Biochemical characterization of enkephalin-like immunoreactive peptides of adrenal glands

The total enkephalin-like immunoreactive peptide content of adrenal glands from dog, cattle, guinea pig and rat was investigated by radioimmunoassay using a (met⁵)-enkephalin antiserum. Dog adrenals contain the highest amount of peptides, cattle and guinea pig adrenals contain lesser amounts, and the rat adrenals had the least amount (0.05% that of the dog). Comparison of the (met⁵)-enkephalin content of the adrenal cortex and medulla with that of whole bovine adrenal gland indicates that the peptides are concentrated in the medulla. Analysis of the chromaffin granules from bovine adrenal medulla indicates this to be the primary storage site for (met⁵)-enkephalin-like peptides. Gel chromatography reveals a molecular heterogeneity of the immunoreactive peptides in all species tested; high molecular weight peptides account for a larger proportion of the immunoreactivity when compared with the low molecular weight peptides.     

Detection of the pro-opiomelanocortin peptide fragments--beta-endorphin and ACTH--in the adrenals of rats and mice by immunohistochemistry

Independent peptide fragments of pro-opiomelanocortin molecule, beta-endorphin and ACTH, have been detected immunohistochemically in the adrenal glands of rats and mice. Immunoreactive beta-endorphin and ACTH have been revealed in the adrenal medulla and reticular zone of the adrenal cortex. beta-endorphin and ACTH distribution patterns in adrenal sections were identical, which is indicative of the linked synthesis of these peptides in the adrenal gland. The data obtained suggest the existence of pituitary-independent mechanisms regulating corticosteroidogenesis in the adrenal gland, involving adrenal pro-opiomelanocortin fragments.     

Characterization of a serine protease that cleaves pro-gamma-melanotropin at the adrenal to stimulate growth.

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-gamma-melanotropin (pro-gamma-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-gamma-MSH, at a specific bond.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

Processing of proenkephalin-A in bovine chromaffin cells. Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.     

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.     

Post-translational incorporation of [35S]sulfate into oligosaccharide side chains of pro-opiomelanocortin in rat intermediate lobe cells

Pro-opiomelanocortin (POMC), the common precursor to beta-endorphin and alpha-melanocyte-stimulating hormone in rat neurointermediate lobe cells, exhibits both charge and size heterogeneity on two-dimensional gel electrophoretograms. Short term [3H]phenylalanine pulse-labeling, and pulse-chase studies, revealed that this heterogeneity is acquired either co-translationally, through the addition of mannose-rich oligosaccharide chains to the nascent protein, or post-translationally, probably during the period of oligosaccharide processing from the high mannose to the complex forms. In this process, radioactive sulfate is incorporated into different glycoprotein variants of POMC. In the presence of tunicamycin, an inhibitor of the N-glycosylation process, [35S]sulfate incorporation does not occur in any of the major variant forms of POMC, thereby preventing the appearance of the most acidic forms on two-dimensional gels. POMC tryptic fragments were separated by high-pressure liquid chromatography. Sulfate incorporation occurred in only two peptides that were also labeled with [3H]glucosamine. Extensive alkaline digestion of these peptides in the presence of sodium borohydride released the sulfate-containing moieties which were separated from free amino acids by gel filtration. Sulfate bearing moieties could also be released by almond emulsin peptide:N-glycosidase digestion. All these results unambiguously show that sulfate moieties preferentially enter asparagine-linked carbohydrate side chains and not amino acid residues of the POMC polypeptide. It is also likely that differential sulfation, conferring unequal amounts of negative charge upon various glycoprotein variants of POMC, is responsible for much of the charge heterogeneity displayed by the prohormone.     

Unrestrained production of proopiomelanocortin (POMC) and its peptide fragments by pituitary corticotroph adenomas in Cushing's disease.

The hallmark of ACTH oversecretion in Cushing's disease is its partial resistance to the normal suppressive effect of glucocorticoids. Because ACTH secretion by the pituitary tumor is not normally restrained ACTH is overproduced with subsequent chronic hypercortisolism. Since peripheral tissues have retained their normal sensitivity to the action of cortisol they appropriately develop the features of Cushing's disease. The question of whether a collection of corticotroph cells, eventually arranged in an adenomatous-like fashion, is a primary pituitary event or is corticotropin-releasing factor driven has had no response so far. Clonal composition of such lesions has been determined by X chromosome inactivation using DNA probes which detect multiallelic polymorphism in females. A monoclonal pattern is found in all macroadenomas. ACTH is co-secreted with other peptide fragments derived from their common polypeptide precursor, proopiomelanocortin (POMC). As a rule POMC processing in pituitary tumors is qualitatively unaltered: plasma values of the N-terminal fragment, the joining peptide, the beta- and gamma-lipotropins, and beta-endorphin all are valid alternate markers of the tumor activity. Tumor POMC peptides including ACTH and its phosphorylated form usually show no peculiar or unexpected molecular forms in contrast with what is often found when POMC expression occurs in a non-pituitary tumor.     

Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum

Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.     

Effects of cell-permeating peptide binding on the distribution of 125I-labeled Fab fragment in rats

The peptides comprising the sequence of HIV-1 Tat protein (positions 48-60), Antennapedia (positions 43-58), and HIV-1 Rev protein (positions 34-50) are known to be cell-permeating. In this study, we examined how the distribution of Fab fragments in rats is affected by conjugation with these peptides. Fab fragment was iodinated by a chloramine-T method and then chemically conjugated with cell-permeating peptide. The complex of 125I-Fab and cell-permeating peptide was administered to male rats intravenously at a dose of 1 mg/kg, and whole-body autoradiography was performed at 4 and 24 h after administration. The patterns of distribution of 125I-Fab exhibited remarkable variation depending on the cell-permeating peptide used. In particular, at 4 h, high concentrations of radioactivity were observed in the spleen, adrenal gland, renal medulla, and liver with Rev peptide-Fab complex, in the liver and spleen with Tat peptide-Fab complex, and in the spleen, adrenal gland, and liver with Antennapedia peptide-Fab complex. Even at 24 h, high concentrations of radioactivity were still observed in the spleen and renal medulla of rat with Rev peptide-Fab complex, and in the spleen and renal cortex of rat with Antennapedia peptide-Fab complex. These findings demonstrate that the patterns of distribution of peptide-125I-Fab complexes can be modulated by selection of cell-penetrating peptides. Moreover, the patterns of retention of peptide-125I-Fab complexes in internal organs also differed at 24 h after administration. These findings provide valuable information for the development of novel antibody pharmaceuticals and therapeutic systems.     

Peptide E and Its Products, BAM 18 and Leu-Enkephalin, in Bovine Adrenal Medulla and Cultured Chromaffin Cells: Release in Response to Stimulation

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys-Arg) typical processing site, would generate two opioid fragments, the amino-terminal fragment BAM 18 and the carboxy-terminal fragment Leu-enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu-enkephalin). Here we present evidence that BAM 18 and Leu-enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys-Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu-enkephalin was found to be present in much greater amounts than was BAM 18-like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu-enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu-enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu-enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.     

Processing of Proenkephalin in Adrenal Chromaffin Cells

The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.     

Peptide E and other proenkephalin-derived peptides are potent kappa opiate receptor agonists

Various proenkephalin-derived peptides such as peptide E and the bovine adrenal medulla peptides BAM-12P and BAM-22P are potent competitors on mu and kappa binding sites in guinea pig brain sections. Moreover, they are all potent agonists in the rabbit vas deferens, a specific kappa opiate receptor bioassay. As described before, dynorphin and some of its fragments are also potent kappa agonists. Our results suggest that not only prodynorphin-derived peptides could act as endogenous kappa ligands but also some proenkephalin-derived peptides such as peptide E.     

Product inhibition of carboxypeptidase H

Carboxypeptidase H is one of several enzymes required for the processing of peptide hormone precursors. In this study, inhibition of carboxypeptidase H by its peptide products was investigated. Carboxypeptidase H activity in bovine adrenal medulla chromaffin granules and rat adrenal medulla homogenate was inhibited by the peptides Met- and Leu-enkephalin, vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone, with oxytocin and ACTH 1-14 having the least effect, at concentrations of 2-20 mM. Inhibition by amidated peptide products (vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone) show that the final products of the precursor processing pathway can regulate carboxypeptidase H. These levels of peptides are similar to known intragranular peptide concentrations indicating that product and feedback inhibition of carboxypeptidase H may play a role in the control of neuropeptide synthesis. The proenkephalin-derived peptides Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, and Met-enkephalin-Arg6-Phe7 competitively inhibited bovine and rat carboxypeptidase H with Ki values of 12.0, 6.5, 7.0, and 5.5 mM, respectively. The significantly greater Ki for Met-enkephalin may reflect the effects of higher intragranular concentration of Met-enkephalin, since one proenkephalin molecule contains four copies of Met-enkephalin and only one copy of each of the other enkephalin peptides. Thus, the products from one multivalent precursor molecule may equivalently inhibit carboxypeptidase H activity. Product inhibition of carboxypeptidase H and perhaps other processing enzymes may serve to limit the maximum peptide concentration within the secretory vesicle.     

An immunohistochemical study of the adrenal medulla of the ox

Summary The adrenal medulla of ox was studied by an indirect immunofluorescent technique using anti-ox chromaffin granule serum. The serum had a weak cross reaction with ox brain stem and splenic nerve. There was a species cross reaction with sheep, pig and horse. Immunoelectrophoresis showed five components in the serum against ox adrenal lysates. The whole adrenal medulla of ox was found to fluoresce by the immunofluorescent technique but not the cortex. The adrenals of sheep, pig and horse behaved similarly using the anti-ox serum. A serum prepared against ox chromogranin-A, the most abundant soluble protein of the chromaffin granules, was also used for immunofluorescence. Again both the adrenaline and noradrenaline storing cells fluoresced, but not the cortex.     

Synthetic modified N-POMC1–28 controls in vivo proliferation and blocks apoptosis in rat adrenal cortex

The identity of the pro-opiomelanocortin (POMC)-derived mitogen in the adrenal cortex has been historically controversial. We have used well-established in vivo models, viz., hypophysectomized (Hyp) or dexamethasone (Dex)-treated rats, to study the effect of the synthetic modified peptide N-terminal POMC (N-POMC_1–28) on DNA synthesis in the adrenal cortex, as assessed by BrdU incorporation and compared with adrenocorticotropic hormone (ACTH). We evaluated the importance of disulfide bridges on proliferation by employing N-POMC_1–28 without disulfide bridges and with methionines replacing cysteines. Acute administration of synthetic modified N-POMC_1–28 distinctly increased DNA synthesis in the zona glomerulosa and zona fasciculata, but not in the zona reticularis in Hyp rats, whereas in Dex-treated rats, this peptide was effective in all adrenal zones. ACTH administration led to an increase of BrdU-positive cells in all adrenal zones irrespective of the depletion of Hyp or Dex-POMC peptides. The use of the ACTH antagonist, ACTH_7–38, confirmed the direct participation of ACTH in proliferation. Two different approaches to measure apoptosis revealed that both peptides similarly exerted a protective effect on all adrenocortical zones, blocking the apoptotic cell death induced by hypophysectomy. Thus, ACTH_1–39 and N-POMC_1–28 have similar actions suggesting that the disulfide bridges are important but not essential. Both peptides seem to be important factors determining adrenocortical cell survival throughout the adrenal cortex, reinforcing the idea that each zone can be renewed from within itself.     

Basic Fibroblast Growth Factor (bFGF) Immunoreactivity Is Present in Chromaffin Granules

Basic fibroblast growth factor (bFGF) has recently been isolated from bovine adrenal glands. Immunohistological data revealed its presence in both adrenal cortex and adrenal medulla. Using immuno-electronmicroscopy, we found that in medullary chromaffin cells bFGF-immunoreactivity is localized in the secretory granules. Immunoreactivity also was observed by electronmicroscopy in isolated granules. Western blot analysis revealed the presence of the typical 18-kDa bFGF and additional immunoreactive materials with molecular masses of approximately 24, 30, and 46 kDa in whole bovine adrenal, and in cortex and medulla. Similar results were obtained with proteins from bovine chromaffin granules, with the following two exceptions: the 46-kDa immunoreactivity was found to be highly enriched when compared with medulla or cortex, and the 18-kDa band could be detected with only an antiserum against a synthetic peptide comprising the 24 NH2-terminal amino acids of bFGF, and not with an antiserum against purified bovine pituitary bFGF. All fractions enriched for bFGF-immunoreactivity showed neurotrophic activity for chick ciliary ganglion neurons, which could be blocked by antibodies. These results demonstrate for the first time the localization and occurrence of bFGF in a cellular secretory organelle, and present further evidence for the existence of higher molecular weight immunoreactive forms of bFGF.