Identiflcation of a Proenkephalin Precursor in Striatal Tissue

Recent studies have supported the suggestion that proenkephalin is the same in both adrenal medulla and brain. However, although previous investigations have characterized enkephalin-containing adrenal intermediates derived from proenkephalin, as yet no such intermediates have been isolated from the brain. This has led to the belief that the processing of proenkephalin in the brain is extremely rapid and enkephalin-containing intermediates do not accumulate. In this investigation Sephacryl-300 gel filtration chromatography of guinea pig striata, extracted in 8 M urea, demonstrated several peaks of both bioactive and immunoreactive enkephalin-like peptides after enzymatic digest (trypsin followed by carboxypeptidase B). Comparable profiles were obtained using rat and bovine striatal tissue. In guinea pig the major species emerging from gel filtration, eluting with an apparent molecular weight of 29,000, represented approximately 9% of the total (methionine) enkephalin immunoreactivity. It had an apparent pI of 5.0 when subjected to chromatofocusing. This species was further characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and nitrocellulose blotting techniques as well as highly specific radioimmunoassays to (Met5)-enkephalin, (Leu5)-enkephalin, and (Met5)-enkephalin-Arg6-Phe7. This species was found to contain these opioid peptides in an approximately 6:1:1 ratio, respectively, and to have an apparent molecular weight of 31,000. It was also indicated that (Met5)-enkephalin-Arg6-Phe7 constituted the C-terminal seven residues of this molecule.     

Is Lysolecithin an In Vivo Constituent of Chromaffin Granules?

Abstract: It was recently claimed that lysolecithin (lysophosphatidylcholine) in chromaffin granules is a postmortem artefact. We have, therefore, determined catecholamine/lysolecithin ratios in adrenal tissues and isolated chromaffin granules. In rat adrenals and bovine medulla the ratios in both tissues and granules were similar. This indicates that even in rapidly frozen rat adrenal glands, sufficient lysolecithin is present in the total tissue to account for its presence in isolated organelles. Owing to the high cortexhedulla ratios such studies cannot be performed with guinea pig or rabbit adrenals. However, isolated chromafh granules from guinea pig, in contrast to a previous study, do contain lysolecithin. We conclude that lysolecithin is an in vivo constituent of chromaffin granules of all species so far investigated.     

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.     

Chloditan-induced changes in lipid peroxidation in the adrenals of dogs and guinea pigs

The effect of adrenocorticolytic drug chloditan (1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane;) on the content and the rate of lipid peroxidation in dog and guinea pig adrenals was studied. In adrenals of dogs and guinea the amount of malondialdehyde (MDA) was increased. In vitro spontaneous lipid peroxidation was delayed in adrenal homogenate of DDD-treated dogs. Spontaneous lipid peroxidation in adrenal homogenate was not decreased by feeding DDD in the guinea pig. DDD consumption did not affect on lipid peroxidation promoted by ascorbic acid and ferrous iron. The accumulation of MDA in dog and guinea pig adrenal homogenates was inhibited by NADPH.     

Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells

Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of [Met5]-enkephalin immunoreactive material (ME-IRM) into the culture medium (1). Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides. The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing [Met5]-enkephalin and [Leu5]-enkephalin. Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts. Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs. Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4. The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation. Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3). Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of [Met5]-enkephalin. The content of [Met5]-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation. We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.     

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.     

Proenkephalin: A general pathway for enkephalin biosynthesis in animal tissues

Guinea pig adrenal, brain, and myenteric plexus have been shown to contain many polypeptides that yield free enkephalins on digestion with trypsin and carboxypeptidase B. The enkephalin-containing polypeptides (ECPs) range from 500 to >20,000 daltons and show similarities in their chromatographic behavior to the ECPs present in the chromaffin granules of the bovine adrenal medulla. Furthermore, the heptapeptide [Met]enkephalin-Arg⁶-Phe⁷, that is now known to represent the carboxyl terminal sequence of the proenkephalin found in bovine adrenal medulla (Gübler et al. (1982) Nature (London), in press), was identified in all three guinea pig tissues. It appears that processing of a proenkephalin similar to the one in adrenal medulla represents a general pathway for enkephalin biosynthesis in animal tissues.     

Identification of pro-opiomelanocortin and the secretion of its peptide fragments in the adrenals of the bull

Immunoreactive alpha-, beta- and gamma-endorphins and beta-lipotropin--C-terminal peptide fragments of pro-opiomelanocortin (POMC)--were discovered and measured by RIA in the bovine adrenal medulla and cortex. These peptides were also discovered in perfusates of the adrenal gland. POMC proper and some intermediate forms of its processing not differing in electrophoretic mobility from the respective molecular forms of hypophyseal POMC were identified in the medulla and cortex of the adrenals by the immunoblotting technique with the use of antiserum to beta-lipoprotein. It is concluded that POMC gene is expressed in the adrenal medulla and cortex and that as a result of POMC processing a noticeable amount of its peptide fragments is formed and secreted in adrenal cells. The authors thus suggest the presence of existence of the pituitary-unrelated mechanisms of adrenal function control with participation of POMC peptides synthesized in the adrenals.     

Helodermin-like peptides in noradrenaline cells of adrenal medulla

Helodermin, a VIP/secretin-like peptide, was first isolated from the venom of the lizard Gila monster. Small amounts of helodermin-like peptides have since been detected in many mammalian tissues. Notably high concentrations were demonstrated in the thyroid gland, and immunocytochemical studies revealed intense helodermin-like immunostaining in thyroid C cells and medullary thyroid carcinoma cells. In the present study, we examined the adrenal gland of mouse, rat and pig for the presence of helodermin-like peptides. Using an antiserum raised against lizard helodermin immunostaining was observed in the noradrenaline-producing cells of the adrenal medulla in all 3 species. Radioimmunoassay revealed high concentrations of helodermin-like peptides in the mouse and rat adrenal. The concentrations in the pig adrenal could not be determined because of a non-parallel dilution curve. Upon high-performance liquid chromatography, the immunoreactive material in extracts of mouse and rat adrenals eluted in one major peak, close to the elution position of lizard helodermin.     

Characterisation of Met-enkephalin(Arg6,Phe7) immunoreactivity in human adrenal and human phaeochromocytoma

The molecular forms of opioid peptides in human adrenal have not been well characterised. These peptides are predominantly derived from the proenkephalin A precursor, which has the sequence of Met-enkephalin(Arg⁶,Phe⁷) as its carboxyl terminus. We have looked in the present study at the subcellular distribution and the molecular form of immunoreactivity to this sequence in post-mortem human adrenal medulla and in phaeochromocytoma. In the human adrenal homogenates, the immunoreactivity distributes on a sucrose gradient in a manner consistent with localisation in chromaffin granules. On chromatography, the immunoreactivity from adrenal medulla is predominantly in the heptapeptide form; the intermediate (3000–4000) molecular weight material is only a minor component of immunoreactivity, in contrast to bovine tissue extracts where this is the major form of immunoreactivity. In the phaeochromocytoma extracts, the heptapeptide sequence again predominates over a minor amount of intermediate sized material. The results are discussed in terms of post-mortem changes, precursor processing and the function of the adrenal medulla.     

Chromatographic characterization of dynorphin and [Leu5]enkephalin immunoreactivity in guinea pig and rat testis

Tissues of the reproductive tract have been shown to contain mRNAs coding for pro-opiomelanocortin (POMC), pro-enkephalin and pro-dynorphin. However, the amounts of immunoreactive opioid peptides in these tissues are low, and in the case of the enkephalins and dynorphin, the molecular species responsible for the immunoreactivities have not been characterized. The chromatographic properties of dynorphin and enkephalin immunoreactivities in extracts of guinea pig and rat testis have therefore been determined. Dynorphin A and dynorphin B immunoreactivity was heterogeneous, with a significant amount attributable to high-molecular-weight forms. About 20% of the dynorphin A immunoreactivity, and about 40% of the dynorphin B immunoreactivity, in guinea pig testis extracts behaved as authentic dynorphin A or B, respectively during fractionation by ion exchange, gel filtration and high-performance liquid chromatography. Both high- and low-molecular-weight forms of [Leu5]enkephalin immunoreactivity were also present, with roughly 50-70% of the immunoreactivity attributable to low-molecular-weight forms. In extracts of guinea pig testis only a small part of this immunoreactivity eluted as authentic [Leu5]enkephalin during high-performance liquid chromatography. In rat testis most of the low-molecular-weight [Leu5]enkephalin immunoreactivity behaved as the authentic peptide. These results confirm that opioid peptides are produced in guinea pig and rat testis, and demonstrate that immunoreactive forms of the peptides similar to those found in brain and pituitary are present in the tissue.     

Corticostatic peptides.

In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14-18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and "supporting" endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.     

Influence of the hormonal forms of ACTH on the pattern of corticosteroid secretion

In the pituitaries of man, monkey, sheep, dog, cat and guinea pig the predominant component of immunoreactive ACTH has a molecular size resembling the usual 1–39 peptide (little ACTH). Cortisol is the predominant glucocorticoid in these species. Rabbit, rat and mouse pituitaries contain a newly described intermediate sized ACTH and corticosterone as their principal glucocorticoid. Bovine pituitaries contain intermediate and little ACTH in about equal amounts and their cortisol: corticosterone ratio is about 1. These findings are consistent with our hypothesis that the hormonal form of ACTH is an important factor regulating the cortisol: corticosterone ratio in mammalian adrenal corticoid secretion.     

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.     

Met5]-enkephalin-like peptides of the adrenal medulla: release by nerve stimulation and functional implications

In adrenal chromaffin cells, various molecular forms of polypeptides cross-reacting with [met5]-enkephalin antisera are stored in granules together with catecholamines and soluble proteins. Splanchnic nerve stimulation increases the release of enkephalin-like peptides from the adrenal gland into the adrenal vein. The release of these peptides appears to be mediated by a cholinergic nicotinic receptor. Fractionation of plasma extracts on Bio-gel P-2 shows the presence of only low molecular weight peptides in the resting condition. The low molecular weight fraction contains mainly [met5]-enkephalin and [leu5]-enkephalin. When the splanchnic nerve is stimulated high and low molecular weight peptides are released and the amount of low molecular weight peptides in plasma is increased. The content of enkephalin-like peptides in adrenal venous plasma was similar in control and reserpinized dogs. Splanchnic nerve stimulation increased the peptide content but not the epinephrine content of plasma in reserpinized dogs. This also caused a fall of arterial blood pressure which could be prevented by pretreatment with naloxone. A decrease in blood pressure was also directly elicited by the injection of [met5]-enkephalin-[arg6-phe7]. The duration of the hypotensive effect of this peptide was prolonged by prior injection of captopril.     

Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy.

A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria.The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction.     

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.     

Somatostatin immunoreactivity in the cat adrenal medulla

Summary Somatostatin-like immunoreactivity was detected within the adrenal gland of the rat using specific monoclonal antibodies. Immunohistochemical studies demonstrated a few somatostatin-immunoreactive nerve fibers within the adrenal medulla. In addition, a large population of chromaffin cells in the cat adrenal medulla displayed intense somatostatin-like immunoreactivity. Similar cells were not observed in rat or guinea pig adrenal glands, although they were found in human material. The somatostatin-positive cells in the cat adrenal medulla often possessed short immunoreactive processes similar to those seen in somatostatin-immunoreactive paracrine cells of the gut. Characterization of the somatostatin-like immunoreactivity of the cat adrenal by high performance liquid chromatography and radioimmunoassay indicated that somatostatin-28 may account for over 90% of the observed immunoreactivity. It is suggested that somatostatin-28 may have a paracrine or endocrine role in the feline adrenal medulla.     

Somatostatin immunoreactivity in the cat adrenal medulla. Localization and characterization

Somatostatin-like immunoreactivity was detected within the adrenal gland of the cat using specific monoclonal antibodies. Immunohistochemical studies demonstrated a few somatostatin-immunoreactive nerve fibers within the adrenal medulla. In addition, a large population of chromaffin cells in the cat adrenal medulla displayed intense somatostatin-like immunoreactivity. Similar cells were not observed in rat or guinea pig adrenal glands, although they were found in human material. The somatostatin-positive cells in the cat adrenal medulla often possessed short immunoreactive processes similar to those seen in somatostatin-immunoreactive paracrine cells of the gut. Characterization of the somatostatin-like immunoreactivity of the cat adrenal by high performance liquid chromatography and radioimmunoassay indicated that somatostatin-28 may account for over 90% of the observed immunoreactivity. It is suggested that somatostatin-28 may have a paracrine or endocrine role in the feline adrenal medulla.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.