Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Dynorphin in bovine adrenal medulla. II. Isolation, partial characterization and biological activity of two distinct molecules

Two highly potent dynorphin-like peptides were isolated from bovine adrenal medulla by successive chromatography of an acid (HCl) extract on Sephadex G-10, carboxymethylcellulose, Sephadex G-50 and partition chromatography on Sephadex G-50. Amino acid analysis of both peptides revealed the presence of 24 amino acids including the composition of dynorphin-(1-13) and differing from each other only by a few residues. Both peptides were shown to have the same activity as dynorphin-(1-13) in the guniea pig ileum assay and reacted as well as dynorphin-(1-13) with a specific antibody (R-31) directed against the synthetic material.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

Summary The coexistence of met5- and leu5-enkephalinlike immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla.

Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections. Four types of primary catecholamine-storing cells were identified. NA1 cells contain cytoplasmic granules up to 0.3 mum in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA2 cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA3 cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA4 cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence. The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA1 cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA2 cells correspond to this latter population (175 nm). NA3 cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA4 cells being in the dimension of 80 nm. Granules in NA1 and NA2 cells show uniformly high density, whereas those in NA3 and NA4 cells display cores of varying density. Granules with moderately dense cores in NA3 and NA4 cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

Diphosphoinositide metabolism in bovine adrenal medulla.

(1) A phosphatidylinositol kinase (EC 2.7.1.67) of a chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue. (2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane. (3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.     

Phosphatidylinositol metabolism in the adrenal medulla

Changes in phosphoinositide metabolism due to muscarinic stimulation of the adrenal medulla are reviewed. Evidence is presented that muscarinic receptors inhibit catecholamine secretion by the bovine gland and that muscarinic agonists do not cause entry of calcium ions. Results are inconsistent with the theory that phosphatidylinositol hydrolysis opens calcium 'gates'. Polyphosphoinositide metabolism is also reviewed and the suggestion made that phosphatidylinositol 4,5-bisphosphate may regulate the activity of the calcium pump ATPase in cells where phosphoinositide-linked receptors promote calcium influx.     

The acetylcholine receptor of the adrenal medulla.

Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [¹²⁵1]α-bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α-bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d-tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α-bungarotoxin. Perfusion experiments showed the acetylcholine receptor to be entirely nicotinic. Stimulation by nicotine was inhibited by atropine and decamethonium, as well as by hexamethonium. Calculated dissociation constants for these antagonist-receptor interactions were in the range of 1 to 3 × 10^?5 m. α-Bungarotoxin failed to inhibit nicotine-stimulated catecholamine release in the perfused adrenal, most likely because of its limited diffusion into the gland.     

The cytoarchitecture of the adrenal medulla in the rat.

The disposition of adrenaline and noradrenaline storing cells in the adrenal medulla has been studied in the rat. It has been demonstrated that no morphological or functional reason exists for the follicular designation of clumps of chromaffin cells in the medulla. No significant difference was found between the outer and inner zone as regards the frequency of NA cells by morphometric and statistical methods. The cytoarchitectural differences between adrenaline- and noradrenaline-storing cell arrangement are described. Special features of the interrelation between noradrenaline storing cells, cortical cells and the connective tissue framework are stressed. The light and electron microscopic appearance of two types of cortical cells, assumed to represent different states of functional activity is described.     

Putative enkephalin precursors in bovine adrenal medulla.

Extracts from bovine adrenal medulla and adrenal medullary chromaffin granules were found to contain three proteins, 20,000, 10,000 and 5,000 approximate molecular weights which yield tryptic peptides with opioid activity. The opioid activity of these peptides was demonstrated with a radioreceptor assay and two radioimmunoassays. The three proteins yield the same active peptides all of which are chromatographically distinct from the tryptic opioid nonapeptide β-LPH 61–69, generated by trypsin digestion of pituitary endorphins and their precursors. Furthermore, these endorphins and their precursors do not appear to be present in the adrenal medulla. These findings further support the hypothesis that the enkephalin biosynthetic pathway is distinct from that leading to β-endorphin.     

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.     

Diagnosis and management of tumors of the adrenal medulla.

The adrenal medulla consists of chromaffin cells, the site of catecholamine biosynthesis. Pheochromocytomas are chromaffin-cell tumors; 80-85 % arise from the adrenal medulla and 15-20 % arise from extra-adrenal chromaffin tissues (paragangliomas). Neuroblastomas are primitive tumors that derive from the same blastic precursor as in pheochromocytomas, and are distributed along the sympathetic nervous system. Pheochromocytomas account for 6.5 % of incidentally discovered adrenal tumors; they are found in 50 % of patients with multiple endocrine neoplasia 2A (MEN 2A) and 5-25 % of patients with von Hippel-Lindau (VHL) syndrome. Neuroblastomas are the most common solid extra-cranial tumors in children, and account for 7-10 % of all tumors. The diagnosis of pheochromocytoma should first be established biochemically by measuring plasma free metanephrines (the measurement of urinary fractionated metanephrines is the second choice). Measurements of homovanillic acid (HVA), norepinephrine and vanilmandelic acid (VMA) in urine are a necessity in patients with suspected neuroblastoma. Anatomical (radiological) imaging with computed tomography (CT) or magnetic resonance imaging (MRI) is necessary for both pheochromocytomas and neuroblastomas. Functional (nuclear medicine) methods are useful for both tumors. Scintigraphy with [123I]-metaiodobenzylguanidine is the specific functional imaging test of first choice; if this is not available, scintigraphy with [131I]-MIBG is the second choice. Other newer specific modalities that have been used for evaluating pheochromocytomas include positron emission tomography (PET) with [18F]-F-fluorodopamine (F-DA) and [18F]-F-dihydroxyphenylalanine (DOPA). These should be used when MIBG scintigraphy is negative. Primary treatment for both types of tumor is surgical; chemotherapy is used for inoperable disease. After successful surgery, survival of patients with benign, sporadic pheochromocytomas is believed to be equal to that of the general population. Depending on the extent of disease and age, patients with neuroblastomas have cure rates of 15-90 %.     

Morphology and histochemistry of the adrenal medulla

Summary Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections.Four types of primary catecholamine-storing cells were identified. NA₁ cells contain cytoplasmic granules up to 0.3 m in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA₂ cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA₃ cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA₄ cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence.The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA₁ cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA₂ cells correspond to this latter population (175 nm). NA₃ cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA₄ cells being in the dimension of 80 nm. Granules in NA₁ and NA₂ cells show uniformly high electron density, whereas those in NA₃ and NA₄ cells display cores of varying density. Granules with moderately dense cores in NA₃ and NA₄ cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.Supported by the Deutsche Forschungsgemeinschaft, grant Nr. Bo 525/1These results were presented in part at 17. Tagung der Deutschen Gesellschaft für Elektronenmikroskopie, Berlin, 21.–26. 9. 1975