Beta-endorphin-like and adrenocorticotropin-like materials in heart tissues of the rat, gerbil, hamster and guinea pig

1. Heart tissues of several rodent species including the rat, gerbil (Meriones unguiculatus), hamster (Mesocricetus auratus) and guinea pig (Cavia porcellus) were extracted with an acetone-water-HCl mixture. An acid acetone powder was obtained by adding a copious volume of acetone to the extract. 2. Rat heart acid acetone powder was subjected to ion exchange chromatography on CM-cellulose. Gerbil heart acid acetone powder was subjected to salt fractionation, gel filtration on Sephadex G-10 and then ion exchange chromatography on CM-cellulose. Hamster and guinea pig heart acid acetone powders were subjected to gel filtration on Sephadex G-25. 3. The fractions were assayed for the ability to stimulate corticosterone production in isolated rat adrenal decapsular (zona fasciculata, zona reticularis and medulla) cells, to displace D-ala2-D-leu5-(tyrosyl-3,5-3H) enkephalin from binding to rat brain membranes, and to inhibit 125I-human beta-endorphin from binding to its antibodies. 4. The widespread occurrence of beta-endorphin-like immunoreactivity among the rat heart CM-cellulose fractions may reflect different species of beta-endorphin. The fraction with the highest beta-endorphin-like immunoreactivity and opiate receptor binding activity was strongly adsorbed on CM-cellulose. 5. In hamster and guinea pig hearts, beta-endorphin-like immunoreactivity and opiate receptor binding activity were distributed among high molecular weight and low molecular weight fractions. 6. In gerbil hearts, opiate receptor binding activity was present in fractions unretarded on Sephadex G-10 (i.e. with a molecular weight greater than 700) as well as in the retarded fractions (i.e. with a molecular weight smaller than 700).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Immunohistochemical localization of 3 beta-hydroxysteroid/delta 5-delta 4-isomerase, tyrosine hydroxylase and phenylethanolamine N-methyl transferase in adrenal glands of sheep fetuses throughout gestation and in neonates.

Immunoreactive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) was localized in adrenal glands of sheep fetuses in cortical-type cells, but not in medullary-type cells, from day 43 of gestation to term and in 2-4-day-old neonates. From day 54 of gestation, the formation of distinct zones within the adrenal cortex was apparent and immunoreactive 3 beta-HSD was found in cortical cells in the zona fasciculata and in groups and cords of cortical cells within the developing medulla, with weak positive staining in the zona glomerulosa. At this stage, most medullary cells were positive for immunoreactive tyrosine hydroxylase, and some of these cells with a juxtacortical distribution also stained positively for immunoreactive phenylethanolamine N-methyl transferase (PNMT). Between days 65 and 130, the adrenal medulla increased in size with little change in the width of the cortex. Organization and zonation of immunoreactive 3 beta-HSD staining cells were evident in the zona fasciculata and in groups of cells in the medulla. Between day 130 and term, uniform immunoreactive 3 beta-HSD staining was found throughout the zona fasciculata, and there was also staining in single cells and small clusters of cells throughout the medulla. At this stage, immunoreactive tyrosine hydroxylase was distributed in most cells throughout the medulla, but in two distinct patterns: cells staining intensely for immunoreactive tyrosine hydroxylase in the central region of the medulla, and cells exhibiting weaker staining for immunoreactive tyrosine hydroxylase localized in a juxta-cortical position. These juxta-cortical cells were also positive for immunoreactive PNMT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for insulin and insulin-like growth factor-I in the human adrenal gland

Human adrenal glands contain high-affinity receptors for insulin and insulin-like growth factor I (IGF-I). Comparative studies with rat, hamster and human adrenal membranes confirmed that IGF-I receptors are most abundant in rat and hamster adrenals, whereas insulin and IGF-I receptors are present in equivalent numbers in human adrenal glands. Covalent crosslinking studies revealed that the human adrenal gland IGF-I receptor binding subunit migrated on dodecyl sulfate polyacrylamide gels with Mr = 135,000, which is identical to the migration of IGF-I receptor binding subunits isolated from other tissues. Autoradiography of frozen human adrenal slices incubated with [125I]insulin showed prominent, displaceable binding of this radioligand to the zona reticularis, zona glomerulosa, vasculature and medulla; in contrast, [125I]IGF-I binding to human adrenal tissue was most prominent in the zona reticularis and negligible in the medullary region.     

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.     

The intermediate filament complement of the retina: a comparison between different mammalian species

We compared the intermediate filament expression of the various cell types in the fully differentiated neural retina from rat, mouse, rabbit, guinea pig, cow, pig, and cat. Many cell types had an intermediate filament complement conserved across species boundaries, such as Müller cells and retinal ganglion cells. In some species (rabbit, guinea pig, and cow), however, we were unable to visualize GFA (glial fibrillary acidic)-positive retinal astrocytes, although such profiles were clearly visible in the remainder. Horizontal cell staining proved to be extremely species-variable. In rat and mouse the processes of these cells were identically displayed with antibodies to vimentin and all three neurofilament triplet proteins. In cow they decorated with antibodies to vimentin and antibodies to the two lower molecular weight neurofilament proteins alone, whereas in pig, rabbit and guinea pig all three neurofilament proteins but not vimentin were present. Finally cat horizontal cells stained for all three neurofilament proteins, some finer processes being additionally stainable with vimentin. A further surprise was the visualization of profiles positive only for the two lower molecular weight neurofilament proteins in the inner nuclear layer of both rabbit and guinea pig retina but not the other species. The implications of these results will be discussed.     

Immunohistochemical localization of adrenal ferredoxin and distribution of adrenal ferredoxin and cytochrome P-450 in the rat adrenal

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P-450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P-450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P-450 are not distributed uniformly throughout the rat adrenal cortex.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

The periodic acid-schiff reaction in the adrenal medulla

Summary The PAS reaction in the adrenal medulla of rat, rabbit, hamster, ox, pig and sheep was investigated. The medullary cells were positive in cryostat sections and potassium dichromate fixed material but not in formaldehyde fixed paraffin sections. The latter result is due mostly to the extraction of PAS positive lipids and loss of PAS positive proteins. No glycogen was detected in the chromaffin cells histochemically. The catechol amines played no part in the PAS reaction unless the fixative contained dichromate. The connective tissue elements were also PAS positive, and the nerve fibres in ox, sheep and pig. Periodate cannot be used to differentiate between adrenaline and noradrenaline storing cells.     

Immunohistochemical studies for multiple peptide-immunoreactivities and co-localization of Met-enkephalin-Arg6-Gly7-Leu8, neuropeptide Y and somatostatin in human adrenal medulla and pheochromocytomas

Three normal human adult adrenal medullas and 12 cases of pheochromocytomas were studied for immunohistochemical localization of various peptides. Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was present in all cases of pheochromocytomas. The normal adrenal medulla showed cells immunoreactive for MEAGL, neuropeptide tyrosine (NPY) and proopiomelanocortin derived N-terminal fragment (NTF). MEAGL and NPY were co-localized in some adrenal medullary cells. Pheochromocytomas showed striking multiple immunoreactivities regardless of histologic types, pleomorphic or organoid. Ten cases showed immunoreactivities for more than two peptides. All cases showed immunoreactivity for MEAGL and 9 cases showed NPY positive cells. Some tumor cells contain both MEAGL and NPY in the cytoplasm. Six cases were positive for somatostatin. Some tumor cells were shown to contain both MEAGL and SS. The appearance of SS and other peptides was considered to be related to the neoplastic transformation of the adrenal medulla.     

An interspecies comparison of gangliosides and neutral glycolipids in adrenal glands

Gangliosides and neutral glycolipids of adrenal glands of mouse, rat, guinea pig, rabbit, cat, pig, cow, monkey, and chicken were analyzed by thin layer chromatography (TLC). The major gangliosides common to all species had lactosylceramide in their core structure. GM3 containing N-acetylneuraminic acid (NeuAc) was the major ganglioside in rat, guinea pig, rabbit, and cat, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major one in mouse, cow, and monkey. GD3 was also detected in all species except mouse and GD3(NeuAc)2 was the major in pig adrenal gland. GM4(NeuAc) was detected in the adrenal glands of guinea pig and chicken but not in those of the other species. In the neutral glycolipid fractions, galactosylceramide, glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide were detected and the proportions of these glycolipids varied among the species. Guinea pig and chicken adrenal glands contained large amounts of galactosylceramide, this being consistent with the presence of GM4 in these two species. Globopentaosylceramide was detected in mouse, guinea pig, cat, and chicken by the TLC-immunostaining procedure.     

Histamine-containing peripheral neuronal and endocrine systems

An immunohistochemical method was developed to detect histamine in tissues. The aim of this study was to reveal the cellular stores of histamine in the gastrointestinal tract, pituitary, and adrenal gland. Histamine-containing nerve fibers were found in both rat and guinea pig gut. The origin of at least some of these fibers in the rat ileum was the submucous ganglion cell layer. In the rat stomach, numerous enterochromaffin-like cells exhibited histamine immunofluorescence, and endocrine cells in the ileum and jejunum contained histamine. Only mast cells contained histamine in the neurohypophysis. A large number of process-bearing cells in the guinea pig but not in the rat adrenal medulla contained histamine. The study shows that histamine is present in peripheral nerves and endocrine cells in addition to mast cells, and may function as a neurotransmitter or hormone.     

Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

Summary The epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.     

Cytochemistry and ultrastructure of the prenatal porcine adrenal medulla

The ultrastructure and cytochemistry of fetal porcine adrenal medullae have been studied at 60, 80, and 100 days of gestation. Adrenal medullae from fetuses at 60 days of pregnancy consisted of norepinephrine cells only. Some cells containing chromaffin granules were seen in the process of mitosis. A few epinephrine cells were present in the outer medullary zone at 80 days at pregnancy, their number increasing by the 100 day of pregnancy. Chromaffin cells containing both norepinephrine and epinephrine storing granules were also present at 80 and 100 days of gestation. Norepinephrine and epinephrine specific granular vesicles in the fetal adrenal medullary cells were smaller than those reported for the adult pig. The general ultrastructural characteristics of the porcine fetal adrenal medulla were similar to those reported for prenatal adrenal medulla of other species.     

Formaldehyde-Induced Fluorescence as a Means for Differentiating Epinephrine Cells from Norepinephrine Cells in Adrenal Medulla

The Falck and Hi Harp technique for the cellular localization of catecholamines by formaldehyde-induced fluorescence was applied to rat, mouse, and Syrian hamster adrenal. Some medullary cells revealed an unexpected orange-brown fluorescence. In guinea pig or rabbit adrenals, which store predominantly epinephrine, orange-brown fluorescence was not readily observed. It was found in Syrian hamsters only at the medullary periphery, where norepinephrine-producing cells are known to occur. Orange-brown fluorescence was depleted by administration of reserpine and intensified by nialamide plus DOPA. The same cell clusters which stained specifically for norepinephrine with ferric ferricyanide were found in adjacent sections to exhibit orange-brown fluorescence. Only by reducing the temperature of the formaldehyde reaction could sections of hamster adrenal showing only yellow-green fluorescence be obtained. These data suggest that the orange-brown fluorescence might result from' polymerization, oxidation, or both, of the isoquinoline produced by the norepinephrine-formaldehyde reaction under conditions slightly more vigorous than optimal and in the presence of high concentrations of norepinephrine     

Immunohistochemical studies on electron transport proteins associated with cythochromes P-450 in steroidogenic tissues II. Microsomal NADPH-cytochrome c reductase in the rat adrenal

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum procued against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase techniques and an indirect fluorecent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

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.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by double-staining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with Mr approximately 46,000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

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.     

Localisation of oxytocin, vasopressin and parts of precursors in the human neonatal adrenal

Being a possible alternative source for the production of vasopressin (AVP) and oxytocin (OXT), a study was undertaken of the fetal adrenal. The concentrations of these peptides within the fetal adrenal turned out to be low, viz., approx. 1 pg/mg in the rat and within the pg/g range in the human. Immunocytochemistry was performed either on conventional autopsy material kept till 12 years in paraffin blocks, or on more recently obtained formalin or glutaraldehyde-paraformaldehyde fixed material. In both types of material staining was good. In order to localize AVP cells, anti-AVP, an antibody against its associated neurophysin (anti-NSN) or an antibody raised against the c-terminal glycopeptide part of the AVP precursor (anti-GP) was used. OXT cells were localized by means of anti-OXT or an auto-antibody of a multiple sclerosis patient (auto-MS) probably recognizing OXT-neurophysin. The antibodies were characterized on human and rat brain material. In the external zone of the definitive cortex, apart from parenchyma cells, anti-AVP, anti-NSN and anti-GP stained fibre-like structures running in the connective tissue septa and around parenchyma cells and the cytoplasma of these cells. Anti-OXT and auto-MS stained droplets in the cytoplasm of the fetal zone cells. Similar distinct staining patterns for AVP and OXT cells were obtained in human anencephalics. These observations show that the peptides are not derived from the fetal brain, but are rather produced in the fetal adrenal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)