Christian Gramsch

Regional distribution of methionine-enkephalin- and beta-endorphin-like immunoreactivity in human brain and pituitary.

Concentrations of methionine-enkephalin- (Met-enkephalin) and beta-endorphin-like immunoreactivities were determined in 33 areas of human brain and pituitary using highly sensitive radioimmunoassays in combination with affinity chromatography for the purification of beta-endorphin. It was found that they have quite different distribution patterns, suggesting the existence of both endorphins in independent systems in the central nervous system. Determination of Met-enkephalin and beta-endorphin immunoreactivities in chronic alcoholics and opiate-dependent subjects revealed no gross changes in comparison to the normal subjects.

Nociceptin/orphanin FQ and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions.

ANTISERA were generated against nociceptin/orphanin FQ, the putative ligand of the opioid receptor-like ORL1 receptor. Dot blot analysis showed that the antibodies selectively detect nociceptin but not dynorphin or other opioid peptides. Immunofluorescent staining of tissue sections revealed dense plexus of nociceptin-immunoreactive nerve fibres and terminals within the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei, locus coeruleus, periaqueductal grey, amygdala, habenula, hypothalamic region and septal area in mice and rats. When adjacent sections were stained either with the nociceptin antibody or the pan-opioid 3-E7 mouse monoclonal antibody, an overlapping distribution was observed in many nociceptive centres including the superficial dorsal horn, sensory trigeminal complex and periaqueductal grey. However, confocal microscopic examination of dual-labelled spinal cord and brain stem sections showed no instances of co-localization of nociceptin and opioid peptides in these regions. Intra- cerebroventricular administration of nociceptin has been shown to induce hyperalgesia. Thus, the present results suggest that nociceptin and opioids are released from different terminals thereby modulating pain signals in opposite ways.

Characterization and localization of immunoreactive dynorphin, α-neo-endorphin, met-enkephalin and substance P in human spinal cord

Abstract By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), α-neo-endorphin (ir-α-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral > lumbar > cervical > thoracic. Concentrations of ir-DYN, ir-α-NEO and ir-SP were 2–10-fold, but of ir-MET 1–2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord. The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter. Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin 1–17 . ir-α-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-α-NEO were also detected in these tissue. These results suggest that dynorphin and α-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.

Immunoreactive dynorphin in human brain and pituitary

The distribution of immunoreactive dynorphin (ir-dyn) has been determined in various regions of human brain and pituitary by use of a highly specific radioimmunoassay. The concentrations of ir-dyn in the substantia nigra (24.5 pmol/g) and hypothalamus were among the highest in the 26 brain areas examined. Substantial amounts were also measurable in other extrapyramidal structures such as the caudate nucleus, pallidus and putamen. Lower concentrations of ir-dyn were detected in the amygdala, hippocampus, periaqueductal gray matter, colliculi, pons, medulla and area postrema, but only low amounts were found in the posterior lobe of the pituitary, while no ir-dyn was detectable in the anterior lobe. By gel permeation chromatography the brain immunoreactivity was shown to consist of 3-4 peaks of apparent molecular weights of about 12,000, 6000, 1800 and less than 1000. It was possible to demonstrate the high opioid potency of 2 of these peaks in the guinea-pig ileum longitudinal muscle bioassay after purification by immunoprecipitation. A comparison of the distribution pattern of ir-dyn revealed some parallels with enkephalin, whereas the distribution of ir-beta-endorphin is quite different.

Immunolocalization of two mu-opioid receptor isoforms (MOR1 and MOR1B) in the rat central nervous system

We have recently shown that the cytoplasmic tail of the rat mu-opioid receptor undergoes alternative splicing giving rise to two isoforms, rMOR1 and rMOR1B. These isoforms exhibit similar pharmacological profiles, however, differ in agonist-induced desensitization of coupling to adenylate cyclase. In the present study, we have raised polyclonal antibodies that specifically detect either rMOR1 or rMOR1B and used these antisera for immunocytochemical localization of the receptor proteins in the rat central nervous system. Prominent MOR1B-like immunoreactivity was found in the external plexiform layer of the main olfactory bulb localized to a dense plexus of dendrites mostly originating from mitral cells and extending into the glomerular layer. MOR1-like immunoreactivity was restricted to the perikarya of mitral cells and to distinct juxtaglomerular cells as well as their processes. While MOR1-, DOR1- and KOR1-like immunoreactivity was absent from the external plexiform layer, high densities of opioid peptides were found in this layer suggesting that MOR1B may be a targeted receptor of these peptides. MOR1-like immunoreactivity was observed in many pain-controlling brain areas including the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei and periaqueductal gray while MOR1B-like immunoreactivity was not detectable in these regions. Taken together, we provide evidence that the mu receptor isoforms, MOR1 and MOR1B, exhibit a strikingly different distribution in that MOR1 appears to be the major isoform widely distributed throughout the central nervous system and MOR1B being predominantly localized to the olfactory bulb.

Binding Characteristics of a Monoclonal β-Endorphin Antibody Recognizing the N-Terminus of Opioid Peptides

Abstract: The present paper describes the isolation and characterization of a clone of hybrid myelomas (3‐E7) secreting a mouse monoclonal antibody to β‐endorphin. An examination of its specificity against a series of human β‐lipotropin fragments and other opioid peptides revealed that the N‐terminus portion of β‐endorphin is the determinant. Complete or almost complete cross‐reactivity was obtained to methionine‐ and leucine‐enkephalin, β‐lipotropin 60–65, and BAM 22; partial cross‐reactivity was seen to dynorphin1–13 and α‐neo‐endorphin, whereas β‐lipotropin, α‐N‐acetyl‐β‐endorphin, Des‐Tyr1‐β‐endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross‐reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for β‐endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of β‐endorphin iodinated with its N‐terminal tyrosine protected by coupling to an antibody. A solid‐phase RIA was developed involving the internally 3H‐labeled monoclonal antibody, which resulted in a 10‐fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.

Differential effects of various opioid peptides on vasopressin and oxytocin release from the rat pituitary in vitro

SummaryDynorphin (1–17), and to a lesser extent, β-endorphin and [Leu]enkephalin (10−6 M each) decreased the spontaneous release of vasopressin (VP) from the rat neurointermediate pituitary in vitro, whereas the oxytocin (OT) release remained unchanged. Naloxone, however, did not significantly alter the spontaneous VP and OT release.Dynorphin (1–17) (10−7 M) increased the electrically evoked release of VP and OT, while 10−6 M had a significant, somewhat less pronounced stimulatory effect only on VP, but not on OT release. The opiate inactive fragment [des-Tyr1]dynorphin (1–17) did not change the evoked VP and OT release, indicating that the dynorphin effect was mediated by opiate receptors. β-Endorphin (10−6 M and 10−7 M) did not alter the evoked VP and OT secretion. 10−6 M [Leu]enkephalin induced a stimulation of the evoked OT, but not VP release; 10−7 M [Leu]enkephalin had no effect, neither on VP nor on OT release.The opiate antagonist naloxone 10−5 M) induced an increase in the evoked VP and, even more pronounced, OT release. In a concentration of 10−6 M, however, naloxone only increased the evoked OT release. When naloxone and dynorphin (1–17) were concomitantly applied, their stimulatory effects on the evoked VP and OT release were additive.Similarly to the effects of naloxone, addition of a monoclonal antibody which binds to the common N-terminal sequence of all endogenous opioid peptides, resulted in a marked increase in the evoked secretion of VP and, to an even more pronounced degree, of OT. Thus, the stimulated VP and OT release seems to be under a tonic inhibitory control of opioids.Electrical stimulation was found to enhance the release of endogenous immunoreactive dynorphin from the neurointermediate pituitary, while the release of immunoreactive β-endorphin was even decreased.In conclusion, the present data indicate a dual role of endogenous opioid peptides in the VP and OT secretion at the level of the posterior pituitary.

Effects of depletion of brain catecholamines during the development of morphine dependence on precipitated withdrawal in rats.

SummaryThe significance of long term depletion of brain catecholamines (CAs) for the development of morphine dependence and for the expression of morphine withdrawal was studied in rats which were implanted with morphine pellets for 10 days. CAs were depleted by inhibition of tyrosine-hydroxylase with alpha-methyl-tyrosine (AMT) or by destruction of catecholaminergic nerve terminals with 6-hydroxydopamine (6-OHDA). In the “acute” experiments these drugs were applied within 24 hrs before precipitation of withdrawal; in the “chronic” experiments drug administration was started before the first implantation and in the case of AMT, continued repeatedly thereafter.With either method, “acute” depletion of brain CAs resulted in reduced intensity of withdrawal. When CAs were kept low through the whole time of morphine exposure and also at the time of withdrawal, the intensity of withdrawal was normal in the case of 6-OHDA administration and only slightly decreased in the case of AMT. When AMT administration was discontinued 40 hrs before precipitation of withdrawal the withdrawal pattern occurred with unchanged intensity.Our experimental data are compatible with the assumption that long lasting depletion of brain CAs is compensated for by induction of neuronal supersensitivity for noradrenaline (NA) and dopamine (DA). While both CAs play an important role in the full expression of the withdrawal syndrome their possible involvement in mechanisms leading to dependence seems to be unlikely although final statements cannot be made by the presented experiments.

Changes in striatal dopamine metabolism during precipitated morphine withdrawal.

Precipitation of withdrawal in morphine tolerant/dependent rats by either naloxone or the partial agonist ZK 48491 caused a significant increase in the contration of striatal DA, which persisted for at least 1 h. During the same time the probenecid-induced accumulation of HVA and DOPAC was reduced in the striatum in relation to probenecid-treated tolerant/dependent controls. 20 min after precipitation of withdrawal by naloxone, the striatal concentration of 3-methoxytyramine was decreased by about 40%, while the activity of the DA metabolizing enzymes, MAO and COMT, remained unchanged. Naloxone-precipitated withdrawal was, further, found to delay the depletion of striatal DA caused by inhibition of synthesis 90 min after alpha-methyl-p-tyrosine treatment. All these results provide evidence for a decreased release of DA from the striatum during precipitated morphine withdrawal.

Extrahypothalamic Corticotropin and α-Melanotropin in Human Brain

The distribution of corticotropin (ACTH) and α -melanotropin ( α -MSH) in human brain was investigated by radioimmunoassay using an antiserum which recognized h-ACTH