Content of beta-casomorphins in milk of women with a history of allergy.

The prevalence of food allergies increased over the past decade. Most symptoms of food allergy appear during the first 2 yr of life. The aim of this study was to determine the beta-casomorphin-5 and -7 (BCMs) in colostrum and milk of 12 breast-feeding women with a history and clinical manifestation of food allergy. The results were compared with the data obtained from a control group of healthy age-matched breast-feeding women. The level of BCM in women with food allergy was constant during lactation, whereas the highest level of opioid peptides was found in colostrums of healthy women with a subsequent rapid decrease in mature milk. These differences in BCMs profile between allergic and healthy breast-feeding women suggest that BCM content in the human milk may be an indicator of allergic conditions.

High levels of endorphin and related pathologies of veterinary concern. A review.

The authors report information about endogenous opioid peptides (EOP), receptors, antagonists and their interference with pain, stress, endocrine and immune system. A relationship between EOP and calcium homeostasis, both at extracellular and intracellular level, has been observed. In vitro, beta-endorphin exerts different actions through calcium channel functionality in epithelial cells. In rat aorta and cerebral cortex: beta-endorphin or Naloxone alternatively influence oocyte maturation through the mu-receptor gene expression and intracellular calcium concentration in granulosa and cumulus cells. Calcium channel block is removed by administrating Naloxone and calcium. In vivo, Naloxone and calcium removes EOP induced apoptosis in granulosa cells; is the most safe therapy in cow's milk fever; allow to remove ovarian follicular cysts. A negative influence of opioids on immune response after vaccination was established; EOP-related metabolic problems in post-partum cows. Abnormal intestinal motility, in which a Ca++ influence is well known, can be removed by Naloxone and calcium administration. Calcium-related function and neuromodulation must be re-evaluated since high level of EOP are involved in many pathologies through their influence on calcium activity. The use of calcium salts and Naloxone offers a safe and supplementary therapeutical possibility, active in any condition of altered endogenous opioids.

Electroacupuncture: mechanisms and clinical application.

Acupuncture is an ancient Chinese method to treat diseases and relieve pain. We have conducted a series of studies to examine the mechanisms of this ancient method for pain relief. This article reviews some of our major findings. Our studies showed that acupuncture produces analgesic effect and that electroacupuncture (EA) is more effective than manual acupuncture. Furthermore, electrical stimulation via skin patch electrodes is as effective as EA. The induction and recovering profiles of acupuncture analgesia suggest the involvement of humoral factors. This notion was supported by cross-perfusion experiments in which acupuncture-induced analgesic effect was transferred from the donor rabbit to the recipient rabbit when the cerebrospinal fluid (CSF) was transferred. The prevention of EA-induced analgesia by naloxone and by antiserum against endorphins suggests that endorphins are involved. More recent work demonstrated the release of endorphins into CSF following EA. In addition, low frequency (2 Hz) and high frequency (100 Hz) of EA selectively induces the release of enkephalins and dynorphins in both experimental animals and humans. Clinical studies suggesting its effectiveness for the treatment of various types of pain, depression, anxiety, spinally induced muscle spasm, stroke, gastrointestinal disorders, and drug addiction were also discussed.

Isolation and structure of a C-terminally amidated nonopioid peptide, amidorphin-(8-26), from bovine striatum: a major product of proenkephalin in brain but not in adrenal medulla.

We have isolated and sequenced a C-terminally amidated peptide from bovine striatum. The peptide was purified to homogeneity by adsorption to XAD-2 resins and four different HPLC steps. Amino acid composition analysis and gas-phase sequence analysis revealed identity of this peptide with residues 8-26 of the proenkephalin-derived opioid peptide amidorphin, which we have recently isolated from bovine adrenal medulla. C-terminal amidation of amidorphin-(8-26) from bovine striatum was demonstrated by its stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the C-terminal amide group as part of the recognition site. The nonopioid peptide amidorphin-(8-26), which lacks the N-terminal [Met]enkephalin sequence of amidorphin, is a major product of the opioid peptide precursor proenkephalin in the brain. In the adrenal medulla, however, where amidorphin occurs in remarkably high concentrations, amidorphin-(8-26) could not be detected. This is indicative of differential post-translational processing of proenkephalin in different tissues. In the brain, as opposed to the adrenal medulla, amidorphin is further processed at the typical cleavage signals of two basic residues, giving rise to the nonopioid peptide amidorphin-(8-26) and, possibly, to the opioid peptide [Met]enkephalin. Thus, proenkephalin in the brain might be considered as a precursor in which an opioid peptide is linked with a nonopioid peptide of possibly different biological function.

The effect of chronic estrogen treatment on immunoreactive beta-endorphin levels in intact female rats.

Intact female rats were treated chronically with estradiol benzoate (EB) until a state of constant estrus (CE) was achieved and maintained. When compared to female rats on the day of estrus, estrogen-treated rats in constant estrus demonstrated a 33% decrease in the concentration of immunoreactive beta-endorphin (IR-BE) in the plasma, and a 45-50% decrease in the content and concentration of IR-BE in the anterior pituitary and hypothalamus. The content and concentration of IR-BE in the neurointermediate lobe of the pituitary were similar in each group. Column chromatography revealed that the reduction in IR-BE in the plasma and anterior pituitary of EB-treated CE female rats appeared to be due to a reduction in peptides coeluting with beta-endorphin and beta-lipotropin, whereas the reduction in IR-BE in the hypothalamus represented a decrease in a peptide which coeluted with beta-endorphin. These data suggest that constant estrus, induced by prolonged treatment of intact female rats with estrogen, resulted in a reduction in central and peripheral levels of IR-BE in these animals as compared to female rats on the day of estrus.

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya.

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.

[A specific and quantitative determination of rat beta-endorphin. Combination of HPLC and RIA].

A specific and quantitative method for the determination of rat beta-endorphin by the combination of HPLC and RIA was developed. Rabbit antiserum against camel beta-endorphin (c beta-E) was raised and used for RIA at the final concentration of 1:10000. The quantitative range estimated from the displacement curve was 0.1-2.0 ng. Cross-reactivities with Met-Enk, Leu-Enk, alpha-MSH, alpha-endorphin, ACTH and human beta-E were less than 0.1, less than 0.1, less than 0.1, less than 0.1, 2 and 100%, respectively. These peptides were separated from each other by reversed phase HPLC with UV254 nm detection, and the minimum detectable dose of c beta-E was found to be 1 microgram. beta-E-like immunoreactivity (beta-ELIR) in the HPLC effluent was determined by RIA. The HPLC-RIA chromatogram of authentic c beta-E exhibited a single peak which coincided with the peak of c beta-E detected by UV, and 80% of the injected c beta-E (1-100 ng) was detected in the c beta-E fraction. The HPLC-RIA chromatogram of rat pituitary, hypothalamus, cerebrospinal fluid and plasma revealed the presence of 1-3 peaks, one of which was observed at the position of c beta-E. The HPLC elution of rat pituitary resolved the material into two peaks of biological activity, one of which coincided with the peak of beta-ELIR at the position of c beta-E. The HPLC-RIA chromatogram of the c beta-E fraction from pituitary obtained by gel-chromatography exhibited three peaks, one of which coincided with c beta-E. These results suggest that beta-ELIR in the c beta-E fraction of the HPLC elution may reflect rat beta-E accurately.

Intracerebral injection of different antibodies against endogenous opioids suggests alpha-neoendorphin participation in control of feeding behaviour.

The mechanism of feeding behaviour of rats was examined. We used antibodies to different opioid peptides in order to reduce the tonic activity of various endogenous opioid peptide systems that may underly appetite. Unilateral microinjection of anti-alpha-neoendorphin antibodies into various areas of the ventromedial hypothalamus (VMH) inhibited food and water intake up to 45% in deprived animals. Injections outside this area failed to affect feeding. Administration of anti-beta-endorphin antibodies into the VMH moderately attenuated appetite. A considerable decrease of food and water intake was observed only upon injection of this antibody into the nucleus periventricularis hypothalami, a region generally believed to be involved with feeding. A marginal reduction of appetite was observed with anti-dynorphin antibodies injected into the VMH. These data may suggest that alpha-neoendorphin is involved in the control of food and water intake in the VMH.

Acupuncture mechanisms in rabbits studied with microinjection of antibodies against beta-endorphin, enkephalin and substance P.

Injection of protein-A purified antibodies against Met-enkephalin and beta-endorphin into the periaqueductal gray matter (PAG) was shown to decrease the analgesic effect of electroacupuncture (EA) in rabbits. Met-enkephalin antibodies were more potent than the beta-endorphin antibodies in causing a statistically-significant effect on electroacupuncture analgesia. Antibodies to Met-enkephalin were also active at the spinal level, whereas antibodies against beta-endorphin were without effect: this is in agreement with a rich enkephalinergic innervation and absence of beta-endorphin-containing fibres in the spinal cord. Substance P, the other neuropeptide of this study, also seems to be important in mediating effects of electroacupuncture. Injection of antibodies into the periaqueductal gray caused decrease of the effect of electroacupuncture whereas intrathecal administration of Fab-fragment substance P antibodies caused a marked potentiation. The demonstration of site specificity of the neuropeptides in mediating analgesia induced by electroacupuncture supports the validity of this experimental approach.

Immunological characterization of endorphins, adrenocorticotropin, and melanotropins in frog hypothalamus.

To gain more information about the nature and regulation of opiomelanocorticotropic peptides in the frog diencephalon, radioimmunological determinations of alpha- and beta-MSH. ACTH, beta- and gamma-endorphins have been performed in hypothalamic extracts. Sephadex G-50 gel filtration revealed a single peak of alpha-MSH-like immunoreactivity (alpha-MSH-LI) comigrating with synthetic alpha-MSH. Two peaks of ACTH-LI were observed; the major one eluting slightly before human ACTH. Using a porcine beta-endorphin antiserum which exhibited 45% cross-reaction (on a molar basis) with ovine beta-LPH, one major peak of beta-endorphin-LI and two additional components were observed in the elution profile; none of these peaks coeluted with synthetic porcine beta-endorphin. No significant beta-MSH or gamma-endorphin-LI was detected. To investigate a possible role of glucocorticoids on the level of opiomelanocorticotropic peptides in frog hypothalamus, 60 male frogs were treated with dexamethasone (300 micrograms/day) during 8 days. Dexamethasone treatment did not modify the chromatographic distribution and the total amount of alpha-MSH-LI and ACTH-LI in hypothalamic extracts. A slight (15%) increase in beta-endorphin-LI was even observed after 8 days of dexamethasone treatment. From these results it is concluded that three classes of opiomelanocorticotropic peptides are present in the frog hypothalamus in the following order of concentration: beta-endorphin-LI greater than alpha-MSH greater than ACTH. In addition, circulating glucocorticoids which significantly reduce the concentration of opiomelanocorticotropic hormones in the distal lobe of the frog pituitary (S. Jégou, M. C. Tonon, F. Leboulenger, C. Delarue, J. Côté, G. Pelletier, and H. Vaudry (1981a). In "Adv. Physiol. Sci.' E. Stark, G. B. Makara, Zs. Acs, and E. Endröczi, eds., Vol. 13, pp. 129-133. Pergamon, Budapest.) do not modify the amount of these peptides in the hypothalamus.

Plasma immunoreactive beta-endorphin and enkephalin concentration in healthy subjects before and after electroacupuncture.

The involvement of the endogenous analgesia systems in the mechanism of analgesia produced by electrical stimulation of the brain or resulting from certain stressful manipulations seems now well-established. The purpose of the present study was to determine whether acupuncture as a method of peripheral sensory stimulation activates, like central stimulation, the endogenous opiate system. Plasma concentrations of ACTH and the best known endogenous opiates: beta-endorphin, met- and leu-enkephalin, were determined before and after standard electroacupuncture stimulation in healthy volunteers. Acupuncture stimulation resulted in a significant (p less than 0.005) decrease of plasma beta-endorphin-like immuno-reactivity (B-EPLI), but plasma ACTH assayed did not change about 5 minutes after acupuncture. The authors conclude that the reaction of the beta-endorphin system to acupuncture (sensory peripheral) stimulation in humans did not involve pituitary hypersecretion, and suggest that the increase of beta-endorphin binding to the tissue receptor sites seems to be responsible for the peripheral (plasma) B-EPLI decrease after acupuncture.

Immunoreactive dynorphin-(1-8) and corticotropin- releasing factor in subpopulation of hypothalamic neurons.

Immunoreactive corticotropin-releasing factor (CRF) and dynorphin-(I-8) were visualized in rat hypothalamus by immunohistofluorescence with specific antibodies. In brains from colchicine-treated, adrenalectomized rats, neuronal perikarya with immunoreactive CRF were observed in the paraventricular nucleus of the hypothalamus. The CRF occurred together with the dynorphin-(1-8). However, the CRF immunoreactivity occurred only in a subpopulation of the dynorphin-(1-8) immunoreactive cells. These findings suggest that there may be a functional interrelationship of CRF with dynorphin-related opioid peptides and provide further evidence that neurons may contain more than one bioactive substance.

Electroacupuncture analgesia blocked by microinjection of anti-beta-endorphin antiserum into periaqueductal gray of the rabbit.

The possible role of beta-endorphin (beta-EP) in mediating the analgesic effect elicited by electroacupuncture (EA) was assessed by injecting antiserum against beta-EP into the periaqueductal gray (PAG) of the rabbit. Two antisera (HO and UA) were raised in rabbits against human beta-EP, each of which recognized human, porcine, bovine, ovine, camel and rat beta-EP, the only difference being that HO recognized rabbit beta-EP while UA did not. The effect of EA analgesia was found to be seriously attenuated by injecting HO into PAG of the rabbit, while UA was ineffective. The results indicated a PAG pool of beta-EP as an important component in mediating EA analgesia.

Immunoassayable beta-endorphin level in the plasma and CSF of heroin addicted and normal subjects before and after electroacupuncture.

The present study was undertaken to evaluate if plasma or CSF beta-endorphin level can be induced to rise during the treatment of heroin addiction by electroacupuncture. Based on the examination of 30 addicts, we obtained no evidence indicating an increase of beta-endorphin level in either the plasma or the CSF after 30 min of acupuncture. In spite of this, the majority of the addicts experienced a reduction of withdrawal symptoms during treatment. Since electroacupuncture may only induce a highly localized secretion of beta-endorphin in the brain, our results cannot unequivocally exclude the possibility that this peptide is involved in mediating the action of acupuncture.