within different regions of brain and pituitary. Moreover, evidence is provided that, in addition to the paired basic amino acids -Lys-Arg- as the "typical" cleavage site for peptide hormone precursors, other cleavage signals also seem to exist for the processing of proenkephalin B.     

Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate

Dynorphins, endogeneous opioid neuropeptides, function as ligands to the opioid kappa receptors and also induce non-opioid effects in neurons, probably related to direct membrane interactions. We have characterized the structure transitions of dynorphins (big dynorphin, dynorphin A and dynorphin B) induced by the detergent sodium dodecyl sulfate (SDS). In SDS titrations monitored by circular dichroism, we observed secondary structure conversions of the peptides from random coil to α-helix with a highly aggregated intermediate. As determined by Fourier transform infrared spectroscopy, this intermediate exhibited β-sheet structure for dynorphin B and big dynorphin. In contrast, aggregated dynorphin A was α-helical without considerable β-sheet content. Hydrophobicity analysis indicates that the YGGFLRR motif present in all dynorphins is prone to be inserted in the membrane. Comparing big dynorphin with dynorphin A and dynorphin B, we suggest that the potent neurotoxicity of big dynorphin could be related to the combination of amino acid sequences and secondary structure propensities of dynorphin A and dynorphin B, which may generate a synergistic effect for big dynorphin membrane perturbing properties. The induced aggregated α-helix of dynorphin A is also correlated with membrane perturbations, whereas the β-sheet of dynorphin B does not correlate with membrane perturbations.     

Ultrastructural demonstration of synaptic connections between calcitonin gene-related peptide immunoreactive axons and dynorphin A(1–8) immunoreactive dorsal horn neurons in a rat model of peripheral inflammation and hyperalgesia

Synaptic contact between dynorphin A(1–8)-like immunoreactive lamina V spinal neurons and calcitonin gene-related peptide-like immunoreactive axon terminals was demonstrated using the immuno-electron microscopic mirror technique in a rat model of peripheral inflammation and hyperalgesia. Adjacent tissue sections were immunocytochemically labeled for either dynorphin A(1–8) or calcitonin gene-related peptide and examined at the electron microscopic level for the presence of synaptic contacts. The results suggest that some opioid neurons which exhibit a dynamic increase in dynorphin peptide associated with peripheral inflammation and hyperalgesia receive direct monosynaptic input from presumptive nociceptive primary afferents.     

Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry

Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson’s disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value.Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1–6; Leu-Enk-Arg), (1–9), (2–13), and (7–13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results.Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1–7) in the cortex and dynorphin B (2–13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1–7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2–13).This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections.     

Regional Distribution of Methionine-Enkephalin-Arg6-Phe7 in the Rat Brain: Comparative Study with the Distribution of Other Opioid Peptides

The distribution of the opioid peptide methionine-enkephalin-arginine6-phenylalanine7 (M-Enk-Arg6-Phe7) has been investigated in various structures of the rat brain by using a highly specific radioimmunoassay (RIA). Immunoreactive M-Enk-Arg6-Phe7 has been further characterized by high performance liquid chromatography. The levels of M-Enk-Arg6-Phe7 in various structures of the rat brain were compared with the levels of several other opioid peptides, including methionine-enkephalin (M-Enk), leucine-enkephalin (L-Enk), dynorphin 1-13, and alpha-neoendorphin, which were also measured by RIA. There was a close relationship between the distribution of M-Enk-Arg6-Phe7 immunoreactive material (ir), M-Enk ir, and L-Enk ir. The distribution of dynorphin 1-13 ir and alpha-neoendorphin ir appeared to be distinct from that of the enkephalin group. These results are in agreement with recent reports on the cloning and sequencing of the c-DNA coding for the prohormones, in which it has been hypothesized that M-Enk-Arg6-Phe7 and M-Enk are synthesized by the same precursor, called proenkephalin, and that dynorphin-related peptides and alpha-neoendorphin arise from a separate precursor, prodynorphin.     

Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates

Abstract: Previously, opioid peptide analogues, β-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via κ-and μ-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µ M , all dynorphins tested and β-endorphin inhibited [³H]thymidine incorporation into DNA by 20–38% in 7-day rat brain cell aggregates. The putative ε-antagonist β-endorphin (1–27) did not prevent the effect of β-endorphin, suggesting that the ε-receptor is not involved in opioid inhibition of DNA synthesis. The κ-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ-receptor subtype. The IC₅₀ value of 0.1 n M estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.     

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.     

Conversion of Leumorphin (Dynorphin B-29) to Dynorphin B and Dynorphin B-14 by Thiol Protease Activity

Dynorphin B (rimorphin) is formed from leumorphin (dynorphin B-29) by the action of a thiol protease from rat brain membranes, in a single step. This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. We have observed that in addition to dynorphin B, dynorphin B-14 is formed from dynorphin B-29. Among the various protease inhibitors tested, none except p-chloromercuribenzensulfonic acid inhibited the formation of the two products. Both temperature and pH had similar effects on the formation of dynorphin B-14 and dynorphin B. The inhibitory potencies of adrenocorticotropic hormone, peptide E, and dynorphin A were virtually identical for the formation of the two products. These results suggest that the same enzyme may be responsible for the formation of dynorphin B-14 and dynorphin B.     

Possible involvement of dynorphin A release via mu1-opioid receptor on supraspinal antinociception of endomorphin-2

It has been demonstrated that the antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated through mu-opioid receptors. Moreover, though endomorphins do not have appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine markedly blocks the antinociception induced by i.c.v.- or i.t.-injected endomorphin-2, but not endomorphin-1. These evidences propose the hypothesis that endomorphin-2 may initially stimulate the mu-opioid receptors, which subsequently induces the release of dynorphins acting on kappa-opioid receptors to produce antinociception. The present study was performed to determine whether the release of dynorphins by i.c.v.-administered endomorphin-2 is mediated through mu-opioid receptors for producing antinociception. Intracerebroventricular pretreatment with an antiserum against dynorphin A, but not dynorphin B or alpha-neo-endorphin, and s.c. pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine dose-dependently attenuated the antinociception induced by i.c.v.-administered endomorphin-2, but not endomorphin-1 and DAMGO. The attenuation of endomorphin-2-induced antinociception by pretreatment with antiserum against dynorphin A or nor-binaltorphimine was dose-dependently eliminated by additional s.c. pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine or a selective mu(1)-opioid receptor antagonist naloxonazine at ultra low doses, which are inactive against mu-opioid receptor agonists in antinociception, suggesting that endomorphin-2 stimulates distinct subclass of mu(1)-opioid receptor that induces the release of dynorphin A acting on kappa-opioid receptors in the brain. It concludes that the antinociception induced by supraspinally administered endomorphin-2 is in part mediated through the release of endogenous kappa-opioid peptide dynorphin A, which is caused by the stimulation of distinct subclass of mu(1)-opioid receptor.     

Nitric oxide synthase inhibitors influence dynorphin A (1-17) immunoreactivity in the rat brain following hyperthermia

Summary.  The possibility that nitric oxide synthase (NOS) inhibitors influence dynorphin immunoreactivity following hyperthermia was examined in a rat model using a pharmacological approach. Previous reports from our laboratory show that hyperthermia induces an upregulation of NOS in several brain regions that seems to be instrumental in causing cell injury. Recent reports suggest that nitric oxide (NO) can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. Since dynorphin is neurotoxic in different animal models of brain or spinal cord injury, it may be that the peptide will contribute to the cell injury in hyperthermia. The present investigation was carried out to determine whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of NOS will influence the peptide distribution in the brain following heat stress. Rats subjected to hyperthermia at 38°C for 4 h in a biological oxygen demand incubator (BOD) resulted in a marked upregulation of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. Pretreatment of rats with two potent NOS inhibitors, L-NAME (30 mg/kg/day, i.p. for 7 days) or L-NMMA (35 mg/kg/day, i.p. for 7 days) significantly attenuated the dynorphin immunoreactivity in the brain. These drugs were also able to reduce hyperthermia induced blood-brain barrier (BBB) permeability, brain edema formation and cell injury. Taken together, our results suggest that (i) hyperthermia has the capacity to upregulate dynorphin immunoreactivity in the brain, (ii) inhibition of NOS considerably attenuates the dynorphin immunoreaction following heat stress and (iii) upregulation of dynorphin is somehow contributing to hyperthermia induced brain damage, not reported earlier. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Chromatographic characterization of dynorphin and [Leu5]enkephalin immunoreactivity in guinea pig and rat testis

Tissues of the reproductive tract have been shown to contain mRNAs coding for pro-opiomelanocortin (POMC), pro-enkephalin and pro-dynorphin. However, the amounts of immunoreactive opioid peptides in these tissues are low, and in the case of the enkephalins and dynorphin, the molecular species responsible for the immunoreactivities have not been characterized. The chromatographic properties of dynorphin and enkephalin immunoreactivities in extracts of guinea pig and rat testis have therefore been determined. Dynorphin A and dynorphin B immunoreactivity was heterogeneous, with a significant amount attributable to high-molecular-weight forms. About 20% of the dynorphin A immunoreactivity, and about 40% of the dynorphin B immunoreactivity, in guinea pig testis extracts behaved as authentic dynorphin A or B, respectively during fractionation by ion exchange, gel filtration and high-performance liquid chromatography. Both high- and low-molecular-weight forms of [Leu5]enkephalin immunoreactivity were also present, with roughly 50-70% of the immunoreactivity attributable to low-molecular-weight forms. In extracts of guinea pig testis only a small part of this immunoreactivity eluted as authentic [Leu5]enkephalin during high-performance liquid chromatography. In rat testis most of the low-molecular-weight [Leu5]enkephalin immunoreactivity behaved as the authentic peptide. These results confirm that opioid peptides are produced in guinea pig and rat testis, and demonstrate that immunoreactive forms of the peptides similar to those found in brain and pituitary are present in the tissue.     

Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin

Intracerebroventricular administration of 20, 40 and 60 nmol of dynorphin (1-13) produced analgesia, as assessed by flinch/jump response to footshock, and hypothermia in the rat. Rats developed tolerance to both the analgesic and thermic effects of the 20 nmol dose of dynorphin. Dynorphin and beta-endorphin showed cross-tolerance with respect to their analgesic but not their thermic effects. Dynorphin and morphine also produced cross-tolerant analgesic effects. Naloxone (10 mg/kg, IP) completely blocked the barrel rolling produced by 20 nmol dynorphin but did not alter its analgesic or thermic effects.     

Neuropeptide processing by single-step cleavage: conversion of leumorphin (dynorphin B-29) to dynorphin B

Dynorphin B (rimorphin) is formed from dynorphin B-29 (leumorphin) by the action of a thiol protease from rat brain membranes. This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. In isotope dilution experiments we find that the radioactivity from radiolabelled dynorphin B-29, which appears in dynorphin B during incubation with the enzyme preparation, is not diminished by addition of a high concentration of dynorphin B-Arg14. Moreover, in pulse-chase experiments, radioactivity that appeared in dynorphin B-Arg14 did not decrease, nor did the radioactivity in dynorphin B increase, after chasing with a high concentration of non-radioactive dynorphin B-29. These results indicate that although some dynorphin B-Arg14 is formed by the impure enzyme preparation, it is not an intermediate in the conversion of dynorphin B-29 to dynorphin B. Thus the formation of dynorphin B does not involve the action of a trypsin-like enzyme followed by removal of arginine-14 by a carboxypeptidase B-like enzyme. It appears that a single enzyme converts dynorphin B-29 to dynorphin B in a single step.     

Spinal Cord Dynorphin Precursor Intermediates Decline During Late Gestation

Abstract: This laboratory has previously reported that the maternal opioid analgesia associated with pregnancy and parturition is mediated, at least in part, by a maternal spinal cord dynorphin/κ opioid system. This analgesia is accompanied by an increase in dynorphin peptides (1–17 and 1–8) in the lumbar spinal cord. Levels of trypsin-generated arginine⁶-leucine-enkephalin (Leu-Enk-Arg)-immunoreactive determinants were also determined and used to reflect the content of dynorphin precursor intermediates. In spinal tissue, the amount of dynorphin A (1–17) contained in the form of precursor is, at a minimum, 10-fold higher than the content of mature dynorphin A (1–17) or dynorphin (1–8). During gestational day 22, the content of dynorphin precursor is reduced significantly (∼50%). The decline in the magnitude of dynorphin precursor intermediates in the spinal cord of pregnant rats vastly exceeds the magnitude of increase in the content of dynorphin peptides (1–17 and 1–8). This difference can best be explained by postulating a corresponding increase in the rate of release of spinal cord dynorphin (1–17). It is suggested that enhanced processing of dynorphin precursor intermediates represents the initial biochemical level of adaptation of spinal dynorphin neurons to increased demands of pregnancy.     

Membrane leakage induced by dynorphins

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors and induce non-opioid excitotoxic effects. Here we show that big dynorphin and dynorphin A, but not dynorphin B, cause leakage effects in large unilamellar phospholipid vesicles (LUVs). The effects parallel the previously studied potency of dynorphins to translocate through biological membranes. Calcein leakage caused by dynorphin A from LUVs with varying POPG/POPC molar ratios was promoted by higher phospholipid headgroup charges, suggesting that electrostatic interactions are important for the effects. A possibility that dynorphins generate non-opioid excitatory effects by inducing perturbations in the lipid bilayer of the plasma membrane is discussed.     

Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Immunoreactive dynorphin in the rat adenohypophysis consists exclusively of 6000 dalton species

Immunoreactive dynorphin in the rat adenohypophysis exhibited an apparent molecular size of approximately 6 kilodaltons (6 kDal) upon characterization by gelfiltration. Essentially no dynorphin-related peptides with a molecular size of dynorphin-(1–17) or dynorphin-(1–8), which constitute the great majority of ir-dynorphin in the rat neurointermediate pituitary and brain, could be detected in the adenohypophysis. The possible presence of putative aggregations of smaller peptides were largely excluded by rechromatography under denaturating conditions in 4 M guanidine-HCL. SDS-gelelectrophoresis revealed that 6 kDal dynorphin consisted of at least three components of similar molecular size. From the predominant form of 6 kDal dynorphin, leucine-enkephalin could be liberated by sequential enzymatic cleavage with trypsin and carboxypeptidase B.