Synthesis and radioreceptor binding activity of turkey beta-endorphin and deacetylated salmon endorphin

Des-acetylated salmon endorphin and turkey beta-endorphin have been synthesized by the solid-phase method. Relative opiate activities in a radioreceptor binding assay are: human beta-endorphin, 100; des-acetylated salmon endorphin, 169; turkey beta-endorphin, 94. Thus, non-mammalian endorphins can show high activity in a mammalian assay system.     

Humoral endorphin: A new endogenous factor with opiate-like activity

Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$ attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.     

Ontogenesis of enkephalin and humoral endorphin in the rat brain

Radioimmunoassay and column chromatography techniques were used to study the postnatal development of two different opioid ligands, humoral endorphin and enkephalin in the rat brain. Similar patterns were observed for both male and female animals during the period examined (from birth to the seventh week of life). Humoral endorphin content of the developing rat brain was found to increase in parallel to enkephalin, exhibiting a ‘lag’ period of 2 weeks. The most dramatic increase in opioid levels was detected during the third week of life; this stage was followed by a gradual change up to the adult levels.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Beta endorphin levels in CSF during methadone maintenance

Recent studies have shown that plasma beta endorphin levels of patients on methadone maintenance are comparable to controls. Furthermore, CSF levels of related peptides in methadone patients also do not differ from controls, although CSF levels of beta endorphin have not been specifically measured. In the current study we compared both CSF and plasma levels of beta endorphin in 11 patients on methadone maintenance for at least 10 months to levels in 13 controls getting spinal anesthesia for surgery. The CSF beta endorphin levels of the methadone maintained patients were significantly higher than the controls (52.3 vs 21.7 pg/ml), while plasma levels of beta endorphin (29.6 vs 31.1 pg/ml) and cortisol (13.8 vs 12.6 micro g/dl) [corrected] did not differ. Covarying for age differences between the samples, slightly increased the magnitude of this difference in CSF beta endorphin levels. Plasma levels of beta endorphin did not correlate with CSF levels, but did correlate with plasma levels of cortisol (r = 0.51, P less than 0.02). These findings supported previous studies of plasma beta endorphin levels. However, the dissociation of beta endorphin levels in plasma and CSF within this patient population was a new finding.     

Identification of endorphin acetyltransferase in rat brain and pituitary gland

An enzyme which N-acetylates beta-endorphin has been identified and characterized in the rat brain and pituitary gland. The beta-endorphin acetyltransferase activity was localized almost exclusively in the intermediate lobe of the pituitary gland. beta-endorphin acetyltransferase activity was also present in the hypothalamus, a brain region which synthesizes beta-endorphin, but not the cerebellum, a region which does not synthesize beta-endorphin. The substrate specificity of beta-endorphin acetyltransferase appeared to be located in the NH2 terminus and midportions of the beta-endorphin sequence. The regional distribution and pharmacological specificity of beta-endorphin acetyltransferase indicate that it is a highly specialized regulatory enzyme. Because N-acetyl-beta-endorphin is nonanalgesic and does not bind to the opiate receptor (Smyth, D.G., Massey, D.E., Zakarian, S., and Finnie, M.D. (1979) Nature (Lond.) 279, 252-254), it appears that the physiological significance of beta-endorphin acetyltransferase is that it can modulate the activity of endorphin secreted from opiomelanotropinergic cells and neurons.     

Binding of synthetic fragments of β‐endorphin to nonopioid β‐endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [³H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (K_d = 2.0 ± 0.1 nM ). The [³H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (K_i = 2.9 ± 0.2 nM ) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met⁵]enkephalin (K_i > 10 µM ). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [³H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the author's name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (K_i = 3.1 ± 0.3 nM ). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [³H]Octarphin was found to bind to macrophages with high affinity (K_d = 2.3 ± 0.2 nM ). The specific binding of [³H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (K_i = 2.4 ± 0.2 and 2.7 ± 0.2 nM , respectively). Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of endorphin exposure at weaning on the endorphin and serotonin content of white blood cells and mast cells in adult rat

Hormonal imprinting takes place perinatally at the first encounter between the developing receptor and its target hormone, resulting in the accomplishment of normal receptor development. In the presence of an excess of target hormone or the absence of it, or an excess of related molecules which can be bound by the receptor, faulty imprinting develops with life-long consequences. In previous experiments neonatal endorphin exposure caused a decrease in endorphin and serotonin content of peritoneal mast cells of adult animals. In the present experiment 25-day-old (weaned) female rats received 2 microg endorphin, and the endorphin as well as serotonin content of adult mast cells and white blood cells was studied by flow cytometry and confocal microscopy. Peritoneal lymphocytes and blood monocytes contained significantly (p<0.01) less endorphin and peritoneal mast cells less serotonin (p<0.07, i.e. of questionable significance) than the untreated control. The results bring attention to the possibility of durable imprinting of differentiating cells later in life and to the durable (possibly life-long) effect of an endorphin excess (perhaps caused by injury) manifested in the change of endorphin and serotonin content of immune cells.     

Persistently low natural killer cell activity and circulating levels of plasma beta endorphin: risk factors for infectious disease

Beta endorphin levels were quantitated in plasma samples obtained from normal subjects (n = 81, 37% males and 63% females, age range 18-45 years) as a component of a prospective study examining the relationship of illness morbidity to natural killer cell activity and psychological indices of stress. The present study was designed to test whether beta endorphin levels contributed additionally to the explanation of illness outcome variance. In the larger study, persistently low NK (LNK) activity was associated prospectively with higher illness morbidity. The findings reported here suggest that the observed LNK activity might be affected by circulating levels of plasma beta endorphin, as lower endorphin levels predicted the LNK pattern, which in turn, predicted higher illness morbidity.     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituaitary

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH₂-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [³H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.     

Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells

The secretion of peptide products derived from pro-ACTH/endorphin was examined with several radioimmunoassays and with polyacrylamide gel analyses of immunoprecipitates of radioactively labeled peptides. In studies using a mouse pituitary tumor cell line the accumulation of each of the four molecular forms of adrenocorticotropic hormone (ACTH) in tissue culture medium was shown to be a linear function of time. No evidence for self inhibition of secretion by accumulated, secreted peptides (i.e., ultra-short feedback) was found. Furthermore, synthetic human ACTH and synthetic camel beta-endorphin did not alter secretion of peptides when added to the culture medium at levels up to 10,000 times physiological. Stimulation of the release of ACTH-, endorphin-, lipotropin-, and 16k fragment immunoreactive material by norepinephrine was fully blocked by cobalt; by this criterion, stimulated release was calcium dependent. All the smaller molecules derived from the pro-ACTH/endorphin common precursor were secreted in equimolar amounts under all circumstances tested, within the precision of these studies (+/- 11%). Norepinephrine and cobalt did not significantly alter the secretion of pro-ACTH/endorphin and ACTH biosynthetic intermediate. The stimulation of secretion by norepinephrine and inhibition of secretion by cobalt was restricted to the lower molecular weight products derived from pro-ACTH/endorphin: glycosylated and nonglycosylated ACTH(1-39); beta-lipotropin, beta-endorphin, and gamma-lipotropin; and 16k fragment.     

Naloxone blockade of acupuncture analgesia: endorphin implicated.

Electroacupuncture in awake mice produced analgesia to noxious heat stimuli causing a 54% increase in latency to squeak. Subcutaneous naloxone completely abolished this acupuncture analgesia implicating endorphin. Naloxone injections in control mice caused a 17% hyperalgesia suggesting that “normal” mice also release endorphin. These results imply that endorphin is released at a low basal rate in “normal” mice, and at a much higher rate during acupuncture.     

Primary sequence of two regions of mouse pro-adrenocorticotropin/endorphin

The amino acid sequences of two previously uncharacterized regions of the mouse anterior pituitary common precursor to adrenocorticotropin (ACTH) and beta-endorphin (pro-ACTH/endorphin) were determined. Portions of the NH2-terminal region of pro-ACTH/endorphin (called the 16K fragment) and the region between ACTH and beta-endorphin (called gamma-lipotropin) were sequenced by Edman degradations of biosynthetically labeled immunoprecipitated proteins and by Edman degradations of purified 16K fragment and beta-lipotropin. With a combination of these two approaches, 29 of the first 34 residues at the NH2-terminal end of the mouse 16K fragment were determined. The NH2-terminal region of the mouse 16K fragment was found to be nearly identical with the homologous porcine and bovine molecules. The complete amino acid sequence of the NH2-terminal region of gamma-lipotropin was determined. In contrast to the highly conserved nature of the 16K fragment, mouse gamma-lipotropin was found to differ substantially from the gamma-lipotropins of other species. Although the NH2-terminal and beta-melanotropin-like regions of the mouse gamma-lipotropin are similar to the corresponding regions of other gamma-lipotropins, the intervening region of mouse gamma-lipotropin is substantially shorter than it is in other gamma-lipotropins. In addition, mouse gamma-lipotropin lacks the pair of basic amino acids that normally mark the proteolytic cleavage site used to produce beta-melanotropin from gamma-lipotropin.     

Thyrotropin releasing hormone antagonizes beta endorphin hypothermia and catalepsy.

Injection of 30 μg β endorphin intraventricularly (ivt) in rats produced an alteration of body temperature, a state of catalepsy, and an increase in antinociceptive latencies. Subsequent ivt injections of 20 μg of thyrotropin releasing hormone (TRH) reversed the ongoing changes in body temperature and catalepsy produced by β endorphin. Since TRH antagonized these effects in hypophysectomized rats, it is implied that these effects of TRH are independent of pituitary-thyroid involvement. In contrast to the above, TRH did $\text{not}$ alter the antinociception produced by β endorphin in either sham-control or hypophysectomized rats. The failure of TRH to antagonize all three of these opiate effects, as well as the inability of TRH to displace bound dihydromorphine from synaptic plasma membranes, suggests that the level of TRH-β endorphin interaction is not at the opiate receptor.     

Subcellular fractionation studies on the post-translational processing of pro-adrenocorticotropic hormone/endorphin in rat intermediate pituitary

The subcellular localization of the post-translational processing steps which occur in the conversion of pro-adrenocorticotropic hormone (ACTH)/endorphin into beta-endorphin-sized molecules in rat intermediate pituitary has been studied. Primary cell cultures were incubated in radioactively labeled amino acids, and a subcellular fraction containing secretory granules was separated from a subcellular fraction containing rough endoplasmic reticulum and Golgi apparatus by centrifugation of homogenates on gradients on Percoll (Pharmacia Fine Chemicals). The radiolabeled beta-endorphin-related material in the granule and rough endoplasmic reticulum/Golgi apparatus fractions was quantitated by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pulse-chase labeling experiment demonstrated that newly synthesized beta-endorphin-related material first appeared in the rough endoplasmic reticulum/Golgi apparatus fraction and after longer incubations (chase) appeared in the secretory granule fraction. After 2 h of chase incubation, about 85% of the beta-endorphin-related material synthesized during the 30-min pulse incubation had been transferred from the rough endoplasmic reticulum/Golgi apparatus to the secretory granule fraction. The conversion of most of the newly synthesized pro-ACTH/endorphin into beta-lipotropin occurred in the rough endoplasmic reticulum/Golgi apparatus fraction, whereas the conversion of most of the beta-lipotropin into beta-endorphin-sized molecules occurred in the secretory granule fraction.     

A preliminary study of beta endorphin during chronic naltrexone maintenance treatment in ex-opiate addicts

Because opioid antagonists acutely produce rises in serum beta endorphin, we studied beta endorphin levels in 21 former opiate addicts chronically taking naltrexone. The mean AM (19.5 pg/ml) beta endorphin level was higher than the AM mean for 39 normals under 40 years old (12.1 pg/ml) (t = 3.2, p less than 0.001); the mean PM level for the naltrexone treated patients was 13.6 pg/ml. Four patients had beta endorphin levels more than 2 S.D. above the mean for the normals (greater than 26.4 pg/ml), and six others had relatively elevated PM levels. Thus, 47% (10/21) had abnormal patterns of beta endorphin levels. We had previously reported abnormally high cortisol levels in these patients, and AM cortisol correlated with AM beta endorphin levels (r = 0.7, p less than 0.001). We concluded that sustained beta endorphin elevations may occur during chronic naltrexone treatment.     

Regulation of hypophysial secretion by endogenous opiates: Humoral endorphin stimulates the release of growth hormone

Humural endorphin, a recently discovered endogenous opioid factor stimulates the release of growth hormone and, to some extent of prolactin, similarly to other endogenous (enkephalin, β-endorphin) and exogenous (morphine) opiates. This stimulatory effect is dose-dependent with peak values at 30 minutes following intraventricular injection to newborn rats. However, in contrast to the other opioid ligands, the effect of humoral endorphin is not blocked in a dose-dependent fashion by naloxone, the potent opiate antagonist. Thus, while moderate doses of naloxone partially inhibit the stimulatory effect, higher doses which completely block morphine, enkephalin and β-endorphin, are ineffective in antagonizing humoral endorphin. This peculiar interaction between naloxone and humoral endorphin resembles the effect of the opiate antagonist on spontaneous release of growth hormone and prolactin, suggesting the involvement of humoral endorphin in the physiological regulation of hypophysial secretion.     

Regulation of endorphin production by glucocorticoids in cultured pituitary tumor cells

When clonal AtT-20 mouse pituitary tumor cells were exposed in culture to physiological concentrations of glucocorticoids for 2 or more days, the intracellular endorphin content was reduced by 50–75%. The steroid specificity is consistent with a glucocorticoid-receptor mediated response. Gel filtration analysis indicates a reduction of both the major and the minor endorphin species present, which resemble β- and α-endorphin, respectively. The results suggest that endorphin synthesis, like corticotropin synthesis, is under negative regulatory control by glucocorticoids in AtT-20 cells and in the corticotropin/endorphin-producing cells of the adenohypophysis.     

Detection of nonopioid β‐endorphin receptor in the rat myocardium

Two selective agonists of nonopioid β‐endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high‐affinity naloxone‐insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [³H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled β‐endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [³H]immunorphin specific binding with the membranes was inhibited by unlabeled β‐endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin and [Leu⁵]enkephalin. Thus, β‐endorphin, immunorphin and octarphin bind to the common high‐affinity naloxone‐insensitive receptor of the rat myocardial membranes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.