Expression of three proopiomelanocortin subtype genes and mass spectrometric identification of POMC-derived peptides in pars distalis and pars intermedia of barfin flounder pituitary.

Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-alpha-MSH-A/B (amino acid sequence of alpha-MSH-A is identical to that of alpha-MSH-B), beta-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide (N-POMC)-A were identified in the PD. Moreover, Des-Ac-alpha-MSH-A/B, alpha-MSH-A/B, beta-MSH-A and -B, N-beta-lipotropin-A, CLIP-A, N-Ac-beta-END-A(1-41) (C-terminally truncated form of N-Ac-beta-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-alpha-MSH-A, CLIP-A, and beta-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of alpha-MSH-A/B and N-Ac-beta-END-A(1-41). These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.

Detection of mRNA transcripts of truncated opiate precursor (POMC) in human cartilage.

In the present study, we have investigated the presence of pro-opiomelanocortin C-terminal fragment derived-peptides in human articular cartilage and cultured chondrocytes. beta-Lipotropin and beta-endorphin were monitored in different cell cultures and biopsies using different techniques. Biopsies were taken from patients undergoing total knee arthroplasty due to osteoarthritis. Both fresh tissue sections and chondrocytes cultured in monolayer were used in the study. Immunohistochemistry, immunocytochemistry, reverse transcriptase-polymerase chain reaction and qualitative Western blots were carried out. The results of the reverse transcriptase-polymerase chain reaction showed transcription of a truncated-form of mRNA for pro-opiomelanocortin in native cartilage and cultured chondrocytes. There was no detection of endogenous production of beta-lipotropin or beta-endorphin in human articular chondrocytes, either in situ or in vitro. Whether pro-opiomelanocortin-derived peptides of non-cartilaginous origin are present in articular cartilage itself still remains unclear.

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry.

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.

Quantitative peptidomics of pituitary glands from mice deficient in copper transport.

We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.

Characterization of beta-endorphin- and alpha-MSH-related peptides in rat heart.

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that beta-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to beta-endorphin- and alpha-MSH-related peptides. Ion exchange HPLC analysis revealed that beta-endorphin(1-31) was further processed to alpha-N-acetyl-beta-endorphin(1-31), which comprised 35.9 +/- 0.1% of total immunoreactivity, and smaller amounts of beta-endorphin(1-27), beta-endorphin(1-26), and their alpha-N-acetylated derivates. The predominant alpha-MSH immunoreactive peptides coeluted with alpha-MSH and N,O-diacetyl-alpha-MSH by reverse-phase HPLC, although small amounts of ACTH(1-13)-NH2 were also present. Thus, multiple forms of beta-endorphin and alpha-MSH are localized in rat heart. beta-Endorphin(1-31) is a minor constituent, however, indicating that nonopioid beta-endorphin peptides predominate.

[Examination of some peptide fractions left over after isolation of insulin from the pancreas. III. Analytical characterization of products by chromatography and electrophoresis]. / Badanie niektórych peptydów wyodrebnionych z pozost alosci po izolowaniu insuliny z trzustki. III. Charakterystyka analityczna produktów chromatografowania i elektroforezy.

The studies of separated and isolated components of Lipotropin-Polfa preparation by determining the contents of nitrogen and qualitative and quantitative composition of amino acids were performed. The component I which constituted 85% of the preparation weight, molecular weight ca. 3500, was separated by means of electrophoresis using polyacrylamide gel, into 3 fractions of polypeptides. The component I proved to have biological activity several times as large as the Lipotropin-Polfa preparation.

Central effects of tumor necrosis factor alpha and interleukin-1 alpha on nociceptive thresholds and spontaneous locomotor activity.

To extend the knowledge on the central effects of cytokines, we studied the effects of tumor necrosis factor alpha and interleukin-1 alpha on nociceptive thresholds and spontaneous locomotor activity in rats. After central administration, both tumor necrosis factor alpha and interleukin-1 alpha significantly (P < 0.001) increase the nociceptive thresholds as measured by the hot-plate test. Tumor necrosis factor alpha, but not interleukin-1 alpha decreases spontaneous locomotor activity evaluated by the Animex test. The increase in nociceptive thresholds induced by tumor necrosis factor alpha or interleukin-1 alpha is not affected by the opiate receptor antagonist naloxone, or antisera against the endogenous opioids beta-endorphin, met-enkephalin or dynorphin. The analgesic effect of tumor necrosis factor alpha is completely antagonized by anti-IL-1 antibodies. Moreover, the cyclooxygenase inhibitor indomethacin does not antagonize the increase of nociceptive thresholds induced by either cytokine.

Beta-endrophin immunoreactivity during human pregnancy.

The human placenta has been implicated as a source of numerous peptide hormones during pregnancy. Since the immunoassay detection of the proopiomelanocortin derived peptide beta-endorphin (beta E) in placental extracts in 1978, it has remained uncertain whether placental beta E immunoreactivity (IR) is 1) secreted into the maternal circulation and 2) opiate receptor active during pregnancy. To elucidate the nature of beta E IR in the placenta, both beta E IR and N-alpha-acetylated beta E (Ac beta E) IR were simultaneously measured in extracts of human pituitaries, placentas, and plasma by two homologous RIAs. Pituitary extracts (n = 6) contained 38 +/- 7 nmol beta E IR per g wet wt tissue (mean +/- SEM), of which only 20 +/- 4 pmol/g were Ac beta E IR. Term placental extracts (n = 19) had 201 +/- 30 fmol/g wet wt total beta E IR and 30 +/- 3 fmol/g wet wt total Ac beta E IR, which comprised 15% of total beta E IR in placental extracts. Total plasma beta E IR rose from 28 weeks gestation (8.5 +/- 0.3 fmol/mL, n = 159) to peak at labor (50 +/- 4 fmol/mL, n = 98; P < 0.01) but total Ac beta E IR was found in only four 28-week (1.7 +/- 0.9 fmol/mL) and 42 labor plasma samples (0.9 +/- 0.1 fmol/mL). Gel filtration chromatography of placental and pituitary extracts showed that while less than 1% of the beta E31-size material was acetylated in the pituitary, up to 60% of the beta E31-size material in placental extracts was acetylated. In pooled third trimester plasma extracts, however, only 4% of the beta E31-size material was acetylated. Furthermore, the ratio of beta E31:beta-lipotropin in pituitary extracts (n = 3) was 0.5; pooled plasma-0.5, and placental extracts (n = 5)-1.2. These data indicate that 1) the placenta extensively N-alpha-acetylates beta E31 destroying its opiate bioactivity while the pituitary does not; 2) beta E IR in pregnant women's plasma is similar to pituitary beta E IR, being mostly nonacetylated and similar in size to beta-lipotropin. These findings are consistent with a pituitary source for the elevated plasma beta E IR found during late pregnancy which may, in turn, be a consequence of elevated plasma concentrations of placentally secreted plasma corticotropin-releasing factor IR present during the third trimester.

The sheep pars tuberalis: an immunohistochemical study. Demonstration of the presence of glycoprotein and lipotropin hormones.

Using indirect immunofluorescence with fourteen different antisera raised against pituitary hormones and peptides, we characterized immunochemically the cells of the sheep pars tuberalis. The presence of LH- and FSH-containing cells, shown in previous studies, was also observed in the present investigation. In addition, we found TSH-containing cells, never observed in sheep, and beta LPH-containing cells. The latter hormone has never been found in any studied species. It appeared that a small amount of perikarya (less than 20%) were immunolabelled and, that the sheep pars tuberalis contained a majority of immunonegative cells as in the guinea-pig rabbit and rhesus monkey. This study may contribute to a better knowledge of the function of the sheep pars tuberalis.

Localization of beta-endorphin in the rat heart and modulation by testosterone.

Chromatographic analysis and radioimmunoassay were used to identify and quantitate beta-endorphin (BE) and beta-lipotropin (B-LPH) in the hearts (devoid of major blood vessels and atria) from intact male rats, castrated male rats, and castrated male rats treated with testosterone propionate (TP). BE and B-LPH in the plasma of these animals were also identified and measured. In comparison to intact animals, castration resulted in a significant elevation in the content of BE in the heart which was reversed by the administration of TP. The content of B-LPH in the heart was not affected by castration or castration in combination with TP. The ratio of BE to B-LPH in the heart of castrated animals was significantly elevated as compared with intact controls. Treatment of castrates with TP returned the ratio of BE to B-LPH to that observed in intact animals. The concentration of BE in the plasma was greater in castrated rats and castrated rats given TP than in intact males, whereas the concentration of B-LPH was diminished in castrated animals given TP. The ratio of BE to B-LPH was greater in castrated animals treated with TP than in castrated and intact animals. The content of BE and B-LPH, as well as the ratios of the two peptides, varied independently in the cardiac tissue and plasma. The present findings indicated that (i) BE and B-LPH are present in cardiac tissue, (ii) the amount of BE and B-LPH in the heart and the ratio of BE to B-LPH appear to be modulated by TP, and (iii) BE and B-LPH detected in the heart was not simply a reflection of the presence of these peptides in the plasma.

[Study of various peptides isolated from residues after extraction of insulin from the pancreas. I. Chemical characteristics of the studied substances]. / Badanie niektórych peptydów wyodrebnionych z pozostalosci po izolowaniu insuliny z trzustki. I. Charakterystyka chemiczna badanego materialu.

Several series of Lipotropin substance produced at "Polfa" were subjected to physiochemical investigations. The contents of dry residue, ash, ribose, protein, total nitrogen, ammonium nitrogen and amino acid nitrogen were determined. Particular series proved to differ in contents of ribose and amino acids. For the purpose of comparison, identical studies were carried out with Lipormone made by "Labor. Choay" (France). Substantial differences were found between the two preparations.

Ectopic ACTH syndrome due to Grawitz tumor.

An extremely unusual case of adrenocorticotropin (ACTH)-producing Grawitz tumor is reported in a 56-year-old female. The clinical feature of the patient was compatible with Cushing's syndrome. The plasma levels of ACTH and cortisol were markedly elevated. At autopsy, a left renal tumor was demonstrated and its histopathological diagnosis was renal cell carcinoma (Grawitz tumor). The adrenal gland was bilaterally enlarged with diffuse hyperplasia of the fasciculate zone. The adenohypophyseal cells were atrophic and showed Crooke's degeneration. The tumor contained extremely high levels of ACTH, beta-lipotropin and beta-endorphin. The presence of large molecular weight forms of ACTH has also been demonstrated by a Sephadex G-50 gel filtration of the tumor extract. We authors believe that this is the first documented case of ACTH-producing Grawitz tumor in the literature.

Beta-endorphin-like immunoreactivity in normal mucosa, muscle layer, adenocarcinoma, and polyp of the colon.

beta-Endorphin-like immunoreactivity was detected in the mucosa and muscle layer of normal colon, adenocarcinomas derived from the colon mucosa, and colon polyps which were histologically confirmed to be adenoma without a focus of carcinoma or with in situ carcinoma. The contents of beta-endorphin-like immunoreactivity in adenocarcinomatous tissue (11.94 +/- 1.77 pmol/g wet wt) and colon polyps without focus of carcinoma (10.71 +/- 1.50 pmol/g wet wt) were found to be significantly higher than those in the mucosal layer (6.86 +/- 0.64 pmol/g wet wt) and muscle layer (8.30 +/- 0.68 pmol/g wet wt) of normal colon. These data suggest that the production of beta-endorphin-like immunoreactivity is specifically increased in some adenocarcinomas and adenomatous polyps and may be related to the alteration of bowel habits. Gel exclusion chromatography of beta-endorphin-like immunoreactivity revealed three peaks corresponding to beta-endorphin, beta-lipotropin, and an immunoreactive form between the two. In the mucosal layer and muscle layer of the colon, a broad major peak was eluted at the position of beta-endorphin, and minor peaks were eluted at the position of beta-lipotropin and between beta-endorphin and beta-lipotropin. In adenocarcinoma and polyp, the peak size corresponding to authentic beta-lipotropin was greater than that of beta-endorphin. This study demonstrated that beta-endorphin-like immunoreactivity existed at a high concentration in some colon adenocarcinomas and polyps whose elution patterns were different from those of normal colon tissue.

[Identification of pro-opiomelanocortin and its C-terminal fragments in the mammalian thymus]. / Identifikatsiia proopiomelanokortina i ego C-kontsevykh fragmentov v timuse mlekopitaiushchikh.

Immunoreactive alpha-, beta-, gamma-endorphins and beta-lipotropin were detected in perfused calf thymus extracts at the following concentrations (fmol/mg) tissue, M +/- m): 1.32 +/- 0.08, 1.53 +/- 0.45, 0.0186 +/- 0.0022 and 0.741 +/- 0.157, respectively. It was demonstrated for all ligands tested that the synthetic peptide and increasing amounts of the extract cause a similar displacement of the corresponding 125I-peptide from its complex with specific antiserum. Using the immunoblotting technique with a highly specific antiserum to bovine beta-lipotropin, the extracts of calf thymus, rat thymocytes and bovine hypophysis were found to contain two polypeptides with Mr of 32 and 14 kD, whose mobility corresponds to that of proopiomelanocortin and beta-lipotropin.

Combined high-performance liquid chromatographic-radioimmunoassay method for the analysis of endorphins, enkephalins and other neurotransmitter peptides.

Assays for beta-endorphin (BE) and its precursors such as beta-lipotropin (LPH) in cerebrospinal fluid (CSF), plasma and some tissues have been difficult because of their low concentrations in limited sample volumes, the non-specificity of most antisera. These problems are compounded by the lack of suitable separation methods. Similar problems exist for the enkephalins, tachykinins and dynorphins, among others. This study reports a high-performance liquid chromatographic (HPLC) separation method in which BE and LPH are well separated from each other and which also separates other neuropeptides of interest. The method uses volatile solvents which do not interfere with radioimmunoassay (RIA). Thus by combining HPLC with RIA the method offers, for the first time, a specific assay method for the endorphin, enkephalin and dynorphin families of peptides which does not suffer from the uncertainties in RIA due to cross-reactivities of antisera. Peptide concentrations obtained from the CSF of a small group of chronic pain patients are also presented.

Immunocytochemical and ultrastructural findings in a mature retroperitoneal teratoma.

Report is made of a mature retroperitoneal teratoma in a 32-year-old man. Investigation of the tumor revealed cells immunoreactive for ACTH, Met-enkephalin, beta-LPH, serotonin, FSH, BPP, S100, Neuron-specific-enolase. These cells were mainly present in the glandular epithelium, lining the cysts of the tumor. Ultrastructurally, neuro-secretory granules were demonstrated in the cytoplasm of the tumoral endocrine cells. At no time did the patient display endocrine symptoms.

Localization and concentrations of beta-endorphin and beta-lipotrophin in human placenta.

Immunohistochemical staining of placental tissue for beta-endorphin immunoreactivity was positive in the syncytiotrophoblast in both early and term pregnancy. Cation-exchange liquid chromatography and radioimmunoassay revealed peaks of beta-endorphin and beta-lipotrophin and a third immunoreactive peak of unknown nature. The concentration of beta-endorphin was higher in the placental tissue than it was in the maternal or cord plasma. beta-Lipotrophin was not detected in all placentae studied. We did not find any effect of gestational age on tissue concentrations of endorphins in the placenta, nor was there any significant difference in the placental endorphin content between placentae collected at elective caesarean section before labour and after spontaneous vaginal delivery.

Cerebral distribution of beta-lipotropin and beta-endorphin in infantile progressive spinal muscular atrophy of Werdnig and Hoffman disease.

The regional distribution's profile of beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) was determined in the brain of an infant who died from Werdnig-Hoffmann's disease. Regional levels of beta-endorphin-like immunoreactivity (beta-ELIR), resulting from beta-EP and beta-LPH, were generally low in comparison to the homologous levels found in victims dying of other diseases.

Use of immobilized exopeptidases and volatile buffers for analysis of peptides by fast atom bombardment mass spectrometry.

beta-Lipotrophin (62-77) or Ac-gastrin releasing peptide was incubated with immobilized carboxypeptidase Y or aminopeptidase M. Subsequent aliquots of each incubation mixture were analysed by fast atom bombardment mass spectrometry using a dithiothreitol/dithioerythritol liquid matrix. The use of immobilized enzymes and volatile buffers for exopeptidase digestions enabled rapid and facile separation of enzyme from digestion products. This approach to mass spectral peptide analysis reduced spectral background arising from a glycerol matrix, buffer salts, or enzyme proteins and contaminants, enabling analysis of as little as 200 picomoles of a suitable peptide.

Hyperprolactinemia associated with clinically silent adenomas: endocrinologic and pathologic studies; a report of two cases.

Pituitary adenomas containing adrenocorticotropic hormone (ACTH) in one case, and ACTH, beta-lipotropin, and beta-endorphin in the other, were demonstrated in two patients who had amenorrhea-galactorrhea and hyperprolactinemia with no manifestation of Cushing's disease. Neither adenoma contained prolactin (PRL). Initial bromocriptine therapy resulted in cessation of amenorrhea-galactorrhea and normalization of PRL levels. However, there was radiologic evidence of tumor enlargement in both patients. After pituitary adenomectomy, the two patients resumed regular menses and normal PRL dynamics. These patients illustrate the need for bromocriptine therapy for possible enlargement of their pituitary adenomas. The diagnosis of silent corticotroph adenoma should be kept in mind.