Characterization of in vitro proteolytic processing of beta-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of beta-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 beta-endorphin related fragments simultaneously was used to study the time-course processing of beta-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between alpha- and gamma-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of alpha/gamma-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that beta-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of alpha/gamma-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

Differential in vitro metabolism of β-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of β-endorphin (βE) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of βE by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (−80°C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment desenkephalin-γ-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of βE 6–21 was also reported. These data suggest that an approach using postmortem human brain is possible in studying β-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

Proteolysis of β-endorphin in brain tissue

The processing of β-endorphin by brain enzymes into peptides related to the behaviorally active γ- and α-type endorphins and the sequence of proteolytic events in the conversion process are described. Multiple enzyme activities contribute to the generation of the peptides with neurotropic activity. It is proposed that the processing into γ- and α-type neuropeptides is a post-secretional event. The enzymes involved may have a key role in the nature and levels of neurotropic β-endorphin fragments in the brain.     

Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase

ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin-related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions.     

Isolation and characterization of alpha-endorphin and gamma-endorphin from single human pituitary glands

alpha-Endorphin and gamma-endorphin, two closely related peptides of the pro-opiomelanocortin family with characteristic biological activities, were purified to homogeneity from single human pituitary glands and chemically identified. Isolation of the peptides was based on size fractionation by Sephadex G-75 chromatography followed by two HPLC steps using reverse-phase and paired-ion reverse-phase systems and was monitored by radioimmunoassay. During the isolation procedure alpha- and gamma-endorphin-sized material behaved chromatographically and immunologically indistinguishably from synthetic alpha- and gamma-endorphin. The amino acid composition and NH2-terminus of isolated peptides demonstrated their identity as authentic alpha-endorphin and gamma-endorphin. Acetylated forms were absent. In addition, evidence is provided that large forms with alpha- and gamma-endorphin immunoreactivity detected during gel filtration are human lipotropin-(1-74) and -(1-75), respectively. The data substantiate that alpha-endorphin and gamma-endorphin exist as endogenous peptides in the human pituitary gland.     

Neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity in the cat hypothalamus

Immunohistochemical studies were conducted on sections of cat hypothalamus in order to determine the distribution of neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity. A large number of neurons in the arcuate nucleus were stained after incubation of sections with antisera to either substance. Analysis of serial sections suggested that each neuron revealed with one antiserum was also revealed with the other antiserum, indicating the co-existence of alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity within these arcuate neurons. In contrast, a more diffuse group of lateral hypothalamic neurons which extended from the retrochiasmatic level to the posterior hypothalamus were stained only with the antiserum directed against alpha-melanocyte stimulating hormone. The present results largely confirm findings in the rat hypothalamus, although the lateral hypothalamic group of alpha-melanocyte stimulating hormone immunoreactive neurons appears to be more extensive in the cat.     

Gamma-endorphin-like peptides in pituitary tissue: evidence for their existence in vivo

Beta and gamma endorphin-like peptides were measured by radioimmunoassay in whole pituitary. Boiling of acetic acid extracts prior to tissue disruption increased the concentration of both beta E- and gamma E-like peptides. The gamma E-like immunoreactivity from the neurointermediate lobe of the pituitary co-eluted with synthetic gamma E upon gel permeation chromatography. Immunoreactivity for beta E-like and gamma E-like peptides in the intermediate lobe of the pituitary was also shown by immunoperoxidase staining. The results suggest that gamma E-like peptides are present primarily in the pars intermedia in vivo and do not arise as artifacts of acid extraction of pituitary tissue.     

Modulation of spontaneous seizures in the mongolian gerbil: effects of beta-endorphin

Intraventricular injection of beta-endorphin (0.1-3 micrograms) into gerbils from the UCLA seizure sensitive strain reduced the incidence and severity of spontaneous epileptiform seizures, both the motor manifestations and the preceding high voltage focal spiking and accompanying seizure activity in the cortical EEG. This "'anticonvulsant" effect of beta-endorphin was prevented by prior administration of naloxone (1 mg . kg-1 IP). These findings suggest that the endogenous opioid peptide may be involved in the normal suppression of the epileptic diathesis in these animals during the interictal periods.     

The role of CYP 1A1 in the in vitro metabolism of pregnenolone by the liver of rainbow trout embryos

We determined the bile acid profiles in bile juice of snake gallbladders by HPLC on a silica gel RP-18 reversed-phase column. Cholic acid and chenodeoxycholic acid were predominant components in three of four snake species. To elucidate the toxic effect of snake bile acids on rats, a synthetic bile acid mixture was prepared mimicking the bile acid composition of a snake Naja naja atra bile juice. Twenty-four male Wistar rats were divided into four groups and treated orally at 3-day intervals with saline (control group) and different doses (1-3x doses) of the bile acid mixture. After treatment, the following parameters increased: the relative ratios of liver and kidney mass to body mass, the concentrations of red blood cell, hemoglobin and hematocrit in the blood, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, plasma urea nitrogen and creatinine in the plasma, and the levels of urine urea nitrogen and creatinine in the urine. Body mass of rats and the levels of Na+, K+, Ca++ in the urine of rats were significantly decreased, especially for groups treated with 2x and 3x doses of the bile acid mixture. Examination of liver and kidney pathology also showed cell enlargement and lesion in cell integrity in treated groups, especially for groups treated with 2x and 3x bile acid mixture, indicating that short-term toxicity of snake N. naja atra bile acids was significant in rats.     

Fronto-cortical regulation of beta-endorphin actions in the rat

Ablation of the frontal neocortex markedly enhanced the antinociceptive and cataleptic actions of beta-endorphin injected into the lateral ventricle of rat brain. This enhanced response was not affected by simultaneous administration of cholecystokinin octapeptide (CCK-8). In sham-operated rats, however, CCK-8 suppressed the effects of beta-endorphin in a dose-related manner. Moreover, ablation of a similar amount of occipital neocortex did neither affect beta-endorphin actions nor the interactions of CCK-8.     

雌激素在大鼠杏仁核与纹状体多巴胺代谢中作用的差异

为探讨雌激素对大鼠杏仁核（amygdala,Amy）与纹状体（striatum,Str）多巴胺（dopamine,DA）代谢的作用,本实验采用离体电化学检测技术--高效液相色谱法（high performance liquid chromatography,HPLC）测定正常雌鼠及经雌激素处理的去卵巢（ovariectomy,OVX）雌鼠Amy和Sr的DA及其代谢产物的组织含量。实验结果显示,OVX雌鼠经雌激素处理后,可引起Amy的DA及其代谢产物含量减少,而Str的DA及其代谢产物含量不受其影响。OVX雌鼠Amy的DA更新率低于正常及雌激素处理的OVX鼠,Amy组织的DA含量约是Str组织的1/6,而更新率是Str的2倍左右。以上结果提示,雌性大鼠血清雄激素浓度可影响其Amy组织的DA代谢及组织含量,而Str的DA组织含量不因雌激素浓度的改变而变化。     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Reduced ACTH, while normal beta-endorphin CSF levels in early epileptic encephalopathies

Since ACTH and the opioids display opposite effects on experimentally-induced seizures, cerebrospinal fluid (CSF) levels of ACTH and beta-endorphin (beta-EP) were measured in 6 children (4-8 months) affected by infantile spasms with hypsarhythmia, an idiopathic early onset encephalopathy, and in 8 age-matched controls. beta-EP levels in the patients (76.3 +/- 14.7 fmol/ml, M +/- SD) did not differ from those in controls (109.8 +/- 42.7) while babies with epileptic encephalopathy showed reduced ACTH levels in the CSF (3.8 +/- 1.5) as compared to controls (9.0 +/- 3.7, p less than 0.01). This resulted in an increased beta-EP/ACTH ratio. Another patient previously treated with ACTH showed a normal CSF level of ACTH (9.0) with a normal beta-EP/ACTH ratio while in clinical and EEG remission. These results are consistent with the hypothesis that some infantile seizures unrelated to brain injuries could originate from an ACTH deficiency at central level and/or an imbalance of neuropeptidergic pathways.     

The effects of MIF-I, beta-endorphin and alpha-MSH on d-amphetamine induced paradoxical behavioral thermoregulation: possible involvement of the dopaminergic system

The neuropharmacological basis for d-amphetamine induced paradoxical behavioral thermoregulation remains unclear. This study examined thermoregulatory behavior of rats in a runway device that housed a heat source at one end and in which locomotion along the length of the runway could be observed. Sprague Dawley rats were pretreated with IP injections of saline, beta-endorphin, MIF-1, or alpha-MSH, with a repeat injection after 30 min. In a second experiment, d-amphetamine was administered as the repeat drug for all Ss. The results showed clear differences for heat-source-on vs. heat-source off. All peptides induced hypermotility, although no differentiated effects for the peptides on d-amphetamine induced paradoxical behavioral thermoregulation were found. These findings are discussed in light of the theoretical possibilities that: (a) a ceiling effect exists; (b) there are separate control systems for maintaining body temperature and another for behavioral thermoregulatory responses, and (c) other neurotransmitters may be involved in such induced paradoxical behavioral thermoregulation.     

Characterization of in vitro proteolytic processing of β-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of β-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 β-endorphin related fragments simultaneously was used to study the time-course processing of β-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between α- and γ-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of α/γ-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that β-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of α/γ-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

γ射线照射对人肌腱组织形态学及羟脯氨酸含量的影响(英文)

目的:研究2.87Mrad剂量γ射线照射后,肌腱组织形态学及羟脯氨酸含量的变化。方法:选取40根人新鲜上肢掌深屈肌腱,-80℃深低温冷冻6周,等分为二:实验组(A组),对照组(B组)。A组肌腱在干冰低温保存条件下行γ射线照射,检测肌腱最终吸收剂量为2.87Mrad。分别行HE、VG染色,在普通光镜和透射电镜下观察其组织形态学变化。用柱前衍生高效液相色谱检测肌腱胶原蛋白水解液中羟脯氨酸含量。结果:①形态学观察发现,同非照射组肌腱相比,照射组肌腱胶原纤维束间隙较大,纤维排列卷曲紊乱,并可见部分断裂。电镜下胶原纤维横纹模糊消失,腱细胞膜溶解消失,核崩解,细胞器减少。②在同等水解条件下,胶原蛋白水解液中照射组羟脯氨酸含量与非照射组相比,有显著差异(P0.05)。结论:2.87Mrad剂量γ射线照射可引起肌腱组织形态结构发生显著改变。在同等水解条件处理下,照射组肌腱胶原蛋白水解液中羟脯氨酸含量显著高于非照射组。     

Action of selected serotonin antagonists on hyperthermia evoked by intracerebrally injected beta-endorphin

Methergoline, an antagonist of cerebral serotonin receptors, has been shown to significantly reduce the rise in rectal temperature (Tre) produced by the intracerebral microinjection of beta-endorphin. In this study the role of serotonin in the increase in Tre elicited by beta-endorphin was further examined using three additional serotonin antagonists. beta-Endorphin was administered twice to rats using a crossover design in which half of the animals were first pretreated with the vehicle solution and half with the antagonist. Serotonin antagonists used were: methergoline, methysergide, cinanserin and cyproheptadine. Although methergoline did cause a marked reduction in the beta-endorphin-induced rise in Tre, neither methysergide, nor cinanserin, nor cyproheptadine produced a marked reduction in the hyperthermia. Since methergoline also interacts with the dopamine receptor, the effect of a dopamine antagonist, haloperidol, on the endorphin-evoked response was also examined. Haloperidol failed to attenuate the rise in Tre. The reason for the apparent discrepancy in the action of these serotonin antagonists is unclear. Further research may reveal distinct subpopulations of serotonin receptors at which these antagonists exert differential effects.     

Measurement of muramic acid in marine sediments by high performance liquid chromatography

Bacterial biomass in marine sediments may be estimated from the amount of muramic acid present. A method for determining muramic acid by high performance liquid chromatography is described, which is simpler and faster than other methods. Muramic acid is released from sediment by acid hydrolysis, and assayed as an o-phthaldialdehyde derivative.     

Thermoregulatory (core, surface and metabolic) responses of unrestrained rats to repeated POAH injections of beta-endorphin or adrenocorticotropin

Repeated preoptic-anterior hypothalamic (POAH) injections of saline and 10 or 25 micrograms/microliters of beta-endorphin or ACTH were given to groups of male Sprague-Dawley rats. One hr after the fifth injection of beta-endorphin or ACTH, each rat received a POAH injection of naloxone HCl (10 micrograms/microliters). Core (Tre-rectal) and surface (Tt-tail) temperatures, metabolic (VO2) and behavioral responses were recorded 30 min before and 60 min after each drug injection. The initial POAH injection of either dose of beta-endorphin produced a hyperthermia. Peak hyperthermia was reduced in the group given 10 micrograms/microliters of beta-endorphin repeatedly. TtS rose after each beta-endorphin injection but temporally lagged Tre increases. Metabolic rate (VO2) was increased with repeated POAH injections of beta-endorphin. Naloxone reduced the elevated Tre seen with beta-endorphin by increasing Tt's further and reducing VO2. POAH administration of ACTH evoked only a slight hyperthermic Tre response, but elevated TtS and VO2S, due to enhanced grooming and explorative behavior. With repeated ACTH injections, TreS did not change from those on the first day as TtS and VO2 remained enhanced. Naloxone reduced VO2 and TtS of the ACTH-treated rats but TreS still were unchanged. Results suggest that the hyperthermia of unrestrained rats given an acute as opposed to repeated POAH beta-endorphin injections is mediated by different effector mechanisms. With the doses used, the slight and unchanging TreS seen with ACTH occurred because this peptide increased heat production due to locomotor activation yet also exaggerated heat loss by vasodilating the peripheral vasculature.     

Peptides in the hemolymph of Hermetia illucens larvae completely inhibit the growth of Klebsiella pneumonia in vitro and in vivo

Extensive use of antibiotics has caused the microbial resistance to rise drastically within the last few decades, and new approaches are therefore needed to develop effective antibacterial substances. In this study, we identified peptide in the hemolymph of Hermetia illucens larvae using reverse-phase chromatography, HPLC and Nano-LC-ESI-MS/MS system. We investigated the antibacterial effect of HP/F9 peptides against Klebsiella pneumoniae in vitro and in vivo. The peptide effectively inhibited the growth of K. pneumoniae in vitro and completely removed K. pneumoniae from the lungs of mice. Importantly, peptides (22,000 Da, HP/F9) successfully reduced lung inflammation upon K. pneumoniae infection. These results indicate that the HP/F9 peptide from H. illucens larva can effectively protect the mouse from K. pneumoniae infection. HP/F9 could be a new candidate for the development of effective antibacterial substance.