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Separation of opioid peptides utilizing high performance liquid chromatography.

Chromatographic procedures have been developed for resolving all of the known enkephalins and endorphins on a single column. The effect of eluant pH on the retention times and separation of the enkephalins and beta-endorphin was determined. By combining these separations with a sensitive radioreceptor assay it is possible to assay all of the opioid peptides in the pituitary gland or in various regions of the brain from individual small laboratory animals.

Amino Acid Sequence↗

The C-fragment of beta-lipotropin: an endogenous neuroleptic or antipsychotogen?

Microinjection of the C-fragment (also called beta-endorphin), which is amino acid sequence 61-91 of the endogenous pituitary hormone, beta-lipotropin (beta-LPH), in the periaqueductal gray of the rat resulted in profound sedation and catalepsy, while microinjection of smaller fragments-that is, methionine-enkephalin [sequence beta-LPH-(61-65)] and its related pentapeptide, leucine enkephalin, and alpha-endorphin [sequence beta-LPH-(61-76)] resulted in attenuated forms of this behavior. This indicates that the C-fragment is an important neuromodulator of the central nervous system. The similarity of this behavior to that seen after systemic administration to experimental animals of exogenous neuroleptics suggests that a disturbance in the bioavailability of this neuropeptide to receptor sites in brain-perhaps due to lack of enzymatic cleavage from the circulating parent hormone, beta-lipotropin--may be an etiological factor in those psychopathological states for which the exogenous neuroleptics exert an ameliorative influence.

Analgesia↗

Effects of neuropeptides on adenohypophyseal hormone response to acute stress in male rats.

The effects of bombesin and other unrelated oligopeptides on hormonal changes induced by stress were studied in conscious adult male rats. Restraint in the cold for 1 h increased plasma corticosterone and PRL levels and decreased GH values but had no effect on LH levels. Bombesin (5 microgram), given intracerebroventricularly (ivt) before stress, inhibited the PRL rise without affecting corticosterone, GH, or LH response. A complete blockade of PRL rise was observed with doses of bombesin ranging from 5 microgram to 100 ng ivt, regardless of the duration (15, 30, 45, or 60 min) or the nature (cold exposure or restraint at room temperature) of the stressor agents. Bombesin was 10(3) more potent as a PRL inhibitor when given ivt than when given iv, and its ivt effect was not reversed by naloxone (1 or 10 mg/kg). Among other unrelated peptides tested (beta-endorphin, neurotensin, substance P, and TRH; 5 microgram ivt), only neurotensin decreased plasma PRL levels in rats subjected to restraint in the cold for 1 h. These results show that in conscious male rats, centrally administered bombesin has a very potent and long acting inhibitory effect on PRL release induced by acute stress. Since a bombesin-like peptide has been found in rat brain, its physiological role in PRL regulation remains to be elucidated.

Animals↗

Ectopic production of high molecular weight calcitonin and corticotropin by human small cell carcinoma cells in tissue culture: evidence for separate precursors.

The DMS-79 continuous line of human small cell lung carcinoma cells, which produces immunoreactive (IR)-corticotropin (ACTH), -lipotropin (LPH), and -beta-endorphin (beta END), was found to produce IR-calcitonin (CT). Two major high molecular weight (HMW) forms of IR-CT were observed after gel exclusion chromatography under denaturing conditions (mol wt. approximately 7,000 and approximately 14,000), as well as a minor HMW IR-CT component (mol. wt. approximately 70,000). None of these IR-CT materials was extracted from DMS-79 medium by affinity chromatography using an ACTH antibody covalently bound to agarose. These results demonstrate ectopic production of HMW forms of CT and ACTH/LPH/beta END by human lung tumor cells in tissue culture, but do not support the existence of a common CT/ACTH/LPH/beta END precursor molecule.

Adrenocorticotropic Hormone↗

Characterization of lipotropin-, corticotropin-, and beta-endorphin-immunoreactive materials secreted in vitro by a human pituitary adenoma responsible for a case of Nelson's syndrome.

A human pituitary adenoma responsible for a case of Nelson's syndrome was maintained in organ culture and the incubation medium was examined with four different RIAs; human corticotropin (ACTH), beta-MSH, lipotropin (LPH), and beta-endorphin (beta-End). All four immunoreactivites (IRs) were present in the medium obtained after 24 h of incubation. Gel exclusion chromatography under denaturing conditions (6 m guanidine HCl) revealed several immunoreactive components. Two components having both human beta-MSH (beta-hMSH) and human LPH (hLPH) IR coeluted with beta-hLPH and gamma-hLPH; a component with beta-hMSH IR but no hLPH IR coeluted with [125I]beta-hMSH; a component with human ACTH (hACTH) IR eluted at the position of hACTH. Sephadex G-50 gel exclusion chromatography revealed that approximately 80% and 20% of human beta-End (beta-hEnd) IR were accounted for by components coeluting with beta-hLPH and beta-hEnd, respectively. These data demonstrate the presence in this incubation medium of materials similar to if not identical with beta-hLPH, gamma-hLPH, hACTH, beta-hMSH, and beta-hEnd; they suggest that all of these peptides may be secreted in the circulation of patients with Nelson's syndrome.

Adenoma↗

Radioimmunoassay of methionine enkephalin and interference by brain factor of immunoreactivity and opiate receptor binding activity.

We developed a radioimmunoassay of methionine-enkephalin (met-enk) which was found to be highly specific to the peptide and 1 pmol was the detection limit. Interference by the following peptides was practically negligible; leucine-enkephalin, LAP- or CNBr-treated met-end, alpha- and beta-endorphins. Morphine and its congeners were totally inert. Measurement of met-enk in rat brain P2 fraction revealed besides met-enk, the presence of the factor which cross-reacted with the antiserum and also appeared to bind with the brain opiate receptor. Though the nature of the factor has not yet been characterized, it is suggested that for qualitative assessment of the tissue level of met-enk, a method to selectively inactivate either the peptide or the factor should be developed first.

Animals↗

ACTH release in pituitary cell cultures. Effect of neurogenic peptides and neurotransmitter substances on ACTH release induced by hypothalamic corticotropin releasing factor (CRF).

The effects of various neurogenic peptides and neurotransmitter substances on the release of ACTH induced by hypothalamic corticotropin releasing factor (HY-CRF) were investigated using monolayer cultured anterior pituitary cells. Test substances were given in combination with 0.05-0.1 hypothalamic extract (HE)/ml, because HE evoked a significant ACTH release and a linear dose response relationship was demonstrated sequentially between 0.0165 HE/ml and 0.5 HE/ml. Relative high doses of lysine-vasopressin showed a slight additive effect on the release of ACTH induced by 0.1 HE/ml. Leu-enkephalin, dopamine, prostaglandin E1 and E2 slightly reduced the release of ACTH induced by HY-CRF, but the inhibitory effect of these substances were not dose-related. Other tested substances including luteinizing hormone releasing hormone, thyrotropin releasing hormone, somatostatin, melanocyte stimulating hormone release inhibiting factor, beta-endorphin, neurotensin, substance P, vasoactive intestinal polypeptide, angiotensin II, norepinephrine, serotonin, acetylcholine, histamine and gamma-amino butyric acid showed neither agonistic nor antagonistic effect on the release of ACTH induced by HY-CRF. These results indicate that the release of ACTH is controlled specifically by HY-CRF and corticosterone, and modified slightly by some other substances such as vasopressin and prostaglandins, and that the effect of most other neurogenic peptides and neurotransmitter substances is negligible or non-physiological at the pituitary level.

Adrenocorticotropic Hormone↗

Adrenocorticotrophin-related peptides in adult and foetal sheep pituitary glands.

Differences in foetal and adult adrenal function may be due to qualitative as well as quantitative changes in the pituitary corticotrophic stimulus. Pituitary glands from adult and foetal sheep were freshly dissected and stored at -70 degrees C until extracted at pH 1.5. The extracts were subjected to chromatography on Sephadex G-100 superfine and fractions were assayed by multiple radioimmunoassays directed against the NH2- and CO2H-terminal sequences of ACTH and lipotrophin (LPH). Peaks corresponding to beta-melanocyte-stimulating hormone (beta-MSH), beta-LHP, gamma-LPH, beta-endorphin and ACTH were identified, with little or no evidence for the presence of alpha-MSH and corticotrophin-like intermediate lobe peptide. Three peaks of large molecular weight material, A. B and C, were identified and their relative proportions shown to be considerably greater in the foetus than in the adult. The immunoassay profile of peaks A and B suggested that they were 'stem hormones' which could give rise to a family of biologically active peptides. Since the 'family tree' which they engender varies according to the stage of development, it is proposed that the changes in the 'trophic family' may explain the different adrenal responses of the foetal and adult sheep.

Adrenocorticotropic Hormone↗

[In vitro biosynthesis of beta-lipotropin in brain tissue].

We have recently developed methods to identify biosynthetized beta-endorphin and beta-lipotropin (beta-LPH) following incubation of Rat pars intermedia with radioactive amino acids. We used the same approach for rat brain tissue. In the striatum we found a peptide similar to beta-LPH while its identification in hypothalamus was less positive. This is the first demonstration of such biosynthesis and it could well be an important step in determining the biosynthetic patterns of cerebral endorphins and enkephalins.

Animals↗

[Comparative activity of endorphins on the opiate receptors].

Displacement of naloxone from membranes of Rat brain by alpha, beta and gamma-endorphins with and without Na+ in the incubating medium has been studied. beta-endorphin shows a higher affinity for the opiate receptors and a stronger agonist property than morphine. alpha and gamma-endorphins have a much lower affinity than morphine and a marked antagonist characteristic. This study suggests the possibility of naturally occurring antagonists of the opiate receptors.

Animals↗

Some recent biochemical findings with possible therapeutic implications for schizophrenia.

Hemodialysis resulted in remission of psychopathological symptoms in eight of ten chronic schizophrenics and successful hemodialysis was associated with a decrease of leucine endorphin levels in the blood. Three endorphins (endogenous peptides) have been isolated from the brain, and among them beta-endorphin was found to be the most potent in inducing behavioral changes in the rat. Nevertheless, neither a positive, nor an inverse relationship between the severity of schizophrenic psychopathology and CSF endorphin concentrations could be borne out by clinical experiments. Most recently, on the basis of an entirely different line of research, the possibility that schizophrenia is a prostaglandin deficiency disease has been raised.

Animals↗

Opiate receptor agonists as modulators of gamma-aminobutyric acid turnover in the nucleus caudatus, globus pallidus and substantia nigra of the rat.

The injection of various doses of morphine, subcutaneously, or of beta-endorphin, intraventricularly, changes the turnover rate of gamma-aminobutyric acid (TRGABA) in the substantia nigra, globus pallidus and nucleus caudatus. The TRGABA decreases in N. caudatus but increases in globus pallidus and substantia nigra. These changes are dose related and can be inhibited by naltrexone. The increased TRGABA in globus pallidus elicited by these opioid receptor agonists may be associated with catalepsy since muscimol, a specific GABA receptor agonist, injected into the globus pallidus causes a dose-related catalepsy. Since this GABA receptor agonist injected into the substantia nigra fails to cause catalepsy, one can exclude that the increase in the TRGABA of substantia nigra elicited by opiate receptor agonists is operative in mediating the catalepsy elicited by opioids.

Animals↗

Human corticotropin (ACTH) radioimmunoassay with synthetic 1--24 ACTH.

A corticotropin antiserum was obtained from rabbits immunized with synthetic 1--24 corticotropin conjugated with bovine serum albumin. The antiserum did not cross react with synthetic alpha-melanotropin or with synthetic beta-endorphin and had a cross reactivity of 0.23% with human beta-lipotropin. We developed a radioimmunoassay with the antiserum obtained, in which we used polyethylene glycol in conjunction with a second precipitating antibody for fast (15-min) separation of antibody-bound and free corticotropin. The assay had a sensitivity of 16 ng/L and was validated on patients with various pituitary and adrenal diseases. From 103 normal subjects, the median value for corticotropin in specimens collected during the morning was 34 ng/L of plasma; the upper 95% confidence limit of the normal range was 98 ng/L.

Adrenal Gland Diseases↗

Biosynthesis and characterization of adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone, and an NH2-terminal fragment of the adrenocorticotropic hormone/beta-lipotropin precursor from rat pars intermedia.

Isolated intermediate lobe cells from 40 rat pituitaries were incubated for 3 h with [35S]methionine + [3H]-phenylalanine, [35S]methionine, [3H]valine, and [3H]leucine. The cell extracts were purified by carboxymethyl-cellulose chromatography (CMC) and the fraction eluting with ovine adrenocorticotropic hormone (ACTH) was further purified either by another CMC under the same conditions or by high performance liquid chromatography (HPLC). Microsequencing of the product from the second CMC allowed the identification of a peptide containing methionine 4 and phenylalanine 7, as expected for the NH2 terminus of ACTH. Purification by HPLC of a similar peptide obtained from the three other incubations gave three main raoactive peaks which were further characterized by their migration rates on polyacrylamide gels, molecular weight, and microsequencing. Results indicated that intact ACTH (residues 1-39) is present in extracts of rat intermediate lobe, but in very small quantities (less than 1% of the beta-endorphin content). ACTH is probably broken down into smaller fragments, e.g. alpha-melanocyte-stimulating hormone (alpha-MSH) (ACTH, 1-13) and corticotropin-like intermediate lobe peptide (CLIP) (ACTH, 18-39). These studies also revealed with existence of a peptide having identical sequence with the (N-1) terminus of the ACTH/lipotropin (LPH) precursor.

Adrenocorticotropic Hormone↗

[Existence of "endorphin cells" in the adenohypophysis of the cat; comparison with "corticotropin cells." Immunocytochemical study].

Indirect immunofluorescence technique with anti-17-39ACTH and anti beta-endorphin sera has allowed us to detect "corticotropic cells" in the anterior and intermediate lobes of the adenohypophysis of the male cat. The corticotropic cells of the anterior lobe are localized in the median zone; they are PAS-positive and appeared intensively coloured in dark blue with the Herlant's tetrachrome. All the cells of the intermediate lobe react with the anti-17-39ACTH serum. Using an anti-beta-endorphin serum, we have observed that all the corticotropic cells of the anterior lobe react; but in the intermediate lobe, only a part of "corticotropic cells" react with the anti-beta-endorphin serum.

Adrenocorticotropic Hormone↗

[Immunocytologic analysis, in rat hypothalamus, of neurons producing peptides related to endorphin, ACTH, MSH and beta-LPH. Comparison with other peptidergic and monoaminergic neurons].

In rat hypothalamus intraventricularly injected with colchicine, the same neurons of the ventral region are stained with I.S. against alpha and beta-endorphin, (1-24) and (17-39) ACTH, alpha and beta-MSH, and beta-LPH. They are distinct from those producing LH-RH, somatostatin, neurophysin, and dopamine. These results suggest that the same neurons elaborate peptides identical with or immunologically related to endorphins, ACTH, alpha-MSH and beta-LPH, probably issued from a common precursor.

Adrenocorticotropic Hormone↗

Specificity of cultured anterior pituitary cells in detecting corticotropin releasing factor(s): the effect of biologically active peptides and neurotransmitter substances on ACTH release in pituitary cell cultures.

Biologically active peptides and neurotransmitter substances were added to anterior pituitary cell cultures to examine the presence of corticotropin releasing factor (CRF)-like activity. Hypothalamic extract (HE) induced significant dose-related increase of ACTH, and the lowest effective dose was 0.01 HE/ml. Other tested substances including luteinizing hormone-releasing hormone, thyrotropin releasing hormone, melanocyte stimulating hormone release inhibiting factor, somatostatin, substance P, neurotensin, beta-endorphin. leu-enkephalin, met-enkephalin, bradykinin, norepinephrine, dopamine, serotonin, acetylcholine, histamine, gamma-amino butyric acid or gamma-hydroxy butyric acid showed no CRF-like activity. Relatively high doses of lysine vasopressin, arginine vasopressin and angiotensin II increased the release of ACTH in pituitary cell cultures, but the maximal ACTH response was markedly less than with HE. These results indicate that cultured anterior pituitary cells are sensitive and fairly specific in detecting CRF(s) comparing with other detecting procedures.

Adrenocorticotropic Hormone↗

The inner dynamics of positive human-animal interactions: Investigating the roles of oxytocin, opioids, dopamine, serotonin and the proteome.

Research on the neuroendocrine basis of positive interactions has predominantly focused on oxytocin (OT), although dopamine (DA) and opioids also play crucial roles. Furthermore, these neurotransmitters are known to interact with each other but have seldom been studied concurrently. In this study, we quantified longitudinal changes in these neurotransmitters using a within-subject, 2 × 2 factorial design by varying human familiarity (familiar versus unfamiliar) and contact type (positive contacts versus ignoring) for 10 min human-pig interaction sessions. We obtained cerebrospinal fluid samples from 10 pigs through a spinal catheter 65 and 5 min before the test and at 10, 30, 60, 120 and 240 min after the start of the test. Samples at various timepoints were analysed for OT, DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), β-endorphin opioids concentrations, and using proteomics to explore novel protein candidates. Additionally, the duration and frequency of physical contacts initiated by the pig toward the human were coded from videos. The test condition (human familiarity × contact type) had a significant effect on the concentration of β-endorphin (F1118.59 = 4.45; P = 0.04) and 5-HIAA (F1,73.47 = 5.02; P = 0.03), and tended to affect the concentration of OT (F1, 119.8 = 3.19; P = 0.08) and DOPAC (F1,70.57 = 3.31; P = 0.07), but pairwise comparisons were not significant or showed complex changes. Specifically, OT was highest when the unfamiliar humans ignored the pig compared to when they delivered positive contacts (t70.6 = 3.31; P = 0.008), and β-endorphin tended to be higher after interacting with the familiar human compared to the unfamiliar humans (60 min post-test; t37.9 = 3.3; P = 0.055). There were only minor differences in the pigs' behaviour, suggesting that pigs seemed to perceive all test conditions as positive. Nevertheless, β-endorphin and OT appear to relate to different aspects of the interactions, possibly familiarity vs. novelty respectively.

Animals↗