PubMed Health⌕ Search

Biomedical subjects

K Unsicker

Publications and source records attributed to K Unsicker.

At least 199 records · Page 11Linked to original sources
PubMed1983

A simple and cheap method for removing glass coverslips from neuronal cultures and relocating identified cells.

A simple and cheap method is described for removing glass coverslips from neuronal cultures grown on collagen and embedded into Epoxy resins for electron microscopy. The method involves scoring the coverslip with a scalpel and rubbing an ice block over the scored coverslip. A thin film of water then runs between the glass and the resin, which expands during cooling and causes the coverslip to spring away. This method is superior to the application of liquid nitrogen, which often causes damage of the embedded tissue.

Adrenal Medulla↗
PubMed1983

Phenotypic plasticity of cultured bovine chromaffin cells. II. Fiber outgrowth induced by elevated potassium: morphology and ionic requirements.

Chromaffin cells isolated from the adult bovine adrenal medulla extend neurite-like processes in culture in response to a variety of agents. In the present study we investigated the effect of chronic depolarization by high potassium on fiber outgrowth and its ionic requirements. Elevated K+ in the culture medium induced process formation of isolated bovine chromaffin cells in a dose-dependent fashion, and so did veratridine. Short-term depolarization by acetylcholine or carbachol caused increased flattening out of the cells, but no outgrowth of neurite-like processes. Formation of processes was accompanied by a significant reduction of endogenous catecholamines in cultured cells after 18 h and 8 days and a relative shift towards storage of primary amines after 8 days. K+-induced fiber outgrowth was dependent on the presence of Ca2+ in the medium and Ca2+-influx into the cells: the effect was inhibited by EDTA, EGTA, CoCl2 and verapamil and mimicked by the Ca2+ ionophore A 23187. Tetrodotoxin, tetraethylammonium, amiloride and ouabain, which interfere with Na+- and K+-fluxes, did not inhibit K+-induced process formation nor did any of them by itself evoke fiber outgrowth. Fiber outgrowth required de novo protein synthesis as shown by the inhibitory effect of cycloheximide. K+-induced formation of processes was not affected by dexamethasone and dbcAMP, both of which inhibit NGF-induced neurite formation of early postnatal rat chromaffin cells in vitro. These results document that chronic depolarization may induce de novo formation of neurite-like processes of bovine chromaffin cells in vitro by a Ca2+- and protein synthesis-dependent mechanism.

Animals↗
PubMed1983

Phenotypic plasticity of cultured bovine chromaffin cells. I. Morphological changes induced by non-chromaffin cells and organ extracts, but not by mouse and bovine nerve growth factor.

Adult bovine chromaffin cells are known to show process outgrowth in culture, similar to that seen with rat chromaffin and pheochromocytoma cells after exposure to nerve growth factor (NGF). To determine whether bovine chromaffin cells respond to NGF or NGF-like factors, dissociated adrenal medullary cells from adult cattle were cultured for up to 4 weeks in the presence or absence of NGF (from mouse submaxillary glands and bovine seminal vesicles, respectively), adrenal non-chromaffin cells and various organ extracts. Chromaffin cells that had been freed from non-chromaffin cells by differential plating and/or gamma-irradiation showed virtually no fiber outgrowth. In the presence of adrenal non-chromaffin cells (NCC), cell-free extracts obtained from these cells or medium conditioned by them, chromaffin cells formed processes. Extracts from bovine seminal vesicles (SVE), but not from other organs including bovine brain, heart, liver, kidney, adrenal glands and male mouse submaxillary glands, also elicited fiber outgrowth. Extension of processes induced by NCC and SVE could not be blocked by administration of monospecific anti-NGF antibodies directed against NGF from mouse submaxillary glands. Purified NGF from mouse submaxillary glands and SVE did not elicited a response. We conclude that adult bovine chromaffin cells in culture show structural plasticity similar to that shown by cultured chromaffin cells from other species, but do not respond to NGF. Neurite outgrowth promoting activities appear to reside with macromolecular constituents of bovine adrenal non-chromaffin cells and SVE.

Animals↗
PubMed1982

Dibutyrylic cyclic AMP and theophylline inhibit proliferation of accessory cells in primary cultures of adrenomedullary cells.

Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast-like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young rat adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bovine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action of dbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.

Adrenal Medulla↗
PubMed1982

Ultrastructural, biochemical, and cell-culture studies of a presumed extraskeletal Ewing's sarcoma with special reference to differential diagnosis from neuroblastoma.

The history of a 6-year-old girl with a tumor originating from thoracic spine and finally becoming resistant to surgery, radio-, and chemotherapy is reported. Tumor-biopsy material was studied by light and electron microscopy, in cell culture, by acetylcholinesterase ultracytochemistry, and by quantitative catecholamine analysis and this led to the rejection of the initial diagnosis of a neuroblastoma. Light microscopy revealed a uniform population of undifferentiated cells incompletely lobulated by broad fibrovascular septa. Using the electron microscope, cells were characterized by large intracellular pools of glycogen, little cytoplasm with an abundance of free ribosomes and a paucity of organelles. A few cells displayed desmosome-like attachment sites. Staining for specific and unspecific acetylcholinesterase was negative with light and electron microscopy, as were the results of catecholamine histofluorescence using the glyoxylic acid method. The latter result was confirmed by the negative outcome of quantitative analyses of dopamine, noradrenaline, and adrenaline with high pressure liquid chromatography nd electrochemical detection in tissue samples. Tumor cells could easily be maintained in culture for up to 4 weeks. None of a variety of treatments that are known to favor expression of neuronal characteristics in neuroblastoma cells (serum withdrawal, nerve growth factor, dbcAMP, dexamethasone) induced morphological differentiation in cultured tumor cells. On the basis of the clinical history, morphology, and of our experiments with tumor cells, the diagnosis of a so-called extraskeletal Ewing's sarcoma is most likely. Our results strengthen the view that a cell biology approach may be valuable in neuroblastoma differential diagnosis.

Acetylcholinesterase↗
PubMed1982

Nerve growth factor requirement of postnatal rat adrenal medullary cells in vitro for survival, aggregate formation and maintenance of extended neurites.

Rat adrenal medullary cells, taken at different postnatal stages (1, 4, 10 and 14 days), were cultured in serum-containing medium with and without nerve growth factor (NGF 2.5 S, 50 ng/ml) to investigate their dependence on NGF for survival and maintenance of extended neurites. It was found that medullary cells depend on NGF for survival at day 1 and, to a lesser extent, at day 4. Neurite-like processes extended in response to NGF treatment breakdown when NGF is withdrawn. NGF-treated adrenal medullary cells displayed a pronounced tendency to form aggregates. It was also shown that plating adrenal medullary cells at higher cell densities, which largely increased the numbers of fibroblast-like cells present, may substitute for NGF with respect to survival.

Adrenal Medulla↗
PubMed1981

Differentiation and transdifferentiation of adrenal chromaffin cells of the guinea pig. II. Adrenal medullary explants grown in tissue culture.

Explants of adrenal medullary tissue taken from newborn guinea pigs were grown in culture for up to two weeks. The explants exhibited sparse outgrowth of neurite-like processes, in contrast to adrenal medullae taken from young postnatal rats or adults guinea pigs that were (i) grown under identical conditions (Unsicker and Chamley 1977) or (ii) transplanted to the anterior chamber of the eye (Unsicker et al. 1981), respectively. Nerve growth factor (10-100 ng/ml, 2.5 S NGF) did not enhance formation of processes. However, electron-microscopic investigations revealed the presence of numerous processes within the explants, which extended from chromaffin cells and were characterized by longitudinally oriented cytoskeletal structures, various population of clear and dense-cored vesicles, varicosities and growth cones. Chromaffin cell bodies largely resembled their in situ-counterparts, but had fewer and smaller storage vesicles than controls. The results are discussed in light of recent findings regarding the potency of NGF and NGF-like growth factors to induce neuronal transdifferentiation of adrenal chromaffin cells.

Adrenal Medulla↗
PubMed1981

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat. Acetylcholinesterase as a means for early discrimination of cell types.

The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80-120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eight postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed. By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining with the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative. It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.

Acetylcholinesterase↗
PubMed1981

High-performance liquid chromatography with electrochemical detection as a highly efficient tool for studying catecholaminergic systems. I. Quantification of noradrenaline, adrenaline and dopamine in cultured adrenal medullary cells.

A method for simultaneous determinations of noradrenaline (NA), adrenaline (A) and dopamine (DA) in tissue is described. After extraction from crude deproteinized homogenates by the batch mode aluminium oxide method individual catecholamines (CA) were separated in a high-performance liquid chromatography (HPLC) system by isocratic elution with pure aqueous buffers from an octadecyl column. These eluates were directly monitored with a new amperometric detector device (Metrohm VA E 641) in a wall-jet flow cell. Sensitivity, specificity, precision and performance of this relatively simple procedure are characterized. Different applications of this assay with relevance to neurobiological research are presented.

Adrenal Medulla↗
PubMed1981

Purification of bovine adrenal chromaffin cells by differential plating.

A method is described for obtaining highly purified cultures of bovine chromaffin cells from crude adrenomedullary cell suspensions. The method is based on the different adhesiveness of chromaffin and non-chromaffin cells to glass and plastic surfaces (differential plating). Crude suspensions isolated by a modified version of the method described by Livett et al. (1979) (cf. Fenwick et al., 1978) contain 74.4 +/- 7.7% (n = 7) chromaffin cells as determined by electron microscopy. Bringing the cells through 5 steps of differential plating results in cultures that are predominantly composed of chromaffin cells (97.5 +/- 0.85%, n = 8). More than 90% of these cells are viable as judged by trypan blue exclusion and by electron microscopy. Cultures obtained by differential plating contain a significantly lower proportion of non-chromaffin cells than primary cultures both after one week (45.5 +/- 1.2% vs 86.7 +/- 4.6%, n = 3) and after two weeks (85 vs 93%), when grown in Falcon flasks with medium 199 and 20% fetal calf serum, but without mitosis inhibitors. Cultures obtained by the method described in this paper may be profitably employed for studying the contribution of non-chromaffin cells to the functions of chromaffin cells.

Adrenal Medulla↗
PubMed1981

Differential effects of cyclic AMP and cholera toxin on nerve growth factor-induced neurite outgrowth from adrenal medullary chromaffin and pheochromocytoma cells.

The extension of neurites from adrenal medullary chromaffin cells and PC 12 cells upon addition of nerve growth factor (NGF) has been proposed to be mediated by cyclic AMP. It is shown here that substances increasing intracellular cyclic AMP levels have a reverse effect on NGF-induced neurite outgrowth of these two related cell types. Hence, cyclic AMP is not generally involved in neurite outgrowth from NGF responsive cells. Furthermore, it is concluded that PC 12 cells cannot always be considered as a suitable model for adrenal medullary chromaffin cells.

Animals↗
PubMed1981

Ultrastructure of growth cones formed by isolated rat adrenal medullary chromaffin cells in vitro after treatment with nerve growth factor.

Growth cones formed by adrenal chromaffin cells from young postnatal rats cultured in the presence of nerve growth factor (NGF) were studied at an electron microscopic level. These growth cones are similar in many respects to those formed by sympathetic neurons in vitro supporting the view that NGF-treated chromaffin cells may undergo neuronal transdifferentiation.

Adrenal Medulla↗
PubMed1980

Effects of cell culture conditions, nerve growth factor, dexamethasone, and cyclic AMP on adrenal chromaffin cells in vitro.

Adrenal chromaffin cells from young rats, newborn guinea pigs, and adult cattle were grown under various cell culture conditions. Nerve growth factor (NGF) elicited the outgrowth of neurite-like processes from rat, but not from bovine and guinea pig, chromaffin cells. Dexamethasone, dibutyryl cAMP, theophylline, and cholera toxin, which are known to augment intracellular cAMP, specifically inhibited neurite outgrowth. We propose that glucocorticoid hormones and mechanisms which act through an increase of cAMP are prerequisites for the acquisition and maintenance of differentiation and expression of an endocrine instead of a neuronal phenotype of adrenal chromaffin cells.

Adrenal Medulla↗