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PubMed1991

Quantitative analysis of exocytosis directly visualized in living chromaffin cells.

Chromaffin cells isolated from the bovine adrenal medulla were observed under a Nomarski microscope through a CCD camera and an image processor. Exocytotic events of individual granules including fusion, extrusion, swelling, omega-figure formation, and membrane retrieval were visualized in individual cells stimulated by acetylcholine or K-rich solution. Initial steps were quicker than 16 ms, and the membrane retrieval was slower than 1-60 s. These findings provided a light microscopic proof for the exocytosis hypothesis as well as a basis for quantification of hormonal release. The technique was used to demonstrate significant secretion induced by a muscarinic agonist.

Acetylcholine↗
PubMed1987

Nerve growth factor effect on adenosine transport in cultured chromaffin cells.

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.

Adenosine↗
PubMed1986

Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells.

Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.

Adrenal Medulla↗
PubMed2000

Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells.

Chromaffin cells from bovine adrenal medulla were examined for the presence of a specific prenylcysteine carboxymethyltransferase by using N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine as artificial substrates and a crude cell homogenate as the enzyme source. From Michaelis-Menten kinetics the following constants were calculated: K(m) 90 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-farnesyl-L-cysteine; K(m) 52 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-geranylgeranyl-L-cysteine. Both substrates were methylated to an optimal extent at the pH range 7. 4-8.0. Methylation activity increased linearly up to 20 min incubation time and was dose dependent up to at least 160 microg of protein. Sinefungin and S-adenosylhomocysteine both caused pronounced inhibition, as also to a lesser extent did farnesylthioacetic acid, deoxymethylthioadenosine and 3-deaza-adenosine. Effector studies showed that the methyltransferase activity varied depending on the concentration and chemical nature of the cations present. Monovalent cations were slightly stimulatory, while divalent metallic ions displayed diverging inhibitory effects. The inhibition by cations was validated by the stimulatory effect of the chelators EDTA and EGTA. Sulphydryl reagents inhibited methylation but to different degrees: Hg(2+)-ions: 100%, N-ethylmaleimide: 30%, dithiothreitol: 0% and mono-iodoacetate: 20%. Due to the hydrophobicity of the substrates dimethyl sulfoxide had to be included in the incubation mixture (<4%; still moderate inhibition at more elevated concentrations). The detergents tested affected the methyltransferase activity to a varying degree. The membrane bound character of the methyltransferase was confirmed.

Adrenal Glands↗
PubMed1987

Influence of hypertonic solutions on catecholamine release from intact and permeabilized cultured chromaffin cells.

Chromaffin cells purified from bovine adrenal medulla and maintained in primary culture were used to study the effects of hyperosmolarity on the nicotine- and high potassium-induced secretory response. A similar study was also performed on cells permeabilized with digitonin and with alpha-toxin from Staphylococcus aureus. Hyperosmolarity does not affect the spontaneous release of catecholamines from either intact cells or permeabilized cells. The nicotine-induced secretion and high potassium-induced secretion from intact cells are inhibited by hypertonic solutions; a 100% inhibition of net release was observed at 660 mOsm (sucrose as osmotic agent). Veratridine- and the cation ionophore X537-A-induced release were both depressed under hyperosmotic conditions. Hyperosmolarity was shown to have reversible effects on the secretory response of intact cells. Finally, hyperosmolarity has intracellular effects on catecholamine release evoked by calcium from both detergent- and alpha-toxin-permeabilized cells. Our data show that hyperosmolarity has multiple effects on the cell membrane and the protein constituents associated with it, but has also a significant effect on intracellular reactions concerned with exocytosis.

Adrenal Medulla↗
PubMed1981

Possible occurrence of Na+-dependent Ca2+ influx mechanism in isolated bovine chromaffin cells.

Chromaffin cells were isolated from bovine adrenals and the effects of experimental manipulations which alter the level of internal Na+ on the release of catecholamines and 45Ca uptake by these cells were investigated. In response to NA+ deprivation both parameters were increased or decreased when internal NA+ was raised or reduced, respectively. The results suggest the existence of Na+-dependent Ca2+ influx mechanism in these cells.

Animals↗
PubMed1977

Identification of small intensely fluorescent (SIF) cells as chromaffin cells in bullfrog sympathetic ganglia.

Small intensely fluoresent (SIF) cells were investigated in the ninth and tenth paravertebral sympathetic ganglia of the bullfrog using histochemical and electron microscopic techniques. Fluorescence histochemistry revealed that clusters of SIF cells are sparsely distributed in the ninth and tenth ganglia; the clusters were usually located in the vicinity of blood vessels. Fluorescent processes were not observed emanating from SIF cells. The clusters stained positively for the chromaffin reaction indicating that SIF cells are chromaffin cells. Ultrastructurally, the SIF-chromaffin cells in the sympathetic ganglia appeared virtually identical to the chromaffin cells in the adrenal gland; this includes two cell types that appear morphologically the same as the epinephrine and norepinephrine containing chromaffin cells in the adrenal gland. Efferent synapses from the SIF-chromaffin cells in sympathetic ganglia to sympathetic ganglia to sympathetic neurons were not observed. The SIF-chromaffin cells in bullfrog sympathetic ganglia did not have the morphological characteristics of interneurons; it is suggested that they may function as extra-adrenal chromaffin tissue.

Adrenal Glands↗
PubMed1992

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins.

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [35S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS-PAGE of the treated cells showed incorporation of label into a broad 97-121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97-121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5'-O-(3-thiotriphosphate). The 97-121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5'-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteolgycans.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Subcellular distribution of acetylcholinesterase forms in chromaffin cells. Do chromaffin granules contain a specific secretory acetylcholinesterase?

The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (Ga1, Ga2 and Ga4), and one non-amphiphilic form (Gna4). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N-linked and O-linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol-specific phospholipases C converted the Ga2 form, but not the other amphiphilic forms, into non-amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. Gna4 was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine-binding sites and Gna4 AChE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of Gna4 during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms.

Acetylcholinesterase↗
PubMed2006

Role of DHEA and growth factors in chromaffin cell proliferation.

Dehydroepiandrostreone (DHEA) is a neuroactive steroid produced by the inner layer of the adrenal cortex close to the adrenomedullary cells. Chromaffin cell growth and proliferation are under the control of insulin-like growth factor II (IGF-II) and basic fibroblast growth factor (bFGF). The aim of the present study was to examine the role of DHEA on chromaffin cell proliferation induced by IGF-II and bFGF. In our model, DHEA significantly decreased IGF-II-induced proliferation by 48.7%, whereas it did not affect the proliferation induced by bFGF. These data suggest that DHEA exerts a paracrine function in the control of chromaffin cell growth.

Cell Proliferation↗
PubMed1996

Similarities and differences in supporting and chromaffin cells in the mammalian adrenal medullae: an immunohistochemical study.

BACKGROUND: The adrenal medulla is a typical paraganglion, having the same origin as the sympathetic ganglia, and contains at least two types of parenchymal cells: chromaffin cells and supporting cells. We previously reported that the extent of cellular association of chromaffin cells with supporting cells was remarkably higher in noradrenaline (NA)-than in adrenaline (A)-cell regions in the adrenal medullae of the rat and pig. METHODS: Cryostat sections of adrenal medullae of nine mammalian species fixed with Zamboni fluid for 24 h were immunostained by ABC methods using antisera to S-100 protein and PNMT. RESULTS: The distribution patterns of A and NA cells in the adrenal medullae were classified into four types. In the chipmunk and rabbit, adrenomedullary chromaffin cells consisted of A cells. S-100-immunoreactive cells were present more frequently in NA- than in A-cell regions in seven species (rat, golden hamster, cat, dog, pig, ox, and horse). These cells sent out cytoplasmic processes and formed a network by immunoreactive elements among NA cells. The cell-association patterns of S-100-positive cells with NA cell were classified into three types. In A-cell regions, only a few S-100-positive cells were seen in most of the species, although the frequency of S-100-labeled cells were exceptionally high in the horse. CONCLUSIONS: The close association of supporting cells with NA cells was commonly found in the adrenal medulla in many mammalian species, irrespective of the proportions and distribution patterns of A cells and NA cells. On the other hand, species differences existed in details of the cellular association between supporting cells and NA cells.

Adrenal Medulla↗
PubMed1985

Requirement for metalloendoprotease in exocytosis: evidence in mast cells and adrenal chromaffin cells.

Exocytosis is initiated by the receptor-mediated influx of calcium that results in fusion of the secretory vesicle with the plasma membrane. We examined the possibility that calcium-dependent exocytosis in mast cells and adrenal chromaffin cells requires metalloendoprotease activity. Metalloendoprotease inhibitors and dipeptide substrates block exocytosis in these cells with the same specificity and dose dependency as that with which they interact with metalloendoproteases. Metalloendoprotease activity is identified in these cells with fluorogenic synthetic substrates, which also blocked exocytosis. Metalloendoprotease activity is highest in the plasma membrane of chromaffin cells. The metalloendoprotease appears to be required in exocytosis at a step dependent on or after calcium entry, since exocytosis initiated by direct calcium introduction in both mast cells and chromaffin cells is blocked by metalloendoprotease inhibitors.

Adrenal Glands↗
PubMed1990

Effects of splanchnic nerve stimulation on the adrenal cortex may be mediated by chromaffin cells in a paracrine manner.

The effects of nerve activation and of the catecholamines epinephrine and norepinephrine on adrenal corticosteroid release were investigated in intact isolated perfused pig adrenals with preserved nerve supply. To study the contact zones of medullary and cortical tissues, porcine adrenals were examined on the histological and ultrastructural levels. Splanchnic nerve activation stimulated in parallel the release of epinephrine (from a basal value of 0.31 +/- 0.11 to 8.13 +/- 0.60 microgram/min) and norepinephrine (from 0.76 +/- 0.68 to 12.94 +/- 3.58 micrograms/min) and the release of the corticosteroids cortisol (from 0.62 +/- 0.19 to 2.00 +/- 0.35 micrograms/min) and aldosterone (from 3.34 +/- 0.59 to 7.53 +/- 1.63 ng sigma in). Also, perfusion of the isolated adrenals with catecholamines provoked a significant release of the corticosteroids. Epinephrine (10(-6) M) stimulated the release of cortisol (from 0.59 +/- 0.31 to 2.66 +/- 0.34 micrograms/min) and aldosterone (from 2.12 +/- 0.42 to 4.68 +/- 0.92 ng/min). Norepinephrine (10(-6) M) stimulated the release of cortisol (from 0.26 +/- 0.07 to 1.28 +/- 0.10 micrograms/min) and aldosterone (from 1.28 +/- 0.37 to 3.57 +/- 0.80 ng/min). Using an immunostaining for synaptophysin, which is specific for neuroendocrine cells, chromaffin cells could be detected within all three zones of the adrenal cortex. The two endocrine tissues appear to be closely interwoven. On the ultrastructural level, medullary cells are in apposition to cortical cells, with close cellular contacts. These results show that the release of corticosteroids cortisol and aldosterone can be stimulated through the sympatho-adrenal system. Taking into consideration the close colocalization of cortical and medullary tissues, this stimulation may be mediated by chromaffin cells in a paracrine manner.

Adrenal Cortex↗
PubMed2000

Generation and initial characterization of conditionally immortalized chromaffin cells.

Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.

Adrenal Glands↗
PubMed1995

Imidazoline binding sites on PC12 cells and bovine chromaffin cells.

Stimulating actions of imidazolines on the adrenal medulla were demonstrated by several laboratories. As only a few data about signal transduction exist, the aim of the present study is to establish a cellular model on which both subtypes of imidazoline receptors are present. Binding studies using [3H]clonidine for I1-sites and [3H]idazoxan for I2-sites were performed on bovine chromaffin cells and a PC12 cell line. The intracellular calcium signal was determined by a Fura-2 signal using a fluorescence microscope. Both subtypes of imidazoline binding sites are present on either crude membrane fractions, purified plasma membranes, and mitochondrial membranes of the adrenal medulla. Although the density of I1- and I2-sites on the plasma membrane fraction is almost equal, on the mitochondrial membrane fraction Bmax of I2-binding sites was double that of the I1-binding number. An increase in intracellular calcium signal could be obtained during stimulation of chromaffin cells by various I1- and I2-receptor modulators. Because a saturation of I2-binding could not be obtained in PC12 cells, adrenal medullary chromaffin cells may be a more suitable model for investigating imidazoline receptor signal transduction.

Adrenal Medulla↗
PubMed2001

Reduction of endogenous TGF-beta does not affect phenotypic development of sympathoadrenal progenitors into adrenal chromaffin cells.

Adrenal chromaffin cells and sympathetic neurons are related, but phenotypically distinct derivatives of the neural crest. Molecular cues that determine the chromaffin cell phenotype have not yet been identified; in contrast to a widely held belief, glucocorticoid signaling is apparently not relevant (Development 126 (1999) 2935). Transforming growth factor-betas (TGF-betas) regulate various aspects of embryonic development and are expressed in the environment of sympathoadrenal (SA) progenitor cells. We have previously shown that neutralization of endogenous TGF-beta from E4 to E8 in the quail embryo significantly increases numbers of adrenal tyrosine hydroxylase-positive cells. Whether endogenous TGF-beta may also be involved in influencing phenotypic development of adrenal chromaffin cells and their SA progenitors has not been analyzed. We now demonstrate that neutralization of endogenous TGF-beta1, -beta2 and -beta3 with a pan-anti-TGF-beta antibody in quail embryos during distinct time windows does not alter phenotypic development of chromaffin cells. In situ hybridizations revealed unaltered expression of neurofilament (NF-160), synaptotagmin I and neurexin I in adrenal glands. Likewise, the NF-associated antigen 3A10, and polyphosphorylated NF epitopes (RT 97) were unaltered. Most importantly, the typical ultrastructure of adrenal chromaffin cells including their large chromaffin secretory granules, a hallmark of the neuroendocrine phenotype, which distinguishes them from sympathetic neurons, was not affected. We therefore conclude that neutralization of endogenous TGF-beta influences chromaffin cell proliferation, but does not interfere with the development of the typical chromaffin cell phenotype.

Adrenal Glands↗
PubMed1993

Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells.

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.

Adrenal Medulla↗