Endorphins do not affect behavioral stress responses in mice.

Effects of endorphins on behavioral stress responses were investigated in mice. For this purpose, we used environment-induced conditioned suppression of motility and forced swimming-induced immobility. The cerebral ventricular administration of alpha-endorphin (2.5-10 nmol), beta-endorphin (0.38-1.5 nmol), or gamma-endorphin (2.5-10 nmol) failed to affect either the environment-induced conditioned suppression of motility or the forced swimming-induced immobility. We have indicated previously that enkephalins attenuate both stress responses and, in contrast, dynorphin potentiates them. These findings indicate that the endorphinergic systems are not responsible for behavioral stress responses and that the role played by endorphins in the present stressful situations may be different from that of enkephalin and dynorphin.     

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.     

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.     

Nicotine-induced alterations in brain regional concentrations of native and cryptic Met- and Leu-enkephalin

The distribution of cryptic forms (larger enkephalin-containing peptides) in neostriatum, hypothalamus, spinal cord T₃-L₁ and neurointermediate lobe of pituitary were determined by radioimmunoassay. Optimal conditions for enzymic hydrolysis of the cryptic enkephalins by trypsin and carboxypeptidase B were established. The proportion of total Met- and Leu-enkephalin represented by native pentapeptide varied markedly among these central nervous system regions. Also, the distributions of native and cryptic Met-enkephalin were distinct from that of Leu-enkephalin. Chromatographic separation by HPLC of immunoreactive Met-enkephalin peptides revealed only two peaks corresponding to Met-enkephalin and Met-enkephalin sulfoxide in rather equal amounts. Hydrolysis of cryptic Met-enkephalin also produced only two HPLC-separable peaks of immunoreactive Met-enkephalin, again corresponding to Met-enkephalin and Met-enkephalin sulfoxide. Bioactivity of cryptic striatal Met-enkephalin after hydrolysis was demonstrated by antinociception and catalepsy in rats following its intracerebroven-tricular injection. Repeated short-term administration of nicotine, 0.1 mg/kg IP six times at 30 min intervals, produced significant increases in native and cryptic Met-enkephalin in striatum, consistent with an increase in neuronal release of Met-enkephalin together with increases in synthesis and processing of proenkephalin A in this brain region. This regimen of nicotine also decreased levels of native Met-enkephalin and of both native and cryptic Leu-enkephalin in neurointermediate lobe, consistent with nicotine-induced release of both proenkephalin A- and prodynorphin-derived peptides from neurointermediate lobe.     

Changes in the processing of pro-dynorphin end products in the substantia nigra during neonatal development

The steady-state levels of pro-dynorphin-related end products were measured in the substantia nigra of the rat at neonatal day 0, 7, 14 and in the adult. At neonatal day 0 there was evidence that pro-dynorphin had undergone posttranslational processing to yield dynorphin A-related products, dynorphin B(1-13) and alpha-neo-endorphin. At this stage the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) was 1:1 and a peak of 4 kilodalton dynorphin A-related immunoreactivity was detected. By neonatal day 7 the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) resembled the adult processing pattern for the substantia nigra. The rapid maturation of the pro-dynorphin system in the substantia nigra is in contrast to the development of the pro-dynorphin system in the posterior pituitary where adult-like processing patterns are not observed until neonatal day 21 (11). Pro-enkephalin products have also been detected in the substantia nigra of the rat (15). At neonatal day 0 and neonatal day 7 the molar ratio of Met-enkephalin to Leu-enkephalin was 4:1. However, in the adult the molar ratio of Met-enkephalin to Leu-enkephalin was 1.4:1 in this terminal field. These results suggest that in the adult or perhaps late in neonatal development some pro-dynorphin end products undergo further proteolytic cleavage to yield Leu-enkephalin.     

Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.     

小鼠神经系统内强啡肽B免疫活性物质及其高压液相层析(HPLC)分析

强啡肽B是一种新发现的阿片肽。本工作以自制的兔抗强啡肽 B血清建立灵敏的放射免疫测定法,测定了小鼠神经系统与垂体内强啡肽 B免疫活性物质的含量,其中以垂体、下丘脑含量最高。 放射免疫测定结合 Sephadex C-50 层析和HPLC分析的结果表明,小鼠脑内强啡肽B免疫活性物质的主要成分是强啡肽 B,另外也包括了一定量的强啡肽-32和另一未知的分子量更大的成分,但不像大鼠还含有强啡肽 B-29。这种种属特异性的意义有待进一步研究。     

Effects of dehydration on pro-dynorphin derived peptides in the neuro-intermediate lobe of the rat pituitary

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.     

Detection of Met-enkephalin in the CNS of the teleosts, Anguilla rostrata and Oncorhynchus kisutch

Acid extracts of the brains of the American eel, Anguilla rostrata, and the coho salmon, Oncorhynchus kisutch, were screened for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography, a peak of Met-enkephalin-related immunoreactivity was detected near the total volume of the column for both species. No higher molecular weight forms of Met-enkephalin-related material were detected, nor were any immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) or alpha-neo-endorphin detected for either species. The enkephalin-sized immunoreactivity was further analyzed by reverse phase HPLC. For both species, a peak of authentic Met-enkephalin was detected. However, Leu-enkephalin, Met-enkephalin-RGL and Met-enkephalin-RF were not detected by RIA in either species. In addition, no novel C-terminally extended forms of Met-enkephalin were detected in either species. Finally, opiate receptor binding activity was only found associated with the peak of immunoreactive Met-enkephalin.     

Dynorphin A immunoreactivity in human cerebrospinal fluid

Dynorphin A immunoreactivity in human cerebrospinal fluid has been characterized. Large quantities of the fluid were fractionated by molecular sieving on a Sephadex G-50 column and analyzed by radioimmunoassay. Active fractions were further analyzed by means of HPLC and an enzyme radioimmunoassay procedure for identification of Leu-enkephalin-Arg6 sequences. Several dynorphin A-active components of varying sizes were identified. However, most immunoreactive material derived from species of higher molecular weight (Mr 3000 and 5000) than authentic dynorphin A (Mr 2000). The Leu-enkephalin core was found to reside in both these large structures.     

Release of a dynorphin A (1-8) degrading enzyme from the spinal cord by intraventricular morphine in the rat

Intraventricular injection of morphine sulfate, 40 micrograms, released an enzyme from the spinal cord into the perfusate which degraded dynorphin A (1-8) and, to a lesser extent, dynorphin A (1-13) in urethane anesthetized rats. The enzyme did not degrade dynorphin A (1-17), Met-enkephalin, Leu-enkephalin, substance P and neurotensin. This dynorphin A (1-8) degrading enzyme was inhibited by aprotinin, thiorphan, and, to a lesser extent, by bacitracin but was not inhibited by bestatin. A kinetic study of the interaction between dynorphin A (1-8) and aprotinin with the enzyme indicated that it is competitive in nature. The pharmacological significance of the findings is still unknown.     

Characterization of opioid receptors on isolated canine gallbladder smooth muscle cells

Smooth muscle cells were isolated from the fundus of the canine gallbladder and examined for the presence of opioid receptors. The cells contracted in a concentration-dependent manner in response to three opioid peptides (Met-enkephalin, dynorphin1-13 and Leu-enkephalin), which are known derivatives of opioid precursors present in myenteric neurons of the gut. The order of potency was Met-enkephalin greater than dynorphin1-13 greater than Leu-enkephalin. The contractile response to opioid agonists was selectively inhibited by opioid antagonists (naloxone and Mr2266) but not by muscarinic, CCK/gastrin or tachykinin antagonists. Equivalent responses to the three opioid peptides exhibited differential sensitivity to preferential antagonists of mu (naloxone) and kappa (Mr2266) opioid receptors consistent with the presence of the three main types of opioid receptors (mu, delta and kappa) on canine gallbladder muscle cells.     

Food deprivation-induced changes in the level of opioid peptides in the pituitary and brain of rat

Following 1-4 days of food-deprivation (FD) male rats were sacrificed. The pituitary and different regions of brain were analyzed for beta-endorphin-like immunoreactivity (beta-EI), dynorphin (dyn) and methionine-enkephalin (ME) content by RIA. Pituitary beta-EI increased by 16, 28 and 43% on days 2, 3 and 4 of FD. In striatum also, beta-EI increased by 140 and 176% on days 2 and 3 of FD. Dyn level in pituitary was not affected but decreased in hypothalamus by 20% and in striatum by 73% on the 4th day of FD. There was a significant decrease (33-55%) in ME levels in striatum, hippocampus and cortex on 4th day of FD. When food-deprived rats were fed for 24 hr, concentration of most of the opioid peptides returned to basal level. These results suggest that FD in rats affects the opioid peptide levels in a differential manner.     

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.     

Comparative effects on blood pressure and heart rate of dynorphin A(1–13) in anterior hypothalamic area, posterior hypothalamic area, nucleus tractus solitarius, and lateral cerebral ventricle in the rat

The objective of this study was to explore the effects of the endogenous opioid peptide dynorphin A(1–13) on the CNS regulation of blood pressure and heart rate. Wistar rats, anesthetized with pentobarbital and halothane, received dynorphin A(1–13) microinjected into the anterior hypothalamus area (AHA), the posterior hypothalamic area (PHA), the nucleus tractus solitarius (NTS), or the lateral cerebral ventricle (ICV). Dynorphin A(1–13), 20 (12 nmol) or 30 μg ICV, produced significant (p < 0.05) reductions in blood pressure and heart rate. Naloxone, 50 μg/kg ICV, completely prevented the blood pressure response and significantly (p < 0.05) blunted the heart rate response to the highest dynorphin concentration, 30 μg ICV (18 nmol). Dynorphin A(1–13), 5 μg, in the NTS significantly (p < 0.05) decreased systolic and diastolic blood pressure and heart rate with the response being evident 10 min and persisting for 30 min after injection. In contrast, the same dose of dynorphin A(1–13) in the AHA produced an immediate, marked, and significant (p < 0.05) decrease in systolic and diastolic blood pressure and heart rate that attained its maximum 1–3 min and returned rapidly towards baseline levels. Dynorphin A(1–13), 5 or 10 μg in the posterior hypothalamic area, was not associated with any change in blood pressure or heart rate. Injection of the diluent at any site was not associated with any changes in blood pressure or heart rate. The maximum change in blood pressure with dynorphin was greater in the AHA than NTS, and the maximum change in heart rate was greater in the NTS than AHA. These data indicate a potential role for dynorphin as a modulator of the CNS regulation of blood pressure and cardiac rate, and this is mediated in part through different areas in the brain that maybe localized to the anterior hypothalamic area and nucleus tractus solitarius but not the posterior hypothalamic area.     

Identification of the tridecapeptide dynorphin B (rimorphin) within perikarya of rat duodenum

Using an immunofluorescence microscopic staining technique, the opioid peptide dynorphin B (rimorphin) was revealed within neuronal cell bodies of the rat duodenum. Dynorphin B immunoreactive perikarya were revealed in the myenteric and submucousal plexus as well as in the longitudinal muscle layer. They were large in diameter and round in shape and they contained a large round nucleus. Because no dynorphin B immunofluorescent nerve fibre and terminal could be noted it might be that dynorphin B is further cleaved by proteases into the bioactive opioid pentapeptide Leu-enkephalin and dynorphin B(6-13). These findings might also indicate that dynorphin B is processed within duodenal perikarya and that it has important physiological roles in the rat duodenum.     

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.     

Effect of prolonged progesterone treatment on the proenkephalin mRNA gene expression and enkephalins concentration in the sheep brain

The opioids modulate reproduction in sheep mostly by inhibiting the activity of the hypothalamo-pituitary-gonadal axis. However, the mechanism by which the negative feedback control systems regulate opioid synthesis and secretion in sheep is still not recognized. As a part of a research dealing with interaction between opioids and steroids, the effect of prolonged administration of progesterone (P4) and opioid receptor agonist or antagonist on the Met-enkephalin synthesis and concentration was examined in sheep brain. Long term P4 treatment significantly decreased the synthesis and the concentration of the opioid peptide in the hypothalamus and pituitary, however, the effect was more pronounced in the hypothalamus. Injections of Met-enkephalin completely or partially reversed the effect of P4. Naltrexone given together with opioid peptide modulated the response to the opioid agonist. The results show that there is an interaction between P4 and endogenous opioids in the central nervous system of cyclic sheep.     

Gonadal steroids and anterior lobe dynorphin in the male rat

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.     

Effects of dynorphin (1-8) on movement: non-opiate effects and structure-activity relationship

Cell bodies in the head of the caudate nucleus that synthesize prodynorphin peptides form a substantial projection to the substantia nigra pars reticulata (SNR). The discovery of this pathway suggested an involvement of prodynorphin products in motor control. The effects of unilateral nigral microinjections of prodynorphin products were tested in an in vivo circling model. Dynorphin (1-8), dynorphin (1-7), dynorphin (1-6), dynorphin (2-17) (des-Tyr-dynorphin), and Leu-enkephalin induced spontaneous contralateral circling at 20 nmol doses. The effect of dynorphin (1-8) was dose dependent and was not blocked by pretreatment with naloxone or WIN 44,441-3. These findings clearly demonstrate the dynorphinergic involvement in nigral motor control which may consist of an opioid and a non-opioid component.