Ascorbate-like factor from embryonic brain. Role in collagen formation, basement membrane deposition, and acetylcholine receptor aggregation by muscle cells.
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Biomedical subjects
C Kalcheim
Publications and source records attributed to C Kalcheim.
Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia.
The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.
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Ciliary ganglia and spinal cord explants release an ascorbate-like compound which stimulates proline hydroxylation and collagen formation in muscle cultures.
A low-molecular-weight factor from embryonic rat brain stimulates collagen production in rat muscle cultures. This effect is associated with increased hydroxylation of proline residues in collagenous proteins produced by the cells. Here, we show that increased hydroxylation (22-and 7.5-fold) was also observed with extracts of rat embryonic spinal cord and extracts of the rat pheochromocytoma cell line PC12. A 5-25-fold stimulation in proline hydroxylation was obtained when muscle cells were cocultured with chick ciliary ganglia or with embryonic rat spinal cord explants. Medium conditioned by rat spinal cord explants also increased prolyl hydroxylation. Incubation of the muscle-nerve cocultures with ascorbate oxidase markedly reduced the observed increase in proline hydroxylation. These results show that the cultured explants release a factor which promotes proline hydroxylation and collagen production by muscle. This factor seems to be ascorbic acid or an ascorbate-like compound.
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Collagen-stimulating factor from embryonic brain has ascorbate-like activity and stimulates prolyl hydroxylation in cultured muscle cells.
Embryonic rat-brain extract contains a collagen-stimulating factor which enhances the production of collagen types I, III, IV and V by cultured rat muscle cells. Here we report on the partial characterization and possible mechanism of action of a low-molecular-mass fraction with ascorbate-like activity isolated from embryonic rat brain extracts. This activity eluted very close to ascorbate when filtered through Bio-Gel P-2 and Sephadex G-10. The peak of biological activity showed properties of a reducing agent. Both the biological and reducing activities were lost when the fraction was treated with the enzyme ascorbate oxidase. This factor enhanced in a time-dependent manner, the secretion of procollagen, pulse-labeled with [3H] proline. Incubation of the muscle cultures with the factor increased by 15-fold the ratio of hydroxyproline to proline residues in secreted macromolecules over controls. A fourfold increase in the above ratio was obtained for the cellular proteins. Crude homogenates from control and factor-stimulated cultures were tested for prolyl hydroxylase activity using [3H](Pro-Gly-Pro)n as a substrate. Cultures treated with the collagen-stimulating factor showed a 5-50-fold increase in prolyl hydroxylation activity compared to controls. No effect on prolyl hydroxylation was found when the factor was added in vitro to either control or stimulated enzyme preparations. Our results suggest that the collagen-stimulating factor contains ascorbate-like activity which promotes the secretion of collagenous proteins by increasing hydroxylation of proline residues in their polypeptide backbone.
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Characterization and localization of collagens synthesized by cultured muscle cells stimulated with collagen-inducing factor from embryonic brain extracts.
A low molecular weight fraction (Mr = 500-1500) was isolated by membrane and gel filtration from rat embryonic brain extract. This fraction was shown to stimulate collagen production in muscle cultures. Procollagen, intermediate collagenous proteins, and collagens of types I, III, and V were identified by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE- and Cm-cellulose chromatography and by selective degradation by pepsin, bacterial collagenase, and cyanogen bromide. Immunofluorescent staining with antisera to collagen types I, III, IV, and V of muscle cultures treated with the collagen-inducing factor revealed a large increase in staining compared to control cultures and a distinct pattern of staining on the surface of the treated myotubes.
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Involvement of collagen in the aggregation of acetylcholine receptors on cultured muscle cells.
The role of collagen macromolecules in the aggregation of acetylcholine receptors on the surface of cultured rat muscle cells was investigated. Both the synthesis and secretion of collagen, as well as acetylcholine receptor aggregation were stimulated by treatment of muscle cultures with an embryonic brain extract. Treatments with cis-hydroxyproline or monensin, which interfere with collagen synthesis and secretion, significantly reduced the number of acetylcholine receptor aggregates detected. Incubation of the cultures with embryonic brain extract induced a decrease in extractability of acetylcholine receptors. This effect could be reversed by treating the cells with bacterial collagenase. In addition, affinity purified antibodies against collagen of types I and IV inhibited the aggregation of acetylcholine receptors by stimulated brain extract. These data suggest that neurotrophic factor(s) present in embryonic nerve tissue have the ability to induce collagen production; the collagen formed plays a role in the formation and/or stabilization of acetylcholine receptor aggregates and possibly also of synaptic connections.
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Aggregation of acetylcholine receptors in nerve-muscle cocultures is decreased by inhibitors of collagen production.
Coculturing of rat embryonic muscle cells with spinal cord explants resulted in the formation of large numbers of acetylcholine receptor aggregates on the myotube surface, compared to those found on muscle cells grown in the absence of nervous tissue. Remarkably fewer receptor aggregates were formed when, upon addition of nerve explants, these cocultures were treated with either cis-hydroxyproline (a specific inhibitor of collagen production) or collagenase. The possibility is raised that collagen participates in the aggregation of acetylcholine receptors and in synapse formation.
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Embryonic brain extract induces collagen biosynthesis in cultured muscle cells: involvement in acetylcholine receptor aggregation.
The involvement of extracellular matrix components in induction of the aggregation of acetylcholine (AcCho) receptors by factor(s) present in embryonic brain extract was investigated. Embryonic brain extract induced a three-fold increase in the number of AcCho receptor aggregates on the surface of cultured myotubes and a 5- to 10-fold increase in the synthesis of procollagen, which was secreted into the medium and converted to collagen. Adult brain extract, embryonic serum, and embryonic liver extract were less active in stimulating both collagen synthesis and AcCho receptor aggregation. A physiological connection between the two processes is suggested, since the number of AcCho receptor aggregates could be reduced to control levels by treating brain extract-stimulated myotubes with purified bacterial collagenase. In addition, stimulation of collagen secretion by ascorbic acid (50 micrograms/ml) promoted a 1.6-fold increase in AcCho receptor aggregation. When ascorbic acid was added together with the brain extract, further increases in both collagen synthesis and AcCho receptor aggregation were observed.
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Goldfish brain extract induces aggregation of acetylcholine receptors on cultured myotubes: increased activity following optic nerve axotomy.
Acetylcholine receptor aggregation-inducing factor(s) is found in goldfish brain extract. The specific activity of the factor(s) is increased during goldfish optic nerve regeneration and peaks at 8 days following the nerve axotomy. It is therefore speculated that this activity is associated with the goldfish visual system plasticity.
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Bisexual behavior in male rats treated neonatally with antibodies to luteinizing hormone-releasing hormone.
For investigating the possibility that the neuropeptide luteinizing hormone-releasing hormone (LHRH) is involved in the process of sexual differentiation, male rat pups were injected on Days 1 and 3 of life with specific antibodies to LHRH (AB-LHRH); control rats were treated with normal rabbit serum (NRS). At maturity, males treated with AB-LHRH were as fertile as controls, and their amount and intromission latencies and the postejaculatory interval were extended only slightly. However, they showed high levels of lordotic behavior, including ear wiggling, when castrated and primed with estrogen or with estrogen plus progesterone. Testosterone propionate, administered neonatally together with AB-LHRH, did not reverse these effects. There was no evidence that neonatal treatment with antiserum to LHRH affected testosterone levels, as measured by radioimmunoassay. Males treated with AB-LHRH and castrated as adults did not respond to estrogen priming by releasing a surge of luteinizing hormone, a result indicating that they did not possess the female type of gonadotropin regulation. These findings indicate that neutralization of endogenous LHRH during neonatal life selectively blocks defeminization of behavior without affecting the process of masculinization.
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