Hormonal regulation of serine dehydratase activity in primary cultures of adult rat hepatocytes

The capacity of the following peptides to stimulate steroidogenesis in suspensions of capsule (largely glomerulosa) and fasciculata/reticularis cells from rat adrenals was studied: ACTH_1–24, ACTH_1–13-amide, α-MSH, γ₁-MSH, γ-MSH precursor, ACTH_4–10, CLIP, and ovine and human β-lipotropin. Only α-MSH and ACTH_1–13-amide stimulated glomerulosa cells alone, without effect on fasciculata/reticularis cells. Like ACTH_1–24 the two samples of β-lipotropin stimulated both capsule and inner zone cell types in a similar manner. Their activity is attributable to slight ACTH_1–39 contamination, as shown by HPLC fractionation. The other peptides lacked any activity. It is likely that the predicted specific glomerulosa stimulant from the pituitary closely resembles α-MSH.     

Peptide hormone degradation by a rat mast cell chymase-heparin complex

Material released from rat mast cells by compound $\text{48}\text{80}$ gave parallel responses using ACTH and β-endorphin radioimmunoassays. However, incubation of these labeled compounds under conditions of radioimmunoassay with released material and chromatography on Sephadex G-25 provided evidence that neither ACTH nor β-endorphin were present in the material released from mast cells, but represented an artifact produced by the presence of a protease. Analysis of the released enzyme on Sephadex G-75 under non-dissociative conditions yielded an active enzyme complex with a M_r > 150,000. Under dissociative conditions, the M_r of the enzyme was 25,000. The dissociated enzyme reassociated with purified rat mast cell heparin to form the high molecular weight complex. Further investigation of pH, substrate and inhibitor specificity showed that the peptide degradation is due to a chymotrypsin-like protease, the previously described mast cell chymase, which is active in degrading β-endorphin, ACTH, and ACTH^1–24.     

Isolation of ACTH1-39,ACTH1-38 and CLIP from the calf anterior pituitary

Calf anterior pituitaries were defatted and homogenized and peptides were adsorbed from the homogenate supernatant onto octadecylsilyl-silica. After elution, the resulting extract was subjected to gradient elution reversed-phase high pressure liquid chromatography (RP-HPLC) using aqueous acetonitrile containing 0.1% ($\text{v}\text{v}$) trifluoroacetic acid (TFA). Radioimmunoassay of column fractions for corticotropin (ACTH) revealed three major areas of immunoreactivity. Each was purified to homogeneity by gradient elution RP-HPLC employing aqueous acetonitrile containing either 0.13% heptafluorobutyric acid ($\text{v}\text{v}$) or 0.1% TFA ($\text{v}\text{v}$). Amino acid analysis and exopeptidase and trypsin digestions revealed the three forms of corticotropin to be ACTH_1–38, corticotropin-like intermediary lobe peptide, (CLIP, ACTH_18–39) and ACTH_1–39. ³H-labeled ACTH_1–39 did not give rise to either ³H-ACTH_1–38 or ³H-CLIP during isolation.     

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.     

The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: structure-activity studies.

Intraventricular administration of synthetic ACTH-like peptides in the rat induces excessive grooming, stretching and yawning. The present study demonstrates that induction of excessive grooming is dose-dependent and independent of the endocrine system. Structure-activity studies show that ACTH_1–24, ACTH_1–16-NH₂, ACTH_1–16, α-MSH and β_p-MSH are equipotent. Although the presence of the sequence ACTH_5–10 in the peptides studies seems of importance in the induction of excessive grooming, it appeared that C-terminal elongation is necessary for the expression of the activity. Administration of [D-Phe⁷] ACTH_4–10 and [D-Phe⁷] ACTH_1–10 results in appreciable grooming activity of the rat. However, substitution of a D-arginine at the 8 position did not alter the activity of ACTH_4–10. The structure-activity relationship of these peptides on grooming activity of the rat is compared to that known for retardation of avoidance extinction. Although some similarities exist, it is concluded that the expression of excessive grooming and retardation of avoidance activity is mediated through different mechanisms.     

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.     

Corticotropin releasing factor stimulates adrenocorticotropin and beta-endorphin release from AtT-20 mouse pituitary tumor cells

Corticotropin releasing factor (CRF) was tested for its ability to stimulate ACTH and β-endorphin secretion from clonal $\text{AtT-20}\text{D16-16}$ mouse pituitary tumor cells. Release of both hormones was stimulated 4 to 5-fold over the basal release at nanomolar concentrations of synthetic CRF. CRF analogues stimulated $\text{ACTH}\text{β-endorphin}$ release with the same order of potency in the tumor cells as in primary cultures of anterior pituitary cells. A 90-min exposure to CRF elicited a 29–35% increase in total ACTH and β-endorphin immunoreactivity in tumor cell cultures. Dexamethasone markedly inhibited CRF-stimulated and basal ACTH and β-endorphin release. $\text{AtT-20}\text{D16-16}$ cells may serve as a good model system for studying the biochemistry of CRF receptor-mediated events involved in $\text{ACTH}\text{β-endorphin}$ release and synthesis.     

Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum.

β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [¹²⁵I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At different time intervals the release of [¹²⁵I]-tyrosine or the change in immunoreactivity of the endorphins was determined. The cSPM preparation displayed both high aminopeptidase and endopeptidase activities. In contrast, human serum mainly contained aminopeptidase activity. The data suggest that functional endorphin metabolism may occur at the synaptosomal plasma membrane. These membranes may potentially be involved in the formation of behaviorally active endorphin fragments.     

Isolation,characterization and opiate activity of β-endorphin from human pituitary glands

The isolation of a 31-amino acid peptide from human pituitary glands has been described. Its amino acid sequence has been proposed to be identical to the sequence of the carboxyl-terminal 31 amino acids of human β-lipotropin. The peptide, designated as β_h-endorphin, possesses significant opiate activity.     

Comparative metabolic clearance rate, volume of distribution and plasma half-life of human beta-lipotropin and ACTH.

An antiserum to β_h-lipotropin (LPH) which does not cross react with β_h-endorphin has been obtained utilizing two different methods of affinity chromatography. This was employed in studies of three normal human subjects in whom the metabolic clearance rate (MCR) apparent volume of distribution (V_d) and fractional rate of disappearance (K_d) of ACTH and β_h-LPH were determined following bolus simultaneous injection of 270 μg highly purified β_h-LPH and 230 μg of synthetic human ACTH. A biphasic disappearance curve was noted for both hormones. $\text{β}{}_{\mathrm{h}}\text{-LPH}$: MCR-0.571, 0.519, and 0.461 L/minute; V_d-30.7, 27.7, 25.0 liters, representing 49, 46 and 35% of body weight; K_d-0.0186, 0.0187, 0.0185 min^?1. $\text{ACTH}$: MCR-0.274, 0.266, and 0.332 L/minute; V_d-6.5, 6.5, 14.5 liters, representing 10.4, 10.8 and 20.4% of body weight; K_d-0.0418, 0.0409, 0.0229 min^?1. The observed larger MCR of β_h-LPH can account for previous observations of basal plasma ACTH/LPH ratios greater than unity, even though these peptides are present in the pituitary in equimolar concentrations.     

Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production

Isolated adrenal cortex cells respond to the addition of ACTH_1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH_1–24, [Lys¹⁷, Lys¹⁸]ACTH_1–8 amide, and ACTH_1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH_1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH_5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH_1–39. The derivative, [Trp(Nps)⁹]ACTH_1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH_11–24 is a competitive antagonist of ACTH_1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces $\text{1}\text{2}$ maximum cyclic AMP production and concentration of cyclic AMP which induces $\text{1}\text{2}$ maximum corticosterone production.     

Influence of β-endorphin on the proliferative and phagocytic activities of peripheral blood cells of men and women from different age groups

It has been found that β-endorphin modulation of lymphocyte proliferative activity in male donors is mainly observed at a relatively young age (in groups aged 20–29 and 30–39 years), it gradually becomes lower with age, and disappears in donors at aged 50–60 years. At the same time, women have a prolonged modulating effect of peptide on proliferation. In women aged 50–59 years, the peptide has a marked promotional effect on spontaneous proliferation at concentrations of 10^?7, 10^?8, and 10^?10 M induced by a suboptimal concentration of phytohemagglutinin (PHA) at 10^?10 M, while in women aged 30–39 years, β-endorphin suppresses PHA-induced proliferative response. In men aged 20–29 years, β-endorphin stimulates the uptake capacity of neutrophils, whereas in those aged 50–59 years, this capacity is suppressed by β-endorphin. In female donors from any age groups, β-endorphin was not found to influence the activity of neurophils.     

Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Solid phase synthesis of porcine α-endorphin and γ-endorphin,two hypothalamic-pituitary peptides with opiate activity

The synthesis by solid phase methodology of α-endorphin (Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-OH) and γ-endorphin (Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH), two morphinomimetic peptides isolated from pig hypothalamus-pituitary extracts, is described. The sequences of these two peptides correspond to residues 61–76 and 61–77, respectively, of porcine β-lipotropin. The two synthetic compounds were shown to have the same physical, chemical and opiate activity as the respective native substances.     

Preparation of tritium labeled beta-endorphin suitable for studying brain receptor interactions

The opioid peptide (porcine) β-endorphin has been tritiated using reductive methylation to prepare a derivative containing mainly [³H]dimethyllysine. The tritiated β-endorphin has a specific activity of 9.8 Ci/mmol and is stable for an extended period of time. The labeled peptide binds reversibly to rat brain membrane preparations with a dissociation constant of 0.4 ± 0.1 nM and a receptor content of 23 ± 2 pmol/g brain. Under the conditions used, there is evidence for only one class of receptors. The technique employed for tritium labeling of β-endorphin should also be applicable to various other peptides including α-endorphin, γ-endorphin, and C′-fragment that have been found in brain and pituitary.     

β-endorphin proteolysis by guinea-pig ileum myenteric plexus membranes: Increased γ-endorphin turnover after chronic exposure to morphine

The influence of chronic morphine exposure $\text{in vitro}$ on the biotransformation of β-endorphin (βE) was investigated using the myenteric plexus-longitudinal muscle of guinea-pig ileum. A membrane preparation was incubated with βE and the degradation of βE as well as the accumulation of several βE fragments in the incubation medium were followed with time. The levels of peptides were determined by specific radioimmunoassays after separation by high-pressure liquid chromatography. It was found that exposure to morphine did not affect the disappearance of βE, but altered the time course of accumulation of βE fragments. In fact, the accumulation of γ-endorphin, α-endorphin and des-tyrosine¹-α-endorphin was enhanced, while that of des-tyrosine¹-γ-endorphin was not changed. Additionally, the disappearance of γ-endorphin appeared to be stimulated by morphine exposure. These data provide evidence that the fragmentation of βE is changed by chronic morphine exposure in such a way that the turn-over of γ-endorphin is increased.     

Immunohistochemical localization of beta-LPH in the human hypothalamus.

In order to establish the presence of β-LPH and to clearly identify the nervous structures containing β-LPH in the human hypothalamus, an immunohistochemical localization of β-LPH was performed in this tissue. The immunohistochemical technique involved use of a specific antiserum to human β-LPH and the peroxidase-antiperoxidase complex. Immunostained neuronal cell bodies were observed in the arcuate nucleus whereas β-LPH-positive nervous fibers could be detected in a large area extending rostro-caudally from the anterior part of the paraventricular nucleus up to the mammillary bodies. Staining was completely abolished by previous immunoabsorption with β-LPH while β-endorphin and ovine γ-LPH_1–47 only partially prevented immunostaining. Although it cannot be excluded that the precursor 31K molecule, β-LPH_1–58 and/or β-endorphin are detected by the immunostaining, it is likely that β-LPH is at least partly responsible for the positive reaction.     

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human β-endorphin to rabbit cerebellar and brain membranes was measured using $\text{[}{}^3\text{H}{}_2\text{-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 × 10⁷ M^?1min^?1 for cerebellum and 2.4 × 10⁷ M^?1min^?1 for brain. Dissociation of tritiated β_h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum, these is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated β_h-endorphin was determined. Ligands with preference for μ, δ, and κ opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward β_h-endorphin sites in both brain and cerebellar membranes.     

Cathepsin D generates gamma-endorphin from beta-endorphin.

A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu⁷⁷-Phe⁷⁸ peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.