Regulation of the rat brain endothelin system by endogenous beta-endorphin

Several lines of evidence indicate that the central endogenous opioid and endothelin (ET) system regulate each other. To explore this idea further, we determined the effect of intracerebroventricular (i.c.v.) administration of anti-beta-endorphin IgG (rabbit) on the expression level of the opioid, corticotropin-releasing hormone and endothelin receptors, and tissue concentration of ET-1. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-beta-endorphin IgG (2.5 microg/microl) on days 1, 3 and 5. On day 6, animals were euthanized and caudate, cortex and hippocampus collected for Western blot analysis. Anti-beta-endorphin IgG down-regulated ET-A receptor protein expression in the caudate (51%), but had no effect on the expression of mu, delta, kappa opioid, ET-B, CRH-1 and CRH-2 receptors in any brain region. Anti-beta-endorphin IgG increased tissue ET-1 levels in the caudate by 30.3%. [35S]GTP-gamma-S binding assays demonstrated that anti-beta-endorphin IgG increased the efficacy of [D-Ala2-MePhe4, Gly-ol5]enkephalin without altering its potency in caudate. Control experiments showed that there was no detectable rabbit IgG in caudate, cortex and hippocampus samples. These results suggest that beta-endorphin in the CSF coordinately regulates ET-1 levels and the ET-A receptor in rat caudate. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between opioid and ET system.     

Regulation of estrogen receptor alpha by estradiol in pregnant and estradiol treated rats

Estrogens play an important role in tissue metabolism through specific regulation of several intracellular pathways. We studied ERalpha regulation in muscle and adipose tissue from pregnant and estradiol treated rats. In both groups, we identified three different ERalpha inmunoreactive proteins (80, 67 and 46 kDa) using total protein extracts. Because it has been showed that estrogens are able to promote rapid effects in several cellular models, we looked for three ERalpha-related proteins at plasma membrane. In skeletal muscle of both groups, we positively identified the three ERalpha-related isoforms in plasma membrane, but in adipose tissue from pregnant we were not able to identify ERalpha67, and in estradiol treated animals ERalpha80 was absent. Taking together, our results showed a tissue-specific regulation of whole-cell ERalpha-related proteins and ERalpha located at plasma membrane, which should be involved in non-genomic actions of 17beta-estradiol. The role of the three ERalpha inmunoreactive proteins is unknown, however, seems probably related to rapid activation of signalling pathways.     

Calcitonin induced increase in ACTH, beta-endorphin and cortisol secretion

The response of ACTH, beta-endorphin and cortisol to calcitonin administration was investigated in 8 subjects with recent fractures of the vertebrae due to postmenopausal or senile osteoporosis (Ost) and in seven normal healthy controls (NC). A significant increase of the three hormones was observed in 13 subjects. The maximum increase was observed between 15 and 60 min.: the cortisol level (microgram/100 ml) rose from 14.3 +/- 1.9 to 24.8 +/- 3.2 (P less than 0.05) in Ost and from 7.7 +/- 0.6 to 21.7 +/- 1.7 (P less than 0.001) in NC, the beta-endorphin (pmol/l) from 5.8 +/- 0.6 and to 21.2 +/- 1.3 in OST (P less than 0.001) and from 5.9 +/- 0.4 to 21.9 +/- 4.5 (P less than 0.01) in NC and the ACTH levels (pg/ml) from 21.3 +/- 5.7 to 61.7 +/- 3.6 (P less than 0.001) in OST and from 30.0 +/- 6.2 to 58.8 +/- 7.5 (P less than 0.05) in NC. The results indicate a possible role of calcitonin in modulating the anterior pituitary function. It also suggests that the analgesic effect of calcitonin might be mediated by the increase of beta-endorphin. The possibility that this analgesic effect of calcitonin is due to its direct binding to the opiate receptors was excluded in the present study by in vitro binding assay.     

Cocaine induced secretion of ACTH, beta-endorphin, and corticosterone

The effect of intraperitoneal administration of cocaine on the concentrations of hypothalamic corticotropin releasing factor like-immunoreactivity (CRF-LI), plasma ACTH, beta-endorphin, and corticosterone was investigated. Groups of rats were injected with 20 mg/kg cocaine HCI or 0.9% NaCl and then killed 0, 10, 20, 30 or 60 minutes later. Hypothalamic CRF-LI, plasma ACTH, beta-endorphin, and corticosterone concentrations were determined by radioimmunoassay. A significant increase in plasma ACTH, beta-endorphin, and corticosterone concentrations was observed after cocaine administration. In contrast, cocaine had no significant effect on hypothalamic CRF-LI concentrations. Intravenous administration of 0.5 and 2.0 mg/kg cocaine to rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine, and pentobarbital elicited a significant increase in plasma corticosterone concentrations. These results demonstrate that cocaine induces the release of ACTH, beta-endorphin, and corticosterone and suggest that this response is mediated at the pituitary level.     

Running elevates plasma beta-endorphin immunoreactivity and ACTH in untrained human subjects

Twenty minutes of submaximal treadmill running was associated with an elevation in plasma levels of beta-endorphin immunoreactivity (P less than 0.02). This increase was greater in men (14.9 +/- 3.4 fmole/ml) than women (2.6 +/- 1.2 fmole/ml)(P less than 0.05). Plasma levels of ACTH and growth hormone also increased after running. ACTH increased more in men (7.8 +/- 1.1 fmole/ml) than in women (1.1 +/-0.44 fmole/ml)(P less than 0.02). There was a similar growth hormone response in both sexes. No correlation can at this time be made with levels in the central nervous system. Changes in plasma levels of beta-endorphin immunoreactivity may be responsible for some of the euphoria and analgesia anecdotally associated with running.     

Regulation of ACTH and cortisol in depression

Disturbances of hypothalamic-pituitary-adrenal regulation are frequently observed in a subgroup of patients suffering from major depression. The mechanism of hypothesized pituitary and hypothalamic involvement in this dysregulation remains relatively uncharacterized. In this paper we investigated the response of adrenocorticotropin (ACTH), as well as cortisol, to dexamethasone inhibition and characterized the dynamic response of ACTH to a one-hour infusion of cortisol in normal subjects and patients suffering from depression. A paradoxical increase in ACTH in response to cortisol is noted in one patient.     

Differential immunostaining of adenohypophysial cells with antisera to ACTH and beta-endorphin

Three antisera, raised in rabbits against human beta-endorphin were denoted Y-10, Y-18 and R-230 and tested for their ability to immunohistochemically stain cells in teh adenohypophysis of the rat and dog. By radioimmunoassay Y-10 was highly specific towards beta-endorphin with minimal cross-reactivity against beta-LPH while Y-18 and R-230 cross-reacted with both beta-endorphin and beta-LPH on an equimolar basis. In the rat pituitary, Y-18 and R-230 antisera stained cells identified as corticotrophs. With the dog, however, beta-endorphin-staining cells in the anterior pituitary and infundibulum did not necessarily co-stain for ACTH-like material. When the beta-endorphin antiserum, Y-10, was applied to rat anterior pituitary tissues, the subsequent positive immunocytochemical staining was associated not only with corticotrophs but also with somatotrophs. The findings are consistent with (a) a differential processing of the 31K pro-opiocortin and (b) the presence in rat somatotrophs of determinants that cross-react immunologically with some beta-endorphin antisera.     

Simultaneous circadian variations of plasma ACTH, beta-lipotropin, beta-endorphin and cortisol

The major objective of this study was to investigate the analogy existing between the typical circadian periodicity of ACTH and that recently described of beta-lipotropin (beta-LPH) and beta-endorphin (beta-EP) plasma levels. The determination of their concentrations, plus cortisol, has been performed on the same plasma samples of 6 healthy volunteers. All hormones were measured by radioimmunoassay. Those of beta-LPH and beta-EP were preceded by a purification of plasma through silicic acid extraction and Sephadex G-75 gel filtration. The highest values (mean +/- SEM) were found in the morning (ACTH: 10.3 +/- 0.9; beta-LPH; 6.3 +/- 0.7; beta-EP: 6.5 +/- 0.5 fmol/ml; cortisol: 378 +/- 30 pmol/ml) and the lowest values in the evening (ACTH: 6:1 +/- 0.7; beta-LPH: 3.3 +/- 0.4; beta-EP: 3.7 +/- 0.6 fmol/ml; cortisol: 130 +/- 23 pmol/ml). Statistical analysis using the Fourier method led to the evidence of a concomitant circadian secretory pattern of the three proopiocortin-related peptides. These results strongly suggest that the phasic secretion of ACTH, beta-LPH and beta-EP underlies a common central control.     

GnRH stimulates ACTH and immunoreactive beta-endorphin release from the rat pituitary in vitro

An in vitro perifusion system was used to investigate the effects of GnRH stimulation on LH, ACTH, and immunoreactive beta-endorphin (i beta-END) release from ovariectomized (1 week) rat anterior hemipituitaries. Either 0, 8 or 80 nM GnRH was administered as a 15 min pulse followed 30 min later by a prolonged 45 min infusion. Both 8 and 80 nM GnRH induced comparable LH release in response to the 15 min as well as the 45 min GnRH stimulation. The initial 15 min exposure to either 8 or 80 nM GnRH did not induce significant changes in ACTH or i beta-END release. In contrast, the subsequent 45 min exposure to 8 nM GnRH induced a significant (p less than 0.01) increase in ACTH release, and the 45 min exposure to 80 nM GnRH induced a significant (p less than 0.01) increase in ACTH as well as i beta-END release. Equimolar (i.e. 8 or 80 nM) GnRH receptor antagonist (ANT) blocked the stimulatory effects of GnRH in all cases. These results demonstrate that GnRH can stimulate not only LH but also ACTH and i beta-END release from ovariectomized rat anterior hemipituitaries in vitro, apparently by a GnRH receptor mediated mechanism independent of actual LH release. Although the time course of these responses appears to be consistent with the hypothesis that GnRH-stimulated gonadotropes release paracrine factor(s) which stimulate corticotrope activity, the mechanism of these responses remains to be determined.     

Synthetic corticotropin-releasing factor stimulates secretion of immunoreactive beta-endorphin/beta-lipotropin and ACTH by human fetal pituitaries in vitro

Synthetic ovine CRF, in an amount approximating that found in pituitary portal plasma of the rat, induced a significant increase in the secretion of both ACTH and immunoreactive beta-endorphin/beta-LPH (i beta-END/LPH) by human fetal hemipituitaries in an in vitro superfusion system. This finding suggests that a molecule similar to synthetic ovine CRF may be a physiologic hypothalamic releasing factor in man.     

Diurnal variations in plasma ACTH, cortisol and beta-endorphin levels in cocaine addicts.

In order to establish possible alterations in the hypothalamic pituitary-adrenal axis and in ACTH-related opioids in cocaine addicts, plasma ACTH, cortisol and beta-endorphin levels were measured throughout the day in 9 cocaine addicts [age: 27 +/- 5 years (mean +/- SE); weight: 72 +/- 6.1 kg, duration of cocaine addiction: at least 2 years] on the day of their admission to a recovery community for drug abusers (first test) and after 15 days of abstinence (second test). Nine normal controls (age: 28 +/- 6 years; weight: 73 +/- 3.2 kg) were tested only once in a similar manner. Blood samples were taken at 06:00, 08:00, 10:00, 12:00, 18:00 and 20:00 h and served for hormonal assays. Urine samples were taken from cocaine addicts at 08:00 h on the experimental day and on the following day. Results of both urine assays were positive for cocaine catabolites, indicating cocaine administration during the day before the experimental test. From the day of their admission in the community (1st experimental day), the patients were forbidden to use cocaine. For 4 days after admission, they were treated with symptomatics to attenuate withdrawal symptoms. Thereafter, the patients underwent a washout period of pharmacological treatments for 10 days before being retested (second test). Urine samples taken at 08:00 h on this second experimental day and on the next day were negative for the presence of drug catabolites. During the first test, cocaine addicts showed higher plasma ACTH, cortisol and beta-endorphin levels than normal controls at all examined time points.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced ACTH, while normal beta-endorphin CSF levels in early epileptic encephalopathies

Since ACTH and the opioids display opposite effects on experimentally-induced seizures, cerebrospinal fluid (CSF) levels of ACTH and beta-endorphin (beta-EP) were measured in 6 children (4-8 months) affected by infantile spasms with hypsarhythmia, an idiopathic early onset encephalopathy, and in 8 age-matched controls. beta-EP levels in the patients (76.3 +/- 14.7 fmol/ml, M +/- SD) did not differ from those in controls (109.8 +/- 42.7) while babies with epileptic encephalopathy showed reduced ACTH levels in the CSF (3.8 +/- 1.5) as compared to controls (9.0 +/- 3.7, p less than 0.01). This resulted in an increased beta-EP/ACTH ratio. Another patient previously treated with ACTH showed a normal CSF level of ACTH (9.0) with a normal beta-EP/ACTH ratio while in clinical and EEG remission. These results are consistent with the hypothesis that some infantile seizures unrelated to brain injuries could originate from an ACTH deficiency at central level and/or an imbalance of neuropeptidergic pathways.     

In vitro secretion of ACTH, beta-endorphin and beta-lipotropin in Cushing's disease and Nelson's syndrome

Tissue from histologically confirmed ACTH cell adenomas in Cushing's disease (CD) and Nelson's syndrome (NS) was gained by transsphenoidal surgery. Combined enzymatic and mechanic agitation of tumor tissue yielded a cell suspension. Aliquots of the cell suspension were transferred to superfusion chambers immediately after isolation and investigated for ACTH and beta-endorphin production. Feedback action of cortisol (CO) and dexamethasone on basal hormone production and on lysine vasopressin (LVP) induced ACTH secretion were studied. Adenomatous tissue and anterior lobe tissue from the same patient in CD could be investigated simultaneously in 4 cases. The paraadenomatous tissue showed depression of basal and LVP-induced ACTH secretion. In all adenomatous tissues investigated there was missing or reduced suppression of basal ACTH secretion by physiological levels of CO. CO not only failed to suppress LVP-induced ACTH secretion but also seemed to enhance LVP stimulation in some experiments. This study confirms former results, that a missing or inversed feedback action or glucocorticoids in adenoma cells is a mechanism involved in the pathological ACTH secretion in CD and NS. Bioassayable and immunoreactive ACTH from media of superfusion and short-term static incubation were compared with beta-endorphin and beta-LPH in an assay detecting these two peptides with equimolar sensitivity. Secretory patterns were basically parallel but great differences showed in quantities of hormones secreted. In addition, Sephadex G-50 gel chromatography was performed to separate beta-endorphin from beta-LPH and to calculate the ratios. These profiles show great variations between different adenomas.     

Regulation of bioactive beta-endorphin processing in rat pars intermedia

Acid extracts of rat pituitary neuro-intermediate lobes have been shown by ion-exchange chromatography and radio-immunoassay to contain predominantly the inactive derivatives of beta-endorphin, alpha, N-acetyl beta-endorphin 1-27 and alpha, N-acetyl beta-endorphin 1-26; the biologically active form, beta-endorphin 1-31, is a minor component. In contrast, it was found that beta-endorphin generated in neuro-intermediate lobe cells in monolayer culture was less processed: the principal peptides related to bioactive beta-endorphin 1-31. When the cultured cells were incubated in the presence of 10(-5) M dopamine or 10(-6) M alpha-ergocryptine there was a marked increase in the degree of proteolysis and acetylation: the processing pattern reverted to that characteristic of the neuro-intermediate lobe in situ, with alpha-N-acetyl beta-endorphin 1-26 and alpha, N-acetyl beta-endorphin 1-27 as the prominent peptides. The results demonstrate that dopaminergic agents can influence the processing of beta-endorphin-related peptides in rat pars intermedia, indicating a new level at which the bioactivity may be regulated.     

Diurnal rhythms of cortisol,ACTH, and beta-endorphin levels in neonates and adults.

To determine whether a diurnal rhythm exists in neonates admitted to neonatal intensive care units where there is continuous artificial lighting and periodic nursing and medical care, plasma cortisol, adrenocorticotropin (ACTH), and beta-endorphin concentrations were measured in two groups of infants and in adult human volunteers. As expected, a diurnal rhythm was seen in adults. A diurnal rhythm was also found for cortisol and endorphin levels in neonates (3 to 4 days postnatally) with minimal stress and in infants who were clinically severely stressed. There was not a significant difference between the morning and afternoon concentrations of ACTH in these infants, but the afternoon concentrations were lower than the morning''s, as would be expected. We found that a diurnal rhythm does exist in neonates within the first few days of postnatal life and that the continuous lighting and medical and nursing interventions do not interfere with this rhythm.     

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta- endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.     

Effect of sandostatin on CRF-stimulated secretion of ACTH, beta-lipotropin and beta-endorphin.

The influence exerted by somatostatin on the secretion of ACTH and opioid peptides has still to be clarified. To gain further information on this issue, we performed in 10 normal volunteers two CRF tests (100 micrograms i.v.) one of which was preceded by s.c. injection of 100 micrograms of the long-acting somatostatin analogue SMS 201-995 (Sandostatin, Sandoz) (SMS), given 30 minutes before CRF. Premedication with SMS markedly inhibited the response of beta-EP to CRF, leaving unchanged the response of beta-LPH, ACTH and cortisol; mean incremental areas of beta-EP were 199.8 +/- 49.31 (SEM) vs 532.9 +/- 95.91 pmol 120 min (P less than 0.01) in the CRF test with and without SMS, respectively. To interpret the selective inhibitory effect of SMS on CRF-stimulated beta-EP secretion, it can be hypothesized that: a) the action of SMS was confined to a population of pituicytes preferentially secreting beta-EP; b) SMS interfered with the processing of POMC inhibiting the formation of beta-EP; c) SMS acted on extrapituitary, possibly peripheral, sources of beta-EP. In conclusion, this study indicates that, in man, somatostatin selectively inhibits the CRF-induced secretion of beta-EP, but not that of ACTH and beta-LPH, by an action that may be exerted at pituitary or extrapituitary level. This is a further example of dissociated secretion of POMC-derived peptides.