Conversion of Des-tyrosine-γ-endorphin by brain synaptic membrane associated peptidases: Identification of generated peptide fragments

Des-tyrosine-γ-endorphin, a β-endorphin fragment with neuroleptic-like properties, was digested with a cSPM fraction of rat brain. A profile of metabolites and a time course of conversion were obtained by HPLC analysis of the digests. Quantitative amino acid analysis and a second HPLC fractionation step which was designed to separate and to identify very similar des-tyrosine-γ-endorphin fragments, combined with dansyl end group determination allowed the characterization of β-LPH 65–77, β-LPH 66–77 and β-LPH 62–73 as main conversion products. In the digests the C-terminal leucyl peptides β-LPH 67–77 and β-LPH 68–77 as well as the N-terminal glycyl peptides β-LPH 62–74 and β-LPH 62–76 were minor components. The data indicate the involvement of several types of peptidase activities in the conversion process. It is suggested that these peptidases have a role in mediating in vivo des-tyrosine-γ-endorphin effects. In addition, this study points to the capacity of the brain to gene-rate small peptides with neuroleptic-like properties.     

Biosynthesis of beta-endorphin by the neurointermediate lobes from rats treated with morphine or alcohol

Rats were rendered tolerant to either morphine or alcohol, by 21- day drug treatment. The neurointermediate lobes (NIL) were removed and incubated with [³H]-phenylalanine for 3 hrs. The biosynthesized pro-opiomelanocortin (POMC), β-lipotropin (β-LPH) and β-endorphin- like peptides (β-EPLPs) were purified from the total protein extract of the NIL by immunoprecipitation with an antiserum to β-endorphin (β-EP), and analyzed by sodium dodecyl sulfate polyacrylamide disc gel elecrophoresis. The β-EPLPs were further characterized by extraction from the gel and microsequencing. The homology of rat POMC to authentic bovine POMC was established by extraction from the gel and peptide mapping of its tryptic digestion products. Furthermore, the β-endorphin like immunoreactivity (β-EPLI) was estimated in the incubation medium and in the NIL extract. The morphine treatment induced a decrease in the degree of incorporation of [³H]- phenylalanine into POMC, β-LPH and β-EPLPs, associated with a decrease in the content of β-EPLI in the NIL extract and in the incubation medium. Alcohol induced an increase in the degree of incorporation of [³H]-phenylalanine into POMC, β-LPH and β-EPLPs, and an increase in the β-EPLI content in the incubation medium, but no change in the β-EPLI in the NIL extract. These results indicate an effect of chronic morphine and alcohol treatment on the biosynthesis and release of β-EPLPs by the NIL.     

Human small cell lung cancer cells express high affinity naloxone-insensitive [125I]β-endorphin binding sites

Previous reports have demonstrated that β-endorphin stimulates the clonal growth of human small cell lung carcinoma (SCLC) cell lines. In this study, the human SCLC lines, NCI-H69, NCI-H345, and NCI-N417, were observed to be highly-enriched in saturable, high-affinity binding sites which are labeled by [¹²⁵I]β-endorphin. In contrast to conventional opioid receptors, [¹²⁵I]β-endorphin SCLC binding was insensitive to naloxone and other mu, delta, or kappa opioid ligands. Further analysis of the NCI-H69 cells demonstrated that specific (naloxone-insensitive) binding was dependent on receptor concentration, reversible, sensitive to sodium ion, but insensitive to the GTP analogue, Gpp(NH)p. These results suggest a role for naloxone-insensitive β-endorphin in modulating SCLC metabolism.     

Characterization of a plasma factor having opiate and immunoactivity like beta-endorphin.

A factor from human plasma having opiate-like activity was characterized in the present study. In addition to its ability to inhibit the binding of [³H]-methionine-enkephalin to opiate receptors, it also cross-reacted with two β-endorphin specific anti-sera. Compared with β-endorphin, the plasma factor had a shorter action on inhibiting the contraction of the guinea pig ileum. By gel-filtration chromatography, the size of this factor was intermediate between that of β-endorphin and methionine-enkephalin.     

Effects of peptides of pituitary origin on the formation of C21 steroid by fetal calf adrenal cells in culture.

Corticotrophic activity of opiate-like peptides was assessed by their ability to stimulate the formation of C₂₁ steroids from [³H] progesterone by three-day old cultures of fetal calf adrenal cells. ACTH_1–39, ACTH_α1–24 and a purified preparation of pituitary ovine β-endorphin caused a marked increase in 17α and 21-hydroxylation while a preparation of pure synthetic porcine β-endorphin gave a minimal stimulation. The activity of the purified ovine β-endorphin preparation could not be accounted for by contamination by ACTH or by a synergistic action between the two peptides. The novel pituitary factor described here may be due to a contaminant of the β-endorphin peak which is different from ACTH_1–39.     

Presence of immunoassayable beta-lipotropin in bovine brain and spinal cord: lack of concordance with ACTH concentrations.

Discrete areas of freshly obtained adult bovine brain were assayed for their content of immunoreactive β-lipotropin (β-LPH), ACTH and β-endorphin. Highest concentrations (pg/100ug protein) of β-LPH were present in hypothalamus (517 ± 81), hippocampus (218 ± 60), central grey rostral mesencephalic level, pons, striatum, and spinal cord (163–258). Lesser concentrations (49–138) were present in other parts of the limbic system, brain stem, cortex and thalamus. Immunoreactive ACTH concentrations were highest in hypothalamus (1702 ± 487) and hippocampus (210 ± 40), with markedly lesser concentrations (5–24) being present in all the other aforementioned areas. Immunoreactive β-endorphin concentrations in hypothalamus were 1990 ± 510, in hippocampus 280 ± 50.     

Comparative metabolic clearance rates of beta-endorphin and beta-lipotropin in humans

In normal human subjects under basal conditions, we have reported that molar concentrations of immunoreactive β-lipotropin (IR-β-LPH) are approximately threefold greater than those of IR-β-endorphin (β-Ep). Following acute stimulation, there is a further two- to threefold disproportionate rise in plasma concentrations of IR-β-LPH as compared to those of IR-β-Ep. To begin to assess the possible factors involved in such altered IR-β-LPH/IR-β-Ep ratios in plasma, the metabolic clearance rate (MCR), volume of distribution (V_d), fractional rate of disappearance (K_d), and half-life ($\text{t}\text{1}\text{2}$) of these peptides were determined by means of bolus injection of highly purified human β-LPH and synthetic human β-Ep in normal human subjects. β-Ep was found to have an MCR and a K_d greater than that of β-LPH, and a shorter $\text{t}\text{1}\text{2}$. These differences, however, although they may in part be contributory, cannot solely account for the greater ratio of IR-β-LPH to IR-β-Ep in plasma, or for the disproportionate rise in plasma concentrations of these peptides after acute stimulation.     

Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with ¹²⁵I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC₅₀ = 1–13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [¹²⁵I]8 and [¹²⁵I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [¹²⁵I]19a/19b and [¹²⁵I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [¹²⁵I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2–3-fold lower uptake in the liver and blood than that observed for [¹²⁵I]MIP-1095. [^125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.     

The effect of running on plasma β-endorphin

Plasma β-endorphin immunoactivity was measured by RIA in 26 trained long distance runners on 35 occasions before and after running. Mean total β-endorphin immunoactivity increased from 11.8 ± 1.8 (SEM) to 17.6 ± 3.1 pg/m1 in 20 runners after an easy run (p = .067), and from 8.2 ± 1.03 to 28.0 ± 6.3 pg/m1 in 15 runners after a strenuous run (p = .008). Total β-endorphin immunoactivity in the plasma extracts of 7 runners before and after the strenuous run was further characterized by Sephadex G-50 chromatography in order to separate β-endorphin from corssreacting β-lipotropin (β-LPH). A rise in β-endorphin and β-LPH concentrations after running was noted in 5 out of 7 runners.     

Presence of immunoreactive β-endorphin in plasma of patients with Nelson's syndrome and Addison's disease

A sensitive radioimmunoassay for β-endorphin (β-EP) has been developed with an antiserum obtained from a guinea pig immunized with β-EP which was contained in crude porcine ACTH preparations (Organon). The minimal detectable quantity of β-EP was 1 pg. This antibody has the same affinity for β-EP and β-LPH on a molar basis, but human ACTH, α-MSH, β-MSH, α-EP, γ-EP, Met⁵-Enkephalin and Leu⁵-Enkephalin failed to displace ¹²⁵I-β-EP from its antibody. Utilizing this radioimmunoassay we have demonstrated the existence of β-EP in plasma from patients with Nelson's syndrome and Addison's disease.     

Generation of methionine and leucine-enkephalin from precursor molecules by cation-sensitive neutral endopeptidase of bovine pituitary

Highly purified preparations of cation-sensitive neutral endopeptidase, from bovine pituitary, and also rabbit brain, generate methionine-enkephalin, from α-endorphin, a peptide containing the amino acid sequence 61–76 of β-lipotropin (β-LPH), The enzyme also catalyzes the hydrolysis of the Leu-Thr bond in the synthetic peptide Tyr-Gly-Gly-Phe-Leu-Thr-2-naphthylamide with the release of leucine-enkephalin and Thr-2-naphthylamide. Neither Met- nor Leu-enkephalin are degraded. The data indicate that the presence of a free N-terminal group of tyrosine inhibits the further degradation of Leu- and Met-enkephalin by the endopeptidase. It is suggested that cation-sensitive neutral endopeptidase is one of the enzymes capable of generating Met- and Leu-enkephalin $\text{in}\text{,}\text{vivo}$.     

Isolation of corticotropin-β-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-β-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824–2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel eletrophoresis, using immunoprecipitated and electrophoretically purified [¹²⁵I]corticotropin-β-lipotropin common precursor as a marker. The preparation was judged homogenous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and β-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-β-lipotropin common precursor possessed specific activities of 116 μg of immunoreactive corticotropin and 210 μg of immunoreactive β-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.     

Design,synthesis, and biological evaluation of radioiodinated benzo[d]imidazole-quinoline derivatives for platelet-derived growth factor receptor β (PDGFRβ) imaging

Several malignant tumors and fibrotic diseases are associated with PDGFRβ overexpression and excessive signaling, making this receptor attractive for molecular targeting and imaging approaches. A series of benzo[d]imidazole-quinoline derivatives were designed and synthesized to develop radioiodinated compounds as PDGFRβ-specific imaging probes. The structure activity relationship (SAR) evaluation of the designed compounds was performed. Among them, 2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (5a) and 4-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (5d) exhibited a relatively high PDGFRβ-TK inhibitory potency, whereas iodinated 5a derivative 5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (8) exhibited a superior inhibitory potency as PDGFRβ inhibitor than iodinated 5d derivative 4-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (11). Furthermore, [¹²⁵I]8 and [¹²⁵I]11 were synthesized and evaluated for PDGFRβ radioligand ability, both in vitro and in vivo. Cellular uptake experiments showed that [¹²⁵I]8 had a higher uptake in BxPC3-luc cells as PDGFRβ-positive cells than [¹²⁵I]11. Incubation of [¹²⁵I]8 after pretreatment of PDGFRβ ligands significantly reduced the uptake of [¹²⁵I]8. In biodistribution experiments using tumor-bearing mice, [¹²⁵I]8 accumulation in the tumor 1?h postinjection was higher than that of the benzo[d]imidazol-quinoline derivative [¹²⁵I]IIQP, used in our previous research. These results indicate that [¹²⁵I]8 could be a promising PDGFRβ imaging agent. Although its clinical application requires further structural modifications, the results obtained in this research may be useful for the development of PDGFRβ-specific radioligands.     

Interaction of synthetic peptide octarphin (TPLVTLFK) with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of non‐opioid β‐endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of [³H]octarphin binding to human T and B lymphocytes separated from normal human blood revealed the existence of one type of high‐affinity binding sites (receptors): K_d 3.0 and 3.2 nM, respectively. Besides unlabeled octarphin, unlabeled β‐endorphin possessed the ability to inhibit the specific binding of [³H]octarphin to Т and B lymphocytes (K_i 1.9 and 2.2 nМ, respectively). Tests of the specificity of the receptors revealed that they are not sensitive to naloxone, α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin. Thus, both T and B lymphocytes from normal human blood express non‐opioid receptor for β‐endorphin. Binding of the hormone to the receptor provides a fragment 12–19. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

β-Endorphin inhibits met-enkephalin breakdown by a brain aminopeptidase: Structure-activity relationships

An aminopeptidase solubilized and isolated from rat brain membranes selectively splits the Tyr¹-Gly² peptide bond of Met-enkephalin. β_h-Endorphin, which is apparently resistant to the aminopeptidase, inhibited the action of this peptidase on Met-enkephalin degradation competitively; the K_i value was 11.5 μM. Arg⁰-β_h-endorphin was found to be 10 times more potent than β_h-endorphin. From further structure-activity data it is concluded that the N-terminal amino group and some residues within region 18–31 of the β-endorphin structure are cooperatively involved in binding to the active site of the aminopeptidase.     

Content of beta-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Radioimmunoassays (RIAs) specific for β-LPH_1–47, β-endorphin, α-MSH and β-MSH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50. When components in extracts of both lobes, eluting at the same position, were measured with the β-endorphin and β-LPH_1–47 RIA systems, marked quantitative differences were seen. The main components reacting with the β-LPH_1–47 system in anterior pituitary extract co-migrated with β-LPH and γ-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than β-endorphin. When the β-endorphin RIA system was used, relatively low amounts of immunoreactive material co-migrating with β-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than β-endorphin was observed in intermediate lobe extract. Some β-MSH was seen in the intermediate lobe. These date indicate that the processing of β-LPH is markedly different in the anterior and intermediate bovine pituitary lobes: β-endorphin immunoreactive material predominates in the intermediate lobe whereas β-LPH and γ-LPH predominate in the anterior lobe.     

The biosynthesis of the thiazole moiety of thiamine in Salmonella typhimurium

The mechanism of biosynthesis of 4-methyl-5-β-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various ¹⁴C-labeled precursors was determined. We found that [Me-¹⁴C]methionine, [2-¹⁴C]methionine, [U-¹⁴C]alanine, and [2-¹⁴C]glycine were not incorporated whereas [2-¹⁴C]-tyrosine was incorporated. Degradation of the 4-methyl-5-β-hydroxyethyl thiazole obtained after [2-¹⁴C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-β-hydroxyethyl thiazole biosynthesis.     

B-ETA-Endorphin immunoreactivity in rat and human blood: radioimmunoassay, comparative levels and physiological alterations.

Antiserum against human β-Endorphin (β_hEP) has been obtained from rabbit. The antiserum, diluted $\text{1}\text{1500}$ bound I¹²⁵ β_h-EP, demonstrating an effective range from 10pM to 10nM. The sensitivity of the assay is 2–3 fmoles. This antibody exhibits 10–15% cross-reactivity with human β-Lipotropin (β_h-LPH). β-EP-like immunoreactivity in rat blood has been detected in unextracted samples when compared to blood from hypophysectomized rats. The whole assay and calibration curves are carried out in plasma from hypophysectomized animals. β-EP-like immunoreactivity can be detected in normal rat plasma (75 ± 15 fmole/ml), and exhibits substantial increases with adrenalectomy (287 ± 32 fmoles/ml). In contrast, samples from five healthy normal human males gave values near the limits of detection of the assay (12 fmoles ± 3.9 per ml of plasma). Such values may be due to cross-reactivity of the antiserum with β_h-LPH or other circulating hormones. In contrast, patients with elevated ACTH production and normal pregnant humans exhibit significantly elevated levels of β-EP immunoreactivity in plasma.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17β-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca²⁺-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10^?11–10^?6 M, showed an inverted U-shape potency in the regulation of Ca²⁺-ATPase activity. At physiologically relevant concentrations (10^?8–10^?9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10^?11–10^?6 M) and 17β-estradiol (10^?12–10^?9 M) caused a dose-dependent increase in the hydrolytic ability of Ca²⁺-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

Avian β-endorphin: Alterations in immunoreactive forms in plasma and pituitary of embryonic and adult chickens

1. A heterologous radioimmunoassay for β-endorphin (β-endo) was established. Plasma and/or extracts of pituitaries from embryonic (days 14.5 and 17.5 of incubation), newly hatched, and adult chickens were chromatographed on a Sephadex G-75 column with 0.1 M acetic acid.2. Embryonic plasma had only a single immunoreactive peak that eluted similar to a β-lipotropin (β-LPH) standard. In contrast, adult plasma had 2 peaks, co-eluting with β-LPH (34%) and β-endo (66%).3. Chromatography of pituitary extracts demonstrated two immunoreactive peaks in both embryonic and adult birds. Although 70% of immunoreactivity eluted with β-endo for embryonic birds, 80% eluted with β-LPH from adults.4. The smaller proportion of β-endo in adult pituitaries may reflect a higher rate of secretion of this hormone into the blood.