Bernd R. Seizinger

Differential effects of acute and chronic ethanol treatment on particular opioid peptide systems in discrete regions of rat brain and pituitary

Acute ethanol treatment induced a significant increase in the tissue levels of immunoreactive (ir-) Met-enkephalin in hypothalamus, striatum and midbrain, but not in hippocampus. Levels of ir-dynorphin, ir-alpha-neo-endorphin and ir-beta-endorphin were not found to be significantly altered in brain and pituitary. Chronic ethanol treatment (by the use of ethanol liquid diet) resulted in a more than 50% decrease of the tissue levels of ir-dynorphin and ir-alpha-neo-endorphin in hypothalamus and hippocampus, while both peptides remained unchanged in midbrain, striatum, adenohypophysis and neurointermediate pituitary. In contrast, ir-met-enkephalin was decreased in striatum and hypothalamus, but unaffected in midbrain and hippocampus. Levels or ir-beta-endorphin remained unchanged in the brain and in the pituitary. However, the de novo biosynthesis of beta-endorphin and its prohormones beta-lipotropin and pro-opiomelanocortin was increased in the intermediate pituitary and to an even more pronounced degree, in the adenohypophysis, after chronic treatment of rats with ethanol liquid diet, nevertheless, the amounts of opiate-active beta-endorphin were found to be reduced in both lobes of the pituitary: In the adenohypophysis, this was due to a retardation of the enzymatic processing of beta-endorphin from its precursor beta-lipotropin, while in the intermediate pituitary the alpha-N-acetylation of beta-endorphin to opiate-inactive alpha-N-acetyl-beta-endorphin was stimulated. In conclusion, acute and chronic ethanol treatment caused selective alterations on different opioid peptide systems within distinct areas of the rat brain and pituitary.

Parallel distribution of immunoreactive α-neo-endorphin and dynorphin in rat and human tissue

Abstract Using antibodies directed against α-neo-endorphin and dynorphin-(1–13) a striking parallelism was found between the regional distribution of the two opioid peptides in various rat and human tissues. In the rat, the highest levels were found in the neurointermediate pituitary, followed by those in the adenohypophysis, the hypothalamus and the striatum. High levels were also found in the spinal cord, whereas the cerebellum contained only small amounts of the immunoreactive peptides. In peripheral tissues, high concentrations of both were detected in the gut and surprisingly, the lungs, whereas other tissues such as the adrenal glands, the kidney and the liver did not contain measurable amounts. The distribution pattern in the human brain was similar to that in the rat. A particularly high concentration of both of the peptides was found in the substantia nigra. In contrast to the rat pituitary, the human pituitary contained only small amounts of the immunoreactive opioid peptides. In all tissues of both species, the levels of immunoreactive (ir-) α-neo-endorphin were somewhat higher than those of ir-dynorphin with the ir-α-neo-endorphin/ir-dynorphin ratio varying between 1 and 3. In response to dehydration, ir-α-neo-endorphin, ir-dynorphin, vasopressin and leu-enkephalin were depleted from the neurohypophysis of the rat indicating a similar regulation of the pools of these peptides in the neurohypophysis. A common biosynthetic origin of ir-α-neo-endorphin and vasopressin, is unlikely, since Brattleboro rats which do not synthesize vasopressin possess levels of ir-α-neo-endorphin in the neurohypophysis comparable to those of control rats. Gelfiltration and HPLC analysis of the immunoreactive components in the rat pituitary lobes revealed that the vast majority of the immunoreactivity in the neurointermediate pituitary coelutes with synthetic mα-neo-endorphin, whereas a substantial part of that in the adenohypophysis appears to comprise a material with a molecular size of about 8000 daltons. There was no evidence for the existence of substantial amounts of β-neo-endorphin in the rat pituitary. The possibility is discussed that the close parallelism in the distribution and secretion of ir-α-neo-endorphin and ir-dynorphin might indicate a common biosynthetic origin for these peptides.

Levels of Dynorphin-(1–13) Immunoreactivity in Rat Neurointermediate Pituitaries Are Concomitantly Altered with Those of Leucine Enkephalin and Vasopressin in Response to Various Endocrine Manipulations

The levels of dynorphin-(1-13), leucine enkephalin, beta-endorphin and vasopressin immunoreactivity (ir-DYN, ir-1-ENK, ir-beta-END, ir-VP) have been determined in the anterior and in the neurointermediate lobes of the pituitary of rats subjected to a variety of manipulations. Dehydration of rats by 5 days enforced inhibition of a 2% solution of NaCl resulted in a significant decrease in the levels of ir-DYN, ir-1-ENK and ir-VP, but not in those of ir-beta-END in the neurointermediate lobe of the pituitary. In contrast, substitution of drinking water by a solution containing 20 microgram/ml dexamethasone for 5 days produced a significant increase in the neurointermediate pituitary content of ir-DYN, ir-1-ENK and ir-VP, whereas levels of ir-beta-END remained unaffected. This treatment, however, resulted in a significant fall in the ir-beta-END content of the adenopituitary without changing levels of ir-DYN in this structure. Adrenalectomy was associated with a significant decrease in the ir-DYN, ir-VP and ir-1-ENK content of the neurointermediate lobe of the pituitary and a pronounced elevation in the ir-beta-END but not ir-DYN content of the adenohypophysis. These observations are indicative that the regulation mechanisms of the functional state of particular endorphins differ between the anterior and neurointermediate lobes of the pituitary. The concomitant alterations in levels of ir-DYN, ir-1-ENK and ir-VP detected suggest that a common or similar mechanism of regulation may exist for these peptides. A common biosynthetic origin, however, appears to be unlikely, since Brattleboro rats which are unable to synthesize vasopressin possess unchanged ir-DYN- and ir-1-ENK- levels in the pituitary.

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

Abstract: The distribution of five major products of proenkephalin B [dynorphin1–17, dynorphin B, dynorphin1–8, α‐neo‐endorphin and β‐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 irdynorphin1–17, 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 dynorphin1–17 was detected in the anterior pituitary. Concentrations of β‐neo‐endorphin were similar to those of α‐neo‐endorphin in the posterior pituitary. In contrast, in all brain tissues α‐neo‐endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of β‐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.

Evidence for the occurrence of the opioid octapeptide dynorphin-(1–8) in the neurointermediate pituitary of rats

Abstract Evidence is provided for the existence of the opioid peptide dynorphin-(1–8) in the neurointermediate pituitary of rats. The octapeptide was isolated by immunoadsorption to antibodies directed against porcine dynorphin-(1–13) followed by a variety of chromatographic separation procedures. The identity of the purified material with dynorphin-(1–8) was indicated by the following criteria: comigration with synthetic dynorphin-(1–8) on gelfiltration chromatography and high-performance liquid chromatography systems and liberation of a peptide with the same chromatographic behavior as leucine-enkephalin after sequential cleavage with trypsin and carboxypeptidase B. Radioimmunological estimations revealed that dynorphin-(1–8) is a major dynorphin-related opioid peptide in the pituitary of rats.

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 greater than caudate nucleus greater than 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.

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

Abstract 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.

Opioid peptides in Huntington's disease: alterations in prodynorphin and proenkephalin system.

The concentrations of dynorphin A1-8 and Met-enkephalin-Arg6-Gly7-Leu8 were measured in the basal ganglia of postmortem brains from patients with Huntingtons disease (HD) and from control subjects. A significant reduction of dynorphin A1-8 concentration was found in caudate nucleus, putamen, external globus pallidus and substantia nigra of HD brains. Levels of Met-enkephalin-Arg6-Gly7-Leu8 were reduced in HD caudate nucleus, putamen, internal and external globus pallidus. These data indicate that both the prodynorphin and proenkephalin opioid peptide system are affected in the basal ganglia in HD.

Continuous social defeat induces an increase of endogenous opioids in discrete brain areas of the mongolian gerbil

Dyads of a victor and a loser of mongolian gerbils (Meriones unguiculatus) coexisted for seven days; isolated animals served as a further experimental group. beta-Endorphin, Met-enkephalin and dynorphin were measured in several brain areas and in the anterior and neurointermediate pituitary. beta-Endorphin and Met-enkephalin were increased in the amygdala of defeated as compared to victorious animals. Met-enkephalin in the hypothalamus and in the striatum were lower in isolated than in coexisting gerbils. Coexistence decreased beta-endorphin in the amygdala and in the hypothalamus as compared to isolation. The results provide biochemical evidence for the role of central endogenous opioid-peptide systems in the physiology of victory and defeat. Dynorphin showed no variation with social conflict and social status.

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.