Endogenous glutamate levels regulate nerve growth factor mRNA expression in the rat dentate gyrus

The levels of nerve growth factor (NGF) mRNA can be regulated in vitro and in vivo in the hippocampal formation by events associated with pharmacological activation of glutamate receptors. In the present study, the level of NGF mRNA in the hippocampal formation was examined following an intrahippocampal injection of 1 nmole fluorocitrate, which temporarily inhibits the astrocyte metabolic activity in vivo. Consistent with previous findings, fluorocitrate treatment significantly increased glutamate levels and decreased glutamine levels in the dentate gyrus as determined by in vivo microdialysis. The increased ratio of glutamate to glutamine was followed by a significant increase in NGF mRNA expression selectively in dentate gyrus granule cells. The effects of increasing glutamate levels were blocked by pretreatment with 50 nmole 2-amino-5-phosphonovalerate (AP5), a competitive antagonist that acts at the N-methyl-D-aspartate (NMDA) glutamate receptor subtype. These findings suggest that NGF mRNA expression is regulated, in part, by changes in endogenous glutamate levels, partially through enhanced excitatory neurotransmission through NMDA receptors.     

Nerve growth factor NT-5 induce increased thermal sensitivity of cutaneous nociceptors in vitro

1. Perfusion of the receptive field (RF) of C- or A delta-fiber nociceptors with nerve growth factor (NGF) in an in vitro preparation of the rat saphenous nerve with functionally attached skin induced a significant decrease in heat threshold without changing mechanical or cold sensitivity. 2. NGF-induced thermal sensitization was absent after saline perfusion and in skin taken from mast-cell depleted animals, hence confirming a role for mast cells in NGF-induced thermal hyperalgesia. 3. Neurotrophin-5 (NT-5) also induced a small but significant reduction in heat threshold without affecting mechanical sensitivity. It is speculated that NT-5 exerts its action either directly on the trkA receptor, as with NGF or alternatively through trkB receptors located on sympathetic efferents or on small diameter afferents.     

Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain

Adrenocorticotropin hormone (ACTH) and adrenal steroids may influence trophic processes operative in neuronal plasticity. Because nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) participate in neuronal trophism, we have investigated whether adrenal steroids induce the expression of these two trophic factors in the rat brain. The systemic administration of dexamethasone (DEX) elicited a rapid (within 3 hr) and sustained accumulation of bFGF and NGF mRNA in the cerebral cortex and hippocampus. Regional studies showed that DEX increases bFGF but not NGF mRNA in the cerebellum, striatum, and hypothalamus. In situ hybridization studies revealed that DEX increases NGF mRNA in superficial layers of the cerebral cortex and in the dentate gyrus of the hippocampus, and bFGF mRNA throughout the brain, suggesting that DEX induces NGF mRNA in neurons and bFGF in glial cells. ACTH administered systemically elicited a temporal and regional induction in NGF and bFGF mRNA similar to that obtained with DEX. Increases in NGF and bFGF mRNAs were also observed after administration of corticosterone and, albeit to a lesser extent, aldosterone, suggesting that the pituitary-adrenocortical axis plays an important role in the regulation of NGF and bFGF expression in the brain. Our data suggest that NGF and bFGF represent a link by which the adrenal cortical system can exert trophic action on the CNS.     

Dexamethasone regulates basic fibroblast growth factor, nerve growth factor and S100beta expression in cultured hippocampal astrocytes

Glucocorticoids regulate hippocampal neuron survival during fetal development, in the adult, and during aging; however, the mechanisms underlying the effects are unclear. Since astrocytes contain adrenocortical receptors and synthesize and release a wide variety of growth factors, we hypothesized that glucocorticoids may alter neuron-astrocyte interactions by regulating the expression of growth factors in hippocampal astrocytes. In this study, three growth factors, which are important for hippocampal neuron development and survival, were investigated: basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and S100beta. Enriched type I astrocyte cultures were treated with 1 microM dexamethasone (DEX), a synthetic glucocorticoid, for up to 120 h. Cells and culture medium were collected and total RNA and protein were measured at 6, 12, 24, 48, 72, 96 and 120 h after the initiation of hormone treatment. Growth factor mRNA levels were measured and quantified using solution hybridization-RNase protection assays and protein levels were quantified using ELISA methods. We report that DEX stimulates the bFGF mRNA levels over the 120-h treatment. In contrast, DEX suppresses NGF mRNA continuously over the same period of treatment. DEX induces a biphasic response in S100beta mRNA levels. In addition, some of the changes in gene expression are translated into parallel changes in protein levels of these growth factors. Our results demonstrate that dexamethasone can differentially regulate the expression of growth factors in hippocampal astrocytes in vitro. This suggests that one of the mechanisms through which glucocorticoids affect hippocampal functions may be by regulating the expression of astrocyte-derived growth factors.     

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

Stimulation of glucocorticoid or beta-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.     

Endogenous tumor necrosis factor and doxorubicin sensitivity in leukemic patients

Sensitivity to doxorubicin (DOX) and expression of intracellular endogenous TNF (enTNF) or manganous superoxide dismutase (MnSOD) activity were found to be inversely correlated in leukemic cells from 19 patients (6 acute lymphoblastic leukemia, 13 acute myeloblastic leukemia). In a case with acquired resistance to chemotherapy which included DOX, enTNF expression and MnSOD activity were increased. Furthermore, in 14 cases treated with a regimen including an anthracycline, 4 cases which failed to respond to chemotherapy showed relatively high amounts of enTNF expression. After TNF antisense cDNA was transfected into leukemic cells isolated from 5 patients, sensitivity of transfectants to DOX increased 1.4- to 2.5-fold. Overall, we found enTNF to act as a resistance factor against DOX in leukemia which may predict response to DOX.     

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.     

Catecholamine-induced mechanical sensitization of cutaneous nociceptors in the rat

C-Fiber mechanoheat (C-MH) nociceptors from the saphenous nerve were studied, in control rats and in rats that underwent surgical sympathectomy. Intradermal injection, alone, of either norepinephrine (NE) or the calcium ionophore, A23187, did not affect mechanical threshold. The combination of A23187 and NE, however, significantly decreased mechanical threshold. In the presence of the alpha 2-adrenergic antagonist, yohimbine, or the cyclooxygenase inhibitor, indomethacin, C-MHs were not sensitized by the combination of NE + A23187. One week after surgical sympathectomy, the number of C-MHs sensitized by NE + A23187 was significantly reduced. In summary, NE appears to sensitize nociceptors indirectly. These data are compatible with the suggestion that a sympathetic postganglionic neuron-dependent release of prostaglandins mediates the sensitization. NE appears to act at an alpha 2-adrenergic receptor, only in the presence of an increased intracellular Ca2+.     

Expression of the low-affinity p75 nerve growth factor receptor in the developing rat pituitary gland

We investigated, by means of in situ hybridization with a digoxigenin-labelled RNA probe, the expression of the low-affinity p75 nerve growth factor receptor (NGFR) in the developing pituitary primordium of the rat. In 13-day pc embryos, intense staining of p75 NGFR mRNA was present in the cytoplasm of all cells of Rathke's pouch. In day-17 pc embryos p75 NGFR expression was present primarily in the cells of the intermediate lobe. In the newborn rat pituitary only very weak staining was observed, predominantly in the intermediate lobe. In neural structures the staining at day 13 pc was comparable to that of day 17 pc. Since p75 expression is seen very early during pituitary development and declines during the time the expression of pituitary hormonal phenotypes are steadily increasing, we suggest that the p75 NGFR expression in Rathke's pouch may play a temporally defined role in the commitment rather than in the differentiation of the various pituitary cell types.     

Effect of yohimbine on nerve growth factor mRNA and protein levels in rat hippocampus

Activation of noradrenergic receptors has been shown to increase expression of nerve growth factor (NGF) gene in brain cells in vitro. The present studies were undertaken to determine if this stimulation was effective in vivo as well. Rats were administered the norepinephrine-releasing drug, yohimbine (YOH), and had their hippocampi assayed for NGF mRNA and protein at various times after the injection. It was found that yohimbine caused a 3-fold increase of NGF mRNA levels at 24 h. Protein levels, however, were unaltered at this time. Thus norepinephrine release in vivo appears to be sufficient for increasing mRNA level but not for translation to protein.     

Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat

A between-side comparison of GABAA receptor subunit expression levels in the globus pallidus and anterior-pole motor thalamic nuclei of rats with an ibotenate lesion of the striatum, and rats receiving a fetal striatal graft in the lesioned area was made by using immunocytochemistry with subunit-specific antibodies, at different times post-lesion or different times post-grafting. At 10 days post-lesion, there was already an increase in the labeling of the alpha 1- and beta 2/3-subunits in the globus pallidus, entopeduncular nucleus and ventrolateral nucleus ipsilateral to the lesion when compared with the contralateral side, while there were no significant changes at the level of the ventromedial nucleus. Labeling of the alpha 2-subunit showed a clear increase in the entopeduncular nucleus compared with the contralateral side at 10 days post-lesion. Similar changes were also observed for the different subunits studied at 30 and 120 days after lesioning. Rats with 20-day old transplants of fetal striatal neurons that were implanted in the ibotenate lesioned striatum at 10 days post-lesioning, continued to show changes in the expression of GABAA receptor subunits, albeit at a lower level than those of ibotenate lesioned rats at similar age post-lesion. However, when examining rats with 70-day old transplants, the ibotenate-lesion induced between-side changes were almost completely compensated. These findings suggest a correlation between the maturation of the grafts and their capability to function in reestablishing neuronal circuits as shown by the reduction of changes in GABAergic transmission induced by ibotenate lesions, as indicated by the reversal of changes in GABAA receptor subunit in several areas of the basal ganglia circuit.     

Negative inotropic effect of basic fibroblast growth factor on adult rat cardiac myocyte

BACKGROUND: Basic fibroblast growth factor (bFGF) is highly expressed in the myocardium in some cardiac disorders, such as ischemia-reperfusion and cardiac allograft rejection. However, whether bFGF has any effects on myocardial contraction is unknown. METHODS AND RESULTS: We examined the effects of bFGF on myocardial contractility using isolated adult rat cardiac myocyte preparations. bFGF exerted a direct negative inotropic effect that was concentration and time dependent. The pretreatment of myocytes with a neutralizing anti-bFGF antibody (100 ng/mL) abolished the negative inotropic effects of bFGF (100 ng/mL). Platelet-derived growth factor (12.5 ng/mL) and transforming growth factor-beta (1 ng/mL) did not exert such effects, which indicated that bFGF-induced negative inotropism was considered to be specific for this growth factor. bFGF decreased the peak intracellular Ca2+ transient by 46% during systole. The enhanced production of nitric oxide was unlikely to be responsible for the bFGF-induced negative inotropic effect. CONCLUSIONS: bFGF, primarily a potent growth promoter, produced acute negative inotropic effects in the adult cardiac myocyte that could have resulted from alterations in intracellular Ca2+ homeostasis. The negative inotropic effect of bFGF may contribute to myocardial dysfunction associated with ischemia-reperfusion injury and heart transplant rejection.     

Indicators of oxidative stress in aging rat brain. The effect of nerve growth factor

INTRODUCTION AND OBJECTIVE: Increased oxidative stress during ageing and the neurodegenerative disorders associated with this has been described. The central nervous system is particularly vulnerable to oxidative damage because of its high energy requirements, high oxygen consumption, high tissue concentration of iron and relatively low levels of some antioxidant systems. Treatment with neurotrophic factors may reverse neurone deterioration and stimulate cholinergic activity in aged rats. It may have a similar neuroprotector effect against damage due to ischaemic reperfusion, hypoglycaemia, inflammation and other pathological conditions involving oxidative stress. In this study we determined some indicators of oxidative stress in rat brains during ageing and evaluated this in response to a plan of treatment with murine nerve growth factor (FCN) for 38 days. MATERIAL AND METHODS: Biochemical techniques were used for determination of oxidative stress indicators. RESULTS AND CONCLUSIONS: We found that with age there was a significant increase in phospholipase A2 and superoxide dysmutase activity and concentration of hipoperoxidases, whilst the concentration of reduced glutathion fell. Catalase activity increased in the hippocampal and striate regions and decreased in the cortex and septal area. There was less oxidative stress in rats treated with FCN. In view of our results, we conclude that the level of oxidative stress increases with ageing, with significant differences between areas of the brain. The region most vulnerable to damage from species reactive to oxygen was the hippocampus, and the protective effect of FCN may be related to potentiation of antioxidant defenses.     

Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain

Neurons and glia are generated throughout adulthood from proliferating cells in two regions of the rat brain, the subventricular zone (SVZ) and the hippocampus. This study shows that exogenous basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) have differential and site-specific effects on progenitor cells in vivo. Both growth factors expanded the SVZ progenitor population after 2 weeks of intracerebroventricular administration, but only FGF-2 induced an increase in the number of newborn cells, most prominently neurons, in the olfactory bulb, the normal destination for neuronal progenitors migrating from the SVZ. EGF, on the other hand, reduced the total number of newborn neurons reaching the olfactory bulb and substantially enhanced the generation of astrocytes in the olfactory bulb. Moreover, EGF increased the number of newborn cells in the striatum either by migration of SVZ cells or by stimulation of local progenitor cells. No evidence of neuronal differentiation of newborn striatal cells was found by three-dimensional confocal analysis, although many of these newborn cells were associated closely with striatal neurons. The proliferation of hippocampal progenitors was not affected by either growth factor. However, EGF increased the number of newborn glia and reduced the number of newborn neurons, similar to the effects seen in the olfactory bulb. These findings may be useful for elucidating the in vivo role of growth factors in neurogenesis in the adult CNS and may aid development of neuronal replacement strategies after brain damage.     

Platelet-derived growth factor B-chain expression in the rat retina and optic nerve: distribution and changes after transection of the optic nerve

To test the possible involvement of platelet-derived growth factor B-chain (PDGF-B) in anterograde and retrograde degenerations of the CNS neurons, we studied the changes of PDGF-B localization and its mRNA expression in the rat retina and optic nerve (ON) after unilateral ON transection, using immunohistochemistry and in situ hybridization. In the control retinas immunoreactivity for PDGF-B and its mRNA expression were localized in the retinal ganglion cells (RGCs) and the nerve fiber layer. After ON transection PDGF-B immunoreactivity in the nerve fiber layer started to decrease on post-injury day 3 or 4. Atrophic changes in the RGCs started on day 5 just after the decrease of PDGF expression, and thereafter the RGC number decreased. In the longitudinal section of the ON rostral to the transected site, swollen axons showed intense PDGF-B immunoreactivity and macrophages, and some glial cells revealed a significant increase in both immunoreactivity and hybridization signals. Based on these findings, we hypothesized that the decrease in PDGF-B in RGCs after axotomy causes the loss of RGCs, and that increased PDGF-B expression in the ON plays a role in the cascade of tissue reactions following ON transection.     

Relationship between DNA synthesis and growth factor expression in primary cultures of adult rat hepatocytes

In this study we employed primary culture of adult rat hepatocytes to verify the effects of two different extracellular matrices (collagen, matrigel) on EGF-stimulated DNA synthesis and c-myc expression. Our results confirm that in adult rat hepatocytes EGF induces DNA synthesis, preceded by a transient increase of c-myc expression, when cells are cultured at low density on collagen. DNA synthesis appears to be in reciprocal relationship with hepatic expression of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-4, suggesting that IGF-I/IGFBPs system is not involved in liver growth.     

Endogenous dopamine regulates phosphate reabsorption but not NaK-ATPase in spontaneously hypertensive rat kidneys

Dopamine's modulatory actions on signal transduction in the spontaneously hypertensive rat (SHR) proximal tubule are blunted; therefore, it was predicted that dopamine does not regulate phosphate (Pi) reabsorption in SHR. To test this hypothesis, dopamine production was inhibited with carbidopa (10 mg/kg ip) 18 h before and during clearance measurements of chronically denervated SHR and Wistar-Kyoto (WKY) rat kidneys. Dopamine excretion decreased 80% from SHR and 85% from WKY rats. Pi excretion decreased 60 to 67%. Plasma Pi and calcium, inulin clearance, and Na excretion did not change. Citrate excretion, which reflects proton secretion by proximal tubules, decreased 72% from WKY rats. Citrate excretion was significantly lower from SHR (5 +/- 10 pmol/min) than from WKY rats (73 +/- 11 pmol/min) and was not altered by carbidopa. Carbidopa, injected 18 and 1 h before kidneys were collected, increased NaK-ATPase in cortical basolateral membranes from WKY rats (27%) but not in membranes from SHR. After the incubation of renal cortical minceates for 15 min with L-DOPA (10(-5) M), there was no change in brush border membrane vesicle uptake of 32Pi, (3H)glucose, or (14C)citrate. Incubation with carbidopa (10(-4) M) increased 32Pi uptake by 11% (P < 0.001) and (3H)glucose uptake by 9% (P = 0.02). (14C)citrate uptake was not increased by carbidopa but was higher in SHR (977 +/- 2 pmol/10 s.mg) than in WKY rats (823 +/- 43 pmol/10 s.mg; P = 0.04). In summary, dopamine produced in WKY rat and SHR proximal tubules decreases Pi uptake by using a signaling process distinct from those that regulate NaK-ATPase and the antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)