Hypoinsulinemic and hyperglycemic effects of beta-endorphin in rabbits

The present study was designed to investigate the in vivo effects of beta-endorphin on plasma levels of glucagon, insulin and glucose in rabbits, and to elucidate some of the mechanisms involved. beta-Endorphin (50 micrograms) injected intravenously into fasted rabbits, decreased plasma levels of insulin (-4.5 +/- 1.3 microU/ml, P less than 0.05) and increased plasma levels of glucose (+2.7 +/- 0.4 mmol/l, P less than 0.05). Similar hypoinsulinemic and hyperglycemic effects were observed for 25 and 2.5 micrograms beta-endorphin in fasted and 50 and 0.5 micrograms beta-endorphin in fed rabbits. beta-Endorphin produced slight and transient increases in plasma levels of glucagon at the highest dose in fed rabbits, only (+80 +/- 9 pg/ml, P less than 0.05). The beta-endorphin-induced hypoinsulinemia was not inhibited by phentolamine, yohimbine, propranolol or atropine, which is in consistency with a direct inhibitory effect of beta-endorphin on the beta-cell in rabbits. The beta-endorphin-induced hyperglycemia was reduced by naloxone (+0.8 +/- 0.1 mmol/l) but not by N-methyl-naloxone (ORG 10908) a peripheral opiate receptor blocking drug (+2.2 +/- 0.2 mmol/l), suggesting a central nervous action on opiate receptors. This central action of beta-endorphin was probably not mediated by catecholamine release or other stimulation of adrenergic or muscarinic receptors, since the beta-endorphin-induced hyperglycemia was not inhibited by phentolamine, yohimbine, propranolol or atropine. These results suggest that the beta-endorphin-induced hyperglycemia was caused, at least in part, by a peripheral inhibition of insulin release and a central stimulation on glucoregulation.     

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.     

beta-Endorphin-induced hyperthermia in the cat

beta-Endorphin was injected into the third cerebral ventricle (ICV) of conscious, unrestrained cats. Hyperthermic response to 50 microgram of this peptide were reduced by 20-100 microgram naloxone given ICV 1 hr later. A dose of 40 microgram beta-endorphin increased body temperature at ambient temperature of 4, 22 and 34 degrees C, with the response being greater the warmer the environment. These results indicate that beta-endorphin acts on a central naloxone-sensitive receptor which is probably the v2 receptor that is activated by low doses of D-Ala2-Met-enkephalinamide to evoke a similar pattern of change in body temperature over a comparable range of ambient temperatures.     

Antinociceptive effects of intrathecally administered human beta-endorphin in the rat and cat

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate and tail-flick response latencies following the injection of human beta-endorphin into the lumbar spinal subarachnoid space through the indwelling catheter. beta-Endorphin was approximately 25 times more potent than morphine on a molar basis. Matching morphine and beta-endorphin doses such that approximately equal submaximal submaximal effects occurred, it was observed that the antinociception produced by beta-endorphin lasted approximately three times longer than that produced by morphine. Experiments with intrathecal injection of beta-endorphin into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by naloxone. In the rats, naloxone administered systemically in doses of 10--100 microgram/kg produced a parallel shift in the dose-response curves of both nociceptive measures suggesting a competitive antagonism. Using a dose ratio analysis, an in vivo pA2 of 7.1 for naloxone was obtained. These data and those derived from previous work based on the pA2 suggest that the interaction of morphine, certain pentapeptides, and beta-endorphin is the same with regard to the spinal opiate receptor population mediating behaviorally defined analgesia.     

gamma-Endorphin generating endopeptidase in rat brain: subcellular and regional distribution

beta-Endorphin is converted into the biologically active fragment gamma-endorphin by an endopeptidase which we term "gamma-endorphin generating endopeptidase". Subcellular and regional distributions of this endopeptidase activity in rat brain were studied by a newly developed assay. After subcellular fractionation of rat brain tissue gamma-endorphin generating endopeptidase activity was predominantly recovered in the cytosolic fraction. A 10 to 15 fold lower activity was present in synaptosomes, mitochondria and synaptic membranes. Hardly any endopeptidase activity was detected in nuclei and myelin. The endopeptidase activity in cytosolic and particulate fraction was found throughout brain, pituitary and spinal cord in a rather homogeneous fashion. Cytosolic activity in all brain parts was 10 to 15 fold higher than the activity in the particulate fraction. It is suggested that rather the beta-endorphin distribution than the endopeptidase is restricting for gamma-endorphin production in certain brain parts.     

Detection from rat pituitary of beta-lipotropin and materials containing opiatelike activity by combined enzymatic radioreceptor assay

Tonin, a proteolytic enzyme isolated from rat submaxillary gland, was allowed to react upon ovine beta-lipotropin (beta-LPH) at 37 degrees C at a variety of pH values and for different lengths of time. Opiatelike activity generated by the reaction was assessed using a radioreceptor assay for beta-endorphin with rat brain homogenate. [3H]naloxone, and beta-endorphin as receptors, tracer, and hormone standard, respectively. Cleavage of beta-LPH with tonin produced a 10-fold increase in opiatelike activity as compared with beta-LPH alone. Digestion of beta-LPH with other enzymes such as renin, cathepsin D, trypsin, and chymotrypsin produced much less opiatelike activity. beta-Endorphin and methionine-enkephalin were not cleaved by tonin. Using this new assay, we were able to detect beta-LPH and materials containing opiatelike activity from rat pituitary extracts after gel chromatography. It is more specific and more sensitive than trypsin digest.     

Identification of beta-endorphin residues 14-25 as a region involved in the inhibition of calmodulin-stimulated phosphodiesterase activity

The inhibition of the calmodulin-mediated stimulation of bovine brain cyclic nucleotide phosphodiesterase activity (3':5'-cyclic adenosine monophosphate 5'-nucleotidohydrolase, EC 3.1.4.17) by the 31-residue opiate peptide beta-endorphin has been investigated. Using conditions in which porcine brain calmodulin (6 nM) is limiting (i.e., to give a 3-fold, Ca2+-dependent stimulation of enzymic activity toward cyclic guanosine monophosphate), the domain of beta-endorphin responsible for the inhibition was mapped by using a series of deletion peptides. beta-Endorphin exhibited an ED50 of several micromolar under the conditions employed, and several amino-terminal deletion peptides were essentially as inhibitory as the parent peptide. Methionine enkephalin and various carboxy-terminal deletion peptides had no demonstrable effect at concentrations of 100-200 microM. Peptides 1-25 and 1-27 (C' fragment) inhibited the calmodulin-dependent activity of phosphodiesterase, but higher concentrations were required than of beta-endorphin. Studies using combined amino- and carboxy-terminal deletion peptides demonstrate that peptide 14-25 was the shortest peptide examined that was capable of inhibiting calmodulin stimulation of phosphodiesterase activity under the conditions used. There was no evidence to indicate that the amino-terminal region comprising residues 1-13 of beta-endorphin contributes to the measured inhibition of calmodulin-stimulated enzymic activity. The circular dichroic spectra of calmodulin, beta-endorphin, and mixtures of the two were obtained, and the ellipticity of the peptide-protein mixtures at 221 nm exceeded that expected by assuming simple additivity. This finding is consistent with a direct interaction of beta-endorphin with calmodulin which seems to lead to enhanced helicity of one or both components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of nonsteroidal anti-inflammatory agents on fibrinolysis and platelet aggregation

The influence of non-steroidal antiphlogistics (NSA, fluor derivatives of phenylanthranilic acid) on fibrinolysis, platelet function, prostaglandin metabolism and pharmacokinetics of indirect anticoagulants was studied in rats and rabbits in vitro and in vivo. NSA were found to shorten the euglobulin lysis time and to enlarge the lysis zones on fibrin plates. They potentiated the fibrinolytic activity of streptokinase and trypsin. Furthermore, they inhibited platelet aggregation induced by arachidonic acid in rabbits. NSA in combination with inhibitors of thromboxane synthetase potentiated inhibition of aggregation. After oral administration, NSA inhibited formation of thromboxane A2 and prostacyclin in rabbits in a dose-dependent manner. At comparatively low doses, thromboxane A2 synthesis was more effectively inhibited than prostacyclin formation. Due to pharmacokinetic interactions NSA enhanced the anticoagulant effect of indirect anticoagulants and accelerated their distribution and elimination.     

An investigation on pro-opiomelanocortin and processed peptides from the teleost fish Prochilodus platensis

Acid extracts of carefully dissected proadenohypophysis (PA) and metaadenohypophysis (MA) of the teleost Prochilodus platensis were subjected to chromatography in Sephadex G-50 after which several pro-opiomelanocortin (POMC) peptides were detected by means of three heterologous RIA systems: alpha-MSH, ACTH and beta-endorphin. Parallelism among extracts displacement curves ranged from 26% to 95% of those of the standard curves for the different systems employed. In PA chromatograms, peaks of ACTH immunoreactivity (IR) were detected at the positions of 30 kilodalton (K), 20K, 9K, a large 4.5K peak and 2K. Only one peak of beta-endorphin IR was detected at 30K. In MA chromatograms, ACTH IR detected similar peaks as in PA runs, but 4.5K peak was much smaller, whereas a large 2K peak roughly coincided with all alpha-MSH detected in the chromatograms. beta-Endorphin IR was detected mainly as a large peak coinciding with synthetic beta-endorphin in MA runs. Bioactivity was detected in both PA and MA 4.5K ACTH peaks, whereas little activity could be demonstrated associated with the 30K, 20K and 9K ACTH IR peaks. Prochilodus PAs and MAs were incubated with tritiated aminoacids and the extracts immunoprecipitated with ACTH, beta-endorphin and N-terminal POMC (N-POMC) antisera. The dissociated complexes were run in SDS polyacrylamide slab gel electrophoresis. The tritiated bands detected confirmed the results obtained with Sephadex chromatography. N-POMC immunoprecipitated peptides were located at 28K, 18K and 9K positions. The first two probably accounted for POMC and the N-POMC/ACTH intermediate respectively; the third corresponded to the mammalian 1-76N-POMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

beta-Endorphin: hyperthermia in mice by intravenous injection

Effects of intravenous beta-endorphin on body temperature and body weight loss were studied in naive and morphine-dependent mice. beta-Endorphin at doses 2.6-25.5 mg/kg injected intravenously caused hyperthermia in naive mice as well as in morphine-dependent mice. In addition, beta-endorphin and morphine reduced body-weight loss during the morphine withdrawal.     

Distribution of opioid peptides in the pituitary: a new hypothalamic-pars nervosa enkephalinergic pathway

The distribution of the endorphins, beta-endorphin and enkephalin (Met5-enkephalin and Leu5-enkephalin), was determined in the pars distalis, intermedia, and nervosa of the rat pituitary using both immunocytochemical and radioimmunological methods. Immunoreactive (ir) beta-endorphin was found in pars distalis and pars intermedia. On gel filtration of the pars distalis extracts, beta-endorphin immunoreactivity was eluted in three peaks corresponding to pro-opiocortin (5%), beta-lipotropin (75%), and beta-endorphin (20%). beta-Endorphin was the only component in the pars intermedia. Enkephalin was found in high amount in the pars nervosa. A new enkephalinergic hypothalamic-pars nervosa pathway was observed. Dehydration experiments on normal rats and analysis of the genetically polyuric Brattleboro rat suggest that this enkephalinergic pathway may modulate neurohypophyseal neurosecretion.     

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.     

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 +/- 2.0 nM, Bmax1 = 40.7 +/- 2.3 pmol/mg protein; Kd2 = 0.25 +/- 0.01 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10(-9)-10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

Antiepileptic Agents Affect Hypothalamic β-Endorphin Concentrations

beta-Endorphin, Met-enkephalin, substance P, and somatostatin concentrations were evaluated in the hypothalami of rats treated either acutely or chronically (15 days) with sodium valproate, diphenylhydantoin, phenobarbital, or ethosuximide. All of these drugs, with the exception of ethosuximide, induced significant decreases in beta-endorphin concentrations after acute treatment, while only sodium valproate induced a decrease after chronic treatment. The acute and chronic effects of sodium valproate were also produced by aminooxyacetic acid, an inhibitor of gamma-aminobutyric acid (GABA) transaminase, while another GABA transaminase inhibitor, ethanolamine-O-sulphate, and THIP, a GABA receptor agonist, were effective after acute administration. Metenkephalin, substance P, and somatostatin concentrations were never affected by the drugs used. The present results, indicating that antiepileptic agents specifically decrease beta-endorphin concentrations, seem to correlate well with the capacity of these agents to blunt the epileptic activity of the peptides tested. Moreover, our data suggest that GABA may be involved in the anticonvulsant-induced reduction of beta-endorphin concentrations.     

Opioid peptides in micellar systems: conformational analysis by CD and by one-dimensional and two-dimensional 1H-NMR spectroscopy

beta-Endorphin has been studied in SDS micelles by one- and two-dimensional nmr spectroscopy (1D and 2D nmr), and to explore the influence of peptide length and composition on the polypeptide structure, the investigation was extended to a number of fragments. The nmr results are compared with those obtained from CD experiments and discussed in terms of a secondary structure that involves the central region of beta-endorphin.     

Distribution of beta-endorphin-related peptides in rat pituitary and brain.

beta-Endorphin, the most potent known naturally occurring analgesic agent, is found in pituitary and brain in company with a series of structurally and biosynthetically related peptides that are essentially devoid of opiate activity. In studies of beta-endorphin it is important to discriminate between the active and inactive forms of the peptide. This review describes the use of chemical and immunological methods for localizing the peptides in the tissues, extracting and resolving the peptides by chromatography, and determining the concentrations of the peptides by radioimmunoassay. These approaches have allowed the distribution of beta-endorphin and its related peptides to be assigned unequivocally in regions of rat pituitary and brain. It has been found that the multifunctional corticotropin-endorphin prohormone can undergo processing by different mechanisms in different tissues, permitting the intrinsic activities of its fragments to be expressed selectively. The different processing patterns can be attributed to the existence of highly specific enzymes, characteristic of individual cells, which regulate the formation of this potent opiate.     

Chimeric derivative of fibrolase, a fibrinolytic enzyme from southern copperhead venom, possesses inhibitory activity on platelet aggregation

Fibrolase, a metalloproteinase isolated from the venom of Agkistrodon contortrix contortrix (southern copperhead snake), is a direct acting fibrinolytic enzyme that has been used to digest occlusive blood clots in animal models. The snake venom enzyme directly degrades fibrin associated with platelet rich blood clots and does not rely on plasminogen activation. Rethrombosis is a serious complication that is experienced in a significant percentage of patients treated with thrombolytic agents to remove occlusive vascular thrombi. The involvement of platelets in the initiation of rethrombosis is well known. Arg-Gly-Asp-(RGD)-containing agents have been shown to inhibit rethrombosis following thrombus dissolution by plasminogen activators. In an effort to create a more effective fibrinolytic enzyme and to target the enzyme to platelet-rich thrombi, thereby decreasing the potential for rethrombosis, a chimeric derivative of fibrolase has been produced. This report describes the construction and biochemical characterization of the chimeric enzyme and an evaluation of its in vitro activities. The chimera was formed by covalently incorporating an RGD-like peptide into fibrolase. The site of peptide attachment was determined to be a single lysine residue remote from the enzymes active site. Covalent modification of fibrolase with the RGD-like peptide did not inhibit either fibrinolytic activity of the enzyme nor platelet aggregation inhibitory activity of the peptide. The chimera not only retained the same level of enzymatic activity as native fibrolase, but also acquired the ability to inhibit platelet aggregation by binding to the fibrinogen receptor (integrin alphaIIbbeta3) on platelets.     

Prophylaxis of recurrent venous thrombosis with long-term enhancement of fibrinolysis.

The effects of 6 months' combined therapy with phenformin and an anabolic steroid were compared in patients with thrombophlebitis migrans (12 patients) and those with superficial thrombophlebitis (15 patients). In both groups of patients an increase in blood fibrinolytic activity, and "capacity" decrease in platelet adhesiveness, plasma fibrinogen, blood lipids, beta lipoproteins as well as serum cholesterol level were found. A statistically significant decrease in frequency of inflammations in patients with thrombophlebitis migrans occurred. In these patients a return of the previously low "fibrinolytic capacity" to normal values was observed. It seems that prolonged activation of fibrinolysis by means of phenformin and an anabolic steriod may be of value in the prophylaxis of venous thrombosis especially thrombophlebitis migrans.     

Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom.

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.     

急、慢性缺氧对大鼠凝血机能的影响

本文讨论了实验性缺氧对大鼠凝血机能产生的影响。实验结果表明,大鼠在模拟8000米高度两小时后,血小板凝聚性、血小板因子3活性明显增加,纤溶活性下降,同时,纤维蛋白原含量和因子X也明显下降。大鼠在模拟7000米经36天间歇性慢性缺氧,血小板计数、血小板凝聚性,血小板因子3活性、纤维蛋白原含量、因子X活性均显著增加,部分凝血活酶时间缩短、纤溶活性下降,明显地出现凝血增强的趋势。本文还讨论了抗缺氧药物复方党参、异叶青兰对人鼠急性缺氧时凝血机能的影响。