Opossum adrenal medulla: II. Differentiation of the chromaffin cell

The ultrastructure of the opossum adrenal medulla was examined in its postnatal development. Maturation of chromaffin cells and genesis of chromaffin vesicles were of particular interest. The primitive sympathetic cell was seen to contain few organelles with no apparent polarity. Initial pheochro-moblasts contained more organelles with some polarity. Endoplasmic reticulum and the Golgi complex increased as the pheochromoblasts matured, which suggested increased synthetic activity. Structures resembling Golgi/endoplasmic reticulum/lysosome (GERL) systems were seen in the pheochromoblasts. It is suggested that some of the components of the chromaffin vesicle may be processed by the GERL while others come directly through the Golgi complex. It is stressed that the developing pheochromoblast in the opossum presents an interesting model in which to study the genesis of the chromaffin vesicle.     

Physiological aspects of exocytosis in chromaffin cells of the adrenal medulla

The adrenal medulla is composed principally of groups of adrenergic and noradrenergic chromaffin cells, with minor populations of small intensely fluorescent cells and ganglionic neurones. Different molecular stimuli evoke distinct secretory events in the gland, involving the release of either adrenaline or noradrenaline together with various neuroactive peptides. The nature of the secretory response can be controlled at a central level or regulated locally within the gland. Specific innervation patterns to the different types of chromaffin cell have been implicated in central regulatory mechanisms, while several explanations for regulating secretion locally have been proposed. The differential distribution of various types of receptors between cell phenotypes, such as muscarinic or nicotinic acetylcholine receptors, histamine receptors, angiotensin receptors and different classes of opiate receptors between the two principal chromaffin cell populations could be involved in local control. In addition exocytosis parameters could be modulated differently in adrenergic and noradrenergic cells by phenotype-specific mechanisms, possibly involving molecules like Growth Associated Protein-43, Synaptosomal Associated Protein-25 isoforms or the p11 annexin subunit. The distribution of the various types of calcium channels is also known to vary between chromaffin cell subtypes. This short review examines possible ways in which specific innervation patterns in the adrenal gland could be programmed and discusses exocytosis mechanisms that could differ between chromaffin cell phenotypes. Data reviewed here suggest that the adrenal medulla should no longer be viewed as a homogeneous entity but as consisting of an ensemble of individual cell subpopulations each with a distinct secretory response that could in part reflect its local history.     

Acute stimulation of chromaffin cell proliferation in the adult rat adrenal medulla

Susceptible strains of rats develop adrenal medullary hyperplasia and neoplasia after long-term administration of the antihypertensive drug reserpine, or of other pharmacologic agents which alter neuroendocrine function. These proliferative lesions are of potential medical importance as a model for familial multiple endocrine neoplasia syndromes, and are of fundamental interest because they might elucidate mechanisms regulating chromaffin cell proliferation during normal development. To study the initiation of the adrenal lesions, chromaffin cell mitoses were counted in adult male rats injected with reserpine or control solvent for 5 days, with the final injection containing colcemid to arrest cells in mitosis. Rare mitoses were observed in mature-appearing epinephrine and norepinephrine cells in control adrenals. Reserpine caused an 8-fold increase in chromaffin cell mitoses in otherwise histologically normal glands, and the mitotic cells after reserpine administration showed marked granule depletion. Reserpine directly depletes catecholamine stores and reflexively increases neurogenic stimulation of chromaffin cells to increase catecholamine synthesis. The findings suggest that signals regulating function also regulate proliferation of mature chromaffin cells, and that prolongation of these signals or superimposed abnormalities may lead to pathologic proliferative states. The reserpine model may be a useful system for elucidating normal and pathologic mechanisms of signal transduction.     

The uptake of ascorbic acid and dehydroascorbic acid by chromaffin granules of the adrenal medulla.

Adrenal chromaffin granules incubated with [¹⁴C]ascorbate or [¹⁴C]dehydroascorbate accumulated radioactively labeled compounds. Dehydroascorbate was generated from ascorbate by including oxidized cytochrome c in the incubation mixture but thin layer chromatography revealed that dehydroascorbate was present even in the absence of cytochrome c. This observation coupled with the fact that radioactively labeled compound was accumulated much faster in the presence of cytochromec than in its absence indicates that dehydroascorbate but not ascorbate is taken up by the chromaffin granules. Uptake of radioactivity was not saturable with ascorbate or dehydroascorbate concentration, and granules which had taken up the radioactively labeled compound lost most of the material in 12 h. In addition 1 mm adenosine 5′-triphosphate--Mg²⁺ + had no effect on either ascorbate or dehydroascorbate uptake. These data indicate that uptake proceeds via an energy independent mechanism. The rate of uptake of dehydroascorbate is 50-fold lower than the rate of catecholamine uptake.Ascorbate but not dehydroascorbate acts as a cofactor for the enzyme dopamine β-hydroxylase in the conversion of dopamine to norepinephrine. Dehydroascorbate taken up by the granules must therefore be reduced in order to be used by dopamine i3-hydroxylase. We suggest that the chromaffin granule electron transport system may be involved in this reduction.     

Recovery of injured adrenal medulla by differentiation of pre-existing undifferentiated chromaffin cells

We described previous that the adrenal medulla recovers rapidly from the injuries due to salinomycin. In the present study we found the recovery to be apparently due to differentiation of pre-existing, primitive undifferentiated, chromaffin cells, rather than mitosis, as evidenced by lack of incorporation of 5-bromo-2-deoxyuridine in the differentiating and/or differentiated chromaffin cells. Electron-dense, striated fibrils observed within the necrotic cells and in the extracellular space were presumed to be intermediate degradation products. Schwann cells actively functioned as phagocytes in this study, and seemed to indirectly stimulate the differentiation of undifferentiated chromaffin cells.     

The topography of gangliosides in the membrane of the chromaffin granule of bovine adrenal medulla

We have studied the topography of the gangliosides of the adrenal chromaffin granules by using neuraminidase to remove sialic acid from membrane gangliosides of intact and ruptured chromaffin granules. Residual sialic acid was then measured to compare the availability of gangliosides on the outer and inner surfaces of the membrane. Measurement of protein sialic acid served as a control since these residues are known to be on the inner surface of the membrane. Prolonged digestion of broken membranes showed that maximally 75% of both lipid and protein-bound sialic acid residues are available to neuraminidase. Prolonged digestion of intact granules produced no measureable loss of sialic acid from either protein or lipid fractions. Comparison of the thin-layer chromatograms of gangliosides extracted from digested and undigested membranes showed no preferential digestion of any component.We conclude that at least 75% of the gangliosides are on the inner leaflet of the membrane and suggest that all of the gangliosides are so located.     

Fine ultrastructure of chromaffin granules in rat adrenal medulla indicative of a vesicle-mediated secretory process

Observation by transmission electron microscopy, coupled with morphometric analysis and estimation procedure, revealed unique ultrastructural features in 25.94% of noradrenaline (NA)-containing granules and 16.85% of adrenaline (A)-containing granules in the rat adrenal medulla. These consisted of evaginations of the granule limiting membrane to form budding structures having different morphology and extension. In 14.8% of NA granules and 12.0% of A granules, outpouches were relatively short, looked like small blebs emerging from the granule surface and generally contained electron-dense material. A proportion of 11.2% of NA granules and 4.9% of A granules revealed the most striking ultrastructural features. These secretory organelles presented thin, elongated, tail-like or stem-like appendages, which were variably filled by chromaffin substance and terminated with spherical expansions of different electron density. A cohort of vesicles of variable size (30–150 nm in diameter) and content was found either close to them or in the intergranular cytosol. Examination of adrenal medullary cells fixed by zinc iodide–osmium tetroxide (ZIO) revealed fine electron dense precipitates in chromaffin granules, budding structures as well as cytoplasmic vesicles. These data indicate that a common constituent is revealed by the ZIO histochemical reaction in chromaffin cells. As catecholic compounds are the main tissue targets of ZIO complexes, catecholamines are good candidates to be responsible for the observed ZIO reactivity. This study adds further to the hypothesis that release of secretory material from chromaffin granules may be accomplished by a vesiclular transport mechanism typical of piecemeal degranulation.     

Substance P and opiate-like peptides in human adrenal medulla

Opiate-like peptides (OLP), substance P (SP) and catecholamines (CA) were measured in 15 human adrenal medullae. Two groups of subjects were investigated. Group a consisted of subjects who died after traffic accidents and Group b consisted of subjects with other causes of death. OLP levels in Group a were only about 13%, and SP and CA levels about 50% of Group b. It is suggested that these differences might be due to massive premortal adrenal medullary discharge rather than post mortem degradation.     

Neurogenic signals regulate chromaffin cell proliferation and mediate the mitogenic effect of reserpine in the adult rat adrenal medulla

The adrenal medulla is innervated by nerve fibers from several sources, which synapse on chromaffin cells and stimulate the secretion of catecholamines. The antihypertensive agent reserpine is known to reflexively increase this neurogenic stimulation by depleting catecholamine stores, and long-term administration of reserpine is associated with adrenal medullary hyperplasia and neoplasia. To determine the role of neurogenic signals in regulating normal and reserpine-stimulated proliferation of chromaffin cells, the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating nuclei was assessed in the adrenal medulla of adult rats. Unilateral adrenal denervation caused a 4-5 fold decrease in chromaffin cell labeling by 5-bromo-2'-deoxyuridine during a 2-week labeling period. Denervation also prevented stimulation of labeling in animals receiving reserpine in their diet. These findings suggest that neurogenic control of cell proliferation may play an important role in the pathogenesis of adrenal medullary hyperplasia and neoplasia, and in the normal development of the peripheral and central nervous systems.     

Unusual cytoplasmic bodies in chromaffin cells of the adrenal medulla of the viscacha (Lagostomus maximus maximus)

Complex structures can be seen associated with large areas of endoplasmic reticulum of the chromaffin cells in the viscacha adrenal medulla. These structures are cylindrical in shape and are formed by chains of globular subunits. A central lumen is observed within which there are bodies resembling chromaffin granules. Parallel cisternae of the endoplasmic reticulum are arranged surrounding these bodies. The functional significance of these structures is unknown.     

Diseases of the adrenal medulla

The adrenal glands are vital in the organism's response to environmental stress. The outer cortex releases steroid hormones: glucocorticoids, mineralocorticoids and sex hormones, which are crucial to metabolism, inflammatory reactions and fluid homeostasis. The medulla is different developmentally, functionally and structurally. It co-releases catecholamines (primarily adrenaline and to some extent noradrenaline) as well as peptides by the all-or-none process of exocytosis from chromaffin granules, to aid in blood pressure and blood flow regulation, with regulated increments during the activation of the sympathetic nervous system. The co-released peptides function to regulate catecholamine release, blood vessel contraction and innate immune responses. Pathology within the adrenal medulla and the autonomic nervous system is primarily because of neoplasms. The most common tumour, called phaeochromocytoma when located in the adrenal medulla, originates from chromaffin cells and excretes catecholamines, but may be referred to as secreting paragangliomas when found in extra-adrenal chromaffin cells. Neoplasms, such as neuroblastomas and ganglioneuromas, may also be of neuronal lineage. We will also briefly discuss the catecholamine deficiency state.     

Immunofluorescence microscopic evaluation of the intermediate filament expression of the adrenal cortex and medulla and their tumors.

Normal adrenal glands (10 specimens) and adrenal gland tumors (58 cases) were immunohistochemically evaluated for different types of intermediate filament (IF) proteins. Some of the normal cortical cells showed cytokeratin positivity, and no positivity was seen for epidermal keratin or other types of IF. In the adrenal medulla, neurofilament positivity was seen in nerve axons, some ganglion cells, and chromaffin cells; and cytokeratin-positive cells could not be detected. Only the vascular and connective tissue elements showed vimentin positivity in both cortical and medullary areas. In half of the cortical carcinomas (13/25), cytokeratin-positive tumor cells were found. Furthermore, vimentin-positive tumor cells were present in 10 of 25 cases, in some of them together with cytokeratin-positive cells. Thus, the results show heterogeneity among the adrenal cortical carcinomas. Interestingly, many benign adrenal cortical tissues and some carcinomas lacked immunoreactivity for all types of IF, suggesting a poorly developed IF system in these tissues. In contrast to adrenal cortical tumors, pheochromocytomas contained neurofilamentlike immunoreactivity. These results reflect the different cellular nature of adrenal cortical and medullary tumors, which apparently can be distinguished from each other with antibodies to intermediate filament proteins.     

Membranes of the adrenal medulla: A comparison of membranes of chromaffin granules with those of the endoplasmic reticulum

The protein and lipid composition of the membrane of the chromaffin granule has been compared with that of an endoplasmic reticulum fraction isolated from bovine adrenal medulla. The endoplasmic reticulum was purified by centrifugation of a microsomal fraction in sucrose and Ficoll density gradients. Only insignificant amounts of chromomembrin B, dopamine β-hydroxylase and cytochrome b₅₅₉, which are characteristic protein components of the chromaffin granule membrane, were present in the endoplasmic reticulum fraction; whereas glucose-6-phosphatase and galactosyltransferase, marker enzymes of membranes of endoplasmic reticulum and Golgi complex respectively, were absent from the membranes of chromaffin granules. The lipid composition of the two types of membranes varies in the relative amounts of lysolecithin, lecithin and sphingomyelin and in the molar ratio of cholesterol to phospholipid. Lysolecithin, which is a major phospholipid of the chromaffin granule membrane, is virtually absent from membranes of the endoplasmic reticulum. Different protein patterns were obtained when these two membrane fractions were solubilized with detergent and subjected to polyacrylamide gel electrophoresis.It is concluded that membranes of the endoplasmic reticulum and those of the chromaffin granules are basically different and therefore that the transport of secretory proteins from their site of synthesis to their site of storage occurs, in the chromaffin cell, by a mechanism that does not involve a mixing of the participating membranes.     

Weight and shape of the human adrenal medulla in various age groups

Summary The weight and shape of the adrenal medulla were studied in 118 adrenal glands obtained at autopsies of 62 patients (38 males and 24 females) between 0 and 52 years of age. In adolescents 15 years and older and adults cases of sudden death only were entered in this study. The weight was calculated using morphometric measurements done on serial sections of the glands. In the new-born, the medulla accounts for less than 1% of the total volume of the adrenal gland. Though there is a rapid growth of the adrenal medulla after birth, the percentage of adrenal medullary volume at all age levels during childhood and adolescence is lower than in adults where it constitutes 9% of the total adrenal volume on the average. This corresponds to an average medullary weight of 0.43 g. In the new-born, the medulla consists of a thin plate made up of immature medulloblasts. Within a few months these are transformed into mature medullary cells; the shape of the medulla soon approaches that of the adult gland: an increase in thickness around the central vein and flat processes into the alae. With increasing age the cortico-medullary border becomes irregular and ragged. Especially in the vicinity of the central vein an intermingling of medullary and cortical cell complexes is found. An unequivocal diagnosis of genuine adrenal medullary hyperplasia can be established only by weighing the dissected medulla or by applying morphometric methods.     

Electrical excitability of cultured adrenal chromaffin cells.

1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro-electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded -50mV. 3. Chromaffin cells were capable of generating all-or-nothing over-shooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 10(-6)g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae.     

The character of the stored molecules in chromaffin granules of the adrenal medulla: A nuclear magnetic resonance study

The organization of the internal contents of bovine adrenal chromaffin granules has been studied using nuclear magnetic resonance spectroscopy. The method permits an examination of the interactions between adenosine triphosphate, metal ions, adrenaline and the chromogranin proteins. Particular advantage accrues when the naturally occurring cations, magnesium and calcium are replaced by manganese cations, since, as shown here, the manganous ion is an effective perturbing probe of structure. The nuclear magnetic resonance spectrum of the protein in the vesicles is compared with that of the isolated proteins.The results show that there is a loose network of interactions between the various components. The major part of the network is the interaction of the chromogranin protein, shown to have approximately a random coil conformation, with the adenosine triphosphate and the adrenaline. The purpose of the organized loose structure would appear to be to lower osmotic pressure without hindering rapid release of the vesicle contents on breaking the membrane.