Effect of dermorphin and morphine on the sympathetic and cardiovascular system of the pithed rat

Dermorphin is a recently discovered opioid peptide which is unique in having a D-amino acid in its sequence. Dermorphin binding sites have been shown in central and peripheral organs and central administered dermorphin produces profound autonomic responses. The purpose of this study was to examine the effect of intravenous dermorphin on heart rate and blood pressure of the pithed rat in basal condition and in response to controlled sympathetic stimulation. Also, since dermorphin is a selective mu-receptor agonist, its effects were compared to morphine, an opiate selective for mu receptors. Dermorphin (0.0001-10 mumol/kg, i.v) or morphine (1-10 mg/kg) had no effect on basal heart rate or blood pressure and failed to modify sympatho-adreno-medullary evoked pressor and tachycardic responses. Furthermore, dermorphin or morphine did not affect the increase in plasma norepinephrine and epinephrine in response to spinal cord stimulation. It is concluded that the dermorphin and morphine have no direct peripheral effects on heart rate or blood vessel tone nor do these mu-receptor agonists have any effect on norepinephrine and epinephrine release from the sympathetic nerves and the adrenal medulla in the rat.     

Dermorphin-like immunoreactivity in guinea pig and rat stomach

Dermorphin is an opioid peptide containing one D-amino acid isolated from amphibian skin. We examined the presence of dermorphin-like immunoreactivity (DMP-IR) in mammalian brain and tissues using an antiserum developed in our own laboratory which works at a dilution of 1:120,000 for Radioimmunoassay (RIA) and can detect 2.1 pg of dermorphin. We were unable to find DMP-IR in guinea pig, rat and toad brain and rat and toad spinal cord either extracted with methanol or with HCl. The guinea pig and rat stomach contained significant amounts of DMP-IR (228.9 pg/mg protein and 97.5 pg/mg protein respectively) when extracted with HCl but not with methanol. The DMP-IR we detected in the stomach is not dermorphin per se. A single peak of DMP-IR was found on Sephadex G-25 gel filtration. When re-chromatographed on reverse phase HPLC, this peak of DMP-IR gave rise to three peaks of DMP-IR.     

Peripheral opioid receptors mediate gastrointestinal secretory and motor effects of dermorphin N-terminal tetrapeptide (NTT) in the dog

Dermorphin N-terminal-tetrapeptide-amide (NTT) increased both basal and pentagastrin- or histamine-induced secretion in conscious dogs chronically implanted with both gastric fistulae and Heidenhain pouches. These excitatory effects were significantly prevented by the opioid receptor antagonists naloxone and N-methyl-levallorphan-methanesulphonate. In conscious dogs fitted with electrodes and strain-gauges in different parts of gastrointestinal tract, a premature phase III of the migrating myoelectric complex (MMC) in the duodeno-jejunum was triggered by NTT, while the activity of the antrum was not significantly modified. Further, the peptide enhanced the contractile activity of both proximal and distal portions of the colon, including a long-lasting period of increased muscle tone on the distal colon. Either naloxone or N-methyl-levallorphan-methanesulphonate completely prevented motor effects of NTT on gastrointestinal tract. It is concluded that NTT displays significant opiate-like activity on gastric acid secretion and intestinal motility of the dog by activating peripheral mu opioid receptors.     

Synthesis and opioid activity of novel tetrapeptides analogous to sequence (1-4) of dermorphin

Seven new tetrapeptides analogous to (1-4) sequence of dermorphin were synthesized and evaluated for their opioid activity. The peptides were synthesized by the solution phase method. Their opioid activity revealed that peptides II and V were the most potent in the analgesia test as well as in the peripheral assays. Peptide II was most active in the guinea pig ileum assay, whereas peptide VI was 2763 times more selective for mu-receptors.     

Met-enkephalin,leu-enkephalin and other opiate-like peptides in human pheochromocytoma tumors

Opiate-like peptides, as measured by radioreceptor assay, were present in 20 samples from 9 human pheochromocytoma tumors obtained at surgery. The variation in peptide content of these samples, found in apparently viable tissue, ranged from 6-fold less to 90-fold higher than the mean value in normal human adrenal medulla. Subcellular fractionation of one tumor homogenate indicated that a large fraction of the opiate-like peptides are stored with catecholamines in chromaffin vesicles. Further analysis of pheochromocytoma opioid peptides revealed the presence of cryptic opioid activity, uncovered by trypsin treatment, and of six well-defined opioid peptide peaks, separated by high performance liquid chromatography. Peaks of opioid activity with high performance liquid chromatography retention times identical to met- and leu-enkephalin account for 43% and 27% of the recovered opioid activity, respectively. The massive stores of opioid peptides present and secreted from pheochromocytoma tumors may be responsible for some of the pleiomorphic symptomatology frequently observed in this disease.     

Opioid peptide release in the rat hippocampus after kainic acid-induced status epilepticus

It has been suggested that kainic acid enhances opioid peptide release. However, no direct evidence exists to support this hypothesis. The main aim of the present study was to determine whether such release occurs in the hippocampus of the rat after status epilepticus induced by kainic acid. Microdialysis experiments revealed significant opioid peptide release in the hippocampus 90-150 min (100%) and 270-300 min (50%) after kainic acid-induced status epilepticus. The peptides released were identified by high-performance liquid chromatography linked to radioimmunoassay as Met-enkephalin, Leu-enkephalin, Dynorphin-A (1-6), and Dynorphin-A (1-8). Reduced extracellular opioid peptide immunoreactivity was detected 28 days after status epilepticus (38% compared with control situation). The present results indicate an important activation of opioid peptide systems by kainic acid-induced status epilepticus. In addition, the reduced hippocampal extracellular opioid peptide levels long-term after kainic acid administration could have important implications for the progressive nature of epileptogenesis.     

Characterization of dermorphin binding to membranes of rat brain and heart

Binding of dermorphin to the two major opioid receptor types, mu and delta, in rat brain membranes was examined by displacement of [3H] [D-Ala2, MePhe4, Gly-(ol)5]enkephalin (DAGO) and [3H]-[D-Ala2,D-Leu5]-enkephalin (DADLE) binding. Affinity of dermorphin binding to mu sites, Kd = 1.24 nM, was almost 3 times greater than that of DAGO, Kd = 3.35 nM. In contrast, the Kd value of dermorphin binding to delta sites was 78 nM only, as compared to Kd = 2.27 nM for DADLE. Dermorphin was ineffective in displacing [3H]ethylketocyclazocine (EKC) binding to kappa receptors after prior blocking of [3H]EKC binding to mu and delta sites. Studies of dermorphin binding to mu sites revealed that the potency of dermorphin increased in the presence of Na+ (+31%) but decreased in the presence of Mn2+ (-81%) or Gpp(NH)p (-44%). Displacement of bound [3H]diprenorphine (DPN) by dermorphin from atrial membranes of the rat heart, left side, was detectable, suggesting the presence of mu sites in this section of the heart.     

Adrenorphin immunoreactivity in rat brain

Adrenorphin is the first C-terminally amidated form of opioid peptides isolated from human pheochromocytoma tumor and is considered to be generated out of proenkephalin A by unique processing. By the highly specific and sensitive radioimmunoassay (RIA) procedure utilizing the antiserum against adrenorphin, combined with high performance liquid chromatography (HPLC), immunoreactive adrenorphin in rat brain was verified to be identical with its authentic peptide. It has been revealed that adrenorphin immunoreactivity distributes widely in rat brain but in the unique pattern distinct from those of other endogenous opioid peptides. Note that immunoreactive adrenorphin was most concentrated in the olfactory bulb, and appreciably in the hypothalamus and striatum. Furthermore, immunohistochemical study has revealed that adrenorphin-immunoreactive structures in hypothalamic region of rat were localized in the neurones of the arcuate nucleus. In addition, adrenorphin-immunoreactive fibre plexus was found in the various regions of the hypothalamus, such as median eminence, periventricular zone and paraventricular nucleus. These indicate that adrenorphin may have a unique physiological function.     

On betaH-Leu5-endorphin and schizophrenia.

A previously unknown peptide, betaH-Leu5-endorphin, has been reported in the dialysates of schizophrenic patients. Accordingly, hemofiltrates from two schizophrenic and two control patients were examined for the presence of betaH-Leu5-endorphin. The opioid peptides were detected by a radioreceptor assay after separation and identification by gel filtration and high-performance liquid chromatography. With a detection limit of 30 pmole/L of hemofiltrate, no betaH-Leu5-endorphin or Met5-endorphin was found in controls or in patients. Whatever the possible involvement of endorphins in schizophrenic behavior, they are not present at detectable levels in the hemofiltrates of two well-characterized schizophrenic patients, thereby casting doubt on a general relationship of Leu-endorphin and schizophrenia.     

Colocalization of immunoreactive proenkephalin and prodynorphin products in medullary neurons of the rat

This study addressed the possible coexistence of products of the proenkephalin and prodynorphin opioid peptide precursors in single neurons of the central nervous system of the rat. Antisera directed against met-enkephalin-arg-gly-leu and against Dyn B were used in immunohistochemical preparations of sections through the rat medulla. Examination of serial three micron frozen sections stained alternately with the two different antisera revealed that the majority of labelled neurons stain with only one of the two antisera. In specific area, however, immunoreactive m-enk and Dyn B could be detected in the same neuron. This was particularly true of the caudal ventrolateral nucleus of the solitary tract, where the two peptides were colocalized in most neurons. Other areas where the two peptides coexist include the midline raphe and the nucleus reticularis paragigantocellularis. These data provide the first evidence for colocalization of different opioid peptide families in single CNS neurons.     

Comparative Distribution of Neurons Containing FLFQPQRFamide-like (morphine-modulating) Peptide and Related Neuropeptides in the Rat Brain

FLFQPQRF-NH2 (F8Famide; morphine-modulating peptide), isolated from bovine brain, is an FMRFamide-like peptide with opioid analgesia modulating effects. In the rat brain, F8Famide is immunohistochemically localized in neurons of the medial hypothalamus and medulla oblongata. Neuropeptide Y (NPY) is structurally related to F8Famide and the mammalian FMRFamide-like immunoreactivity (LI) was once thought to be due to an NPY-like peptide. We compared the anatomical distribution of F8Famide-LI with the localization of enkephalin- and NPY-LI-containing structures in the rat brain to find out if NPY or enkephalins coexist with F8Famide-LI. Cryostat sections of colchicine-treated Wistar rat brains were incubated with specific antisera against F8Famide, NPY, YGGFMRGL (Met-enkephalin-Arg-Gly-Leu), or YGGFMRF (Met-enkephalin-Arg-Phe) raised in rabbits. The immunoreactivity was visualized by the peroxidase - antiperoxidase or immunofluorescence method. The light microscopic mirror method was applied to study the colocalization of F8Famide and NPY. The F8Famide-immunoreactivity was concentrated in smaller areas of medial hypothalamus and nucleus of the solitary tract than that of enkephalins and NPY. In all brain areas, the distributions of F8Famide-, enkephalin- and NPY-immunoreactive neurons were distinct. F8Famide-, NPY- and enkephalin-LI-containing nerve terminals were seen in the nucleus of the solitary tract and in the lateral parabrachial nucleus. These results show that the neuronal systems containing F8Famide-, enkephalin- or NPY-LI are anatomically separate in all brain regions. However, there are terminal areas in which more than one type of these immunoreactivities are detected. These results have anatomical correlation with pharmacological reports, suggesting modulatory functions for these peptides on regulation of blood pressure, feeding behaviour and endocrine functions.     

Immunocytochemical localization of enkephalins in the brain of the African lungfish,Protopterus annectens,provides evidence for differential distribution of Met-enkephalin and Leu-enkephalin

The distribution of various opioid peptides derived from proenkephalin A and B was studied in the brain of the African lungfish Protopterus annectens by using a series of antibodies directed against mammalian opioid peptides. The results show that both Met-enkephalin- and Leu-enkephalin-immunoreactive peptides are present in the lungfish brain. In contrast, enkephalin forms similar to Met-enkephalin-Arg-Phe, or Met-enkephalin-Arg-Gly-Leu, as well as mammalian α-neoendorphin, dynorphin A (1–8), dynorphin A (1–13), or dynorphin A (1–17) were not detected. In all major subdivisions of the brain, the overwhelming majority of Met-enkephalin- and Leu-enkephalin-immunoreactive cells were distinct. In particular, cell bodies reacting only with Leu-enkephalin antibodies were detected in the medial subpallium of the telencephalon, the griseum centrale, the reticular formation, the nucleus of the solitary tract, and the visceral sensory area of the rhombencephalon. Cell bodies reacting only with Met-enkephalin antibodies were found in the lateral subpallium of the telencephalon, the caudal hypothalamus, and the tegmentum of the mesencephalon. The preoptic periventricular nucleus of the hypothalamus exhibited a high density of Met-enkephalin-immunoreactive neurons and only a few Leu-enkephalin-immunoreactive neurons. The distribution of Met-enkephalin- and Leu-enkephalin-immunoreactive cell bodies and fibers in the lungfish brain showed similarities to the distribution of proenkephalin A-derived peptides described previously in the brain of land vertebrates. The presence of Met-enkephalin- and Leu-enkephalin-like peptides in distinct regions, together with the absence of dynorphin-related peptides, suggests that, in the lungfish, Met-enkephalin and Leu-enkephalin may originate from distinct precursors. J. Comp. Neurol. 396:275–287, 1998. © 1998 Wiley-Liss, Inc.     

Purification by HPLC of a large peptic fragment of myelin basic protein

A method is described for the separation of two large peptic peptides of myelin basic protein (residue numbers, 1–88 and 89–169) by high performance liquid chromatography. These peptides had previously been purified by gel filtration and ion-exchange chromatography. Although the peptides were judged to be pure by gel electrophoresis, it was found that peptide 1–88 stimulated proliferation and enhanced transfer of experimental allergic encephalomyelitis with guinea pig peritoneal exudate cells sensitized to peptide 89–169. This result suggested that peptide 1–88 was contaminated with a fragment of peptide 89–169 that contained the encephalitogenic site, residues 113–122. Further purification of peptide 1–88 by high performance liquid chromatography was monitored by gel electrophoresis and an antibody-blotting technique, as well as by cellular proliferation and transfer. After purification by high performance liquid chromatography, the main fraction of peptide 1–88 no longer stimulated cellular proliferation or transfer of experimental allergic encephalomyelitis, nor did it react with antibody to peptide 89–169 on antibody blots.     

Gabapentin modifies extracellular opioid peptide content in amygdala: a microdialysis study

Opioid peptide release was monitored in the amygdala and hippocampus of freely moving rats following a single oral administration of gabapentin using microdialysis. Extracellular opioid peptide levels were elevated above basal levels in the amygdala within the first 60 (54%) and 90 min (68%) after gabapentin administration. Levels returned to basal conditions 120 min following the treatment. No significant changes were detected in the hippocampus. The majority of immunoreactive material recovered from the amygdala following gabapentin administration was identified as Leu-enkephalin and Met-enkephalin by high performance liquid chromatography (HPLC) analysis. It is proposed that the enhanced opioid peptide release in the amygdala induced by gabapentin might be involved with the antiepileptic effects as well as with some adverse events produced by this drug.     

Low affinity inhibition of opioid receptor binding by FMRFamide

The ability of the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) to inhibit the binding of opioid-receptor radioligands to mammalian neural tissue was examined. Rabbit brain membrane preparations were exposed to tritiated dihydromorphine and ethylketocyclazocine in the presence of various concentrations of FMRFamide. FMRFamide inhibited the specific binding of both ligands in a dose-related manner, suggesting that the neuropeptide can inhibit binding to at least two subtypes of opioid receptors (mu and kappa). These data are consistent with the recent proposal that FMRFamide, or the immunoreactive FMRFamide-like material in mammalian brain, spinal cord, and gastrointestinal tract, can act as an endogenous opioid antagonist. However, the low binding affinity of FMRFamide might suggest an alternative mechanism for FMRFamide antagonism of opioid action in vivo.     

Quantitative EEG and autonomic patterns of synthetic peptides related to dermorphin.

The effects of dermorphin on EEG and autonomic variables are compared with the effects of 2 analogues and 2 homologues, all administered intracerebroventricularly in the rabbit. Dermorphin was the most effective in modifying all considered parameters: increase of cortically derived and calculated total power, bradycardia, respiratory depression and hypothermia. The dibenzylated heptapeptide was essentially inactive. The electrocortical pattern induced by the administration of L-dermorphin suggests a functional correlation between the amino acid D-ala 2 and the effects on EEG. Comparison between the effects produced by the N-terminal tetrapeptide and pentapeptide led us to hypothesize that amino acid Tyr 5 may be specifically involved in inducing the autonomic effects.     

The proenkephalin-A-derivative Met-enkephalin-Arg-Gly-Leu is not present in the feline species

No opioid octapeptide Met-enkephalin-Arg-Gly-Leu was detected either in the brain or in the adrenal gland of the cat using a specific radioimmunoassay. Whereas it was possible to determine the Met-enkephalin and Leu-enkephalin contents. The Met-enkephalin versus Leu-enkephalin concentration ratio was around five in each area of the brain assayed. The presence of authentic Met-enkephalin and Leu-enkephalin was confirmed by high performance liquid chromatography analysis. All in all, these data seem to indicate that the cat proenkephalin is partly different from that previously analysed in mammalian species including humans, rats and cows.     

Distribution and development of proenkephalin-like immunoreactivity in the Lumbar spinal cord of the chicken

Met5-enkephalin- (Met-ENK), Leu5-enkephalin-, Met5-enkephalin-Arg6-Phe7-, metorphamide- and BAM 22P-like peptides could be detected in the lumbar spinal cord of the chicken by immunocytochemistry and/or high performance liquid chromatography. However, a peptide identical to Met5-enkephalin-Arg6-Gly7-Leu8 could not be detected in the lumbar spinal cord of the chicken using an antiserum that was capable of detecting the octapeptide in mammalian tissues. Nerve fiber- and terminal-like processes containing proenkephalin-derived peptides were concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of processes containing proenkephalin-derived peptides occurred in the medial and lateral motor columns of the ventral horn. The level of total radioimmunoassayable Met-ENK in the lumbar spinal cord of the chicken embryo increased more than 1000-fold between day 4.5 and day 18. A schedule of curare administration that had previously been shown to prevent naturally occurring somatic motoneuron death in the chicken lumbar spinal cord resulted in a two-fold increase in total radioimmunoassayable Met-ENK in the lumbar spinal cord.     

Variation in H-2Kk peptide motif revealed by sequencing naturally processed peptides from T-cell hybridoma class I molecules

Class I major histocompatibility complex (MHC) molecules interact with a diverse array of self and foreign peptides, displaying them on the cell surface and providing an extracellular indication of intracellular invasion. The most clearly defined role for these class I/peptide complexes is to cause effector responses upon binding to antigen-specific receptors of cytotoxic T cells. We have characterized the mouse thymoma/rat Vβ8.2+ T-cell hybridoma C14/BW12-12A1 by fluorescence-activated cell sorting analysis and have used immunoaffinity chromatography to purify class I molecules from these cells. The peptides bound to the class I molecules were fractionated by high-performance liquid chromatography and sequenced. Self-peptide mixtures eluted from the mouse H-2K^k class I allele revealed a dominant primary sequence motif, with a carboxyl-terminal residue that appeared to be invariantly valine and a secondary or auxiliary anchor residue at position 2 that could be either glutamate or proline. The majority of naturally processed peptide ligands appeared to be octamers. Although peptides eluted off H-2K^k molecules from tissue derived from a number of different inbred mouse strains also appeared to be octamers, others have reported that isoleucine is the dominant carboxyl-terminal residue. Thus, different cell types displayed distinct differences in naturally processed peptides bound by the same class I alleles. The variation in naturally processed peptides loaded onto the same class I allele most likely reflects the nature of the pool of peptides within the cell available for loading class I molecules. © 1996 Wiley-Liss, Inc.