The effect of naloxone administration on pregnancy-associated seizures

Pregnant mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The possibility that the well-known increase in beta-endorphin concentration which accompanies pregnancy was involved in this effect was examined by testing whether naloxone administration could block the increased seizure susceptibility. Pregnant female, control female and male C3H mice were treated with 5-50 mg/kg naloxone 5 min before flurothyl seizure testing. Naloxone markedly increased clonic seizure susceptibility in all three groups at a dose of 50 mg/kg, but had little effect at lower doses. In contrast, naloxone had differential effects on myoclonic seizures in pregnant and control female mice, being anticonvulsant in the controls, but proconvulsant in the pregnant mice. A role for endogenous opiates is unlikely in mediating clonic seizures in pregnant mice, but may be involved in myoclonic seizures.     

The Posttranslational Processing of β-Endorphin in Human Hypothalamus

beta-Endorphin is posttranslationally processed to six derivatives, which, although structurally similar, produce distinctly different biological effects. beta-Endorphin 1-31 is a potent opioid receptor agonist, but beta-endorphin 1-27 exhibits antagonist properties, and beta-endorphin 1-26 and the alpha-N-acetyl derivatives of all three peptides lack opioid receptor activity. In the present study, we identified the beta-endorphin peptides synthesized in human hypothalamus using cation exchange HPLC. First, we tested whether postmortem changes occur by storing rat hypothalami at 4 degrees C. This demonstrated that relative amounts of the six beta-endorphin forms did not change for up to 24 h, although total beta-endorphin immunoreactivity significantly declined after 6 h. HPLC analysis of human hypothalami revealed that beta-endorphin 1-31 was the principal form, constituting 58.4 +/- 5.4% of total immunoreactivity. Substantial amounts of beta-endorphin 1-27 (13.4 +/- 1.2%) and beta-endorphin 1-26 (13.1 +/- 1.6%) were also present, but alpha-N-acetylated forms were quantitatively minor, each comprising approximately 5% of total beta-endorphin. A similar processing pattern occurred in preoptic and suprachiasmatic areas of the hypothalamus. These results show that, despite differences in primary sequence, beta-endorphin is processed similarly in both rat and human hypothalamus. Opiate-active beta-endorphin 1-31 is the principal form in both species.     

Regulation of bioactive beta-endorphin processing in rat pars intermedia

Acid extracts of rat pituitary neuro-intermediate lobes have been shown by ion-exchange chromatography and radio-immunoassay to contain predominantly the inactive derivatives of beta-endorphin, alpha, N-acetyl beta-endorphin 1-27 and alpha, N-acetyl beta-endorphin 1-26; the biologically active form, beta-endorphin 1-31, is a minor component. In contrast, it was found that beta-endorphin generated in neuro-intermediate lobe cells in monolayer culture was less processed: the principal peptides related to bioactive beta-endorphin 1-31. When the cultured cells were incubated in the presence of 10(-5) M dopamine or 10(-6) M alpha-ergocryptine there was a marked increase in the degree of proteolysis and acetylation: the processing pattern reverted to that characteristic of the neuro-intermediate lobe in situ, with alpha-N-acetyl beta-endorphin 1-26 and alpha, N-acetyl beta-endorphin 1-27 as the prominent peptides. The results demonstrate that dopaminergic agents can influence the processing of beta-endorphin-related peptides in rat pars intermedia, indicating a new level at which the bioactivity may be regulated.     

Effects of dynorphin A(1-13) and of fragments of beta-endorphin on blood pressure and heart rate of anesthetized rats.

The effect on blood pressure and heart rate of central administration of dynorphin A(1-13) and of beta-, gamma-, and alpha-endorphin related peptides was studied in urethane-anesthetized rats. Intracerebroventricular (i.c.v., 0.1-10 micrograms) administration of beta-endorphin resulted in a dose-dependent, naltrexone-reversible hypotension and bradycardia. N-terminally modified fragments of beta-endorphin did not reduce blood pressure and heart rate. On the other hand, a dose of 10 micrograms of beta-endorphin(1-27), which lacks the four C-terminal amino acid residues of beta-endorphin, induced a fall in blood pressure and had a biphasic effect on heart rate. These responses, however, were resistant to pretreatment with naltrexone. None of the fragments of beta-endorphin smaller than beta-endorphin(1-27) affected blood pressure when administered i.c.v. in a dose of 10 micrograms. A small transient bradycardia was observed after i.c.v. administration of 10 micrograms of beta-endorphin(1-26), alpha, and gamma-endorphin. The naltrexone-reversible bradycardic response of alpha- and gamma-endorphin was not present in des-tyrosine- and des-enkephalin-alpha- and gamma-endorphin and also not in alpha-endorphin(10-16) and gamma-endorphin(10-17). Upon i.c.v. administration (0.1-50 micrograms) a dose-dependent, naltrexone-reversible decrease in blood pressure and heart rate was induced by dynorphin A(1-13). The present data indicate a hypotensive influence of beta-endorphin, beta-endorphin(1-27), and dynorphin A(1-13), whereas other fragments of beta-endorphin had little or no effect on the cardiovascular parameters investigated.     

Interleukin 1 potentiates agonist-induced secretion of beta-endorphin in anterior pituitary cells

Interleukin 1 (IL-1) has been shown to potentiate the release of beta-endorphin induced by secretagogues, including corticotropin releasing factor (CRF) and phorbol ester (TPA), in the mouse AtT-20 pituitary tumor cell line (Fagarasan et al., PNAS, 1989, 86, 2070-2073). In cultured rat anterior pituitary cells, pretreatment with IL-1 caused only a small increase in beta-endorphin release but significantly potentiated CRF-and vasopressin-stimulated beta-endorphin secretion. Vasopressin stimulates the secretion of beta-endorphin in normal pituitary cells but not in AtT-20 cells. However, treatment of AtT-20 cells with IL-1 induced the expression of vasopressin-mediated beta-endorphin release; this effect of IL-1 was reduced after depletion of protein kinase C by prolonged treatment with TPA. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in AtT-20 cells after depletion of protein kinase C, and after treatment with staurosporine. These findings indicate that treatment with IL-1 amplifies receptor-mediated responses to the major physiological secretagogues in normal corticotrophs, and initiates a secretory response to vasopressin in AtT-20 cells.     

beta-Endorphin-(1-27) inhibits the spinal beta-endorphin-induced release of Met-enkephalin

The effect of intraventricular beta-endorphin-(1-27) on the spinal release of Met-enkephalin induced by intraventricular beta-endorphin was studied using the intrathecal superfusion technique in urethane anesthetized rats. Intraventricular injection of beta-endorphin at a dose of 15 micrograms released Met-enkephalin from the spinal cord. This release of Met-enkephalin induced by beta-endorphin was significantly reduced by beta-endorphin-(1-27), 60 micrograms, injected intraventricularly. Injection of beta-endorphin (1-27) itself did not cause any release of Met-enkephalin. The finding is in line with the previous report that beta-endorphin (1-27) inhibited the analgesia induced by beta-endorphin.     

Examination of the requirement for an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides

In our approach to beta-endorphin modeling, we have proposed that the biological properties of the natural peptide are determined by the combination of three basic structural units: a highly specific opiate recognition sequence at the NH2 terminus (residues 1-5) connected via a hydrophilic peptide link (residues 6-12) to a potential amphiphilic helix in the COOH-terminal residues 13-31. In the alpha-helical conformation the hydrophobic domain twists around the length of the helix and covers almost one-half of its surface. The other distinctive features of the helix include its basicity and the two aromatic residues Phe18 and Tyr27. In contrast to previous models we have studied, peptide 4 is a "negative" model in the sense that it was designed and examined in order to determine how the lack of a well defined amphiphilic structure affects the biological properties of beta-endorphin. For this purpose, peptide 4 retains the three structural units previously postulated for beta-endorphin, but the amino acids of the 13-31 region are arranged in such a way that no definite continuous hydrophobic zone could be formed in an alpha- or pi-helical conformation of this region. In aqueous buffered solutions, peptide 4 showed almost the same amount of alpha-helical structure as beta-endorphin, with a slight tendency toward less helicity in 50% aqueous 2,2,2-trifluoroethanol. In rat brain homogenate, peptide 4 was degraded slightly slower than beta-endorphin, in contrast to the apparently much higher stability of previous models under the same conditions. With regard to opiate receptor binding, peptide 4 was twice as potent as beta-endorphin in mu-receptor assays but half as potent in delta-receptor assays. The opiate potency of peptide 4 on the guinea pig ileum was higher than that of beta-endorphin. In contrast, in the rat vas deferens assay, which is very specific for beta-endorphin, the potency of peptide 4 was very low and could be shown not to be mediated by the same opiate mechanism or by the same opiate receptor. A comparison of these results with those of previous model peptides provides further evidence for the importance of an amphiphilic helical structure in beta-endorphin residues 13-31, which determines the resistance to proteolysis of the natural molecule and contributes to the delta- and mu-opiate receptor interaction. The amphiphilicity of this helical structure must also be essential for high opiate activity on the rat vas deferens (epsilon-receptors), whereas no such structural requirement appears to be necessary for interaction with the opiate receptors on the guinea pig ileum.     

Regulation of IL-1beta and IL-8 production by mu-, delta-opiate receptors agonists in vitro

The beta-endorphin 10(-7-)-10(-11) M in LPS (lypopolisaccharide) presence and in spontaneous cultures promoted the IL-1beta production in mixed leukocyte fraction. LPS-induced IL-8 production in leukocyte fraction was inhibited by beta-endorphin 10(-7), 10(-11) M. The enchasing effect of beta-endorphin on IL-1beta production was not blocked by naloxone and naltrindole. The inhibitory effect of beta-endorphin on IL-8 production was blocked by naloxone and naltrindole. In mononuclear and neutrophile fractions beta-endorphin and delta-agonist DADLE enchased IL-1beta production in spontaneous and LPS-stimulating cultures, when IL-8 production inhibited beta-endorphin and delta-agonist DADLE only in LPS presence. No effect of mu-agonist DAGO were observed on IL-1beta production, whereas LPS-induced IL-8 secretion in neutrophile fraction inhibited by DAGO.     

Competitive binding of dynorphin-(1-13) and beta-endorphin to cerebroside sulfate in solution

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).     

In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

Stimulation of insulin secretion by beta-endorphins (1-27 & 1-31)

Synthetic human beta-endorphin potentiates insulin secretion by the isolated perfused rat pancreas when glucose is present in the perfusate at concentrations of either 125 or 200 mg/dl, whereas it fails to exert any effect on insulin secretion in the presence of a substimulatory concentration of 100 mg/dl. Similar potentiation of insulin secretion occurred in response to the 1-27 fragment (beta-endorphin1-27) of beta-endorphin. This transient potentiation lasts only 3 to 4 minutes, whereupon secretion returns toward control levels. Thus beta-endorphin produces only a transient spike-like secretory profile similar to the first phase of glucose-induced insulin secretion and it fails to produce any chronic insulin secretory response comparable to the second phase of insulin secretion. The insulinotropic effect of beta-endorphins occurred at concentrations varying from 0.1 to 5.0 ug/ml. These data suggest that beta-endorphin and beta-endorphin1-27 potentiate insulin secretion via a common beta cell opioid receptor, and that beta-endorphin may exert a paracrine control of insulin secretion. However, any such regulation appears to be via short-term alterations in the secretory process per se.     

Pineal mediation of the thermoregulatory and behavioral activating effects of beta-endorphin

Intraventricular administration of the opioid peptide, beta-endorphin to goldfish altered their body temperatures and activity levels. Low doses (0.5-5.0 pg g-1 body weight) of beta-endorphin significantly increased behaviorally selected body temperatures while higher doses (15 pg g-1) decreased the preferred temperatures selected in horizontal thermal gradients. There was a significant day-night rhythm in the extent of these effects. These thermoregulatory effects could be blocked and reversed by systemic administration of the opiate antagonist, naloxone, supporting mediation of the thermoregulatory effects at opioid receptors. In addition, administration of naloxone by itself significantly decreased preferred temperature. Removal of the pineal gland significantly increased the preferred temperatures selected by goldfish and eliminated the thermoregulatory effects of beta-endorphin administration in both the day and the night. The behavioral activity effects of beta-endorphin were dependent on the thermal conditions. In fish held at a constant temperature (20 degrees C) beta-endorphin caused a dose-dependent increase in activity, while in individuals held in thermal gradients administration of beta-endorphin had no effects on activity. In both situations naloxone caused a decrease in activity levels. Pinealectomy also eliminated the behavioral activating effects of beta-endorphin, though it had no apparent effects on the actions of naloxone. These results indicate that the pineal gland is involved in the mediation of the thermoregulatory and behavioral activating effects of beta-endorphin. Speculations are made as to the possible mechanisms of action of the pineal gland in mediating the effects of opioid neuropeptides.     

Binding characteristics of dermorphin, and [dermorphin1-7]-beta c-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin (beta c-EP) analog in which residues 1-7 correspond to the dermorphin sequence ([Dermorphin1-7]-beta c-EP) have been investigated with respect to their receptor binding characteristics using human and camel beta-EP as reference peptides. Tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin, ethylketocyclazocine and human beta-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel beta-endorphin was the most potent peptide in all experiments. [Dermorphin1-7]-beta c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala2, D-Leu5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human beta-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine (potency relative to camel beta-EP, 1-4%) but it is more potent (9%) in competition with tritiated human beta-endorphin. Dermorphin and the [Dermorphin-1-7]-beta c-EP appear to interact preferentially with mu opiate receptors.     

Assessment of plasma opioid peptides, beta-endorphin and met-enkephalin, at the end of an international nordic ski race

Plasma met-enkephalin, beta-endorphin, cortisol and lactic acid concentrations were measured in seventeen volunteer male subjects at rest and after a long-distance nordic ski race. Immediately after the race, mean plasma met-enkephalin did not show any significant change, but significant rises in beta-endorphin, cortisol and lactic acid were noted in all skiers. The change in beta-endorphin with exercise was significantly related to the change in cortisol (r = 0.68; p less than 0.001) and to the change in plasma lactic acid (r = 0.60; p less than 0.001). Furthermore, the experienced skiers training over 150 km X week-1 of nordic ski had significantly faster skiing times in this event and showed greater beta-endorphin, cortisol and lactic acid levels than the recreational skiers who trained for 20 km X week-1. Our results imply that the changes in plasma beta-endorphin depend on the intensity of exercise. However the significance of higher levels of skiing training or previous nordic ski experience in the release of beta-endorphin is expected and cannot be excluded.     

beta-endorphin: synthesis and biological activity of shortened peptide chains

Three analogs of beta-endorphin have been synthesized by the solid-phase method: betac-endorphin-(1--5)-(28--31), betac-endorphin-(6--31) and betah-endorphin-(1--5)-(16--31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than beta-endorphin. These data suggest that the entire beta-endorphin molecule is necessary for full in vivo analgesic activity.     

Human beta-endorphin. Immunoreactivity of synthetic analogs with various chain lengths.

Immunoreactivity of synthetic human beta-endorphin analogs with various chain lengths has been examined using a specific radioimmunoassay. It was found that beta-endorphin-(1--21) and analogs of shortened chain exhibit no immunoreactivity, whereas beta-endorphin-(1--15) possesses significant in vitro opiate activity. It appears that immunoreactivity of beta-endorphin resides in the COOH-terminal segment of residues (22--31). The data also show the lack of correlation between opiate and immunological activities of beta-endorphin.     

Infusion of beta-endorphin improves insulin resistance in fructose-fed rats.

In an attempt to probe the effect of beta-endorphin on insulin resistance, we used Wistar rats that were fed fructose-rich chow to induce insulin resistance. Insulin action on glucose disposal rate (GDR) was measured using the hyperinsulinemic euglycemic clamp technique, in which glucose (variable), insulin (40 mU/kg/min), and beta-endorphin (6 ng/kg/min) or vehicle were initiated simultaneously and continued for 120 min. A marked reduction in insulin-stimulated GDR was observed in fructose-fed rats compared to normal control rats. Infusion of beta-endorphin reversed the value of GDR, which was inhibited by naloxone and naloxonazine each at doses sufficient to block opioid mu-receptors. Opioid mu-receptors may therefore be activated by beta-endorphin to improve insulin resistance. Next, soleus muscle was isolated to investigate the effect of beta-endorphin on insulin signals. Insulin resistance in rats induced by excess fructose was associated with the impaired insulin receptor (IR), tyrosine autophosphorylation, and insulin receptor substrate (IRS)-1 protein content in addition to the significant decrease in IRS-1 tyrosine phosphorylation in soleus muscle. This impaired glucose transportation was also due to signaling defects that included an attenuated p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) and Akt serine phosphorylation. However, IR protein levels were not markedly changed in rats with insulin resistance. beta-endorphin infusion reversed the fructose-induced decrement in the insulin-signaling cascade with increased GDR. Apart from IR protein levels, infusion of beta-endorphin reversed the decrease in protein expression for the IRS-1, p85 regulatory subunit of PI3-kinase, and Akt serine phosphorylation in soleus muscle in fructose-fed rats. The decrease in insulin-stimulated protein expression of glucose transporter subtype 4 (GLUT 4) in fructose-fed rats returned to near-normal levels after beta-endorphin infusion. Infusion of beta-endorphin may improve insulin resistance by modulating the insulin-signaling pathway to reverse insulin responsiveness.     

Lymphocyte-mediated regulation of beta-endorphin in the myenteric plexus

Lymphocytes are antinociceptive and can modulate visceral pain perception in mice. Previously, we have shown that adoptive transfer of CD4+ T cells to severe combined immune-deficient (SCID) mice normalized immunodeficiency-related visceral hyperalgesia. Pain attenuation was associated with an increase in beta-endorphin release by T cells and an upregulation of beta-endorphin in the enteric nervous system. In this study, we investigated the relationship between T cells and opioid expression in the myenteric plexus. We examined opioid peptide and receptor expression in the myenteric plexus in the presence and absence of mucosal T cells. We found a positive association between T cells and beta-endorphin expression; this was accompanied by a downregulation of the micro-opioid receptor (MOR). In vitro, T helper (Th) type 1 and type 2 cytokine stimulation of CD4+ T cells or isolation of T cells from in vivo Th-polarized mice did not increase T cell release of beta-endorphin or the induction of beta-endorphin expression in the myenteric plexus. However, exogenous beta-endorphin did upregulate beta-endorphin expression, and both cycloheximide and naloxone methiodide inhibited peptide upregulation. Therefore, our results suggest that nonpolarized CD4+ T cells release beta-endorphin, which, through an interaction with MOR, stimulates an upregulation of beta-endorphin expression in the myenteric plexus. Thus, we propose that the mechanism underlying lymphocyte modulation of visceral pain involves T cell modulation of opioid expression in the enteric nervous system.     

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.     

Beta-endorphin-(1-27) antagonizes beta-endorphin-induced hypothermia in mice

The effects of beta-endorphin and beta-endorphin-(1-27) on the body temperature of mice was studied at an ambient temperature of 10 degrees C. Intracerebroventricular injection of beta-endorphin (0.25-4 micrograms) and beta-endorphin-(1-27) (0.61 to 10 micrograms) caused a dose-related hypothermia. The duration of hypothermia induced by beta-endorphin-(1-27) was shorter than that induced by beta-endorphin. The hypothermia induced by 2 micrograms of beta-endorphin was attenuated by 5 micrograms of beta-endorphin-(1-27). Our results indicated that beta-endorphin-(1-27) is a partial agonist which produces a small degree of hypothermia and an antagonist which blocks the beta-endorphin-induced hypothermia.