Biomedical subjects

K Unsicker

Publications and source records attributed to K Unsicker.

At least 163 records · Page 9Linked to original sources

PubMed1989

The neuro-endocrine ambiguity of sympathoadrenal cells.

This introductory article presents a survey of the principle cellular constituents of the sympathoadrenal section in the peripheral autonomic nervous system, their development and plasticity and factors that govern the expression of particular morphologic and transmitter phenotypes. The article focuses on results obtained in cell culture studies with isolated chromaffin cells that have permitted the analysis of molecular signals possibly serving as environmental cues during the development of sympathoadrenal cells.

Adrenal Glands↗

PubMed1989

Survival and neuritic growth of sympathoadrenal (chromaffin) precursor cells in vitro.

Chromaffin precursor cells from embryonic rat adrenal glands were isolated at 16.3 and 20.3 days of gestation and purified by centrifugation on density gradients. Approximately 50% of the cells of both age groups that had attached to the culture substratum by 12 hr survived during a 4-day culture period in the absence of exogenous trophic factors. Nerve growth factor (NGF) and a C6 glioma-cell-conditioned medium (C6-CM) had no or a very moderate promoting effect on survival. The glucocorticoid dexamethasone (DEX) supported the survival of 70-80% of the cells that otherwise would have died. Spontaneous neuritic growth of the sympathoadrenal precursor cells was significantly more pronounced with cells isolated at embryonic day (E) 16.3 than at E20.3. NGF had a significantly smaller promoting effect on neurite ougrowth at E16.3 than at E20.3. C6-CM induced neurite outgrowth from 25% (E16.3) and 35% (E20.3) of the surviving cells. DEX (10(-6) M) completely abolished spontaneous neuritic growth and partially suppressed C6-CM-mediated fiber outgrowth. These data underscore the importance of glucocorticoids for the maintenance and development of an endocrine morphologic phenotype of sympathoadrenal precursors. They suggest that the cells may be initially driven by growth factors other than NGF into a neuronal direction and that they lack NGF-responsiveness and -dependence during the early stages of their development.

Adrenal Medulla↗

PubMed1989

Laminin and other basal lamina proteins with neurite promoting activity in medium conditioned by C6 glioma cells.

Neurite promoting activities (NPFs) are essential factors in neuronal differentiation. Some of them are associated with proteins of the extracellular matrix (ECM). C6 cells, a rat glioma cell line, release NPF activities into the cell culture medium. We used antibodies against ECM-proteins for enrichment and partial characterization of these activities. Results show that, (1) C6 cells express and release laminin; (2) the C6-laminin consists of 260 kD chains only and is therefore different from typical basal lamina laminin (220 and 440 kD chains), but comparable to other laminins of glial origin (chains in the 200 kD range only); (3) C6-laminin partially purified by affinity chromatography shows NPF-activity; (4) laminin concentration in C6 cell-conditioned medium is not sufficient to account for the total neurite promoting activity of the medium, and (5) in addition to laminin C6 cells express and release fibronectin and possibly type IV collagen.

Animals↗

PubMed1989

Immunocytochemical localization of basic fibroblast growth factor in bovine adrenal gland, ovary, and pituitary.

We studied the distribution of basic fibroblast growth factor (bFGF) immunoreactivity in bovine adrenal gland, ovary, and pituitary, using a polyclonal anti-bFGF antibody. In the adrenal gland, the inner layers of the capsule, the zona glomerulosa of the cortex, and the chromaffin cells of the adrenal medulla were intensely stained. In the ovary, follicular epithelial cells of growing follicles and granulosa cells of mature follicles showed strong bFGF-like immunoreactivity. Endocrine cells of the pituitary anterior and intermediate lobes displayed a positive immunoreaction. Blood vessels, including endothelial and smooth muscle cells, as well as stromal cells in all three organs studied, were not stained. This distribution pattern of bFGF immunoreactivity is only partially compatible with the established mitogenic role of this protein, and suggests a wider spectrum of bFGF functions.

Adrenal Glands↗

PubMed1988

Calcium-dependence of chromogranin A-catecholamine interaction.

Major components of the secretory organelle of bovine adrenal medullary cells, the chromaffin vesicles, are the acidic protein chromogranin A, catecholamines and Ca2+. The binding of Ca2+ to chromogranin A has been established. To study the interaction between chromogranin A and catecholamines and its dependence on Ca2+ we immobilized chromogranin A to a newly raised monoclonal antibody. It is shown that chromogranin A can bind (i) about 0.5 mol catecholamines per mol in a non-calcium-dependent manner and (ii) about 5 mol per mol in the presence of calcium. These results further support the notion that chromogranin A may act as a secretory granule-condensing protein.

Adrenal Medulla↗

PubMed1988

Reciprocal age-dependent pattern of two neuronal markers, tetanus toxin and neuron-specific enolase, in postnatal rat sensory and sympathetic neurons.

Tetanus toxin (TT) and antibodies to neuron-specific enolase (NSE) were used to study the developmentally regulated expression of two neuronal markers in isolated and short-term cultured rat sensory and sympathetic neurons at different postnatal ages. The results show a reciprocal pattern of TT binding and NSE-immunoreactive neurons during postnatal maturation: there is a gradual transition from TT-positive, NSE-negative to TT-negative, NSE-positive phenotypic expressions. We conclude that TT may serve as a neuronal marker for newborn, anti-NSE antibodies as markers for adult neurons, and a combination of both for adolescent neurons.

Adrenergic Fibers↗

PubMed1988

Immunocytochemical demonstration of fibroblast growth factor in cultured chick and rat neurons.

The presence of fibroblast growth factor (FGF) was investigated by immunocytochemistry in cultured neuronal cells derived from the peripheral (PNS) and the central nervous system (CNS) of chick and rat embryos. Polyclonal antimouse FGF antibodies, which cross-react with basic and acidic FGF, were used in the peroxidase immunocytochemical staining method. FGF immunoreactivity was found in neurons. Staining intensity in chick and rat brain neuronal cells increased during the culture period, reached a maximum after 6-8 days, and subsequently declined. Embryonic chick ciliary and dorsal root ganglionic (DRG) neurons as well as DRG neurons from newborn rat displayed intense FGF immunoreactivity at 12 and 48 hr in culture. Nonneuronal cells were not stained. The data demonstrate for the first time that cultured neurons from the CNS and PNS contain FGF.

Animals↗

PubMed1988

Catecholaminergic nerves in the embryonic chick ovary: co-localization with beta 2-adrenoceptor-bearing steroidogenic cells.

The present study investigates the innervation of the embryonic chick ovary with regard to (i) development and compartmentalization of catecholaminergic nerves, and (ii) presence of adrenoceptors on steroidogenic target cells of catecholaminergic nerve terminals. Catecholaminergic nerve fibers visualized by glyoxylic acid-induced histofluorescence first appeared at embryonic day (E) 13. From E15 through E21 the density of fluorescent aminergic nerves increased markedly in parallel with the concentration of catecholamines and numbers of nerve bundles and single axons seen at the electron-microscopic level. Catecholaminergic nerves were confined to the ovarian medulla and closely associated with interstitial cells. Nerve terminals approached interstitial cells up to a distance of 20 nm and, in their majority, exhibited uptake of the false adrenergic transmitter 5-hydroxydopamine. Although adrenaline amounted to 14% of the total catecholamine content at E21, adrenaline immunoreactivity was only detected in adrenal chromaffin cells, but not in nerve fibers or cell bodies within the ovary. Interstitial cells structurally matured between E15 and E21 as documented by an increase of smooth endoplasmic reticulum and tubular mitochondria. Monoclonal antibodies mAB 120 and BRK 2 raised against avian beta 1- and mammalian beta 2-adrenergic receptors revealed the presence of beta 2-adrenoceptor-like immunoreactivity on the surface of interstitial cells, but not on any other cell type. The results are consistent with the notion of a dense adrenergic innervation of the embryonic chick ovarian medulla and its steroidogenic interstitial cells, and suggest the chick ovary as an excellent model for elucidating the functional role of a neural input to steroidogenic cells during development.

Animals↗

PubMed1988

Promotion of survival and neurite outgrowth of cultured peripheral neurons by exogenous lipids and detergents.

Gangliosides, in particular the monosialoglycosphingolipids Gtet 1 (GM1), have previously been implicated in the mediation of neuronal rescue and restitutional axonal growth, both in vitro and subsequent to brain and peripheral nerve lesions. In the present study it is shown that the bis-sialosyl gangliosides Gtet2b and Gtet3b, but not the gangliosides Gtet2a and Gtet1, promote the survival of dissociated dorsal root ganglion (DRG) neurons cultured from Embryonic Day (E) 8 chicks (DRG8) almost to the same extent as nerve growth factor (NGF). Ciliary ganglion (CG) neurons from E8 chicks (CG8) and DRG10 neurons were virtually not supported suggesting considerable specificity in terms of neuronal targets and developmental stages being addressed. Moreover, a variety of other lipids including cerebroside (Cb), dipalmitoylphosphatidylcholine (DPPC) and -serine (DPPS), sulfatide (Sf), and sphingomyelin (Sm) were tested for putative survival promoting activity toward chick CG, DRG, and lumbar sympathetic ganglion (SG11) neurons. At the highest concentration employed (2.5 x 10(-5) M), Sm, DPPC, and DPPS maintained between 45 and 65% of the plateau survival with CG8 (maximally supported by ciliary neuronotrophic factor (CNTF], DRG8, and DRG10 neurons, and 30 to 40% with SG11 neurons. Cb supported CG8 neurons at about 55% of the plateau value achieved with CNTF, but had hardly any effect on the other neuron populations tested. Control experiments using highly enriched neurons and serum-free conditions assured that the effects were unlikely to be mediated by serum components or nonneuronal cells. A variety of detergents, in particular Triton X-100, also promoted the survival of CG8 and DRG10 neurons. Ganglioside Gtet1, Sm, and Triton X-100 shifted the NGF titration curve for DRG10 neurons between 6- and 15-fold in a dose-dependent manner suggesting synergisms between NGF and lipids for neuronal maintenance. These results document the neuronotrophic potency of certain gangliosides, a heterogeneous group of structurally unrelated lipids, and detergents. The mechanisms by which these agents modulate neuronal survival still await clarification.

Animals↗

PubMed1988

Neuronotrophic factors released by C6 glioma cells.

Glial cells have been shown previously to release factors that promote survival of central and peripheral neurons [neuronotrophic factors (NTFs)]. We have investigated the release of NTFs by C6 cells, a rat glioma cell line, under different modes of conditioning. Media conditioned in the presence or absence of serum [C6 cell conditioned media (C6CMs)] were analyzed using biological, biochemical, and immunological assays. We report that (a) nuclear and cytoskeletal proteins were not present in C6CMs, indicating that C6CM proteins result from release by C6 cells rather than from cell death; (b) C6CM contained 1-3 micrograms protein/ml, corresponding to a secretion rate of about 0.5 pg protein per cell and day; (c) C6CM contained the neurite-promoting factor laminin and low amounts of nerve growth factor; (d) the presence of fetal calf serum in the culture medium was essential for synthesis and release of NTFs; and (e) our C6CM contained at least three NTFs differing by their temporal secretory patterns and three NTFs differing by biochemical properties, indicating that C6 cells produce and secrete six different NTFs. Within these, nerve growth factor seems to be the only established NTF.

Animals↗

PubMed1987

Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia.

Sciatic nerve transection performed on adult rats caused neuronal losses after 4 weeks in the L4-6 dorsal root ganglia (DRG) of 35% as compared to the unlesioned side. Nerve growth factor (NGF) administered at a single dose of 6000 Biological Units in silicone tubes fixed to the proximal nerve stump completely prevented these cell losses. Basic fibroblast growth factor (bFGF) also protected DRG neurons, but at the concentrations applied (6000 Trophic Units, tested on embryonic chick ciliary ganglion neurons), failed to maintain cell numbers identical to unoperated side. Our data indicate that NGF and bFGF protect adult sensory neurons from lesion-induced death. Preliminary results suggest that local accumulation of neurotrophic activities at the proximal nerve stump elicited by NGF and, possibly bFGF, might be involved in the beneficial effects of these proteins on the maintenance of axotomized sensory neurons.

Animals↗

PubMed1987

Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve.

Basic and acidic fibroblast growth factors (FGF) were implanted next to the proximal stump of the transected optic nerve of adult rats, in order to assess whether these molecules have neurotrophic activity in vivo. Of the 119,973 +/- 2484 (S.E.M.) retinal ganglion cells present in retinae of unoperated control rats, 11,375 +/- 2413 (S.E.M.) remained at 30 days after transection of the optic nerve in control operated rats. After implantation of gel foam soaked in basic FGF, the number of retinal ganglion cells surviving at 30 days after axotomy tripled (36,387 +/- 3270 (S.E.M.], after acidic FGF, it increased almost 4-fold (40,916 +/- 5405 (S.E.M.]. These results indicate that FGF has neurotrophic activity in the adult central nervous system, and that this molecule is able to rescue adult retinal ganglion cells from axotomy induced cell death. It remains to be shown whether FGF acts directly on retinal ganglion cells or indirectly via glial cells or other cells.

Animals↗

PubMed1987

Neuron-enriched cultures of adult rat dorsal root ganglia: establishment, characterization, survival, and neuropeptide expression in response to trophic factors.

It is unknown whether adult dorsal root ganglion (DRG) neurons require trophic factors for their survival and maintenance of neuropeptide phenotypes. We have established and characterized neuron-enriched cultures of adult rat DRGs and investigated their responses to nerve growth factor (NGF), ciliary neuronotrophic factor (CNTF), pig brain extract (PBE, crude fraction of brain-derived neuronotrophic factor, BDNF), and laminin (LN). DRGs were dissected from levels C1 through L6 and dissociated and freed from myelin fragments and most satellite (S-100-immunoreactive) cells by centrifugation on Percoll and preplating. The enriched neurons, characterized by their morphology and immunoreactivity for neuron-specific enolase, constituted a population representative of the in vivo situation with regard to expression of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) immunoreactivities. In the absence of trophic factors and using polyornithine (PORN) as a substratum, 60-70% of the neurons present initially (0.5 days) had died after 7 days. LN as a substratum did not prevent a 30% loss of neurons up to day 4.5, but it subsequently maintained DRG neurons at a plateau. This behavior might reflect a cotrophic effect of LN and factors provided by non-neuronal cells, whose proliferation between 4.5 and 7 days could not be prevented by addition of mitotic inhibitors of gamma-irradiation. CNTF, but not NGF, slightly enhanced survival at 7 days on either PORN or LN. No neuronal losses were found in non-enriched cultures or when enriched neurons were supplemented with PBE, indicating that non-neuronal cells and PBE provide factor(s) essential for adult DRG neuron survival. Proportions of SP-, SOM-, and CCK-immunoreactive cells were unaltered under any experimental condition, with the exception of a numerical decline in SP cells in 7-day cultures with LN, but not PORN, as the substratum. Our data, considered in the context of recent in vivo and vitro studies, suggest that a combination of trophic factors or an unidentified factor, rather than the established molecules NGF, CNTF, and BDNF, which address embryonic and neonatal DRG neurons, are required for the in vitro maintenance of adult DRG neurons.

Animals↗

PubMed1987

Age-dependent differences in 125I-nerve growth factor binding properties of rat adrenal chromaffin cells.

Nerve growth factor (NGF) has been shown to influence survival, morphology, and transmitter phenotype of young postnatal rat chromaffin cells in vitro. Significant differences in NGF responses of chromaffin cells from newborn rats compared to 8-10-day-old ones have been reported. For this reason we studied equilibrium binding and dissociation kinetics of 125I-NGF on newborn (D1) and 10-day-old (D10) rats. Under equilibrium conditions no differences were found between the two cell types, with respect to dissociation constant (approximately 2.5 X 10(-9) M) and receptor number (10-22,000 per cell). In dissociation experiments D10 chromaffin cells exhibited two classes of NGF receptors, similar to those found in other NGF-responsive cells. From fast receptors 125I-NGF was released rapidly both at 4 degrees C and at 37 degrees C, whereas dissociation from "slow" receptors was observed only at 37 degrees C. The slow receptor class was not found on D1 cells. Instead, more than 50% of specifically bound 125I-NGF did not dissociate in the presence of excess unlabeled NGF at 37 degrees C. These age-dependent differences seem to indicate regulatory developmental changes in NGF-binding properties of rat chromaffin cells.

Adrenal Medulla↗

PubMed1987

Survival, morphology, and catecholamine storage of chromaffin cells in serum-free culture: evidence for a survival and differentiation promoting activity in medium conditioned by purified chromaffin cells.

Adult bovine and young rat chromaffin cells cultured in serum-free medium were examined for their survival and differentiation following exposure to various additives, trophic agents and conditioned media. Adrenal chromaffin cells dissociated from 8 day old rats were maintained by dexamethasone, NGF and CNTF or without any additives in an N1-supplemented medium in similar numbers as in serum-containing medium for up to 6 days. Neuritic growth elicited by NGF or CNTF was enhanced in the absence of serum. Medium conditioned by purified bovine chromaffin cells improved cell survival and caused neurite outgrowth in a dose-dependent manner. The activiti(es) was sensitive to heat and trypsin and not blocked by the addition of anti-NGF antibodies. Bovine chromaffin cell survival was reduced by 30% when cells were maintained for one week in the absence as compared to the presence of serum. Addition of insulin, the N1 supplement, dexamethasone or dbcAMP single or in combinations improved the survival to different extents. A combination of insulin (5 micrograms/ml) and dexamethasone (5 X 10(-6) M) proved to be optimal in this respect. However, these supplements failed to restore the cellular catecholamine, noradrenaline and adrenaline contents to levels seen in the presence of serum. This was also true for a chromaffin cell-conditioned medium, which improved survival without elevating the catecholamine contents. Conditioned medium, however, partly restored a more physiological adrenaline-noradrenaline-ratio.

Animals↗

PubMed1987

Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons.

Embryonic and neonatal neurons require specific trophic supplements for their survival and the induction of transmitter-synthesizing enzymes in vivo and in vitro. Acidic and basic fibroblast growth factor (aFGF, bFGF) and the closely related astroglial growth factors AGF-1 and AGF-2 were studied for putative neurotrophic functions using dissociated, highly neuron-enriched cultures from chick and rat peripheral ganglia and central nervous system tissues. Embryonic chick ciliary ganglion neurons were the only peripheral neurons that responded to bFGF and AGF-2 by enhanced survival equivalent to that obtained with ciliary neurotrophic factor. Half-maximal effects were achieved with bFGF at 360 pg/ml or AGF-2 at 3 ng/ml. Small effects seen with aFGF could be potentiated by adding heparin at 1 microgram/ml. bFGF, but not ciliary neurotropic factor, also promoted neuron survival after the factor was bound to polyornithine and laminin. Both AGF-2 and ciliary neurotropic factor induced choline acetyltransferase activity during 48 hr. AGFs and FGFs also enhanced the long-term survival of embryonic chick spinal cord neurons, including motoneurons that had been retrogradely labeled with rhodamine isothiocyanate. These results demonstrate the potency of a class of mitogenic growth factors as neurotrophic agents for embryonic ciliary ganglion and spinal cord neurons--adding to the emerging evidence that mitogenic and neuronal growth factors are not strictly separate entities.

Animals↗

PubMed1987

Neuroblastoma cells contain a trophic factor sharing biological and molecular properties with ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a protein supporting the in vitro survival of a characteristic spectrum of embryonic chicken and rat peripheral neurons. High-speed supernatants of extracts from two neuroblastoma (NB) cell lines--the mouse C 1300 N2a and the human IMR 32--mimic the effects of CNTF on identical target neurons. Promotion of survival is dose-dependent with an ED50 of 80 micrograms (IMR 32) and 140 micrograms (C 1300 N2a) of protein per ml and saturable at plateau values for surviving neurons identical to those achieved with purified CNTF. Small amounts of a CNTF-like material are also detectable in medium conditioned by NB cells. The activity is destroyed by heat and trypsin and not blocked by antibodies to (mouse) nerve growth factor. Unlike the neurite-promoting and neuronal-survival modulating agent laminin, it cannot be depleted on poly(L-alpha-ornithine)-coated plastic surfaces. NB IMR 32 cell extracts were electrophoresed using NaDodSO4/PAGE and transferred to nitrocellulose. Ciliary ganglion neurons seeded on the blotting paper in culture medium lacking CNTF ("cell blot") exclusively survive on two distinct bands with apparent molecular masses of 24 and 48 kDa. Twenty-four kilodaltons is the molecular mass of a CNTF purified from rat sciatic nerve. These results suggest that NB cells may contain a CNTF-like protein and provide further evidence that neurons may store neurotrophic factors. Purified (chicken) CNTF failed to affect proliferation and neurite growth of NB cells. The biological relevance of CNTF for NB cells, therefore, remains to be elucidated.

Animals↗

PubMed1987

Differential effects of nerve growth factor and ciliary neuronotrophic factor on catecholamine storage and catecholamine synthesizing enzymes of cultured rat chromaffin cells.

The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.

Adrenal Medulla↗