Regulation of nerve growth factor synthesis/secretion by catecholamine in cultured mouse astroglial cells

The nerve growth factor (NGF) synthesis/secretion by cultured mouse astroglial cells was modulated by catecholamine. In quiescent cells, epinephrine (EN) and dopamine (DA) markedly increased the NGF content in the conditioned medium (CM). Conversely, EN, DA, and norepinephrine (NE) decreased the NGF content in growing cells. Cholinergic agonists, metacholine and carbamylcholine, slightly increased the NGF content in quiescent cells, but showed no effects on growing cells. Other neurotransmitters tested had no effects on either growing or quiescent cells. These results suggest that catecholamine is one of the molecules responsible for regulation of NGF synthesis/secretion in the mouse brain.     

Catecholamines increase nerve growth factor mRNA content in both mouse astroglial cells and fibroblast cells

Previous studies have shown that catecholamines increase the nerve growth factor (NGF) content in medium conditioned by mouse L-M fibroblast cells and mouse astroglial cells. In this study, the NGF mRNA levels in these cells were measured by Northern blot analysis. In astroglial cells treated with epinephrine (EN), the cellular NGF mRNA level increased prior to accumulation of NGF in the culture medium. 3-Hydroxytyramine (DA) and norepinephrine (NE) also increased the cellular NGF mRNA content. An increased level of NGF mRNA elicited by EN was also observed in mouse L-M cells. These results indicate that catecholamines enhance NGF synthesis of L-M fibroblast cells and astroglial cells by increasing the cellular content of NGF mRNA. The present results also indicate that the effects of catecholamines are not mediated by adrenergic receptors.     

Biological and biochemical properties of nerve growth factor synthesized by mouse S-180 cells in culture

Nerve Growth Factor (NGF), a protein involved in the maintenance and differentiation of sensory and sympathetic neuronal cells [1], is synthesized by several different types of cells in culture [2-7]. In this paper, the biochemical and biological properties of NGF synthesized by a mouse S-180 sarcoma cell line were examined. These cells do not appear to produce the 7S-NGF molecule, a form of NGF found in high concentrations in the mouse submandibular gland [8]. The 7S-NGF is comprised of three distinct protein subunits named beta-NGF, alpha and gamma [9]. Although the S-180 cells do not produce 7S-NGF, the cells do synthesize one of the component subunits of 7S-NGF, the beta-NGF subunit. Biological, electrophoretic, immunological and molecular weight criteria were used to establish that the beta-NGF synthesized by the S-180 cells is very similar to the submandibular gland beta-NGF. The S-180 beta-NGF was bound to an unidentified binding component(s) which is not immunologically similar to either the alpha- or gamma-subunit. The functional significance of this interaction is not known.     

Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae

The DNA coding for human nerve growth factor (hNGF) was chemically synthesized and introduced into Saccharomyces cerevisiae. Expression and secretion of hNGF was obtained by use of the yeast phosphoglycerate kinase-encoding gene promoter and the pre-pro sequence of the yeast alpha-mating factor. Immunoblotting with antiserum raised against a protein A-hNGF fusion protein, allowed the detection of an immunoreactive material secreted into the culture medium. A preparation from the culture medium, partially purified by ion-exchange column chromatography, stimulated neurite outgrowth from rat pheochromocytoma PC12h cells.     

Release of nerve growth factor by human glial cells in culture

Non-transformed human glial cells obtained from brain biopsies (lines U-787 CG, U-1169 CG and U-1508 CG) release to their culture medium a factor which, in bioassay, induces neurite outgrowth in spinal and sympathetic embryonic chick ganglia. The neurite-stimulating activity, which was enhanced after pressure dialysis of glial-conditioned medium, is inhibited by specific antiserum prepared to mouse βnerve growth factor (NGF). The glial factor was partially purified by gel filtration on Sephadex G-200 of concentrated, serum-containing, conditioned medium. The activity eluted close to a molecular weight of 30000, as did mouse NGF run under identical conditions. Ion-exchange chromatography on DEAE-Sepharose CL-6B and flat-bed electrofocusing of conditioned medium showed the activity to be associated with a heat-labile entity having an isoelectric point of about 4.1. All purified preparations were blocked by anti(mouse)-βNGF. The results demonstrate the existence of a human glial NGF which in several respects resembles the mouse submandibular gland NGF.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Synthesis and secretion of beta-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Secretion of nerve growth factor by central nervous system glioma cells in culture

Bacteriophage immunoassays, radioimmunoassays, and biological assays have been used to measure levels of NGF in media conditioned by rat C-6 glioma cells in culture. By all three criteria, these cells secrete a macromolecule which is indistinguishable from mouse submandibular gland NGF.     

Synthesis of nerve growth factor in rat glioma cells

We have analyzed the incorporation of radioactive amino acids by rat C6 glioma cells into material precipitable with anti-β-nerve growth factor (NGF). We show that amino acids are incorporated into a protein the size of β-NGF which is immunologically related to NGF and which has peptides similar to those of mouse β-NGF. Several lines of evidence obtained in this study support the hypothesis that NGF is produced by the proteolytic cleavage of a higher molecular weight precursor. This evidence includes kinetic studies, demonstration of higher molecular weight material (24,000) immunologically related to NGF, and in vitro processing of the 24,000 MW protein to material of the approximate size of β-NGF.     

Human fibroblast cells synthesize and secrete nerve growth factor in culture.

Using our enzyme immunoassay system developed for recombinant hNGF, we examined the synthesis and secretion of human NGF (hNGF) by human fibroblast (WS-1) cells. The amount of the factor secreted by WS-1 cells increased linearly and a significant amount of NGF was detected in the conditioned medium of WS-1 cultures. WS-1 NGF showed properties identical to those of recombinant human NGF in immunoreactivity and molecular weight. An increase in cell density or the withdrawal of serum from the culture medium caused a drastic decrease in the rate of NGF secretion. These results suggest that WS-1 cells are able to synthesize and secrete hNGF in culture and that the synthesis/secretion is regulated in a growth phase-dependent manner.     

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transforming growth factor related to epidermal growth factor is expressed by fetal mouse salivary mesenchyme cells in culture

Fetal mouse salivary mesenchyme cells secrete a protein with an apparent MW of 15 Kd that is immunologically related to epidermal growth factor (EGF). Conditioned medium collected from these cells in culture stimulates the growth of primary mouse mammary epithelial cells cultured within collagen gels, competes for binding to EGF receptor sites on these mammary epithelial cells and stimulates the anchorage-independent growth of normal rat kidney fibroblast cells within soft agarose. Prior immunoprecipitation of salivary mesenchyme cell conditioned medium with anti-EGF antibodies effectively removes or attenuates all of these effects confirming that an EGF-like factor is involved in these responses.     

Human nerve growth factor: Lack of immunocrossreactivity with mouse nerve growth factor

Human β-nerve growth factor (hNGF) was purified from term human placenta. The biological potency of hNGF in the chick dorsal root ganglion assay did not differ significantly from that of mouse NGF (mNGF). Molecular weight determinations of mNGF and hMGF were also similar. No immunological crossreactivity was noted between hNGF, at a concentration of 100 μg/ml, and mNGF in a radioimmunoassay for mNGF using 6 different antisera to mNGF. hNGF shares several properties with mNGF but is immunological distinct. The results of studies in man using antisera to mNGF should be interpreted with caution.     

Sodium and potassium uptake in primary cultures of proliferating rat astroglial cells induced by short-term exposure to an astroglial growth factor

Primary cultures of rat astroglial cells were maintained in a serum-free medium. After 8–10 days of cultivation the cells were exposed to an astroglial growth factor (AGF2) for short periods (1–120 min). Subsequently, uptake of²²Na⁺ and⁴²K⁺ into control and AGF2-pretreated cells was studied. Assay of the Na⁺ and K⁺ values in the cells was also performed by atomic absorption spectrometry. Treatment of rat astroglial cells with AGF2 resulted in a significant increase of the uptake of both Na⁺ and K⁺ depending on the duration of the exposure period. To reach the maximum increase of cation uptake, 6–10 min and 30 min of AGF2 pretreatment were needed for Na⁺ and K⁺, respectively. Amiloride blocked this increase of Na⁺ and K⁺ uptake elicited by AGF2 pretreatment, but the control cells were amiloride resistant. Treatment with AGF2 increased the ouabain sensitivity of the K⁺ uptake as that: 10^–4 M ouabain inhibited K⁺ uptake of the AGF2-treated cells to the same degree as 5×10^–3 M ouabain with the control cells. The Na⁺ uptake of AGF2-treated cells, however, exhibited no relevant changes in the presence of ouabain. A significant part of the AGF2-induced K⁺ uptake could be inhibited by both ouabain and amiloride, but a ouabain-resistant and amiloride-sensitive component also was revealed. The furosemide sensitivity of both Na⁺ and K⁺ uptake into cultured astroglial cells was also significantly increased by AGF2. Our findings suggest that short-term exposure of cultured glial cells to AGF2 induces these very early ionic events: 1) The appearance of a relevant amiloride-sensitive Na⁺/H⁺ exchange, and as a consequence of increased Na⁺ entry into the cells, secondary activation of the ouabain-sensitive K⁺ uptake via the Na⁺,K⁺-pump. 2) A direct effect of AGF2 on the Na⁺,K⁺-pump assembly in the membrane, resulting in increased Na⁺ sensitivity of the inner pump sites and enhanced ouabain sensitivity of the external K⁺-binding sites. 3) An increase of ouabain-resistant but amiloride- or furosemide-sensitive Na⁺ and K⁺ uptake.Some of the results reported here were presented as a lecture at a Symposium on Na⁺/H⁺ exchange of the Second European Congress on Cell Biology, Budapest, Hungary, 1986.     

Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture

Primary cultures of normal astroglia started from the cerebral hemispheres of neonatal rats took up dopamine (DA) and norepinephrine (NE) in the concentration range of 10^?7 to 10^?4M and metabolized each to their respective principal central nervous system products by the actions of both catechol-0-methyl transferase and monoamine oxidase. At 10^?7M, uptake of ³H labelled DA and NE was inhibited by omission of Na⁺, addition of ouabain or lowered temperatures. Uptake at 10^?4M was considerable but was Na⁺-independent. Only Na⁺-independent uptake was seen in primary cultures started from the meninges of neonatal rats. These data suggest that astroglial cells in the CNS have a high affinity uptake system for catecholamines, and such uptake is followed by catecholamine metabolism.     

Synthesis and secretion of β-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, β-nerve growth factor (βNGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature βNGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Calcium homeostasis and nerve growth factor secretion from vascular and bladder smooth muscle cells

Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output.     

Comparison of 7S nerve growth factor and nerve growth factor I from mouse submandibular glands

7S nerve growth factor (7S NGF) and nerve growth factor I (NGFI) are NGF-containing protein complexes isolated from mouse submandibular glands by different protocols, and reports suggest that the molecules differ chemically. In this study, we compared the molecular properties and subunit compositions of the two proteins. Purified 7S NGF and NGFI electrophoresed to identical positions on polyacrylamide gels in nondissociating buffers, with electrophoretic mobilities indistinguishable from that of unpurified NGF in salivary gland extracts. Ultraviolet absorption curves were identical, and sedimentation coefficients were similar (7.3 +/- 0.25 S for 7S NGF; 7.2 +/- 0.2 S for NGFI) as determined by sedimentation velocity analysis. By sedimentation equilibrium analysis, molecular weights of 135 000-140 000 were obtained for both complexes at protein concentrations in the centrifuge cell greater than 85 micrograms/mL; when protein concentrations within the centrifuge cell ranged from approximately 30 to 100 micrograms/mL at equilibrium, both complexes dissociated. Molecular weight values determined by gel filtration on Bio-Gel P300 and Sephadex G200 resins were similar for both proteins, and the values determined on Sephadex agreed with those obtained by ultracentrifugation. The subunit compositions of the complexes were also similar as determined by nonequilibrium isoelectric focusing, NGFI being composed of proteins that migrated to positions identical with those of the alpha, beta, and gamma subunits of 7S NGF. Furthermore, the stoichiometry of the subunits was similar in the two complexes as determined by radioimmunoassays to each of the subunits and by densitometric analysis of electrophoretic gels. Both methods showed that the complexes contain approximately 2 mol of the alpha and gamma subunits per mole of beta-NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain-derived growth factor is a chemoattractant for fibroblasts and astroglial cells

Bovine brain-derived growth factor (BDGF), a 16-17 kDa protein with biochemical properties resembling brain-derived acidic fibroblast growth factor (acidic FGF) and endothelial cell growth factor, was found to have potent chemotactic activity for bovine ligament fibroblasts, human skin fibroblasts and rat astroglial cells, maximal at 100-200 pg/ml. The chemotactic activity was completely blocked by protamine sulfate (5 ug/ml), an inhibitor of receptor-binding and mitogenic activity of BDGF. BDGF did not stimulate migration of human monocytes. These results indicate that the effects of BDGF 'in vivo' might extend to mesenchymal cell recruitment.