Biomedical subjects
Publications and source records attributed to C H Li.
Peptide E (PE), an opioid peptide from adrenal medulla, is known to possess very low analgesic potency when injected intracerebroventricularly in mice. The present study showed that PE analgesia was increased by co-injection with protease inhibitors (bacitracin and bestatin).
Six female adult Macaca mulatta monkeys were made dependent upon morphine sulfate and were implanted with a chronic indwelling needle in the lateral ventricle of the brain for sterile intraventricular injections. Both beta-endorphin and morphine, in a dose dependent manner given intraventricularly suppressed the signs of 14 hour acute morphine abstinence. On a molar basis, beta-endorphin was more active than morphine in suppressing the signs of morphine abstinence. When given intravenously in much larger doses, beta-endorphin was ineffective in contrast to morphine which was effective in suppressing abstinence.
Seven human corticotropin-secreting adenomas causing Cushing's disease or Nelson's syndrome were maintained in long-term culture. Pooled media from the individual adenomas were analyzed for the composition of their secretory products. From a radioimmunoassay (RIA) with 100% cross-reactivity for human beta-endorphin (beta h-EP) and beta-lipotropin (beta h-LPH), immunoreactive beta h-EP (IR X beta h-EP) was found to be the predominant secretory product after Sephadex G-50 analysis in 4 cases (40-80% of total IR), immunoreactive beta h-LPH (IR X beta h-LPH) predominated in 1 case, and both were equipresent in 2-cases. IR X beta h-EP was further purified by high-performance liquid chromatography (HPLC) and analyzed in 4 cases with ion-exchange chromatography on SP-Sephadex C-25 and a RIA which completely cross-reacts with beta h-EP, [N alpha-Ac]-beta h-EP, beta h-EP-(1-27) and [N alpha-Ac]-beta h-EP-(1-27). In all cases, the IR X beta h-EP was the main component (40-70%); the remaining IR material was attributable partially to [N alpha-Ac]-beta h-EP or other, less defined immunoreactive material. In 3 cases, enough IR X beta h-EP material was available for HPLC and to perform a radioreceptor assay using tritiated beta h-EP as primary ligand. The displacing potency of these preparations relative to synthetic beta h-EP was related to the content of the immunoreactive component eluting in the position of synthetic beta h-EP.
We studied the cerebrospinal fluid (CSF) and plasma concentration-time profiles of morphine, methadone, and beta-endorphin after lumbar epidural or intrathecal injection in 17 patients with cancer. After epidural injection, all three drugs reached peak levels in lumbar CSF within 34 minutes that were 50 to 1300 times higher than free drug concentrations in plasma. The rate of decline of CSF levels correlated with drug lipid solubility (methadone [t1/2 = 73 minutes] greater than morphine [126 minutes] greater than beta-endorphin [317 minutes]). Plasma levels were comparable with those after intragluteal injection of the same dose. In four patients given intrathecal morphine or methadone, CSF at the C1-2 level contained high levels of morphine as early as 1 hour after injection, but levels of methadone were lower or undetectable. Three of 17 patients reported improved analgesia initially, but none were improved at 2 weeks after chronic therapy. We conclude that analgesia induced by intrathecal or epidural morphine injections is caused by drug acting at both spinal and supraspinal sites. The use of spinal opiates such as morphine is of limited value in patients whose pain is not adequately managed by high systemic doses of morphine-like drugs.
The potent opioid peptide beta-endorphin is found in the brain and pituitary with two related fragments, beta-endorphin-(1-27) and beta-endorphin-(1-26). The fragments retain substantial opioid-receptor binding activity but are virtually inactive analgesically. beta-Endorphin-(1-27) inhibits beta-endorphin-induced and etorphine-induced analgesia when coinjected intracerebroventricularly into mice. Antagonism by competition at the same site(s) is suggested from parallel shifts of the dose-response curves of etorphine or beta-endorphin in the presence of beta-endorphin-(1-27). Its potency is 4-5 times greater than that of the opiate antagonist naloxone. beta-Endorphin-(1-26) does not antagonize the antinociceptive action of etorphine or beta-endorphin in doses up to 500 pmol per animal.
Two additional peptides with inhibin-like activity have been isolated from human seminal plasma. One consists of 52 amino acids and the other, 92 amino acids. They are designated alpha-inhibin-52 and alpha-inhibin-92. Sequence analyses show that the NH2-terminal 31 amino acids of alpha-inhibin-52 are identical to the structure of the inhibin-like peptide previously reported [ILP-(1-31), now designated alpha-inhibin-31], and the COOH-terminal 52 amino acids of alpha-inhibin-92 are identical to the structure of alpha-inhibin-52. The amino acid sequence of alpha-inhibin-92 is: (sequence in text) Bioassay data in mouse pituitaries in vitro show that alpha-inhibin-52 is 3.4 times more active and alpha-inhibin-92 is greater than 40 times more active than alpha-inhibin-31 in suppressing follitropin-release. Radioimmunoassay data indicate that alpha-inhibin-52 and alpha-inhibin-92 have only 60% immunoreactivity.
Antisera raised in rabbits to a synthetic peptide consisting of 31 amino acids with a sequence identical to inhibin-like peptide (ILP) isolated from human seminal plasma afford a highly specific and sensitive radioimmunoassay. Synthetic ILP completely displaces antiserum binding of radioiodinated [Tyr4]-ILP, with half maximal displacement at 36 fmoles ILP/tube. ILP, [Tyr4]-ILP and ILP-(9-31) had essentially equal potency, while ILP-(1-25), ILP-(1-23) and ILP-(1-16) had reduced potency. No cross reactivity was found among a variety of peptide hormones and proteins. Human seminal plasma displaces 50% of [125I-Tyr4]-ILP at dilutions equivalent to 50-250 pl/tube, corresponding to immunoreactive ILP concentration of 0.5-2.5 mg/ml.
Two human retinoblastoma cell lines (Y79 and McA) were evaluated for the presence of binding sites for human beta-endorphin (beta h-EP). Using tritiated beta h-EP (3H-beta h-EP) and synthetic beta-EP analogues, it was possible to demonstrate binding sites for 3H-beta h-EP with an ED50 of 3.5 nM in Y79 cells and 8 nM in McA cells respectively. The non-opioid segment [beta h-EP-(6-31)] retained about 20% relative potency in Y79 and 40% in McA in displacing the tritiated hormone when compared with beta h-EP. Camel beta-EP had a relative potency of less than 1% and beta h-EP-(1-27) was inactive in both cells in doses as high as 4 microM. Taken together with previous reports on similar binding sites in human neuroblastoma and glioblastoma cell lines, it appears that cell lines of neural origin have binding sites for the COOH-terminal of human beta-EP.
The isolation of two peptides similar in amino acid composition to that of human beta-lipotropin is presented. Peptide patterns after enzymatic digestions of these two peptides by Staphylococcus aureus protease and by trypsin were nearly identical. Paper electrophoresis and amino acid analyses of acidic peptides generated from the enzymatic digestions of these two peptides indicate that there is an amide difference between the two peptides. It is proposed that this amide difference is in amino acid residue number nine, and that one is the human beta-lipotropin and the other its [Gln9] analog.
Three synthetic analogs of human beta-endorphin (beta h-EP) (I, [Gln8, Gly31]-beta h-EP-Gly-Gly-NH2; II, [Arg9,12,24,28,29]-beta h-EP and III, [Cys11,26, Phe27, Gly31]-beta h-EP), which have been shown to possess potent inhibiting activity to beta h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were beta h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: beta h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.
Rat brain membranes were incubated in N-ethylmaleimide (NEM, 0.5-1.0 mM) in the presence and absence of various concentrations of morphine, Leuenkephalin and human beta-endorphin (beta h-EP). After sufficient washing, the binding of dihydromorphine (DHM), [D-Ala-D-Leu]-enkephalin (DADLE) and tritiated beta h-EP was 10-40% above that of membranes treated with NEM alone. There was no additive effect of morphine and Leu-enkephalin with respect to their effect on recovery of beta h-EP binding. Evaluation of beta h-EP as protecting ligand proved to be difficult since preincubation completely inhibits subsequent DHM and DADLE binding unless a more extensive washing protocol is employed. A protocol for washing beta h-EP preincubated membranes using a Tris-phosphate buffer of pH 6 containing 150 mM NaCl, 20 mM MgCl2 and 10% glycerol was used to recover enough binding potential to evaluate the effects of beta h-EP preincubation towards NEM treatment. Preincubation with beta h-EP itself at 0.1-1.0 microM did not result in any increased recovery of opiate binding, in contrast to the findings with the other two ligands.
Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.
Reactions of human beta-endorphin, corticotropin and their synthetic analogs with leucine aminopeptidase have been investigated. The results confirmed previous findings that beta-endorphin is resistant to the aminopeptidase action whereas corticotropin is not. Beta-endorphin-(1-5) is completely digested by the enzyme while beta-endorphin-(1-17) is resistant. In contrast, the NH2-terminal 7 residues in corticotropin are removed readily by leucine aminopeptidase. This is confirmed by the observation that human corticotropin-(7-38) is not hydrolyzed by the enzyme. This contrasting behavior of the two hormones toward leucine aminopeptidase may be related to differences in their conformational structures.
Opioid receptor selectivity of several beta-endorphin (beta-EP) analogs which antagonize beta-EP-induced analgesia has been assessed using partially selective binding assays. Although the apparent affinity dissociation constant of beta-EP in these assays varies from 0.2 to 360 nm, the potency of beta-EP antagonists relative to beta-EP remains largely unchanged. It is unlikely that differences in receptor affinities can account for the antagonist properties of these analogs in vivo.
Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical.
Growth hormone (GH) deficiency in rats is associated with decreased ovarian steroidal responsiveness to gonadotropins, possibly through a reduction in the production of the GH-dependent Somatomedin C/insulinlike growth factor I (SM C/IGF I). We have investigated the direct effects of synthetic SM C/IGF I on gonadotropin-stimulated ovarian steroidogenesis in vitro. Granulosa cells were cultured in a serum-free medium for 48 h in the presence of follicle-stimulating hormone (FSH), with or without SM C/IGF I. FSH dose-dependently increased both estrogen and progestin production. Concomitant treatment with SM C/IGF I led to a dose-dependent augmentation of progestin secretion over the full range of FSH doses tested, by a maximum of 2.3- to 2.6-fold. FSH-stimulated estrogen was enhanced by up to 2.4-fold but only at low doses of FSH. SM C/IGF I-enhanced progestin production was associated with increased pregnenolone production and 3 beta-hydroxysteroid dehydrogenase activity, whereas augmented estrogen production appeared to be due to enhanced aromatase activity. The actions of SM C/IGF I, at physiologically relevant concentrations were correlated with increased extracellular cAMP accumulation and cellular protein content but were independent of any change in cell number or viability. In contrast to SM C/IGF I, the closely related peptide multiplication-stimulating activity decreased estrogen production while increasing progestin metabolite accumulation. The present results indicate that the GH-dependent peptide SM C/IGF I may play a role in ovarian development by enhancing gonadotropin-stimulated granulosa cell steroidogenesis.
beta-Endorphin recently was proposed as a possible physiological stimulus of aldosterone secretion based on studies in animals. Since human beta-endorphin (beta h-endorphin) does not contain the ACTH-(4-10) homology common to other ACTH-related neuropeptides that stimulate aldosterone, its mechanism of stimulation might differ from that of the other peptides. In the present study, we infused beta h-endorphin into six normal subjects under carefully controlled conditions at dosage levels several orders of magnitude higher than endogenous levels. No increase in plasma aldosterone was found in these subjects ingesting a normal sodium intake despite the fact that other biological actions of beta h-endorphin were manifest. By contrast, an equimolar infusion of ACTH-(1-24) caused a significant increase in plasma aldosterone. These studies do not support a significant role for beta h-endorphin in control of aldosterone secretion in man and are consistent with the concept that the ACTH-(4-10) amino acid sequence, common to ACTH, beta-lipotropin, gamma-lipotropin, beta MSH, and alpha MSH, is a major determinant of their aldosterone-stimulating capacity.
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