Increased Nerve Growth Factor mRNA Research Articles

Synthesis and secretion of Nerve Growth Factor is regulated by Nitric Oxide in bladder cells in vitro under a hyperglycemic environment

Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2′-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.

Treatment with platelet-derived growth factor (PDGF) and rock inhibitors is related to declined nerve growth factor (NGF) signaling in an experimental model of rat pulmonary hypertension.

Recent studies reveal that nerve growth factor (NGF) plays a critical role in the pathobiology of pulmonary hypertension (PH). The aim of the present study is to clarify the relationship between NGF signaling and treatment with PDGF or ROCK inhibitors in an animal model of PH. Lung tissues were obtained from animals with monocrotaline (MCT)-challenged PH which had been administered long term imatinib, fasudil or statin. Reversal of disease was indicated by decreases in right ventricle pressure (RVP) and hypertrophy. NGF expression was examined at the mRNA and protein levels using quantitative real-time PCR reaction and ELISA. MCT significantly increased NGF mRNA and protein content in lung tissue. ROCK inhibitor (fasudil) and PDGF inhibitor (imatinib) caused significant decreases in NGF mRNA and protein content when administered alone, with no further effects noted when used in combination. The beneficial reversal of MCT-mediated effects in PH caused by PDGF or ROCK inhibition may be also partially mediated by decreased NGF signaling.

Human Tryptase Cleaves Pro-Nerve Growth Factor (Pro-NGF): HINTS OF LOCAL, MAST CELL-DEPENDENT REGULATION OF NGF/PRO-NGF ACTION

Several factors regulate nerve growth factor (NGF), which is formed from pro-NGF by intracellular and extracellular enzymatic cleavage. The close proximity between mast cells expressing the protease tryptase and NGF-producing smooth muscle-like peritubular cells in the testes of infertile patients led us to examine whether tryptase is among those factors. Human peritubular cells express functional tryptase receptors (PAR-2). Recombinant enzymatically active β-tryptase increased NGF levels in the culture medium of primary human peritubular cells, but the peptide agonist for PAR-2 (SLIGKV) did not. Neither tryptase nor the peptide increased NGF mRNA levels. To test whether the increase in NGF is due to enzymatic activity of tryptase acting on pro-NGF, supernatants of peritubular cells and synthetic pro-NGF were treated with tryptase. Results of Western blot studies indicate enzymatic cleavage of pro-NGF by active tryptase. Heat-inactivated tryptase or SLIGKV was not effective. Mass spectrometry analysis of in vitro cleavage products from recombinant tryptase and synthetic pro-NGF revealed multiple cleavage sites within the pro-NGF sequence. The results also indicate the generation of mature NGF and smaller NGF fragments as a result of tryptase action. Thus, tryptase-secreting mast cells in the vicinity of pro-NGF/NGF-secreting cells in any human tissue are likely able to alter the ratios of pro-NGF/NGF. As NGF and pro-NGF have different affinities for their receptors, this indicates a novel way by which mast cells, via tryptase, can modify the microenvironment in human tissues with regard to neurotrophin actions.

TRPV1 and TRPA1 Function and Modulation Are Target Tissue Dependent

The nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) families of growth factors regulate the sensitivity of sensory neurons. The ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential channel, subfamily A, member 1 (TRPA1), are necessary for development of inflammatory hypersensitivity and are functionally potentiated by growth factors. We have shown previously that inflamed skin exhibits rapid increases in artemin mRNA with slower, smaller increases in NGF mRNA. Here, using mice, we show that, in inflamed colon, mRNA for both growth factors increased with a pattern distinct from that seen in skin. Differences were also seen in the pattern of TRPV1 and TRPA1 mRNA expression in DRG innervating inflamed skin and colon. Growth factors potentiated capsaicin (a specific TRPV1 agonist) and mustard oil (a specific TRPA1 agonist) behavioral responses in vivo, raising the question as to how these growth factors affect individual afferents. Because individual tissues are innervated by afferents with unique properties, we investigated modulation of TRPV1 and TRPA1 in identified afferents projecting to muscle, skin, and colon. Muscle and colon afferents are twice as likely as skin afferents to express functional TRPV1 and TRPA1. TRPV1 and TRPA1 responses were potentiated by growth factors in all afferent types, but compared with skin afferents, muscle afferents were twice as likely to exhibit NGF-induced potentiation and one-half as likely to exhibit artemin-induced potentiation of TRPV1. Furthermore, skin afferents showed no GDNF-induced potentiation of TRPA1, but 43% of muscle and 38% of colon afferents exhibited GDNF-induced potentiation. These results show that interpretation of afferent homeostatic mechanisms must incorporate properties that are specific to the target tissue.

Synthesis and evaluation of brain-targeted chemical delivery systems for the neurotrophomodulator 4-methylcatechol.

Since various 4-alkylcatechols stimulate nerve growth factor (NGF) biosynthesis both in-vitro and in-vivo, delivery of these agents to the brain may provide beneficial effect for the treatment of neurodegenerative diseases such as Alzheimer's. Several dihydropyridine-pyridinium salt type redox chemical delivery systems (CDS) of 4-methylcatechol (4-methylcatechol) were prepared as potential brain selective targetry forms for 4-methylcatechol. After preliminary evaluation by in-vitro stability studies in various buffer solutions and biological media, a selected CDS was further investigated in the rat to determine its in-vivo distribution. Selective and sustained delivery of the compound of interest to the rat brain was achieved. Furthermore, the NGF stimulatory activity in the rat brain after peripheral administration of the selected CDS was evaluated by measuring the levels of pre-pro-NGF mRNA in the rat hippocampus and frontal cortex, by dot blot hybridization and analysis. Results showed the peripheral administration of the CDS to achieve a 1.7-fold increase in NGF mRNA compared to control in the rat hippocampus, and an approximately 1.4-fold increase in the frontal cortex.

Cytokine mRNA induction by interleukin-1β or tumor necrosis factor α in vitro and in vivo

Hypothalamic and cortical mRNA levels for cytokines such as interleukin-1β (IL1β), tumor necrosis factor alpha (TNFα), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are impacted by systemic treatments of IL1β and TNFα. To investigate the time course of the effects of IL1β and TNFα on hypothalamic and cortical cytokine gene expression, we measured mRNA levels for IL1β, TNFα, interleukin-6 (IL-6), interleukin-10 (IL-10), IL1 receptor 1, BDNF, NGF, and glutamate decarboxylase-67 in vitro using hypothalamic and cortical primary cultures. IL1β and TNFα mRNA levels increased significantly in a dose-dependent fashion after exposure to either IL1β or TNFα. IL1β increased IL1β mRNA in both the hypothalamic and cortical cultures after 2–6 h while TNFα mRNA increased significantly within 30 min and continued to rise up to 2–6 h. Most of the other mRNAs showed significant changes independent of dose in vitro. In vivo, intracerebroventricular (icv) injection of IL1β or TNFα also significantly increased IL1β, TNFα and IL6 mRNA levels in the hypothalamus and cortex. IL1β icv, but not TNFα, increased NGF mRNA levels in both these areas. Results support the hypothesis that centrally active doses of IL1β and TNFα enhance their own mRNA levels as well as affect mRNA levels for other neuronal growth factors.

Triptolide Upregulates NGF Synthesis in Rat Astrocyte Cultures

Triptolide (T10), an extract from the traditional Chinese herb, Tripterygium wilfordii Hook F (TWHF), has been shown to attenuate the rotational behavior induced by D: -amphetamine and prevent the loss of dopaminergic neurons in the substantia nigra in rat models of Parkinson's disease. To examine if the neuroprotective effect is mediated by its stimulation of production of neurotrophic factors from astrocytes, we investigated the effect of T10 on synthesis and release of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in rat astrocyte cultures. T10 did not affect the synthesis and release of either BDNF or GDNF. However, it significantly increased NGF mRNA expression. It also increased both intracellular NGF and NGF level in culture medium. These results indicate that the neuroprotective effect of T10 might be mediated, at least in part, via a stimulation of the production and release of NGF in astrocytes.

Brain distribution of cytokine mRNA induced by systemic administration of interleukin-1β or tumor necrosis factor α

Brain cytokine mRNA levels are impacted by systemic cytokines. For example, systemic interleukin-1β (IL1β) increases brain IL1β mRNA; subdiaphragmatic vagotomy blocks this effect. To localize which brain regions respond to intraperitoneal cytokines, we measured mRNA levels in selected brain regions for a variety of cytokines and growth factors, IL1β, TNFα, interleukin-6 (IL-6), interleukin-10 (IL10), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Relative to saline administration, IL1β increased IL1β, TNFα and IL6 mRNAs in the nucleus tractus solitarius (NTS), hypothalamus, hippocampus and somatosensory cortex (SSctx), but did not induce any changes in IL10. TNFα also increased TNFα and IL1β mRNAs in the hypothalamus, hippocampus and SSctx. TNFα increased TNFα, IL1β and IL10 mRNAs in the NTS, but did not induce any changes in IL-6 mRNA. In the amygdala, IL1β enhanced IL6 mRNA and TNFα increased IL1β mRNAs. In the insular cortex, IL1β enhanced IL6 mRNA and TNFα increased IL1β mRNA. TNFα administration increased NGF mRNA in the SSctx but decreased NGF and BDNF mRNA levels in the insular cortex. Both IL1β and TNFα decreased BDNF mRNA in the amygdala. We also verified the IL1β-induced increases in TNFα mRNA within the NTS using in situ hybridization. These results support the hypothesis that somnogenic doses of IL1β and TNFα enhance their own mRNA levels as well as affect mRNA levels for other sleep-promoting substances.

Dexamethasone enhances upregulation of nerve growth factor mRNA expression in ischemic rat brain

Nerve growth factor (NGF) is well-established as a trophic factor that plays a crucial role in neuroregeneration and plasticity after brain insults. Dexamethasone (DEX), a powerful glucocorticoid steroid, has long been used in the clinical management of neurological disorders. We examined the relationship between NGF and DEX after an ischemic insult to the brain. In situ hybridization was used to measure NGF mRNA expression in the rat hippocampus after 20 min of transient forebrain ischemia. Immunostaining for NGF protein was performed using the avidin-biotin peroxidase method. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was also used to study the astrocyte reaction in the hippocampal CA1 area. Ischemic brain from rats not treated with DEX had a 2 and 3 fold increase in NGF mRNA compared to sham-operated rats at 4 and 6 h after ischemia, respectively. The NGF mRNA expression returned to basal levels 12 h to 7 days post-ischemia. Treatment with DEX potentiated the ischemia-induced increase of NGF mRNA to 4 times that of sham-operated rats at 6 h following reperfusion and NGF protein expression was similarly elevated. Additionally, the number of GFAP positive astrocytes in the CA1 region in the ischemic rats was markedly increased. These data suggest that DEX may play a role in modulating NGF mRNA expression in the hippocampal neuronal response to brain ischemia.

Synergistic effect of dexamethasone and β-adrenergic receptor agonists on the nerve growth factor gene transcription

Activation of β-adrenergic receptor (βAR) increases the synthesis of nerve growth factor (NGF) in the brain and in C6-2B glioma cells. However, in the brain, the βAR-mediated increase in NGF expression appears to require the presence of glucocorticoids, suggesting that NGF promoter may be sensitive to cAMP and glucocorticoid-dependent transcription factors. We tested this hypothesis by exposing C6-2B glioma cells to dexamethasone (DEX) in combination with agents that increase cAMP levels and examining the DNA binding activity of two cAMP-dependent transcription factors that regulate NGF expression: cAMP responsive element binding protein (CREB) and CCAAT/enhancer binding protein δ (C/EBPδ). Electrophoretic mobility shift assays revealed that the β 2AR agonist clenbuterol (CLE) or high levels of cAMP elicited a time-dependent increase in C/EBPδ binding activity as well as phosphorylated CREB (P-CREB). When DEX, which per se showed little effect on these transcription factors, was combined with CLE, dibutyryl cAMP or isoproterenol, enhanced induction of P-CREB and C/EBP binding activity as well as NGF mRNA was observed. Moreover, the increase in NGF mRNA in the presence of DEX was prolonged compared to that obtained by CLE or other cAMP inducing agents alone. In fact, NGF mRNA levels remained significantly elevated at least for 24 h. These studies suggest that the synergistic effect of DEX on the induction of NGF mRNA may include the ability of this glucocorticoid to potentiate the βAR-mediated induction of transcription factors.

NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines.

The sympathetic nervous system plays a central role in lipolysis and the production of leptin in white adipose tissue (WAT). In this study, we have examined whether nerve growth factor (NGF), a target-derived neurotropin that is a key signal in the development and survival of sympathetic neurons, is expressed and secreted by white adipocytes. NGF mRNA was detected by RT-PCR in the major WAT depots of mice (epididymal, perirenal, omental, mesenteric, subcutaneous) and in human fat (subcutaneous, omental). In mouse WAT, NGF expression was observed in mature adipocytes and in stromal vascular cells. NGF expression was also evident in 3T3-L1 cells before and after differentiation into adipocytes. NGF protein, measured by ELISA, was secreted from 3T3-L1 cells, release being higher before differentiation. Addition of the sympathetic agonists norepinephrine, isoprenaline, or BRL-37344 (beta(3)-agonist) led to falls in NGF gene expression and secretion by 3T3-L1 adipocytes, as did IL-6 and the PPARgamma agonist rosiglitazone. A substantial decrease in NGF expression and secretion occurred with dexamethasone. In contrast, LPS increased NGF mRNA levels and NGF secretion. A major increase in NGF mRNA level (9-fold) and NGF secretion (<or=40-fold) in 3T3-L1 adipocytes occurred with TNF-alpha. RT-PCR showed that the genes encoding the p75 and trkA NGF receptors were expressed in mouse WAT. These results demonstrate that white adipocytes secrete NGF (an adipokine), NGF synthesis being influenced by several factors with TNF-alpha having a major stimulatory effect. We suggest that NGF is a target-derived neurotropin and an inflammatory response protein in white adipocytes.

Integrins regulate neuronal neurotrophin gene expression through effects on voltage-sensitive calcium channels

Integrin adhesion receptors regulate gene expression during growth and differentiation in various cell types. Recent work, implicating integrins in functional synaptic plasticity, suggest they may have similar activities in adult brain. The present study tested if integrins binding the arginine–glycine–aspartate (RGD) matrix sequence regulate neurotrophin and neurotrophin receptor gene expression in cultured hippocampal slices. The soluble RGD-containing peptide glycine–arginine–glycine–aspartate–serine–proline (GRGDSP) increased neurotrophin mRNA levels in transcript- and subfield-specific fashions. Integrin ligand effects were greatest for brain-derived neurotrophic factor transcripts I and II and barely detectable for transcript III. In accordance with increased nerve growth factor mRNA levels, GRGDSP increased c- fos expression as well. In contrast, growth-associated protein-43, amyloid precursor protein and fibroblast growth factor-1 mRNAs were not elevated. Ligand effects on brain-derived neurotrophic factor transcript II and c- fos mRNA did not depend on the integrity of the actin cytoskeleton, neuronal activity, or various signaling pathways but were blocked by L-type voltage-sensitive calcium-channel blockers. These results indicate that in mature hippocampal neurons integrin engagement regulates expression of a subset of growth-related genes at least in part through effects on calcium influx. Accordingly, these synaptic adhesion receptors may play the same role in maintaining an adult, differentiated state in brain as they do in other tissues and changes in integrin activation and/or engagement may contribute to dynamic changes in neurotrophin expression and to neuronal calcium signaling.

Interleukin-10 and nerve growth factor have reciprocal upregulatory effects on intestinal epithelial cells.

The intestinal mucosa is in a constant state of controlled inflammation, but the processes whereby this occurs are poorly understood. The aims of this study were to look at the role of IL-10 and nerve growth factor (NGF) in intestinal epithelial cell regulation. The human colon epithelial cell lines T84, HT-29, and CACO-2 were used. RT-PCR, flow cytometry analysis, and immunohistochemistry were applied to measure the cytokine changes in epithelial cells induced by recombinant cholera toxin and its B subunit, IL-10, and NGF. Cholera toxin B subunit caused selective dose-dependent increased mRNA for IL-10 in T84 cells and the protein in T84, HT-29, and CACO-2 cells. IL-10 dose dependently selectively increased NGF mRNA in T84 cells and intracellular protein synthesis in all three epithelial cell lines. The effect of NGF was reciprocal, selective, and dose dependent because it increased mRNA for IL-10 and IL-10 synthesis. Our results suggest that the epithelium may actively participate in downregulation through innate mechanisms involving IL-10 and NGF. The reciprocal interaction suggests for the first time that NGF may be involved in local downregulation by mucosal epithelium and thus may play a potent protective role in response to injury, by prevention of undue inflammation.

Brain-Derived Neurotrophic Factor Increases in the Uninjured Dorsal Root Ganglion Neurons in Selective Spinal Nerve Ligation Model

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two major members of the neurotrophin family. Using immunohistochemistry and in situ hybridization histochemistry, we examined the effect of L5 spinal nerve ligation (SPNL), a neuropathic pain model, on the expression of BDNF in the uninjured L4 dorsal root ganglion (DRG). After L5 SPNL, both immunoreactivity for BDNF and the hybridization intensity for BDNF mRNA increased mainly in the small- and medium-sized neurons. The percentage of BDNF mRNA-expressing neurons increased in the ipsilateral L4 DRG compared with the contralateral DRG from the third to 28th day after ligation. A significantly greater number of BDNF-immunoreactive neurons were observed in the ipsilateral L4 DRG than contralateral side 14 d after ligation. To test the contribution of BDNF to the thermal hyperalgesia produced in this model, we intrathecally injected anti-BDNF antibody at third day after ligation. This treatment clearly attenuated thermal hyperalgesia for a few hours. Almost all BDNF mRNA-expressing neurons coexpressed trkA, a high-affinity NGF receptor, mRNA. The percentage of BDNF mRNA-expressing cells of trkA cells significantly increased in the ipsilateral L4 DRG 14 d after ligation. Furthermore, we examined the contribution of NGF on this phenotypic change using ELISA, Northern blot analysis, and anti-NGF antibody. NGF content in the ipsilateral L4 DRG linearly increased and reached a statistical significant level 14 d after L5 SPNL. Moreover, at this time point, the increase in NGF mRNA was observed in the ipsilateral L5 DRG and sciatic nerve, but not in the ipsilateral L4 DRG or L4 spinal nerve. Local application of anti-NGF antibody to the L4 spinal nerve beside the L5 spinal nerve-ligation site prevented the development of thermal hyperalgesia for 5 d after ligation. Our data suggest that BDNF, which increased in the uninjured L4 DRG neurons, acts as a sensory neuromodulator in the dorsal horn and contributes to thermal hyperalgesia in this neuropathic pain model. The contribution of locally synthesized NGF to thermal hyperalgesia was also demonstrated. These dynamic alterations in the expression and content of BDNF and NGF in the uninjured DRG neurons might be involved in the pathomechanisms of neuropathic pain.

Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury

Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.

Pharmacological modulation of nerve growth factor synthesis: a mechanistic comparison of vitamin D receptor and β 2-adrenoceptor agonists

Increasing nerve growth factor (NGF) in the PNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were undertaken to compare two compounds, a vitamin D 3 analogue (CB1093) with minimal calcaemic effects, and clenbuterol, a long-acting β 2-adrenoceptor agonist, both of which induce NGF synthesis in vivo. Clenbuterol caused significant increases in both NGF mRNA and protein in 3T3 cells; with maxima at 10 nM and at 8–12 h exposure. Effects of clenbuterol on NGF mRNA were antagonized by propranolol. Mobility shift assays on whole cell extracts showed that clenbuterol increased AP1 binding in 3T3 cells prior to increasing NGF synthesis. Clenbuterol was without effect on NGF mRNA levels in L929 cells, whereas CB1093 caused significant increases in both NGF mRNA and protein levels in both 3T3 and L929 cells. Stimulation was almost maximal at 24 h exposure and was sustained for at least 72 h. The magnitude of the increase was much greater in L929 (700% increase) than in 3T3 cells (80%). Binding to the vitamin D nuclear receptor (VDR), which acts as a transcription factor itself, was increased as early as 30 min after exposure to of CB1093 and maintained up to 24 h. Increased VDR binding preceded increased NGF mRNA. A 150% increase in AP-1 binding was also evident. This study demonstrates that CB1093 and clenbuterol stimulate NGF levels in vitro and that AP-1 binding could be a commonality between the mechanism of NGF induction of these two compounds.

Increased nerve growth factor mRNA in lateral calf skin biopsies from diabetic patients.

This study set out to establish a novel procedure for the measurement of human nerve growth factor (NGF) messenger ribonucleic acid (mRNA) and to use this method to measure NGF expression in skin biopsies from control subjects and from patients with early neuropathies. NGF mRNA levels were related to functional measures of the competence of NGF-responsive nerves. mRNA levels were measured by competitive reverse transcription with polymerase chain reaction amplification (cRT-PCR). Functional correlates of this observation were assessed by indices of thermal sensitivity--mediated by C-fibres, whose phenotype is regulated by NGF. NGF mRNA was increased in skin biopsies from 19 diabetic patients (5.12+/-3.88 (SD)) compared with samples from eight controls (1.57+/-0.95; P=0.001). Diabetic patients showed significantly (P < 0.001) diminished detection of cool and warm stimuli compared to age matched control group (n=24), but there were no differences in detection of heat as pain, or correlation with NGF mRNA levels. These findings suggest abnormally increased expression of NGF in diabetic neuropathy, which may represent a compensatory mechanism for impaired phenotype in NGF-responsive neurones.

Dexamethasone Induces Hypertrophy of Developing Medial Septum Cholinergic Neurons: Potential Role of Nerve Growth Factor

Glucocorticoid hormones influence neuronal plasticity during development; however little is known about the mechanisms of this trophic activity. Because glucocorticoids increase nerve growth factor (NGF) synthesis in selected brain areas and NGF plays a role in the development of basal forebrain cholinergic neurons, we tested the hypothesis that glucocorticoids may foster maturation of the cholinergic phenotype during postnatal development via the induction of NGF biosynthesis. The synthetic glucocorticoid dexamethasone (DEX) was injected systemically (0.5 mg/kg, s.c.) once a day for 1 week in 7-d-old (P7) rats. DEX elicited an increase in NGF mRNA and protein levels in the cerebral cortex and hippocampus as well as specific NGF responses, such as TrkA tyrosine phosphorylation in the septum, choline acetyltransferase (ChAT) and p75 neurotrophin receptor (p75NTR) immunoreactivity, and a relative number of cholinergic neurons in the medial septum. To examine whether the effect of DEX is age-related, we treated 1- and 14-d-old rats with DEX for 1 week. DEX increased NGF expression in rats treated from P1 to P8 but not in those treated from P14 to P21. The age-related increased expression of NGF correlated with the induction of ChAT immunoreactivity in the medial septum. Moreover, in the spinal cord, neither NGF nor ChAT levels were increased by DEX, suggesting that the glucocorticoid-mediated changes seen in the basal forebrain are associated with specific NGF responses. Our data suggest that by increasing NGF levels, glucocorticoids may play a role in the maturation of postnatal cholinergic neurons.

Increased nerve growth factor mRNA stability may underlie elevated nerve growth factor secretion from hypertensive vascular smooth muscle cells

Altered nerve growth factor (NGF) regulation has been linked to the pathophysiology of hypertension. Vascular smooth muscle cells from an inbred hypertensive, but normoactive rat strain (WKHT) secreted NGF at a greater rate than from a hyperactive, normotensive strain (WKHA). Exposure to phorbol ester increased NGF secretion rates from WKHT by 400–800% but not from WKHA vascular muscle. NGF secretion rates from both WKHT and WKHA vascular cells were elevated by co-application of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) by 300–1000%. This response was partially attenuated by actinomycin D, an inhibitor of RNA transcription. These results suggest that regulation of NGF production does not occur solely at the level of transcription and post-transcriptional mechanisms operate. Analysis of NGF mRNA stability in the two strains following PDGF and TGF-β1 treatment showed that NGF mRNA in WKHT had a half-life of 126.2±11.68 min while in WKHA vascular smooth muscle cells, the half-life was 47.33±11.98 min. In addition to increased NGF mRNA stability in WKHT vascular muscle, these cells have an increased translational efficiency of NGF protein; elevated synthesis of NGF protein per unit NGF mRNA. Differences in signaling pathways may result in increased NGF mRNA stability and translational efficiency that may account for the elevated NGF protein in WKHT vascular smooth muscle cells.

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain

Stimulation of glucocorticoid or β-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2–3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.