Chromaffin cell mitogenesis by neurturin and glial cell line-derived neurotrophic factor.

Neurturin and glial cell line-derived neurotrophic factor are novel mitogens for normal adult rat chromaffin cells in vitro. These neurotrophic factors differ from the previously described adult chromaffin cell mitogens, nerve growth factor and basic fibroblast growth factor, in that their effects are potentiated by depolarization and activation of protein kinase C. Neurturin and glial cell line-derived neurotrophic factor signal via the receptor tyrosine kinase, ret, but may also act independently of ret. Both depolarization and phorbol esters act synergistically with neurturin to up-regulate ret protein expression in chromaffin cell cultures, suggesting a mechanism for potentiation of mitogenesis. However, a direct role for ret in mitogenesis has not been established. Stimulation by neurturin causes increased phosphorylation of extracellular signal-regulated kinases 1 and 2 in cultured chromaffin cells, and mitogenesis is prevented by inhibitors of their phosphorylation. Inhibitors of phosphatidylinositol 3-kinase also prevent mitogenesis.The present findings suggest the hypothesis that neurotrophic factors and neurally derived signals might cooperatively regulate chromaffin cell proliferation in vivo in the rat. In addition, trans-synaptic stimulation might provide a route by which epigenetic factors could influence the development of adrenal medullary hyperplasia in humans with hereditary multiple endocrine neoplasia syndromes 2A and 2B by affecting expression and/or activation of ret.     

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.     

Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma

Ret is a developmentally regulated tyrosine kinase involved in formation and maintenance of the nervous system. Ret mutations predisposing to pheochromocytomas and medullary thyroid carcinomas occur in multiple endocrine neoplasia (MEN) syndromes 2A and 2B. Biochemical studies have demonstrated overexpression of Ret mRNA and protein in pheochromocytomas compared to normal adrenal medulla. However, the cellular distribution of Ret in the normal human adrenal and in hyperplastic lesions that antecede pheochromocytomas are unclear. The present investigation was undertaken to resolve the histological distribution of Ret in the normal human adrenal, in pheochromocytomas evolving from adrenal medullary hyperplasia in MEN2A and in sporadic pheochromocytomas. Ret expression was studied by immunohistochemistry using both a polyclonal and a monoclonal antibody, with confirmation by immunoblotting of representative cases. Only occasional cells stained for Ret in the normal adrenal, consistent with the distribution in adult adrenals of other species. Heterogeneous, progressively increased Ret expression was observed during the evolution of pheochromocytomas. In both normal and neoplastic adrenal, the most intense immunoreactivity was observed in cells with neuron-like features. Our finding that Ret is not expressed at high levels in the early stages of disease suggests that elucidation of mechanisms that regulate Ret expression is required for understanding the pathobiology of MEN2A. The association of high-level Ret expression with neuronal morphology suggests that the variable overexpression of Ret in pheochromocytomas might in part be an epiphenomenon, reflecting the known phenotypic plasticity of these tumors.     

Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia

Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.     

Different responses to mitogenic agents by Adult rat and human chromaffin cells in vitro

Adrenal medullary hyperplasia and pheochromocytomas occur frequently in laboratory rats, both in the courseof aging and in response to prolonged administration of a variety of drugs and other substances. In contrast, these lesions are rare in humans. Rat chromaffin cells proliferate throughout life, but the proliferative capacities of human chromaffin cells are unknown. To determine whether the difference in prevalence of adrenal lesions might be correlated with differences in cell proliferation, adrenal medullary cells from 3 patients undergoing radical nephrectomy were maintained in vitro for up to 2 weeks in control medium or in the presence of nerve growth factor (NGF) and/or tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. Both NGF and TPA are known mitogens for neonatal and adult rat chromaffin cells. At intervals, the cultures were pulsed for up to 36 hours with bromodeoxyuridine (BrdU) to label S-phase nuclei. They were then fixed and consecutively stained for BrdU and for tyrosine hydroxylase, to confirm that labeled cells were chromaffin cells. Cells from adult female F344 rats were similarly maintained. Human chromaffin cells labeled with BrdU were extremely rare (less than 0.1 %) under all culture conditions, and effects of NGF or TPA could not be demonstrated. Rat chromaffin cells showed little or no labeling with BrdU in control medium but, in contrast to their human counterparts studied, showed marked increases in the percentages of labeled cells in the presence of NGF (37% ± 3%), TPA (7% ± 1%), or both (31% ± 3%). The apparently lower responsiveness of human chromaffin cells to mitogenic signals, or responses to different types of signals, may contribute to the lower frequency of adrenal medullary hyperplasia and pheochromocytomas in humans compared to rats.     

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 ret-Activating Ligand GDNF Is Differentiative and Not Mitogenic for Normal and Neoplastic Human Chromaffin Cells In Vitro

Activating mutations of the receptor tyrosine kinase, ret, are associated with multiple endocrine neoplasia type 2A (MEN 2A). However, the mechanisms leading to tumor development are unclear. Glial-derived neurotrophic factor (GDNF) activates wild-type ret via interaction with a second receptor, GFR a-l. We have utilized GDNF to stimulate normal and neoplastic chromaffin cells in order to ask whether ret activation is mitogenic. Cells from three normal adult adrenal medullas, one sporadic pheochromocytoma, and three MEN-2A pheochromocytomas were labeled with bromodeoxyuridine (BrdU) for 12 d in the presence or absence of GDNF or nerve growth factor (NGF), which is known to stimulate neurite outgrowth, but not proliferation in human chromaffin and pheochromocytoma cell cultures. Responses to GDNF and NGF were comparable, except for two MEN-2A pheochromocytomas that responded minimally to GDNF and robustly to NGF. These tumors responded to GDNF biochemically, as measured by phosphorylation of mitogen-activated protein kineses, despite their weak morphological responses. Our findings suggest that activation of ret may not be sufficient to produce chromaffin cell hyperplasia or neoplasia directly by stimulating cell proliferation. However the possibility that altered cell-cell or cell-substrate interactions might cause responses to become differ entiative rather than proliferative in vitro has not been ruled out. We also demonstrate, for the first time, that at least some human pheochromocytomas with an MEN-2A ret mutation respond to a normal ret ligand. This responsiveness could be mediated by a remaining normal ret allele or by other mechanisms.     

Adrenergic Differentiation and Ret Expression in Rat Pheochromocytomas

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27^Kip1, which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27^Kip1 was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long–Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27^Kip1 does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains.     

Histogenesis of the human adrenal medulla. An evaluation of the ontogeny of chromaffin and nonchromaffin lineages.

The authors previously evaluated the expression of a panel of chromaffin-related genes during histogenesis of the human adrenal medulla. In these studies, chromaffin and nonchromaffin adrenal neuroblasts were identified. To better characterize these nonchromaffin neuroblasts, the authors evaluated two additional markers: HNK-1, an antibody recognizing the migratory neural crest cell; and S-100, a protein expressed by sustentacular cells of the adrenal medulla. HNK-1 immunoreactivity was found in both chromaffin and nonchromaffin cell types at different times during development, marking the nonchromaffin lineage during the second trimester of gestation as well as the chromaffin lineage in the neonatal period. In addition, S-100 expression was noted in some nonchromaffin neuroblasts, and sustentacular cells were first identified at approximately 28 weeks of gestational age. These data suggest a model of human adrenal medullary histogenesis that incorporates the chromaffin, ganglionic, and sustentacular lineages known to constitute the adult adrenal medulla.     

Pheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile

Pheochromocytomas are adrenal medullary tumors that typically occur in adult patients, with increased frequency in multiple endocrine neoplasia type 2, von Hippel-Lindau disease, familial paraganglioma syndromes and neurofibromatosis type 1 (NF1). Pheochromocytomas arise in adult mice with a heterozygous knockout mutation of exon 31 of the murine Nf1 gene, providing a mouse model for pheochromocytoma development in NF1. We performed a microarray-based gene expression profiling study comparing mouse pheochromocytoma tissue to normal adult mouse adrenal medulla to develop a basis for studying the pathobiology of these tumors. The findings demonstrate that pheochromocytomas from adult neurofibromatosis knockout mice express multiple developmentally regulated genes involved in early development of both the CNS and peripheral nervous system. One of the most highly overexpressed genes is receptor tyrosine kinase Ret, which is known to be transiently expressed in the developing adrenal gland, down-regulated in adult adrenals and often overexpressed in human pheochromocytomas. Real-time polymerase chain reaction validated the microarray results and immunoblots confirmed the overexpression of Ret protein. Other highly expressed validated genes include Sox9, which is a neural crest determinant, and Hey 1, which helps to maintain the progenitor status of neural precursors. The findings are consistent with the recently proposed concept that persistent neural progenitors might give rise to pheochromocytomas in adult mouse adrenals and suggest that events predisposing to tumor development might occur before formation of the adrenal medulla or migration of cells from the neural crest. However, the competing possibility that developmentally regulated neural genes arise secondarily to neoplastic transformation cannot be ruled out. In either case, the unique profile of gene expression opens the mouse pheochromocytoma model to new applications pertinent to neural stem cells and suggests potential new targets for treatment of pheochromocytomas or eradication of their precursors.     

Neuropeptide Y in the adrenal gland: characterization, distribution and drug effects

The occurrence of neuropeptide Y-like immunoreactivity in the adrenal gland was investigated by means of radioimmunoassay, chromatography and immunohistochemistry. The adrenal levels of neuropeptide Y-like immunoreactivity varied considerably between species with lowest amounts in the rat and highest in the cow where immunoreactivity was observed in chromaffin cells and nerve fibres in the capsule and cortex. The distribution of neuropeptide Y-like immunoreactivity in the cow medulla overlapped that of enkephalin-like immunoreactivity. Chromatographic characterisation of rat and cow adrenal extracts showed that the majority of the neuropeptide Y-like immunoreactivity was similar in molecular weight and solubility properties to porcine neuropeptide Y. Rat adrenal contained additional material some of which may represent oxidised neuropeptide Y. The administration of insulin and reserpine to rats in vivo showed that the turnover of adrenal neuropeptide Y-like immunoreactivity is regulated by the splanchnic nerve. Splanchnic activation following insulin-induced hypoglycaemia elicited a 60% depletion of neuropeptide Y-like immunoreactivity 2 h post insulin injection. A smaller (maximum 40%) depletion of neuropeptide Y-like immunoreactivity was measured 24 h after reserpine injections which may be explained by splanchnic activation. Five days after reserpine injections the levels of adrenal neuropeptide Y-like immunoreactivity were increased to 200% of control levels and remained slightly elevated at 25 days. Adrenal enkephalin-like immunoreactivity showed similar but not identical changes following reserpine. The reserpine-induced elevation in neuropeptide Y-like immunoreactivity at the 5-day time point was abolished in rats with a chronic bilateral splanchnectomy. This evidence indicates that neuropeptide Y may be considered as a new adrenal medullary hormone.     

Catecholamine-synthesizing enzymes and chromogranin proteins in drug-induced proliferative lesions of the rat adrenal medulla.

Both epinephrine (E) and norepinephrine (NE) cells in the rat adrenal medulla are able to proliferate in response to pharmacologic stimulation. However, previous biochemical studies have suggested that drug-induced or spontaneous pheochromocytomas in rats are almost invariably NE-producing. To resolve these apparently conflicting data, immunocytochemical techniques were utilized to establish functional profiles of adrenal medullary lesions classified as pheochromocytoma or nodular hyperplasia in rats treated chronically with a phosphodiesterase inhibitor which induced pheochromocytomas. Sixteen of 17 pheochromocytomas and all hyperplastic nodules stained positively for tyrosine hydroxylase and dopamine beta-hydroxylase, consistent with an ability to produce NE. No lesion of either type stained for phenylethanolamine N-methyltransferase, consistent with an inability to produce epinephrine. Lesions of both types showed variable staining for chromogranin proteins. The findings indicate that qualitative functional differences cannot be used to discriminate hyperplastic nodules from small pheochromocytomas in rats. Some lesions currently classified as hyperplastic nodules might in fact be small pheochromocytomas. Others might represent diffuse hyperplasia within pre-existing islands of NE-cells in a background of hyperplastic epinephrine-cells.     

Adrenal medulla calcium channel population is not conserved in bovine chromaffin cells in culture

During the stress response adrenal medullary chromaffin cells release catecholamines to the bloodstream. Voltage-activated calcium channels present in the cell membrane play a crucial role in this process. Although the electrophysiological and pharmacological properties of chromaffin cell calcium channels have been studied in detail, the molecular composition of these channels has not been defined yet. Another aspect that needs to be explored is the extent to which chromaffin cells in culture reflect the adrenal medulla calcium channel characteristics. In this sense, it has been described that catecholamine release in the intact adrenal gland recruits different calcium channels than those recruited during secretion from cultured chromaffin cells. Additionally, recent electrophysiological studies show that chromaffin cells in culture differ from those located in the intact adrenal medulla in the contribution of several calcium channel types to the whole cell current. However there is not yet any study that compares the population of calcium channels in chromaffin cells with that one present in the adrenal medulla. In order to gain some insight into the roles that calcium channels might play in the adrenal medullary cells we have analyzed the alpha1 subunit mRNA expression profile. We demonstrate that the expression pattern of voltage-dependent calcium channels in cultured bovine chromaffin cells markedly differs from that found in the native adrenal medulla and that glucocorticoids are only partially involved in those differences. Additionally, we show, for the first time, that the cardiac isoform of L-type calcium channel is present in both bovine adrenal medulla and cultured chromaffin cells and that its levels of expression do not vary during culture.     

Adrenal medullary hyperplasia. A morphometric analysis in patients with familial medullary thyroid carcinoma.

The syndrome of familial medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid hyperplasia is inherited as an autosomal dominant trait, and is characterized by development of bilateral and multicentric thyroidal and adrenal medullary tumors. One of the earliest manifestations of adrenal medullary hyperfunction in patients with this syndrome is an increased ratio of epinephrine to norepinephrine in urine. In order to define the morphologic correlates of these early catecholamine abnormalities in a large kindred with familial MTC, a morphometric analysis based on a point-counting system to asses adrenal medullary volume was undertaken. These studies clearly revealed adrenal medullary hyperplasia as reflected by a two- to three-fold increase in medullary volume and weight as compared to age- and sex-matched controls. The increase in total medullary mass resulted from diffuse and multifocal modular proliferations of adrenal medullary cells primarily within the head and body regions of the glands. These results support the hypothesis that the pheochromocytomas in patients with familial MTC may, in fact, represent extreme degrees of nodular hyperplasia of the medulla.     

Adrenal medullary hyperplasia: A clinicopathologic study of four cases

This study presents the clinicopathologic findings in four new cases of adrenal medullary hyperplasia. The patients presented with episodic hypertension frequently associated with palpitations headache, and diaphoresis. All four had elevated urinary catecholamine levels during attacks, and were thought clinically to have a pheochromocytoma. In each case laparotomy revealed a diffusely enlarged adrenal gland without a discrete tumor. Histologic examination of the adrenals demonstrated a diffuse or diffuse and nodular expansion of the medulla confirmed by morphometric study. Of the four patients, three underwent unilateral and one bilateral adrenalectomy. Two patients who underwent unilateral adrenalectomy have been free of symptoms for three years. Thus, it would appear that adrenal medullary hyperplasia may occur unilaterally or asynchronously in the two glands. The bilaterally adrenalectomized patient has had persistent attacks, suggesting that the stimulus to adrenal medullary hyperplasia may possibly affect other chromaffin tissues. On the basis of our cases and a review of the literature, we propose the following criteria for the diagnosis of adrenal medullary hyperplasia: a clinical history of episodic attacks suggesting pheochromocytoma (generally with associated increased urinary catecholamine levels), an adrenal gland showing diffuse expansion of the medulla into the alae or tail of the gland with or without nodule formation, a medulla composed of enlarged cells with or without pleomorphism, and, most important, an increased medulla/cortex ratio, together with an increased calculated medullary weight asddetermined by morphometric analysis.     

Rodent and primate adrenal medullary cells in vitro: phenotypic plasticity in response to coculture with C6 glioma cells or NGF

Summary In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C₆ glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C₆ glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64–82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histofluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.     

Reserpine increases opiate-like peptide content and tyrosine hydroxylase activity in adrenal medullary chromaffin cells in culture

Primary cultures of adrenal medullary chromaffin cells maintained in a serum-free medium retain high levels of both catecholamines and opiate-like peptides. Addition of reserpine (100nM) to the culture medium results in the exponential loss of cellular catecholamines (t_½ = 1.5days) and in an elevation of opiate-like peptide content and tyrosine hydroxylase activity without altering total cell protein content. The maximum increase in opioid activity as a result of reserpine treatment averaged 230% of untreated cell levels and was reached by 2–3 days after initiation of treatment. A similar time-course was observed for the elevation of tyrosine hydroxylase activity. The increases of opiate-like peptides and tyrosine hydroxylase induced by reserpine are blocked by inclusion of actinomycin D or cycloheximide in the culture medium, suggesting that both messenger ribonucleic acid and protein synthesis are required for the induction.These data suggest that synaptic activation is not the only long-term regulator of opiate-like peptide and catecholamine biosynthesis in the adrenal medulla and that the biosynthesis of opiate-like peptides, which are components of chromaffin vesicles, and tyrosine hydroxylase, the cytoplasmic, rate-limiting enzyme in catecholamine biosynthesis, are coordinately regulated.     

Rat adrenal medulla: levels of chromogranins, enkephalins, dopamine beta-hydroxylase and of the amine transporter are changed by nervous activity and hypophysectomy

The levels of several constituents of chromaffin granules were determined in rat adrenal medulla after treatment with insulin or reserpine and after hypophysectomy. Insulin treatment induced increased levels of enkephalins, dopamine beta-hydroxylase, the amine carrier and of chromogranin B whereas catecholamines, cytochrome b-561 and chromagranin A remained slightly below control levels. Administration of reserpine led to similar changes with the exception of the enkephalins which increased much less. After hypophysectomy, chromogranin A was reduced to low levels; dopamine beta-hydroxylase, the amine carrier and catecholamines showed an intermediate reduction, whereas the other components remained at control levels. We can conclude that nervous stimulation of the adrenal medulla (by insulin and reserpine) induces the synthesis of enkephalins, dopamine beta-hydroxylase, the amine carrier and chromogranin B whereas the level of chromogranin A apparently depends on corticosteroids. Thus, major components of the secretory content and the membranes of chromaffin granules are regulated separately, which allows the biogenesis of chromaffin granules with a significantly altered composition.     

Distribution of chromogranin and S100 protein in normal and abnormal adrenal medullary tissues

The distribution of chromogranin and S100 protein was studied in 30 adrenal pheochromocytomas and 19 normal adrenal medullary tissues. Immunostaining in the tumors was compared with staining in sections of histologically normal medullae. Chromogranin showed diffuse cytoplasmic staining in all chromaffin cells. Chromogranin staining was consistently more intense in normal medullae, while less intense staining was present in most tumors from all four groups. S100 protein was present in the cytoplasm and nuclei of sustentacular cells surrounding chromaffin cells and in nerve branches. Many S100 protein-positive cells were present in normal medullae, in the two hyperplastic medullae, and in pheochromocytomas from patients with multiple endocrine neoplasia, type 2. Very few sustentacular cells were present in the other pheochromocytomas. These results indicate that S100 staining may be helpful in separating pheochromocytomas in patients with multiple endocrine neoplasia, type 2 disease from benign and malignant sporadic tumors.