Reiner Westermann

Basic Fibroblast Growth Factor in the Adrenal Gland

The importance of trophic agents for the development and maintenance of neurons and their presence in mesenchyme‐derived neuronal target organs such as muscle is well exemplified by the protein nerve growth factor (NGF) and its synthesis in target areas of sympathetic and sensory nerves. Stringent conceptualization of target organ‐regulated neuronal maintenance would imply that neurons were able to provide trophic support to their presynaptic counterparts. We present data suggesting that basic fibroblast growth factor (bFGF), a mitogen and trophic factor for several neuron populations in vitro, may be such a protein involved in retrograde trophic neuron‐neuron interaction. Basic FGF or a closely related protein is present in the adrenal medulla and its sympathetic neuron‐like chromaffin cells. A polyclonal antibody specific for bFGF recognizes an 18 kD band in Western blots of bFGF‐enriched bovine adrenal medulla extracts and immunostains isolated bovine chromaffin cells. This antibody also blocks the bFGF‐like activity present in adrenal medullary extracts and chromaffin granule extracts that both promote in vitro survival of embryonic chick ciliary ganglionic neurons. Furthermore, like bFGF, the soluble proteins of bovine chromaffin granules are mitogenic for cultured bovine aorta endothelial cells. Electrothermal unilateral destruction of the adrenal medulla causes the disappearance of 25% of Nissl‐stained neurons in the ipsilateral intermediolateral column (IML) of the spinal cord between levels Th7 and L1, which contains the preganglionic neurons projecting to the adrenal medulla. Substitution of the adrenal medulla by gel foams soaked with bFGF prevents neuron losses in the IML. The effects are specific in that NGF and cytochrome C are ineffective. Our results suggest that bFGF is located in chromaffin cells and maintains target‐deprived autonomic spinal cord neurons, thus possibly acting as an interneuronal trophic messenger in vivo.

Basic Fibroblast Growth Factor (bFGF) Immunoreactivity Is Present in Chromaffin Granules

Abstract: Basic fibroblast growth factor (bFGF) has recently been isolated from bovine adrenal glands. Immunohistological data revealed its presence in both adrenal cortex and adrenal medulla. Using immuno‐electronmicroscopy, we found that in medullary chromaffin cells bFGF‐immunoreactivity is localized in the secretory granules. Immuno‐reactivity also was observed by electronmicroscopy in isolated granules. Western blot analysis revealed the presence of the typical 18‐kDa bFGF and additional immunoreactive materials with molecular masses of ∼ 24, 30, and 46 kDa in whole bovine adrenal, and in cortex and medulla. Similar results were obtained with proteins from bovine chromaffin granules, with the following two exceptions: the 46‐kDa immunoreactivity was found to be highly enriched when compared with medulla or cortex, and the 18‐kDa band could be detected with only an antiserum against a synthetic peptide comprising the 24 NH2‐terminal amino acids of bFGF, and not with an antiserum against purified bovine pituitary bFGF. All fractions enriched for bFGF‐immunoreactivity showed neurotrophic activity for chick ciliary ganglion neurons, which could be blocked by antibodies. These results demonstrate for the first time the localization and occurrence of bFGF in a cellular secretory organelle, and present further evidence for the existence of higher molecular weight immunoreactive forms of bFGF.

Basic fibroblast growth factor (bFGF), a multifunctional growth factor for neuroectodermal cells

Summary Basic fibroblast growth factor (bFGF), a heparin-binding mitogen for mesoderm-derived cells, also acts as a mitogen, differentiation inducing and maintenance factor for many neuroectodermal cells including glial cells, neurons, paraneurons, and their tumor counterparts. The molecule is expressed in several types of neuroectodermal cells in vitro and in vivo. Furthermore, bFGF occurs in many neuronal target tissues, and can prevent ontogenetic as well as lesion-induced neuron death. Thus, in terms of its wide range of functions, bFGF is apparently more than a ‘classical’ neurotrophic factor. Some of its essential features, such as regulation of expression, local availability and transport in the nervous system remain to be studied.

Neuronotrophic Factors Released by C6 Glioma Cells

Abstract: 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 μg 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.

Inhibition of Growth and Induction of Differentiation of Glioma Cell Lines by Oncostatin M (OSM)

The neuropoietic cytokines of the interleukin-6 family are a group of structurally and functionally related polypeptides. We studied the effect of the multifunctional neuropoietic cytokines, including oncostatin M (OSM), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), on anaplastic glioma cell lines. Growth and morphology of the glioma cell lines were affected differently. While IL-6 and LIF exerted no or only small minor morphological changes and growth retardation, OSM induced a marked change in morphology and a strong suppression of growth. OSM treated cells were characterized by enlargement and the formation of multiple, thin processes thus resembling mature cultured astrocytes. The growth inhibitory effects were dose dependent with a maximum exerted by addition of 50 ng/ml OSM. The inhibition of DNA synthesis by OSM could be abolished by antibodies blocking either the activity of OSM or the OSM-receptor component, gp130.

High molecular weight forms of basic fibroblast growth factor recognized by a new anti‐bFGF antibody

An antibody against basic fibroblast growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30–33 and 46 kDa in inununoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti‐bFGF antibody raised against a synthetic peptide comprising the 24 N‐terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.

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.

Basic fibroblast growth factor promotes transmitter storage and synthesis in cultured chromaffin cells.

We have studied the effects of basic fibroblast growth factor (bFGF), which occurs in the adrenal medulla, on the survival, morphological phenotype, storage capacity for catecholamines and induction of the synthesizing enzymes tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) of cultured chromaffin cells from young postnatal rats. Basic FGF (40 ng/ml), like nerve growth factor (NGF; 40 ng/ml) prevented a drastic numerical decrease of chromaffin cells over a 4-day culture period, but, in contrast to NGF, did not induce neurite outgrowth, unless the cells were maintained for 7 days. Basic FGF was also more effective than NGF in maintaining the initial storage capacity for catecholamines, and even increased it under certain culture conditions (laminin instead of polyornithine, or 200 ng instead of 40 ng/ml). Basic FGF and NGF did not induce TH and PNMT activities beyond their initial levels, but partially prevented the reduction of TH activity seen after 4 days in culture. Based on the present data and the previously reported greater in vitro survival and transmitter stability of older chromaffin cells, which contain bFGF, and the relative instability of young postnatal chromaffin cells, which express no or very low levels of bFGF until 8 days postnatally, but respond to it, we hypothesize that bFGF is an important autocrine/paracrine maintenance factor for adult chromaffin cells.

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.

Neuroectodermal Cells: Storage and Release of Growth Factors

Neurons and glial cells, both derived from the embryonic neuroectoderm, are the principal cellular constituents of the nervous system. Homo- and heterologous cell - cell adhesion, metabolic and trophic couplings between these partners play key roles for development, maintenance and regenerative processes of neural systems.