Nerve Growth Factor Pretreatment Research Articles

NGF induces pulmonary arterial hyperreactivity through Connexin 43 increased expression

Nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. To determine the mechanisms of NGF-induced PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Expression of NGF, its TrkA receptor and Cx43 were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from control donors or from patients suffering from idiopathic pulmonary arterial hypertension (iPAH). Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and surface biotinylation, after NGF treatment. Gap junction activity was assessed by FRAP microscopy. Contractions of rat PA were induced by phenylephrine (PHE), with or without NGF pre-treatment. The role of Cx43 in NGF-induced rat PA hyperreactivity was evaluated ex vivo by use of 43 Gap26, a Cx43 blocking peptide, or in vivo by use of an anti-Cx43 siRNA. TrkA expression was increased in hPASMC from iPAH patients compared to control hPASMC. NGF increased Cx43 expression, plasma membrane localisation and phosphorylation, with a higher sensitivity in iPAH cells compared to controls. In both cell types, this increase was abolished with K252a, a TrkA kinase inhibitor. Activity of Cx43-dependent GAP junctions was also increased after NGF treatment, with iPAH hPASMC being more sensitive to NGF than control cells. NGF-induced PA hyperreactivity to PHE was inhibited after treatment with 43 Gap26 or with anti-Cx43 siRNA. NGF, through activation of its TrkA receptor, increases Cx43 expression and plasma membrane localisation in hPASMC. This mechanism enhances Cx43-dependent GAP junction activity and contributes to PA hyperreactivity. Moreover, NGF pathway is upregulated in iPAH hPASMC compared to control hPASMC, and iPAH cells are more sensitive to NGF than control cells regarding Cx43 increased expression and Cx43-dependent GAP junction increased activity.

NGF protects endothelial cells from indomethacin-induced injury through activation of mitochondria and upregulation of IGF-1

Background/aimsEndothelial cells (ECs) lining blood vessels are critical for delivery of oxygen and nutrients to all tissues and organs and play a crucial role in the regeneration of blood vessel following tissue injury. ECs are also major targets of injury by a variety of noxious factors [e.g., ethanol and nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin, diclofenac], especially in gastric mucosa that has direct exposure to these agents. In this study, we investigated whether nerve growth factor (NGF) can protect gastric microvascular ECs (GECs) from injury by indomethacin (INDO) and the mechanisms involved. MethodsGECs were isolated from rat gastric mucosa and pre-treated with either vehicle or NGF (100ng/ml) for 30min to 4h followed by treatment with vehicle or 0.25mM INDO for 4h. Studies1) cell viability using Calcein AM live cell tracking dye, 2) mitochondrial structure and function using MitoTracker, molecular probe that stains mitochondria in live cells in a manner dependent on mitochondrial membrane potential (MMP), 3) in vitro angiogenesis – endothelial tube formation on Matrigel, 4) expression and subcellular localization of NGF receptor, TrkA, and 5) expression of IGF-1 protein. ResultsTreatment with INDO reduced GEC viability and in vitro angiogenesis and induced mitochondrial injury and MMP depolarization. NGF pre-treatment protected GECs from INDO-induced injury preventing both INDO-induced MMP depolarization and reduced in vitro angiogenesis. The NGF high affinity receptor, TrkA, was localized in GECs to both cell membrane and mitochondria. NGF treatment of GECs also resulted in increased IGF-1 protein expression. Conclusions1) NGF protects GECs against IND-induced injury. 2) Mitochondria are major targets of both INDO-induced injury and NGF afforded protection of GECs. 3) TrkA expression in the mitochondria of GECs indicates that the protection afforded by NGF is partly mediated by its direct action on mitochondria. 4) NGF prevents MMP depolarization and increases expression of IGF-1 protein in GECs. These studies indicate that NGF may play a protective role against injury to GECs; and, that maintenance of mitochondrial structure and function is one of the mechanisms.

Nerve growth factor pretreatment inhibits lidocaine‑induced myelin damage via increasing BDNF expression and inhibiting p38 mitogen activation in the rat spinal cord.

The present study aimed to investigate the effect of exogenous nerve growth factor (NGF) pretreatment on demyelination in the spinal cord of lidocaine-treated rats, and explored the potential neuroprotective mechanisms of NGF. A total of 36 rats were randomly assigned to three groups (n=12 per group): Sham group; Lido group, received intrathecal injection of lidocaine; NGF group, received intrathecal injection of NGF followed by intrathecal injection of lidocaine. Tail-flick tests were used to evaluate neurobehavioral function. Ultrastructural alternations were analyzed by transmission electron microscopy. Immunofluorescence was used to examine the expression of myelin basic protein (MBP) and brain-derived neurotrophic factor (BDNF). ELISA was used to determine serum levels of MBP and proteolipid protein (PLP). Western blotting was used to detect the expression of phosphorylated mitogen activated protein kinase (MAPK). NGF pretreatment reduced lidocaine-induced neurobehavioral damage, nerve fiber demyelination, accompanied by a decrease in MBP expression in the spinal cord and an increase in MBP and PLP in serum. In addition, NGF pretreatment increased BDNF expression in the spinal cord of lidocaine-treated rats. Furthermore, NGF pretreatment reduced p38 MAPK phosphorylation in the spinal cord of lidocaine-treated rats. NGF treatment reduces lidocaine-induced neurotoxicity via the upregulation of BDNF and inhibition of p38 MAPK. NGF therapy may improve the clinical use of lidocaine in intravertebral anesthesia.

Nerve growth factor and proNGF simultaneously promote symmetric self-renewal, quiescence, and epithelial to mesenchymal transition to enlarge the breast cancer stem cell compartment.

The discovery of cancer stem cells (CSCs) fundamentally advanced our understanding of the mechanisms governing breast cancer development. However, the stimuli that control breast CSC self-renewal and differentiation have still not been fully detailed. We previously showed that nerve growth factor (NGF) and its precursor proNGF can stimulate breast cancer cell growth and invasion in an autocrine manner. In this study, we investigated the effects of NGF and proNGF on the breast CSC compartment and found that NGF or proNGF enrich for CSCs in several breast cancer cell lines. This enrichment appeared to be achieved by increasing the number of symmetric divisions of quiescent/slow-proliferating CSCs. Interestingly, in vitro NGF pretreatment of MCF-7 luminal breast cancer cells promoted epithelial to mesenchymal transition in tumors of severe combined immunodeficient mice. Furthermore, p75(NTR), the common receptor for both neurotrophins and proneurotrophins, mediated breast CSC self-renewal by regulating the expression of pluripotency transcription factors. Our data indicate, for the first time, that the NGF/proNGF/p75(NTR) axis plays a critical role in regulating breast CSC self-renewal and plasticity.

Nerve growth factor protects the ischemic heart via attenuation of the endoplasmic reticulum stress induced apoptosis by activation of phosphatidylinositol 3-kinase.

Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway.Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses.Results: NGF pretreatment significantly improved the recovery of post-ischemia cardiac hemodynamics. Reduced creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity and cTnI levels, as well as decreased myocardial apoptosis ratio were observed in the NGF group. The improvement of NGF on recovery of cardiac function and alleviation of myocardial injury were completely abolished by K252a or LY294002. GRP78, caspase-12 and CHOP were highly expressed in ischemic myocardium, while NGF significantly inhibited the overexpression of these proteins which were involved in ER stress-induced myocardial apoptosis. NGF pretreatment also induced phosphorylation of Akt. When the activation of PI3K/Akt pathway is blocked by LY294002, the NGF induced suppression of the apoptosis-related proteins expression was reversed.Conclusions: NGF pretreatment may protect the ischemic heart via inhibition of the ER stress-induced apoptosis; this pro-survival effect is mediated by PI3K/Akt pathway.

The inhibitory action of the antimigraine nonsteroidal anti-inflammatory drug naproxen on P2X3 receptor-mediated responses in rat trigeminal neurons

Enhanced nociceptive firing in trigeminal ganglion neurons is a likely reason for migraine pain. In experimental migraine-like conditions induced by the calcitonin gene-related peptide (CGRP), P2X3 receptors abundantly expressed in trigeminal neurons are highly responsive to the excitatory action of extracellular ATP. In this study, we tested whether naproxen, a common antimigraine medicine, could affect the function of P2X3 receptors in the presence or absence of the algogen nerve growth factor (NGF), the level of which is elevated in patients with chronic migraine. We used calcium imaging and patch clamp recordings from rat trigeminal neurons, which were activated by a relative specific P2X3 agonist α,β-meATP or by high potassium-induced depolarization. In the absence of NGF, naproxen dose-dependently (0.1–1 mM) reduced intracellular calcium transients elicited by α,β-meATP. Naproxen also led to a slight, but significant, reduction in calcium transients induced by potassium ions, indicating the involvement of voltage-gated calcium channels. The inhibitory action of 1 mM naproxen was enhanced after NGF pretreatment, suggesting that P2X3 receptors in sensitized neurons are more susceptible to inhibition by high doses of this nonsteroidal anti-inflammatory drug (NSAID). Using patch clamp recordings from HEK293 cells expressing P2X3 receptors, we tested the direct action of naproxen on P2X3 receptor-mediated membrane currents. In clinically relevant concentrations of 0.5 mM, naproxen produced a use-dependent blocking effect on ATP receptors. Kinetic analysis suggests that naproxen inhibited P2X3 receptors via facilitation of fast desensitization, which determines current decay in the continuous presence of the agonist. In summary, we present a novel fast mechanism for the antimigraine action of naproxen, which can act in synergy with the cyclooxygenase inhibition to attenuate headaches.

A study of the protective effects of lidocaine and nerve growth factor pretreatment on cerebral ischemia/reperfusion injury in gerbils

To study the protective effects of lidocaine and nerve growth factor (NGF) pretreatment on cerebral ischemia/reperfusion (I/R) injury. Fifty-four Mongolian gerbils were randomly divided into nine groups: normal group (Group A), lidocaine control group (Group L), NGF control group (Group N), lidocaine pretreatment groups at the point of 12, 24, 48 hours (named respectively Group L12, L24, L48), NGF pretreatment groups with NGF given 12, 24, 48 hours before injury (named respectively Group N12, N24, N48), with 6 animals in each group. Except Group A, the carotid artery on both sides were occluded for 20 minutes, then they were released to allow reperfusion. Alteration of apoptosis was observed with terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and the expression of Bcl-2 and Bax was assessed by immunohistochemistry method. No apoptosis, Bcl-2 and Bax were found in Group A. In lidocaine or NGF pretreatment group, the expression of Bcl-2 (Group L12: 36.60 + or - 3.31, Group L24: 34.73 + or - 1.82, Group L48: 65.17 + or - 1.53; Group N12: 35.70 + or - 1.18 , Group N24: 37.30 + or - 3.86, Group N48: 62.77 + or - 2.91) was obviously increased comparing with control group (Group L: 24.53 + or - 1.48, Group N: 25.43 + or - 1.85), but the number of the apoptosis (Group L12: 32.87 + or - 0.99, Group L24: 31.90 + or - 4.14, Group L48: 24.50 + or - 0.70; Group N12: 32.80 + or - 1.27, Group N24: 32.83 + or - 1.30, Group N48: 23.30 + or - 0.86) and Bax expression (Group L12: 33.47 + or - 1.21, Group L24: 33.70 + or - 1.20, Group L48: 24.67 + or - 2.09; Group N12: 32.17 + or - 2.21, Group N24: 31.97 + or - 1.79, Group N48: 23.27 + or - 1.20) were significantly decreased comparing with the control group (the number of the apoptosis: Group L 67.43 + or - 3.92, Group N 67.80 + or - 3.82; Bax: Group L 59.73 + or - 1.32, Group N 59.37 + or - 1.54, P<0.05 or P<0.01). In lidocaine and NGF pretreatment groups, the number of cell apoptosis and expression Bax were significantly lower at 48 hours than those at 12 hours or 24 hours, but the cell expression Bcl-2 was obviously higher (all P<0.05). However, there was no difference between lidocaine and NEG pretreatment groups at each time point in regard of the number of cell apoptosis, expression of Bcl-2 and Bax. Lidocaine and NGF pretreatment before cerebral I/R can alleviate I/R injury to the cerebral tissue. The protective effect was most obvious when the treatment was given 48 hours before ischemia. The mechanism may be related with an increase in expression of Bcl-2 as well as a decrease in Bax level.

A comparative study on the protection effect of nerve growth factor and edaravone pretreatment against cerebral ischemia/reperfusion injury

To compare the protective effect of nerve growth factor (NGF) and edaravone (a free radical scavenger) pretreatment against cerebral ischemia/reperfusion (I/R) injury to nerve cells. Cortical neurons of Sprague-Dawley (SD) mouse aged shorter than 24 hours were cultured for 7 days, then they were randomly divided into control group, I/R group, NGF of 10, 50, 100 microg/L pretreatment groups and 100 mumol/L edaravone pretreatment group. In pretreatment groups the cells were pretreated with drugs correspondingly. After culturing for 24 hours, glutamate of 200 mumol/L was given into the culture of all groups, except control group, for half an hour. Then culture medium in all groups were renewed with ordinary culture medium, and cultures were continued for 24 hours. The survival rate (by methyl thiazolyl tetrazolium assay), the content of lactate dehydrogenase (LDH, by spectrometry) and the rate of apoptosis (by flow cytometric) were determined. The cellular shape and ultrastructure were observed by hematoxylin-eosin (HE) staining and electronic microscopy correspondingly. The survival rate of nerve cells in NGF groups and edaravone group was significantly higher than that in I/R group [(0.21 + or - 0.04)%, (0.23 + or - 0.04)%, (0.21 + or - 0.04)%, (0.24 + or - 0.04)% vs. (0.19 + or - 0.04)%]. The content of LDH in culture medium and the rate of apoptosis in NGF groups and edaravone group were lower than those in I/R group (P<0.05 or P<0.01). The release rate of LDH in each group was (0.50 + or - 0.06)%, (0.46 + or - 0.07)%, (0.50 + or - 0.02)%, (0.43 + or - 0.06)% vs. (0.56 + or - 0.03)%, respectively. The rate of apoptosis in each group was (10.77 + or - 1.07)%, (10.38 + or - 0.70)%, (13.34 + or - 0.57)%, (9.99 + or - 0.77)% vs. (14.52 + or - 0.77)%, respectively. The cellular shape and ultrastructure of nerve cells in NGF groups and edaravone group were affected much less than that of I/R group. NGF of 50 microg/L pretreatment group gave the best effect among three groups. There was no significant difference between NGF 50 microg/L pretreatment group and edaravone pretreatment group. NGF and edaravone pretreatment 24 hours before cerebral I/R give protective effects against cerebral I/R injury. The protective effects are best in NGF of 50 microg/L pretreatment group and edaravone pretreatment group, and there is no significant difference between them.

Effects of NGF pretreatment on cerebral injury in gerbils

Objective To investigate the effects and underlying mechanisms of different doses of nerve growth factor(NGF) pretreatment on neuron apoptosis and the expressions of the apoptosis-related protein, Bcl-2 and Bax, in the gerbil cerebral prefrontal cortex following global cerebral ischemia-reperfusion(I/R) injury. Methods Fifty-four gerbils were randomly divided into five groups, group C: sham operation( n = 6); group I/R( n = 12), group L( n = 12): low-dose NGF+I/R, group M( n = 12): medium-dose NGF+I/R and group H ( n = 12): high-dose NGF+I/R. Groups I/R, L, M and H were further divided into 2 subgroups according to the duration of reperfusion(24 h and 72 h). The global cerebral I/R injury model was induced by bilateral carotid artery occlusion for 20 min, followed by removal of the clamps to permit reperfusion. In groups L, M and H, NGF was injected into the lateral ventricle 24 h prior to ischemia. Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL) and immunohistochemical staining were performed to detect neuron apoptosis and the expressions of Bcl-2 and Bax protein in the cerebral cortex, respectively. Results In the I/R group and NGF pretreatment groups(L, M and H groups), reperfusion for 72 h caused higher percentages of both neurons exhibiting apoptosis and Bax positive cells( P < 0.01), but lower percentages of Bcl-2 positive cells compared with the corresponding 24 h reperfusion groups( P < 0.05). All NGF pretreatment groups exhibited lower percentages of neurons exhibiting apoptosis and Bax positive cells but a higher percentage of Bcl-2 positive-cells relative to the I/R group( P < 0.01). Moreover, the high-dose NGF pretreatment group had a greater decreased neuronal apoptosis and Bax protein expression and increased Bcl-2 protein expression than either the low-or medium-dose groups. Conclusion Neuron apoptosis participates in the progression of cerebral ischemia-reperfusion injury. The protective effect of NGF pretreatment against ischemia-reperfusion-induced neuron apoptosis seems to be both time- and dose-dependent. This anti-apoptosis mechanism may be associated with upregulation of Bcl- 2 protein expression and downregulation of Bax protein expression.

Nerve growth factor blocks thapsigargin‐induced apoptosis at the level of the mitochondrion viaregulation of Bim

This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (−3, −7 and −9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bimel mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax.

Chlorpyrifos Affects Phenotypic Outcomes in a Model of Mammalian Neurodevelopment: Critical Stages Targeting Differentiation in PC12 Cells

The organophosphate insecticide chlorpyrifos (CPF) adversely affects mammalian brain development through multiple mechanisms. To determine if CPF directly affects neuronal cell replication and phenotypic fate, and to identify the vulnerable stages of differentiation, we exposed PC12 cells, a model for mammalian neurodevelopment, to CPF concentrations spanning the threshold for cholinesterase inhibition (5–50 μM) and conducted evaluations during mitosis and in early and mid-differentiation. In undifferentiated cells, exposure to 5 μM CPF for 1–3 days reduced DNA synthesis significantly without eliciting cytotoxicity. At the same time, CPF increased the expression of tyrosine hydroxylase (TH), the enzymatic marker for the catecholamine phenotype, without affecting choline acetyltransferase (ChAT), the corresponding marker for the cholinergic phenotype. Upon exposure to nerve growth factor (NGF), PC12 cells developed neuritic projections in association with vastly increased TH and ChAT expression accompanying differentiation into the two phenotypes. CPF exposure begun at the start of differentiation significantly reduced ChAT but not TH activity. In contrast, when CPF was added in mid-differentiation (4 days of NGF pretreatment), ChAT was unaffected and TH was increased slightly. Thus, CPF exerts stage-specific effects, reducing DNA synthesis in the undifferentiated state, impairing development of the cholinergic phenotype at the start of differentiation, and promoting expression of the catecholaminergic phenotype both in undifferentiated and differentiated cells. CPF administration in vivo produces deficits in the number of neurons and cholinergic function, and because we were able to reproduce these effects in vitro, our results suggest that CPF directly influences the phenotypic fate of neuronal precursors.

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model.

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38alpha and beta, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.

Nerve growth factor stimulates diacylglycerol de novo synthesis and phosphatidylinositol hydrolysis in pheochromocytoma cells

Induction of neurite outgrowth by treating pheochromocytoma cells (PC12 cells) with nerve growth factor (NGF) is associated with major increases in cellular levels of diacylglycerol (DAG), an essential and probably limiting precursor in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) syntheses. To identify the sources of this DAG we examined the effects of NGF treatment on the conversion of [ 3 H ]oleic acid (OA) or [ 3 H ]glycerol to [ 3 H ]glycerolipids, and the turnover of these products in PC12 cells. In kinetic studies on [ 3 H ]OA incorporation, most of the radioactivity in the cells initially was free [ 3 H ]OA; then it appeared predominantly as [ 3 H ]DAG and, eventually, as large amounts of [ 3 H ]phospholipids (PLs). In NGF pre-treated cells, the increases in the levels of [ 3 H ]DAG (which were most prominent) and PLs were similar to those in unlabeled DAG and PLs. These effects of NGF could be partially blocked by an inhibitor (triacsin C) of long chain acyl-CoA synthetase. NGF pre-treatment also significantly enhanced the incorporation of [ 3 H ]glycerol into lipids, a pathway for de novo synthesis of glycerolipids. In studies on the degradation of [ 3 H ]OA-labeled lipids, the disappearance of [ 3 H ]OA-labeled neutral lipids exhibited an initial rapid phase and a subsequent stable phase. NGF treatment transiently promoted the hydrolysis of [ 3 H ]PI to [ 3 H ]DAG. These data suggest that the increases in DAG levels observed in PC12 cells exposed to NGF derive mainly from de novo synthesis and, to a lesser and transient extent, from the hydrolysis of [ 3 H ]PI.

Enhancement of Peroxynitrite-Induced Apoptosis in PC12 Cells by Fibroblast Growth Factor-1 and Nerve Growth Factor Requires p21Ras Activation and Is Suppressed by Bcl-2

Extracellular trophic factors can regulate whether cells subjected to oxidative stress will survive to proliferate or else undergo cell death. We have previously shown that about 35% of undifferentiated PC12 cells undergo apoptosis 18 h after exposure to peroxynitrite and that pretreatment with nerve growth factor (NGF) protects PC12 cells through activation of phosphatidylinositol (PI) 3-kinase. In contrast, pretreatment with acidic fibroblast growth factor (FGF-1) approximately doubled apoptosis. We report here that NGF added immediately after peroxynitrite treatment no longer protected against apoptosis, but instead enhanced apoptosis to the same extent as FGF. We further investigated which signaling pathways were involved in increasing the level of apoptosis. Overexpression of Bcl-2 blocked the increased apoptosis caused by NGF and FGF-1, but Bcl-2 did not prevent the induction of apoptosis by peroxynitrite alone. The increase in apoptosis caused by the trophic factors was also blocked by the expression of a dominant negative p21Ras mutant. Activation of PI 3-kinase by NGF pretreatment completely protected against both the enhanced apoptosis induced by FGF-1 pretreatment and NGF posttreatment and the apoptosis induced by peroxynitrite alone. Our results indicate that the enhancement of peroxynitrite-induced apoptosis caused by NGF and FGF-1 is dependent on the stimulation of a proapoptotic pathway involving p21Ras that can be suppressed by Bcl-2.

Nerve growth factor maintains regulation of intracellular calcium in neonatal sympathetic neurons but not in mature or aged neurons.

We examined the effects of nerve growth factor on the regulation of intracellular calcium levels of superior cervical ganglion neurons in terms of postnatal maturation and ageing. Rat superior cervical ganglion neurons from three age groups (neonatal: 0 to one-day-old, young adult: three to six-month-old, and aged: more than 24-month-old) were dissociated and cultured in the presence or absence of 100 ng/ml of nerve growth factor. Intracellular free calcium levels ([Ca 2+] i) were measured using the fura-2 microfluorometry. Nerve growth factor treatment increased the resting [Ca 2+] i of neonatal neurons, although it had no effect on those of mature and aged neurons. We further examined the effects of nerve growth factor on the transient increase of [Ca 2+] i induced by methacholine (0.1 mM caffeine (20 mM) or high-potassium medium (40 mM K +). Nerve growth factor pre-treatment significantly increased the population of neonatal superior cervical ganglion neurons which responded to methacholine, whereas almost all young adult and aged neurons responded to methacholine regardless of pre-treatment of nerve growth factor. Caffeine induced a cyclic alteration of [Ca 2+] i (oscillation) in 45% of the neonatal superior cervical ganglion neurons when they were maintained without nerve growth factor, but nerve growth factor treatment suppressed the oscillation to 10% of neurons. In contrast to neonatal neurons, all of the young adult and aged neurons showed only a transient increase of [Ca 2+] i in response to Caffeine independent of nerve growth factor treatment. There was no significant effect of nerve growth factor on K + depolarization-induced [Ca 2+] i elevations at any of the ages studied. Nerve growth factor did not substantially alter the pattern of the transients induced by these three agents. Our results indicate that exogenous nerve growth factor is necessary to maintain normal acetylcholine receptor-mediated [Ca 2+] i responses as well as Ca 2+-induced Ca 2+ release from intracellular calcium storage in neonatal superior cervical ganglion neurons. In mature superior cervical ganglion neurons, Ca 2+ homeostasis becomes independent of exogenous nerve growth factor, and Ca 2+ homeostasis and its independency are well preserved in aged neurons.

Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.

Expression of integrin alpha 1 beta 1 is regulated by nerve growth factor and dexamethasone in PC12 cells. Functional consequences for adhesion and neurite outgrowth.

PC12 cells respond to nerve growth factor by differentiating into sympathetic neuron-like cells that employ the integrin alpha 1 beta 1 to attach to, and extend neurites on, substrata coated with collagen or laminin. In one PC12 subline, PC12i, prolonged treatment with nerve growth factor results in a marked increase in synthesis of alpha 1 subunits and in the level of alpha 1 mRNA, with a corresponding increase in alpha 1 beta 1 expressed on the cell surface. These changes are accompanied by substantial increases in initial cell attachment to collagen and in the fraction of neurite-bearing cells and average neurite length. Integrin beta 1-subunits are constitutively expressed, so that alpha 1 synthesis controls the amount of alpha 1 beta 1 heterodimer expressed on PC12i cells. Acidic fibroblast growth factor also induces alpha 1 beta 1 in PC12i cells, with consequent enhancement of neurite outgrowth; treatment with epidermal growth factor or dibutyryl cyclic AMP does not have these effects. Another subline, PC12c, expresses high levels of alpha 1 mRNA and alpha 1 protein constitutively. With or without nerve growth factor pretreatment, these cells adhere well to collagen and a majority extend neurites when replated in the presence of nerve growth factor. Dexamethasone treatment of PC12c cells reduces expression of alpha 1 mRNA and alpha 1 protein, with consequent reduction in attachment to collagen. In both sublines, then, there is a direct relationship between the level of a specific matrix receptor and cell-matrix adhesion. Moreover, our results suggest that induced expression of this matrix receptor is an essential aspect of the regulation of neurite extension by nerve growth factor in PC12i cells.

Effect of Nerve Growth Factor (NGF) on Survival of Superior Cervical Ganglion (SCG) Transplanted into the Third Ventricle in Rats

Effect of short-term exposure to nerve growth factor (NGF) on neuron survival of superior cervical ganglion (SCG) transplanted into the third ventricle in rats and that on neurite outgrowth of SCG neurons in a tissue culture system were examined. Because SCG taken from 3 week-old rats exhibited a high survival rate in transplantation and they were highly sensitive to NGF in culture, they were used in the short-term NGF treatment experiment. SCG from 3 week-old rats were preincubated with NGF for 30 min at room temperature, and then they were cultured for 2 days or grafted for 14 days. Short-term exposure to NGF at the concentration of 10 μg/ml enhanced the survival of transplanted SCG neurons. The same concentration of NGF enhanced neurite outgrowth of SCG neurons in culture. These results suggest that short-term NGF pretreatment could increase the efficiency of neuronal transplantation.

The role of transcription-dependent priming in nerve growth factor promoted neurite outgrowth

Studies were carried out with clonal PC12 rat pheochromocytoma cells to test and elaborate the “priming” model for the mechanism by which nerve growth factor (NGF) promotes neurite outgrowth. The rate of appearance, initial extent of elongation, and insensitivity to inhibitors of transcription for neurites regenerated by passaged PC12 cells were all found to increase progressively as a function of time of NGF pretreatment. Cells pretreated (or “primed”) with NGF were thus able to rapidly regenerate long processes even when RNA synthesis was blocked. The long-term, progressive nature of neurite outgrowth and priming were not entirely accounted for by cell cycle effects. These observations were thus consistent with a mechanism in which NGF promotes process outgrowth by specifically stimulating cells to synthesize and progressively accumulate substances which are required for neurite production and elongation. This process appeared to require RNA transcription and the accumulation of specific rather than total cell constituents. Priming occurred for cells grown in suspension or in the presence of inhibitors of microtubule assembly. Priming and neurite outgrowth were suppressed at high cell density and were not elicited by exposure to depolarizing agents. Examination of the properties of NGF-promoted neurite outgrowth by cultured neurons suggests that priming may occur during development of the normal nervous system. It is also conceivable that self-priming may be a feature of growth factors in addition to NGF.