Metorphamide, a C-terminally amidated opioid peptide, in human adrenal and human phaeochromocytoma

There appears to be only one possible site for the production of an amidated peptide in the human proenkephalin sequence; this will give rise to the peptide named metorphamide. Since amidation of peptides is commonly an activation step in the synthesis of regulatory peptides, we have examined the levels and form of immunoreactivity to metorphamide in human post-mortem adrenal and phaeochromocytoma extracts. In three out of four post-mortem adrenal extracts, and in each of the two phaeochromocytoma extracts examined, there was 3-4 times more immunoreactivity to the carboxy-terminus of pro-enkephalin, Met-enkephalin(Arg6,Phe7), than to metorphamide. The metorphamide immunoreactivity was shown in each extract to measure only the amidated octapeptide according to gel exclusion and reverse-phase chromatography data. The implications for processing of proenkephalin in human adrenal are indicated.     

Large-molecular-weight somatostatin in human adrenal medullary tissue

Three human catecholamine-secreting adrenal medullary tumours, identified as phaeochromocytoma, were found to contain 774, 168, and 78 pmol/g of somatostatin-like immunoreactivity (SLI), compared to 40 pmol/g in a sample of normal human adrenal medulla. Sephadex-G50 gel-filtration chromatography of extracts from these tissues revealed SLI eluting in the position of somatostatin-14, somatostatin-28, and a peak eluting with a mol. wt. of about 5 K. After digestion of eluted material with clostridiopeptidase B, the predominant form of adrenal medullary SLI was found to elute in the position of a 7-K polypeptide.     

Met-Enkephalin [Arg6,Phe7] Immunoreactivity in Bovine Caudate and Bovine Adrenal Medulla

Abstract: A radioimmunoassay specific for the COOH-terminus of Met-enkephalin [Arg⁶,Phe⁷] and a separate assay specific for the COOH-terminus of Met-enkephalin are described. Immunoreactivity by these two assays was compared in bovine caudate and bovine chromaffin granule preparation after Sephadex G75 chromatography in 50% acetic acid. When the assays were applied to the chromatography fractions of the bovine caudate extract, the majority of the immunoreactivity was found in the fractions corresponding to the heptapeptide and the pentapeptide respectively. When the chromaffin granule chromatography fractions were assayed, both of the radioimmunoassays showed that most reactivity was in several peaks in the larger molecular weight fractions. The major peak for the Met-enkephalin [Arg⁶,Phe⁷] assay had an apparent molecular weight of 2800, while with the Met-enkephalin assay the dominant peak of immunoreactivity had an apparent molecular weight of 10,000. The presence of authentic Met-enkephalin [Arg⁶,Phe⁷] in both caudate and chromaffin granule extracts was confirmed by reverse-phase chromatography of the previously sized fractions. It appears then that the processing of precursors of opioid peptides is directed, in the caudate, to the synthesis and storage of the enkephalins and of Met-enkephalin [Arg⁶,Phe⁷]; in the adrenal medulla the major products of precursor processing are a variety of polypeptides of larger sizes.     

Secretion of [Met]Enkephalyl-Arg6-Phe7-Related Peptides and Catecholamines from Bovine Adrenal Chromaffin Cells: Modification by Changes in Cyclic AMP and by Treatment with Reserpine

Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.     

Novel Opioid Peptide Amidorphin: Characterization and Distribution of Amidorphin-Like Immunoreactivity in Bovine, Ovine, and Porcine Brain, Pituitary, and Adrenal Medulla

We have recently isolated from bovine adrenal medulla a novel C-terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species, authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N-terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high-molecular-weight form of amidorphin was detected. These findings are indicative of a tissue-specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C-terminal fragments in the brain and pituitary.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

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.     

Existence of Met-enkephalin-Arg6-Gly7-Leu8 with Met-enkephalin,Leu-enkephalin and Met-enkephalin-Arg6-Phe7 in the brain of guinea pig,rat and golden hamster

High performance liquid chromatography (HPLC) coupled with specific radioimmunoassays for methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-E-Arg⁶-Gly⁷-Leu⁸), methionine-enkephalin (Met-E), leucine-enkephalin (Leu-E) and methionine-enkephalin-Arg⁶-Phe⁷ (Met-E-Arg⁶-Phe⁷) has demonstrated that Met-E-Arg⁶-Gly⁷-Leu⁸ exists together with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ in the brain of guinea pig, rat and golden hamster. The content of Met-E-Arg⁶-Gly⁷-Leu⁸ was comparable to those of Leu-E and Met-E-Arg⁶-Phe⁷, whereas that of Met-E was the highest among the four opioid peptides. These results are compatible with the recent studies on the nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla, which reveal that this precursor molecule contains four copies of Met-E and one copy each of Leu-E, Met-E-Arg⁶-Phe⁷ and Met-E-Arg⁶-Gly⁷-Leu⁸. The co-existence of Met-E-Arg⁶-Gly⁷-Leu⁸ with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ suggests that their biosynthetic pathway in the brain is similar to that in the adrenal medulla.     

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.     

Structural identification, subcellular localization and secretion of bovine adrenomedullary neuromedin C [GRP-(18-27)

Bombesin-like immunoreactivity (BLI) was purified from acid (HCl) extracts of bovine adrenal medulla. High performance liquid chromatography (HPLC) on a mu-Bondapak C18 column revealed the presence of five molecular forms of BLI, one coeluting with synthetic gastrin releasing peptide (GRP), the mammalian counterpart of amphibian bombesin, one coeluting with neuromedin C, one coeluting with neuromedin B and the two other ones coeluting with the oxidized forms of neuromedins B and C. The material corresponding to neuromedin C was purified to homogeneity and its amino acid composition and sequence corresponded to those expected for neuromedin C. HPLC analysis on an analytical SP-5PW column of subcellular extracts of bovine adrenal medulla indicated that neuromedin C is almost exclusively localized in secretory granules. The neuropeptide function of neuromedin C and/or other BLI peptides at this level was supported by the stimulatory effect of carbamylcholine (500 microM) on the release of BLI (4.5-fold increase over the basal release of 19 fmol/5 min) from perfused bovine adrenal glands.     

Demonstration of a neuropeptide Y-like immunoreactivity in human phaeochromocytoma extracts

Using a porcine neuropeptide Y directed radioimmunoassay it was shown that acid extracts of human phaeochromocytoma tumour tissue contained a neuropeptide Y-like peptide. Further fractionation and purification of this immunoreactivity showed that this human neuropeptide Y-like immunoreactivity was closely similar in molecular size and separation characteristics to porcine neuropeptide Y. The possible contribution of neuropeptide Y to the hypertension characterizing human phaeochromocytoma is discussed.     

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.     

PGE2: Opposite effect on catecholamine release from phaeochromocytoma and adrenal medulla

The effect of PGE₂ on catecholamine release from human adrenal medulla and phaechromocytoma was studied in slices incubated . In each of 3 normal human adrenal medullae PGE₂ (10⁻⁷ M) caused a significant inhibition of the release of catecholamines in incubation. In each of 3 phaeochromocytomas studied PGE₂(10⁻⁷ M) caused a significant increase of catecholamine release in incubation. The possible relevance of this mechanism to the regulation of catecholamine release in phaeochromocytoma is discussed.     

Characterization of opioid peptides from maternal and fetal sheep adrenal glands

Enkephalin immunoreactive material from adrenal glands was characterized both in maternal and fetal sheep at various gestational ages. Whole gland extracts from both maternal and fetal sheep contained three major peaks of Enk immunoreactivity corresponding to apparent molecular weights of 10,000, 2800, and less than 1200 daltons. The majority of maternal adrenal Enk immunoreactivity was found in medullary tissue, although cortex also contained low but detectable amounts. This was also the case in newborn lambs and 139 day fetuses, where adrenal cortex was sufficiently developed to allow extraction and quantitation of opioid material. In fetuses at mid-gestation (70-80 days), adrenal medullary Enk immunoreactivity was approximately 75% of maternal values. Met-Enk and Leu-Enk content in 139 day fetal medulla were 70 and 76% of maternal values respectively, while newborn Met- and Leu-Enk medullary content were similar to maternal values. The molar ratio of Met-Enk to Leu-Enk was approximately 4:1 in both maternal and fetal adrenal medulla, and 2:1 in adrenal cortex, suggesting different synthetic processing of opioid peptides in the two tissues. The early appearance of significant levels of adrenal medullary Enk immunoreactivity and subsequent development paralleling that of catecholamines suggest a predominant role for adrenal enkephalins in regulation of fetal cardiovascular function early in gestation.     

Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum

An antiserum was generated against a synthetic peptide corresponding to amino acids 95-117 of bovine proenkephalin, and a sensitive radioimmunoassay was developed. Comparison of the reactivities of the synthetic peptide, its specific cleavage products, and other synthetic peptides showed that the important immunological determinant was contained within residues 101-109 of bovine proenkephalin (-Gly-Gly-Glu-Val-Leu-Gly-Lys-Arg-Tyr-). Radioimmunoassay of fractions after gel filtration of bovine adrenal medullary chromaffin granule lysate showed three pools of immunoreactivity: pool 1 (Mr 20,000-30,000), pool 2 (Mr 10,000-20,000), and pool 3 (Mr approximately 5,000). Further characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that the antiserum recognized 27-, 20.5-, 16.5-, and 5.6-kilodalton enkephalin-containing proteins. The radioimmunoassay was also used to detect proenkephalin-like material in extracts of rat adrenal and regions of rat brain and spinal cord following gel filtration. Immunoreactivity from the rat adrenal chromatographed predominantly as high molecular weight material (Mr 31,500-43,500), whereas material in regions of rat brain showed a broader molecular weight distribution (Mr 4,000-43,500). This indicated differences in the processing of proenkephalin between rat adrenal and brain tissue. Differences were also seen in the molecular weight profile of immunoreactivity in different brain regions, most noticeable in the case of striatum and hypothalamus, suggesting regional differences in processing. Based on quantitation of higher molecular weight immunoreactive proenkephalin-like material and free Met-enkephalin immunoreactivity in different brain regions, it was apparent that extensive processing of proenkephalin occurs in brain. We concluded that antisera against proenkephalin-(95-117) recognize a wide range of intermediates in the processing of proenkephalin in both bovine adrenal medulla and rat adrenal, brain, and spinal cord, making it a useful tool for further studies concerned with the expression and post-translational processing of proenkephalin.     

Met-enkephalyl-Arg6-Phe7 immunoreactivity in a human neuroblastoma cell line: effect of dibutyryl 3':5'-cyclic AMP and reserpine

The carboxy terminal part of the proenkephalin A sequence is the 31 amino acid peptide B, which has as its final seven amino acids the sequence of the opioid peptide Met-enkephalyl-Arg6-Phe7. Using a radioimmunoassay which recognises both these peptides we have investigated the relative amounts of peptide B and Met-enkephalyl-Arg6-Phe7 in a human neuroblastoma cell line. We show that these cells contain peptide B-like immunoreactivity but not its heptapeptide fragment. This may be due to lack of proteolytic activity cleaving Met-enkephalyl-Arg6-Phe7 from its precursor, peptide B. On treatment with dibutyryl cyclic AMP the level of immunoreactivity approximately doubles, due to increased amounts of peptide B-like immunoreactivity. Treatment with reserpine, which increases conversion of peptide B to the heptapeptide in bovine chromaffin cells in culture does not stimulate the accumulation of Met-enkephalyl-Arg6-Phe7 in the human neuroblastoma cells. The results are discussed with respect to peptide processing.     

Immunoreactive Met-enkephalin Arg6 in rat brain, and bovine brain, gut, and adrenal

Antibodies directed against the Met-enkephalin-related hexapeptide, Met-enk Arg6, have been used in radioimmunoassays in the characterization of material in rat brain, and bovine striatum, colon, and adrenal medulla. Met-enk Lys6 reacted 0.27 relative to Met-enk Arg6, but Leu-enk Arg6 and C-terminal extensions or deletions of Met-enk Arg6 showed less than 0.02 immunoreactivity. In rat brain, the concentration of Met-enk Arg6-like immunoreactivity was less than 20 pmol X g-1 in all regions, but after trypsinization of tissue extracts there were up to 80-fold increases in immunoreactivity as a result of cleavage of C-terminally extended forms. The tryptic product eluted as Met-enk Arg6 on gel filtration. In control extracts of rat brain there were at least three immunoreactive forms of Met-enk Arg6; one eluted in the position of the hexapeptide standard on gel filtration and HPLC while the others had properties of N-terminally extended forms. In bovine striatum and colon the hexapeptide-like material predominated; but in bovine adrenal extracts, there were relatively low concentrations of the hexapeptide and, instead, the dominant immunoreactive forms corresponded to two components that were probably N-terminally extended variants. Trypsin again produced marked increases in immunoreactivity. HPLC studies indicated that Met-enk Arg6Phe7- and Met-enk Arg6Gly7Leu8-like immunoreactive peptides were important substrates in bovine brain for the production of hexapeptide immunoreactivity after trypsin. The differences in the patterns of immunoreactive forms in bovine adrenal, colon, and brain are consistent with tissue variations in the pathways of posttranslational processing of the precursor molecules.     

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut,pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B

Summary GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420–493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1–17 and 20–38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry.In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3±18 pmol GAWK 1–17/g wet weight of tissue (mean±SEM) and 12.4±2.9 pmol GAWK 20–38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1–17 2071.8±873.2 and GAWK 20–38 1292.7±542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B.Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.