A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids

Adrenal medullary endocrine (chromaffin) cells and sympathetic neurons both derive from the neural crest. We have found that the embryonic adrenal medulla and sympathetic ganglia are both initially populated by precursors expressing neural-specific genes. By birth, however, the medulla consists largely of chromaffin cells. In primary culture, the medullary precursors have three developmental fates: in NGF they continue to mature into neurons and survive, whereas in glucocorticoid they either extinguish their neuronal properties and exhibit an endocrine phenotype, or else continue to develop into neurons but then die. These data suggest that, in vivo, the adrenal medulla develops through both the glucocorticoid-induced differentiation of bipotential progenitors and the degeneration of committed neuronal precursors, which have migrated into the gland.     

p60c-src activity detected in the chromaffin granule membrane

Using monoclonal antibodies specific for p60c-src we have detected high levels of this kinase in adrenal medullary chromaffin tissue and in highly purified chromaffin granule (secretory vesicle) membranes. An immune complex kinase assay was applied to fractions of adrenal medullary tissue resolved on sucrose density gradients. Thirty-seven per cent of the total tissue p60c-src activity was found in association with chromaffin granule or granule membrane markers. Localization of a significant fraction of total cellular p60c-src activity to this secretory vesicle membrane suggests that the kinase may function in the regulation of neurotransmitter release.     

Immunocytochemical localization of NCAM and catecholamine-synthesizing enzymes in rabbit intra- and extra-adrenal chromaffin tissue

Summary The expression of the neural cell adhesion molecule, chromagranin A, and catecholamine-synthesizing enzymes (tyrosine hydroxylase and phenylethanolamineN-methyl transferase) in adrenal medulla and para-aortic bodies (paraganglia) of the adult rabbit, was studied by immunofluorescence. The specificity of the neural cell adhesion molecule antibody employed was demonstrated on rabbit tissue by immunoblotting. Neural cell adhesion molecule was found to be expressed not only by adrenal medullary cells but also by extra-adrenal chromaffin cells present in para-aortic bodies. These paraganglionic cells were as intensely immunolabelled for chromagranin A as adrenal medullary chromaffin cells. They were also labelled for the catecholamine-synthesizing enzymes tested here. However, their levels of the adrenalin-synthesizing enzyme phenylethanolamineN-methyl transferase were lower than those of medullary chromaffin cells.     

Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.     

Glial-cell-line-derived neurotrophic factor induces nerve fibre formation in primary cultures of adrenal chromaffin cells

Neurotrophic factors, such as nerve growth factor (NGF), have been shown to promote the differentiation of neural crest neuroblasts into sympathetic neurons, whereas glucocorticoids promote the endocrine phenotype of adrenal medullary chromaffin cells. This pluripotency is preserved to some extent in adult chromaffin cells, with NGF and other neurotrophic factors influencing the differentiation of these cells. In this study, the effects of glial cell line-derived neurotrophic factor (GDNF) on explanted chromaffin tissue have been investigated. The localization of mRNAs corresponding to the two components of the GDNF receptor, GDNF family receptor alpha 1 (GFRalpha1) and Ret, were demonstrated in adult adrenal medullary ganglion cells. GFRalpha1 mRNA was expressed in explanted chromaffin tissue at levels dependent on the presence of serum in the medium but decreased on the addition of blocking antibodies against transforming growth factor beta (TGFbeta). However, TGFbeta1 (1 ng/ml) did not upregulate GFRalpha1 mRNA expression when added to serum-free medium. GDNF induced neurite formation from chromaffin cells, as measured by the ratio of neurite-bearing versus total number of chromaffin cells in primary cultures of adult adrenal medulla. The most potent dose inducing neurites from chromaffin cells was 100 ng/ml GDNF. However, this dose was not as efficient as that seen when chromaffin cells were stimulated with NGF (100 ng/ml). Thus, adrenal medullary cells express mRNAs for the GDNF receptor components Ret and GFRalpha1, increase their expression upon being cultured in serum-containing medium and respond to GDNF treatment with an increase in the number of cells that develop nerve processes.     

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.     

Survival of dissociated adrenal chromaffin cells of rat and monkey transplanted into rat brain

Summary Adrenal chromaffin cells from adult rats and monkeys were mechanically dissociated and implanted into the striatum of adult rats by stereotaxic injection. Rat chromaffin cells survived (5%) and showed differentiation by forming processes 1 h-28 days after implantation. Monkey chromaffin cells survived for 48 h but showed very little formation of processes. The method presented allows rapid nonenzymatic dissociation and transplantation of adrenal medullary cells.     

Comparative Incorporation of Proenkephalin-Derived Peptides, Chromogranin A, and Dopamine β-Hydroxylase into Chromaffin Vesicles

The incorporation of enkephalin-containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse-labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met-enkephalin sequences in gradient fractions determined. 35S-ECPs were incorporated into particles suggestive of immature vesicles within 1-2 h after radiolabeling. Vesicle maturation, measured by co-equilibration of 35S-ECPs and total ECPs in the gradients, was complete within 9-12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S-labeled dopamine beta-hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.     

Mouse adrenal chromaffin cells can transform to neuron-like cholinergic phenotypes after being grafted into the brain

The development of neuron-like cholinergic immunophenotypes by adrenal chromaffin cells was studied in 10-week-old mouse adrenal medullary grafts. Fragments of chromaffin tissue were implanted into mouse hippocampus, and antibodies specific for neurofilaments (NF), neuron-specific enolase (NSE), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and phenylethanolamine-N-methyltransferase (PNMT) were applied to the grafts. Adrenal medulla grafts survived well and most of the transplanted cells were either round or polygonal. A minority of chromaffin cells elaborated an intermediate or sympathetic neuron phenotype. Chromaffin cells showed pronounced immunoreactivity for NSE in their perikarya and axon-like processes: immunoreactivity for NF was only found in a few processes. In adjacent immunohistochemically stained sections, the transplanted cells stained for ChAT and AChE. At the electron-microscope level, the immunohistochemical reactions for the two acetylcholine-related enzymes were mainly located on the endoplasmic reticulum and in cell processes. Immunoreactivity for PNMT was found to decline in transplanted chromaffin cells below that of normal adrenal medulla. These observations suggest that, in adrenal medullary grafts implanted into the hippocampus, chromaffin cells are endowed with neuron-like cholinergic immunophenotypes.     

Enhancement of Ca2+-induced catecholamine release by the phorbol ester TPA in digitonin-permeabilized cultured bovine adrenal chromaffin cells

The phorbol ester, 4 beta-phorbol 12-myristate acetate (TPA), increased the extent of catecholamine release induced by Ca2+, without affecting the basal release response in digitonin-permeabilized chromaffin cells. This finding is consistent with the hypothesis that protein kinase C has a role to play in stimulus-secretion coupling in the bovine adrenal medullary chromaffin cell.     

The isolation and characterization of the methyl acceptor protein from adrenal chromaffin granules

A methyl acceptor protein (MAP), which serves as a substrate for adrenal medullary protein carboxymethylase (PCM, E.C. 2.1.1.24), has been isolated from a hypotonic lysate of adrenal chromaffin granules. The isolated MAP was shown to be distinct from the adrenal chromaffin granule protein, dopamine β-hydroxylase (DBH). The properties of MAP, including its amino acid composition, were comparable to those reported for chromogranin A, a major acidic protein found in adrenal chromaffin granules.     

Neural Transplantation for Parkinson's Disease

1. Neural transplantation is one promising approach for the treatment of Parkinson's disease. Fetal substantia nigra cells are a good source of dopamine, but in order to avoid ethical and immunological problems, adrenal medullary chromaffin cells have been investigated as an alternative source.2. Grafted adrenal medullary chromaffin cells can provide dopamine as well as several neurotrophic factors that affect dopaminergic neurons in the brain.3. We review experimental studies for application of neural transplantation techniques in Parkinson's disease, including immunological studies, cryopreservation, microvasculature, donor tissue, and direct gene delivery studies performed in our laboratory. Our clinical experience and new approach involving a polymer-encapsulated cell grafting procedure are also described.     

Distinct populations of macrophages in the adult rat adrenal gland: a subpopulation with neurotrophin-4-like immunoreactivity

Macrophages are widely distributed in lymphohaemopoietic and many other mammalian tissues, where they are mainly involved in host defence mechanisms, phagocytosis, wound repair, and secretion of growth factors. Increasing evidence suggests that secretory products of macrophages can influence adrenal gland functions. In the present study, we have used specific antibodies to ED1 (cytoplasmic antigen), ED2 (membrane antigen), ED8 (membrane antigen), and OX-6 (MHC class II/membrane antigen) as markers for macrophages to examine their distribution within the adult rat adrenal gland. ED2 and OX-6 recognize distinct subpopulations of adrenal gland macrophages, whereas macrophages immunoreactive (-ir) for ED1 and ED8 could not be detected. OX-6-ir macrophages were most numerous in the cortical reticularis and glomerulosa zones, while only few cells were found in the zona fasciculata and in the adrenal medulla. Macrophages immunoreactive for ED2 were restricted to the adrenal medulla. The majority of these macrophages were associated with vascular sinuses or chromaffin cells. By double-immunolabelling we found that most of ED2-ir medullary macrophages contain neurotrophin-4 (NT-4)-like ir. Attempts to clarify whether macrophages take up NT-4 from NT-4-ir chromaffin cells indicated that medullary macrophages are immunonegative for chromogranin A and neuropeptide Y, two major secretory products of chromaffin cells. In situ hybridizations and immunofluorescence showed expression of the neurotrophin receptor TrkA, but not TrkB in the adrenal medulla. In vitro studies indicated that NT-4, similar to nerve growth factor, can induce c-fos-ir in chromaffin cells. We conclude that chromaffin cells are putative targets for adrenal medullary NT-4, whose functions remain to be clarified.     

Protein phosphorylation and the exocytosis-like interaction between isolated adrenal medullary plasma membranes and chromaffin granules

The interaction between isolated adrenal medullary plasma membranes and chromaffin granules has been proposed as a cell-free model for exocytosis. Phosphorylation experiments showed that isolated chromaffin granules as well as isolated plasma membranes contain protein kinases and phosphate accepting membranous proteins. Upon joint incubation however, the chromaffin granule-located proteins are preferentially phosphorylated. β-ν-methylene-ATP, a non-hydrolysable analogue, was able to reduce both the plasma membrane-induced release of the soluble chromaffin granular content and the phosphate incorporation into the protein fraction. The results of these experiments on a cell-free model system fit in the hypothesis originating from work on several types of intact cells that the exocytotic event is linked with protein phosphorylation.     

Embryologic development of rat adrenal medulla in transplants to the anterior chamber of the eye

The morphological development and plasticity of embryonic and postnatal rat adrenal medullary cells were studied in homologous adrenal grafts to the anterior chamber of the eye. The eyes of recipient rats were adrenergically denervated 10 days prior to grafting by extirpation of the superior cervical ganglion in order to increase levels of NGF and NGF-like activities in the iris. Grafts taken at the 15th day of embryonic development (E15), i.e., at the beginning of immigration of medullary progenitor cells into the adrenal cortical anlagen, contained no cortical or mature medullary cells after 2 weeks in oculo. Numerous sympathoblastic cells, however, were located at the anterior surface of the iris. E 16 and E 17 transplants showed abundant mature cortical tissue after 2 weeks. Small groups of medullary cells with the ultrastructural characteristics of mature pheochromoblasts or young chromaffin cells were interspersed among cortical cells without forming a discrete medulla. Neuronal cells were exclusively found outside the cortical cell mass. Sympathoblasts grew at the surface of the iris, while young sympathetic nerve cells, which were invested by Schwann cells and received synaptic axon terminals, were embedded into the stroma of the iris. Grafting of E 21 adrenals yielded very similar results except that, in a few instances, young chromaffin cells were located outside the cortex and sympathetic nerve cells were seen to be in close contact with cortical cells. In transplants of adult medullary cells typical mature adrenaline and noradrenaline cells were clearly distinguishable after 8 weeks even in the absence of cortical cells. The only indication of phenotypical changes in these cells was the formation by some of them, of neuritic processes which could be visualized in glyoxylic acid-treated whole mounts of irises. These results are compatible with the idea that embryonic adrenal medullary cells have the environmentally controlled potential to develop along the neuronal or endocrine line, but could also be interpreted in terms of a selection of a specific subpopulation with predetermined potentialities by a specific microenvironment. Moreover, these results suggest that increasing differentiation of medullary cells is accompanied by progressive restrictions in their genetic program, which eventually prevent full transdifferentiation of mature chromaffin into neuronal cells.     

Reserpic acid as an inhibitor of norepinephrine transport into chromaffin vesicle ghosts

Reserpic acid, a derivative of the antihypertensive drug reserpine, inhibits catecholamine transport into adrenal medullary chromaffin vesicles. Since it does not affect the membrane potential generated by the H+-translocating adenosine triphosphatase but inhibits ATP-dependent norepinephrine uptake with a Ki of about 10 microM, reserpic acid must block the H+/monoamine translocator. Because reserpic acid is much more polar than reserpine, it does not permeate the chromaffin vesicle membrane, nor is it transported into chromaffin vesicle ghosts in the presence of Mg2+-ATP. Although it inhibits norepinephrine transport when added externally, reserpic acid does not inhibit when trapped inside chromaffin vesicle ghosts. Therefore, reserpic acid must bind to the external face of the monoamine translocator and should be a good probe of the translocator's structural asymmetry.     

Relationship Between the Regulation of Enkephalin-Containing Peptide and Dopamine β-Hydroxylase Levels in Cultured Adrenal Chromaffin Cells

Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin-containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine beta-hydroxylase, were examined. Catecholamine-depleting drugs, such as tetrabenazine, and cyclic nucleotide-elevating drugs, including forskolin, 8-bromo-cyclic AMP, and theophylline, increase chromaffin cell enkephalin-containing peptide levels but fail to increase dopamine beta-hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin-containing peptides and dopamine beta-hydroxylase. The increased amounts of enkephalin-containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density-gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin-containing peptides, but that after treatment with catecholamine-depleting or cyclic nucleotide-related agents enkephalin-containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.     

Human neuroblastoma tumor cell lines correspond to the arrested differentiation of chromaffin adrenal medullary neuroblasts

We have examined the hypothesis that nonhematopoietic malignancies may contain cells corresponding to those which occur during the differentiation of tissue precursors. Neuroblastoma, an embryonal tumor of the adrenal medulla, was studied because of its well described ability to differentiate both in vivo and in vitro. We examined the expression of four genes during development of the human adrenal medulla: tyrosine hydroxylase, chromagranin A, pG2, and beta-2-microglobulin. The sequential expression of these genes by adrenal neuroblasts marks successive stages during maturation of the chromaffin lineage. We also observed a population of neuroblasts during adrenal medullary development that did not express any of these four genes, suggestive of adrenal medullary cells differentiating along nonchromaffin lineage(s). We then evaluated 27 neuroblastoma cell lines for the expression of these genes and found that 24 expressed chromaffin markers, with 19 of these mimicking the pattern of gene expression found during development. Three cell lines did not express tyrosine hydroxylase, chromogranin A, or pG2, consistent with either a very undifferentiated neural crest cell or maturation along a nonchromaffin lineage. These data indicate that neuroblastoma tumor cells correspond to adrenal neuroblasts arrested during morphogenesis of the adrenal medulla and raise the possibility that malignant transformation of cells at different stages of tissue maturation may contribute to the diversity that characterizes tumors of solid tissues.     

神经生长因子启动哮喘神经-内分泌-免疫网络功能失衡

神经生长因子是一种对神经生长、分化起到营养作用的肽类,其在哮喘发病过程中被认为是连接神经-内分泌-免疫网络的桥梁,作用机制可能如下:a.神经生长因子引起气道神经解剖结构和功能变化,促进气道神经末梢合成和释放递质,有助于气道重构的形成;b.神经生长因子能够增强变应原特异性IgE抗体的表达,促进肥大细胞、嗜酸性粒细胞、淋巴细胞等在气道聚集,诱导其释放炎症介质,改变免疫应答平衡状态;c.神经生长因子可能启动肾上腺髓质细胞冗余性,使其向神经细胞转变,导致髓质细胞内分泌功能削弱,使肾上腺素合成、释放和再摄取功能障碍,最终导致循环中肾上腺素达不到维持气道舒张状态所需水平.     

Vasoactive intestinal peptide and substance P increase levels of enkephalin-containing peptides in adrenal chromaffin cells

The neuropeptides substance P and vasoactive intestinal peptide (VIP), reported to exist in the splanchnic nerve terminals innervating the adrenal medulla, elevate the levels of enkephalin-containing peptides (ECPs) in cultured bovine adrenal medullary chromaffin cells. Cellular ECP stores were increased over 48 hr by 72 and 46 percent, respectively, following incubation with 5 microM VIP or 10 microM substance P, maximally effective concentrations. The results suggest that VIP and substance P may be trans-synaptic modulators of chromaffin cell ECP stores.