Differential in vitro metabolism of beta-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of beta-endorphin (beta E) 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 beta E 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 degrees 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 des-enkephalin-gamma-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of beta E was also reported. These data suggest that an approach using postmortem human brain is possible in studying beta-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

In vivo and in vitro effects of beta-endorphin on glucose metabolism in the rat

The effects of beta-endorphin (beta-Ep) on plasma glucose levels in rats and on glucose metabolism in isolated rat liver cells were examined. Intravenous injection of beta-Ep (5 micrograms/100 g BW) into ether-anaesthetized rats resulted in prompt and sustained hyperglycaemia with increases in the plasma glucagon and somatostatin levels and decrease in the plasma insulin level. When liver cells isolated from fed rats were incubated in the presence of beta-Ep at concentrations of 6 X 10(-8) M to 6 X 10(-7) M, glucose release into the medium increased within 15 min in a dose-related manner. Time course experiments showed that beta-Ep increased the level of cyclic AMP within 3 min. Significant increase in gluconeogenesis in liver cells isolated from fasted rats was also observed on addition of 10(-7) M beta-Ep in the presence of 10 mM L-lactate. These results suggest that the hyperglycaemia induced by beta-Ep may be caused, at least in part, by the effects of beta-Ep on releases of pancreatic hormones and glucose production in liver cells.     

Intrinsic regional differences in androgen receptors and dihydrotestosterone metabolism in human preadipocytes.

Androgens play an important role in regulating the central obesity that is a strong risk factor for cardiovascular disease and insulin resistance. This study confirms that androgen receptors are present in subcultured human preadipocytes, with androgen receptor gene expression and saturable specific dihydrotestosterone binding, dissociation constant 1.02 - 2.56 nM and maximal binding capacity 30.8 - 55.7 fmol/mg protein. There was an intrinsic regional difference in androgen receptor complement, with more androgen receptors in visceral than in subcutaneous preadipocytes. Dihydrotestosterone was metabolised by human preadipocytes, with more androstanediol produced by subcutaneous than visceral preadipocytes. While dihydrotestosterone metabolism was insufficient to explain the regional variation in androgen binding, both of these differences would reduce the androgen responsiveness of the subcutaneous preadipocytes compared with visceral preadipocytes. There were no gender differences in androgen binding or metabolism. While the direct effects of androgens on human PAs remain uncertain, these regional differences suggest that AR-mediated regulation of certain PA functions influences adipose tissue distribution.     

The regional metabolism of 5-hydroxytryptamine in mouse brain in vitro.

The relative rates of formation of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) from exogenous 5-hydroxytryptamine, showed regional variations when examined in homogenates of seven separate areas of mouse brain. 5-HTOL production was highest in the cerebellum, and lowest in the corpus striatum, whereas the production of 5-HIAA was greatest in the hypothalamus. Addition of NADPH was shown to increase the formation of the alcohol catabolite in whole brain homogenates. The production of 5-HTOL decreased in the brain homogenates of mice which had previously been injected with phenytoin sodium or oxypertine, with the latter also causing a fall in overall 5-HT metabolism.     

Central metabolism of beta-endorphin in different species of temperature acclimated rodents

The purpose of this study was to characterize the central metabolism/processing of beta-endorphin in wild, desert rodents and to study the effect of heat and cold acclimation on central metabolism/processing and beta-endorphin half-life. This report suggests that a shift occurs in the processing of beta-endorphin in desert rodents acclimated to heat and cold leading to an accumulation of gamma-type endorphins. The data support a significant difference in beta-endorphin half-life between the rodents and the laboratory white rat.     

In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

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.     

Ethanol treatment alters beta-endorphin metabolism by purified synaptosomal plasma membranes

Ethanol administration has been shown to affect beta-endorphin (beta-E) levels in most brain areas. Chronic ethanol treatment has also lead to changes in the levels of Met- and Leu-enkephalin which may be due to recent finding that enkephalin A activity is significantly altered. To determine if proteolytic enzymes responsible for beta-E metabolism at the pSPM are also altered, we studied the effect of chronic ethanol (7% v/v; 8 days) administration on in vitro central beta-E metabolism in male C57/BL mice. Purified SPM was time-course incubated with beta-E (20 microM) for 30-120 min and subjected to HPLC analyses for determination of beta-endorphin and related fragments. Chronic ethanol significantly reduced the half-life for beta-E at the pSPM (T1/2 = 50/min) versus controls (T1/2 = 100.4 min). Chronic ethanol also caused significant accumulation of the behaviorally active alpha- and gamma-type endorphins formed at the pSPM. These results suggest that chronic ethanol treatment leads to an increase in the activity of peptidases responsible for beta-E metabolism at pSPM leading to an increased formation of both alpha- and gamma-type endorphins which may affect alcohol related behaviors.     

Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.     

Investigation of beta-endorphin reception in preimplantation development of a mouse embryo in vitro

The effect of β-endorphin on 2-, 4-, and 8-cell embryo development in vitro was studied. It is shown that the hormone has no effect on a 2-cell embryo development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number of blastocyst formations increases in the presence of 0.1 μM β-endorphin in embryo cultured medium, and the number of blastocysts with abnormal structure decreases. The effect of the hormone on the change of intracellular concentration of Ca²⁺ ions in 2-, 4-, and 8-cell mouse embryos has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase and phospholipase activity blockers, and naloxone on the change of intracellular concentration of Ca²⁺ ions in the early mouse embryo in the presence of β-endorphin has also been studied. It is shown that 2-cell embryos have opioid and nonopioid β-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopioid β-endorphin receptors. It is also shown that the effect of β-endorphin in the early mouse embryo through nonopioid receptors occurs with the participation of intracellular Ca²⁺ and adenylate cyclase signaling system.     

Sex differences in prostaglandin metabolism.

Prostaglandin E2 (PGE2), PGF2alfa and PGD2 were synthetized from [1-¹⁴C]-arachidonic acid by rat kidney medulla microsomal fraction. The formation of each prostaglandin was significantly less in female animals than in males. The rate of inactivation of [³H]-PGF2alfa by kidney cortex cytosol was almost linear with the time of incubation during the first 30 min. The production of PGF2alfa metabolite (13,14-dihydro-15-keto PGF2alfa) was higher in male rats than in females.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.