Detection of nerve growth factor mRNA in the developing chicken embryo

Nerve growth factor (beta NGF) is a protein supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. A DNA probe derived from a genomic clone coding for chicken NGF was used to study NGF mRNA levels during development. NGF mRNA was detected in the chicken embryo as early as day 3.5 of incubation. The level of NGF mRNA in total embryo increased four-fold until day 8, remained high until day 12, and subsequently decreased. No corresponding peak in NGF mRNA expression was found in heart and brain measured separately. Instead these organs showed increased NGF mRNA levels after hatching. The highest levels of NGF mRNA in the day-8 embryo were found in skin and eye (in particular cornea, but also iris, sclera-choroid and neural retina) suggesting a correlation between sensory innervation and this early peak of NGF expression.     

Regulation of alpha3 nicotinic acetylcholine receptor subunit mRNA levels by nerve growth factor and cyclic AMP in PC12 cells

To investigate the effects of nerve growth factor (NGF) and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor subunit alpha3 mRNA, we used PC12h cells, PC12 cells expressing dominant-negative Ras protein, and the parental PC12 cells. PC12h cells have NGF-responsive tyrosine hydroxylase activity. Expression of dominant-negative Ras protein prevents the signaling through the Ras-mitogen-activated protein kinase cascade. The morphological changes of the parental PC12 cells in response to NGF and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPTcAMP), a cell-penetrating cAMP analogue, were similar to those of PC12h cells. NGF up-regulated the alpha3 mRNA level in PC12h cells and down-regulated the alpha3 mRNA level in the parental PC12 cells. Expression of dominant-negative Ras protein and an inhibitor of mitogen-activated protein kinase kinase inhibited the effects of NGF on alpha3 mRNA level. CPTcAMP down-regulated the alpha3 mRNA level in all three PC12 cell lines. An inhibitor of protein kinase A inhibited the CPTcAMP-induced down-regulation of alpha3 mRNA. The alpha3 mRNA down-regulation required prolonged treatment with CPTcAMP even after cAMP response element binding protein phosphorylation was decreased. Membrane depolarization with high K+ had no effect on the alpha3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate alpha3 mRNA levels in PC12 cells.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.     

Regulation of nerve growth factor synthesis and release in organ cultures of rat iris

We studied the synthesis and release of nerve growth factor (NGF) in cultured rat iris with a two-site enzyme immunoassay by measuring the time course of NGF levels remaining in the iris and relased into the medium up to 72 h. For up to 3 h, the NGF levels in the iris did not change significantly. After that, they increased to a maximal level of 350 +/- 30 pg NGF/iris at 19 h, which is 200 times higher than the in vivo content. Between 20 and 72 h in culture, the NGF level decreased to 130 +/- 10 pg NGF/iris, whereas general protein synthesis did not change during that time period. Maximal rate of NGF production (203 pg NGF/h/iris) was seen between 9 and 12 h in culture. In the medium, NGF levels were first detectable after 6 h. Levels then increased with a time course similar to that seen within the iris, reaching a maximal level of 1,180 +/- 180 pg after 19 h in vitro, and then did not significantly change for up to 48 h. The NGF production of the densely sympathetically innervated dilator was three times higher than that of the predominantly cholinergically innervated sphincter. The NGF production was blocked by inhibitors of messenger RNA synthesis (actinomycin D) and of polyadenylation (9-beta-D-arabinofuranosyladenine) as well as by inhibitors of translation (cycloheximide). Monensin, which interferes with the transport of proteins through the Golgi apparatus, decreased NGF levels to 8-12% of controls in the medium, suggesting that the Golgi apparatus is involved in the intracellular processing of NGF.     

Expression of acidic fibroblast growth factor mRNA in the developing and adult rat brain.

We have localized acidic fibroblast growth factor (aFGF) mRNA in the developing and adult rat brain using in situ hybridization histochemistry. Prenatally, hybridization to aFGF mRNA was observed throughout the brain, with the strongest signal associated with cells of the developing cortical plate. Postnatally, labeling was localized to specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer and dentate granule cells was observed and became progressively more intense with maturation. Labeling was also observed in both the external and internal granule cell layers of the developing cerebellum. Pyramidal cells of the neocortex as well as neurons of the substantia nigra and locus ceruleus also express aFGF. This pattern persists into adulthood, although the intensity of the labeling is significantly reduced in the adult brain. These patterns of hybridization correlate with specific developmental events and suggest that aFGF plays a significant role in both central nervous system development and neuronal viability in the adult brain.     

Regulation of kynurenic acid levels in the developing rat brain

Summary Several brain-specific mechanisms control the formation of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the adult rat brain. Two of these, dopaminergic neurotransmission and cellular energy metabolism, were examined in the brain of immature (postnatal day 7) rats. The results indicate that during the early postnatal period cerebral KYNA synthesis is exceptionally amenable to modulation by dopaminergic mechanisms but rather insensitive to fluctuations in cellular energy status. These findings may be of relevance for the role of KYNA in the function and dysfunction of the developing brain.     

The synthesis of nerve growth factor (NGF) in developing skin is independent of innervation

The arrival of sensory fibers in developing mouse skin has been demonstrated to coincide precisely with the initiation of nerve growth factor (NGF) synthesis in the skin (Davies et al., 1987). This temporal correlation suggested that the arrival of sensory fibers might initiate NGF synthesis in their target tissues. Here we have eliminated the sensory and motor neurons projecting to the chick leg by the removal of the neural primordia in 3-day-old embryos. The levels of mRNA NGF of intact and denervated leg skin were identical, indicating that the developmental regulation of NGF synthesis in the skin of chick embryos is independent of its innervation.     

Regulation of nerve growth factor synthesis in mouse submaxillary glands by testosterone.

—It has long been known that the activity of nerve growth factor (NGF) in extracts obtained from the male mouse submaxillary gland is higher than in extracts from the female gland, and that the activity present in female glands can be increased by testosterone treatment. This communication presents a study of the mechanism of the testosterone effect. Of several different steroids administered to female Swiss–Webster mice only testosterone propionate led to increased gland NGF activity. The increase did not appear to be due to an enhancement of the activity of pre-existing molecules on sympathetic nerve fiber outgrowth, or due to an altered affinity for the specific antibodies used in the estimation of NGF content, but appeared rather to be due to an accumulation of NFG molecules. The kinetics of change in the male gland NGF content upon castration and secondary testosterone propionate stimulation was analyzed by application of the plateau principle. The rate of loss of NGF from this organ was not measureably different between the castrate and testosterone propionate stimulated state. On the other hand, there was estimated to be a 10-fold difference in the rate of input between the basal and steroid stimulated state. Tracer amounts of radioiodine labelled NGF administered i.v. was not accumulated by the gland, and there is no evidence for uptake of this protein from the circulation. We, therefore, infer that the increased NGF concentration in male submaxillary glands is due to a 10-fold increase in the rate constant of synthesis.     

Acetylcholine supersensitivity in the rat heart produced by neonatal sympathectomy

Effects of neonatal sympathectomy with antiserum to nerve growth factor or 6-hydroxydopamine on the acetylcholine sensitivity of the rat left atria were investigated. Sensitivities to acetylcholine of atria from immunologically and chemically sympathectomized rats were much higher than that of control at 4 weeks of age. These results suggest possible involvement of the sympathetic nervous system in regulation of cardiac cholinergic sensitivity.     

Localization of nerve growth factor receptor mRNA in the rat basal forebrain within situ hybridization histochemistry

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.     

Regulation of ornithine aminotransferase mRNA levels in rat kidney by estrogen and thyroid hormone

The regulation of the mitochondrial matrix enzyme, ornithine aminotransferase, by estrogen and triiodothyronine (T3) in rat kidney was examined using a cloned cDNA probe and in vitro translation of poly(A+) RNA. After a single, acute dose of either 17 beta-estradiol or T3, the rate of enzyme synthesis and the levels of translatable and hybridizable ornithine aminotransferase mRNA all increase in parallel. Levels of hybridizable mRNA were estimated by hybridization of randomly 32P-labeled RNA to filter-bound plasmid DNA. Maximal levels of induction by estrogen and T3 were about 15- and 3-fold, respectively. Lag times of at least 5 h and less than 3 h were observed for induction by T3 and estrogen. T3 and estrogen exert a synergistic effect in increasing ornithine aminotransferase mRNA levels. 16 h after T3 administration and 24 h after estrogen administration, a 1.6- and 13-fold increase in mRNA levels were observed. Both of these treatments in combination for the indicated time periods resulted in a 21-fold increase in ornithine aminotransferase mRNA. From the mRNA accumulation curves, half-lives of 10 to 14 h and 12 to 16 h were estimated for the mRNA after estrogen and T3 induction, respectively. These similar half-lives suggest that an increase in the rate of mRNA production is primarily responsible for the induction observed after estrogen administration.     

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.