Ng/ml Nerve Growth Factor Research Articles

Bovine Lactoferrin Promotes Neurite Outgrowth in PC12 Cells via the TrkA Receptor.

Lactoferrin (LF) is a multifunctional protein abundant in breast milk that modulates the functions of neural stem cells. Recent studies have demonstrated the efficacy of bovine LF (bLF) in mitigating behavioral changes; however, the molecular mechanisms on the nervous system have not yet been elucidated. The presented study aimed to characterize the molecular mechanisms of bLF on nerve extension in PC12 cells. PC12 cells were treated with 0.01-1000 µg/mL of bLF, and cell viability was determined using the cell counting kit-8 assay after treatment for 24 h. Morphometric evaluation was performed after 24 or 72 h of treatment with 50 ng/mL nerve growth factor (NGF) or 100-500 µg/mL bLF. The molecular mechanisms were investigated using Western blotting and real-time quantitative PCR. Cell viability was significantly decreased after treatment with 600-1000 µg/mL bLF for 24 h compared with the control group. Morphometric evaluation revealed neurite outgrowth after 72 h of NGF treatment, with a significant increase in neurite outgrowth after treatment with 250 µg/mL bLF. The phosphorylated p44/42 expression ratio peaked at 5 min and persisted for up to 10 min. Quantitative real-time PCR revealed a significant decrease in MAP2 expression. Our findings suggested that bLF enhanced PC12 cell neurite outgrowth to a similar extent as NGF. These effects are thought to be mediated via the TrkA receptor and activated by the phosphorylated ERK signaling pathway. Therefore, this study demonstrates that bLF promotes neurite outgrowth via a pathway similar to that of NGF.

Mage-D1 binding to activated p75NTR positively regulates mineralization of rat ectomesenchymal stem cells in vitro

To detect the binding of Mage-D1 with activated p75NTR and explore their role in regulating mineralization of ectomesenchymal stem cells (EMSCs). EMSCs were isolated from the tooth germs of embryonic SD rats (19.5 days of gestation) by tissue explant culture and were identified for surface markers using flow cytometry. The cultured cells were divided into blank control group, 100 ng/mL nerve growth factor (NGF) stimulation group, and lentivirus-mediated Mage-D1 interference (SH-Mage-D1) group. Proximity ligation assay was used to detect the binding of Mage-D1 with activated p75NTR in the EMSCs, and the binding strength was compared among the 3 groups. Alizarin red staining and ALP staining were used to observe mineralization of the induced cells. The expressions of ALP, Runx2, OCN, BSP, OPN, Msx1 and Dlx1 at both the mRNA and protein levels were detected using RT-PCR and Western blotting. The isolated EMSCs expressed high levels of cell surface markers CD44, CD90, CD29, CD146, and CD105 with a low expression of CD45. The results of proximity ligation assay showed that the binding of Mage-D1 with activated p75NTR in the cells increased over time, and the binding strength was significantly greater in NFG-treated cells than in the cells in the other two groups (P < 0.05). Alizarin red staining and ALP staining of the induced cells showed that the changes in the mineralization nodules were consistent with those of ALP activity. The cells treated with 100 ng/mL NGF exhibited significantly increased expressions of ALP, Runx2, OCN, BSP, OPN, Col1, Msx1 and Dlx1 as compared with the cells in the other two groups (P < 0.05). Mage-D1 directly binds to activated p75NTR in embryonic rat EMSCs to positively regulate the mineralization of the EMSCs.

Human umbilical cord mesenchymal stem cells differentiate into neuron-like cells after induction with B27-supplemented serum-free medium

To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells (HUCMSCs) to differentiate into neuron- like cells after induction with B27- supplemented serum- free medium. HUCMSCs at passage 4 were cultured for 14 days with serum-containing medium (SCM) (group A), SCM supplemented with 20 ng/mL nerve growth factor (NGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (group B), serum-free medium (SFM) (group C), or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF. The culture medium were changed every 3 days and the growth of the neurospheres was observed using an inverted microscope. The cell markers were analyzed with flow cytometry and the expressions of nestin, neuron- specific enolase (NSE), neurofilament heavy polypeptide (NEFH), and glial fibrillary acidic protein (GFAP) were quantified by quantitative real-time PCR (qRT-PCR) and Western blotting. Before induction, HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29 (99.5%), CD44 (49.6%) and CD105 (77.7%). Neuron-like cells were observed in the cultures on days 7, 10, and 14, and the cell differentiation was the best in group D, followed by groups C, B and A. In all the 4 groups, the cellular expressions of nestin and GFAP gradually lowered while those of NEFH and NSE increased progressively. The expressions of GFAP, NEFH, nestin and NSE were significantly different between group A and the other 3 groups (P < 0.001 or 0.05). B27-supplemented SFM effectively induces the differentiation of HUCMSCs into neuron- like cells, and the supplementation with cytokines (NGF and bFGF) strongly promotes the cell differentiation.

Comparative analysis of three different protocols for cholinergic neuron differentiation in vitro using mesenchymal stem cells from human dental pulp

ABSTRACTA decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer’s disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of β-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 µg/ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 µM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 µM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuron-like morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.

Suppressive Activity of Adiponectin on the Development of Allergic Rhinitis in Mice.

Adiponectin is accepted as playing pivotal roles in the development of allergic rhinitis (AR) through modulation of production of inflammatory mediators. Although it is also well known that neuropeptides, especially substance P (SP), function in the development and persistence of clinical conditions of AR, the influence of adiponectin on neuropeptide production is not well understood. The present study was designed to examine the influence of adiponectin on the production of SP both in vivo and in vitro. PC-12 cells (1×104 cells) were stimulated with 10.0 ng/ml nerve growth factor (NGF) for 2 h and then with 10.0 ng/ml capsaicin in the presence of different concentrations of adiponectin. After 72 h, culture supernatants were obtained, and SP levels were measured with enzyme-linked immunosorbent assay (ELISA). The influence of adiponectin on the total number of neurites developed per PC-12 cell and on the percentage of PC-12 cells with outgrowing neurites was also examined 24 and 72 h after the start of culture, respectively. In the second part of the study, BALB/c mice were sensitized intraperitoneally with 1.0 μg of ovalbumin and then challenged with intranasal ovalbumin. At 7 days following sensitization, these mice were treated with different doses of adiponectin intranasally in a volume of 5.0 μl. Nasal allergy-like symptoms, which were induced by bilateral application of 0.1 % OVA (5.0 μl), were assessed by counting sneezing and nasal rubbing behavior for 10 min immediately after nasal ovalbumin challenge. SP levels in nasal lavage fluid obtained 6 h after nasal ovalbumin challenge were examined by ELISA. Treatment of NGF-stimulated PC-12 cells with adiponectin suppressed SP production, which was induced by capsaicin stimulation. The minimum concentration of adiponectin that caused significant suppression was 7.5 ng/ml. On the other hand, adiponectin did not affect the total number of neurites and the percentage of PC-12 cells with outgrowing neurites, even at 1,000 ng/ml. Intranasal instillation of adiponectin into ovalbumin-sensitized mice at more than 10.0 ng/ml, but not 5.0 ng/ml, significantly inhibited the appearance of SP in nasal secretions, which was increased by intranasal challenge with ovalbumin. Adiponectin also suppressed the development of nasal allergic-like symptoms, sneezing and rubbing behavior, when ovalbumin-sensitized mice were treated intranasally with adiponectin at more than 10.0 ng/ml. The present results strongly suggested that adiponectin suppresses the production of SP and results in improvement of the clinical conditions of AR.

Overexpression of NTRK1 Promotes Differentiation of Neural Stem Cells into Cholinergic Neurons.

Neurotrophic tyrosine kinase type 1 (NTRK1) plays critical roles in proliferation, differentiation, and survival of cholinergic neurons; however, it remains unknown whether enhanced expression of NTRK1 in neural stem cells (NSCs) can promote their differentiation into mature neurons. In this study, a plasmid encoding the rat NTRK1 gene was constructed and transfected into C17.2 mouse neural stem cells (NSCs). NTRK1 overexpression in C17.2 cells was confirmed by western blot. The NSCs overexpressing NTRK1 and the C17.2 NSCs transfected by an empty plasmid vector were treated with or without 100 ng/mL nerve growth factor (NGF) for 7 days. Expression of the cholinergic cell marker, choline acetyltransferase (ChAT), was detected by florescent immunocytochemistry (ICC). In the presence of NGF induction, the NSCs overexpressing NTRK1 differentiated into ChAT-immunopositive cells at 3-fold higher than the NSCs transfected by the plasmid vector (26% versus 9%, P < 0.05). The data suggest that elevated NTRK1 expression increases differentiation of NSCs into cholinergic neurons under stimulation of NGF. The approach also represents an efficient strategy for generation of cholinergic neurons.

A synthetic analog of verbenachalcone potentiates NGF-induced neurite outgrowth and enhances cell survival in neuronal cell models

This study uses NeuroScreen-1 (NS-1) cells, a derivative of pheochromocytoma (PC12) cells, to examine neurite outgrowth induced by a novel synthetic verbenachalcone derivative, DSRB20-022 (C22). We treated NS-1 cells with varying concentrations of C22 in the presence of 2 ng/mL nerve growth factor (NGF). A dose-dependent effect of C22 was observed at concentrations of 2 μM and above, resulting in significant enhancement of NGF-dependent neurite outgrowth in NS-1 cells. C22 did not exhibit neuritogenic activity in the absence of NGF, but promoted a concentration-dependent increase in neurite-bearing cells without inducing cytotoxicity. Cell viability assays showed that C22 and the parent compound verbenachalcone (VC) are neuroprotective and enhanced survival of NS-1, PC12, and the murine neuro-2A (N2a) cell lines under conditions of serum deprivation. The results show that augmentation of NGF-induced neurite outgrowth by C22 in NS-1 was dependent on MAP kinase. Furthermore, the neuroprotective function of C22 and VC was accompanied by suppression of caspase-3/7 activation. However, C22 and VC exerted their antagonistic effects on caspase-3/7 activation through potentially different mechanisms of action.

A novel inducible tyrosine kinase receptor to regulate signal transduction and neurite outgrowth

Nervous system growth factor gene delivery can promote axonal growth and prevent cell death in animal models of CNS trauma and neurodegenerative diseases. The ability to regulate growth factor expression or signaling pathways downstream from growth factor receptors remains a desirable goal for in vivo gene transfer. To achieve precise pharmacological modulation of neurotrophin activity, we have generated a chimeric trkA receptor (ItrkA) by fusing the entire intracellular domain of the trkA high-affinity NGF receptor to two intracellular, modified FK506 binding domains for the synthetic small molecule dimerization ligand AP20187. Rat PC12 cells were transduced with lentiviral vectors containing ItrkA and green fluorescent protein (GFP; via an internal ribosome entry site). Treatment of ItrkA-expressing PC12 cells with AP20187 induced neurite outgrowth and differentiation in a time- and dose-dependent fashion, with a half-maximal response at a concentration of 1 nM AP20187. Seventy percent of cells responded to AP20187 by day 3. Western blots demonstrated that AP20187 treatment resulted in phosphorylation of Erk1/2 and Akt in ItrkA-transduced PC12 cells but not in nontransduced, naïve cells. Phosphorylation levels were comparable to levels obtained with 50 ng/ml nerve growth factor (NGF). In addition, ItrkA lentiviral transduction of primary E15 dorsal root ganglion neurons significantly increased neurite growth three- to fourfold in the presence of AP20187 compared with control GFP transduced and naïve neurons. These results demonstrate that small ligand-induced dimerization of the intracellular domain of trkA can efficiently simulate the biological activity of NGF and provide a means to regulate intracellular neurotrophin receptor signaling.

Docosahexaenoic Acid Enhances Iron Uptake by Modulating Iron Transporters and Accelerates Apoptotic Death in PC12 Cells

The effect of docosahexaenoic acid (DHA; 22:6 n-3) on Fe(2+)-mediated and/or H(2)O(2)-mediated oxidative stress (OS) was investigated in a PC12 pheochromocytoma cell line in the presence or absence of 50 ng/ml nerve growth factor (NGF). DHA-supplemented cells showed enhanced Fe(2+)-induced cell damage as evident by increased lipid peroxides formation (10-fold) and reduced neutral red (NR) dye uptake in a NGF-independent fashion. DHA caused a nearly 10-fold increase in free iron uptake in NGF-treated cells and doubled iron uptake in nondifferentiated cells. DHA-enrichment induced an elevation in the transferrin receptor protein in the nondifferentiated cells whereas NGF-treatment led to a substantial increase in the ubiquitous divalent metal ion transporter 1 (DMT-1) as detected by mRNA levels using qRT-PCR. The mechanism of action of DHA to accelerate cell death may be associated with the externalization of amino-phosphoglycerides (PG) species of which, increased ethanolamine plasmalogen levels, may be essential for cell rescue as noted in NGF-treated PC12 cells.

Neuronal Conditioning Medium and Nerve Growth Factor Induce Neuronal Differentiation of Collagen-Adherent Progenitors Derived from Human Umbilical Cord Blood

The aim of the study was to isolate and characterize a population of neuronal progenitors in the human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction, for in vitro manipulation towards neuronal differentiation. Selection of the HUCB neuronal progenitors (HUCBNPs) was based on the neuronal prerequisite for adherence to collagen. Populations of collagen-adherent, nestin-positive (94.8+/-2.9%) progenitors expressing alpha1/2 integrin receptors, as revealed by Western blot and adhesion assay using selective antagonists, were isolated and survived for more than 14 days. In vitro differentiation of the HUCBNPs was achieved by treatment with 10% human SH-SY5Y neuroblastoma cell-conditioning media (CM) supplemented with 10 ng/ml nerve growth factor (NGF). Some 83+/-8.2% of the surviving progenitors acquired a neuronal-like morphology, expressed by cellular outgrowths of different lengths. About 35+/-6% of the HUCBNPs had long outgrowths with a length/cell diameter ratio greater than 2, typical of developing neurons. The majority of these progenitors, analyzed by immunocytochemistry and/or RT-PCR, expressed common neuronal markers such as microtubule-associated protein 2 (MAP-2; 98.5+/-2%), neurotrophin receptor (TrkA; 98.5+/-0.06%), neurofillament-160 (NF-160; 94.2+/-1%), beta-tubulin III (89.8+/-4.2%) and neuron specific enolase (NSE). Combined CM and NGF treatment induced constitutive activation of the mitogen-activated protein kinases ERK2 (36-fold vs control), p38alpha (nine-fold vs control) and p38beta (23-fold vs control), most likely related to survival and/or differentiation. The results point to operationally defined conditions for activating neuronal differentiation of HUCBNPs ex vivo and emphasize the crucial role of neuronal CM and NGF in this process.

TTLL7 Is a Mammalian β-Tubulin Polyglutamylase Required for Growth of MAP2-positive Neurites

Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for alpha-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.

Modeling and synthesis of non-cyclic derivatives of GPI-1046 as potential FKBP ligands with neurotrophic properties

Prompted by the therapeutic potential of the neuroimmunophilin FK506-binding protein (FKBP) ligand, GPI-1046, in the treatment of nerve injuries and neurodegenerative diseases, a novel series of non-cyclic derivatives of GPI-1046 were designed and synthesized. Computer modeling analysis revealed that these relatively linear derivatives could energy-favorably bind to FKBP12 with an analogous binding mode to GPI-1046. The neurotrophic activity of the target compounds was assessed in chick dorsal root ganglion (DRG) cultures. As a result, 6 out of 11 test compounds at either or both concentrations of 1 pM and 100 pM significantly promoted neurite outgrowth in DRGs in the presence of 0.15 ng/ml nerve growth factor (NGF). Compound 5c at 100 pM exhibited the greatest neurotrophic effect in promoting both the number and length of neurite processes. However, in the absence of exogenously added NGF, all test compounds, including GPI-1046, failed to afford any positive effect on DRGs. This study suggests the intriguing potential of these compounds for further investigation.

Cyclic phosphatidic acid elicits neurotrophin-like actions in embryonic hippocampal neurons.

Cyclic phosphatidic acid (cPA; 1-acyl-sn-glycerol-2,3-cyclic phosphate) is an analog of the growth factor-like phospholipid mediator lysophosphatidic acid (LPA). As brain tissue is the richest source of cPA we tested its effects on hippocampal neurons from day 16/17 embryonic rat cultured in a serum-free medium. Nanomolar concentrations of cPA elicited a neurotrophic effect and promoted neurite outgrowth that exceeded that of 50 ng/mL nerve growth factor (NGF). Pertussis toxin, the LPA1/LPA3 receptor-selective antagonist dioctylglycerol pyrophosphate, the myristoylated inhibitory pseudosubstrate peptide of protein kinase A (PKI), Wortmannin and PD98059 abolished the neurite-promoting effect. cPA elicited a sustained activation of extracellular signal-related kinases (ERK) 1/2 and Akt. Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, reduced cPA-induced enhancement of neurite outgrowth. In B5P cells, a clonal cell line of PC12 cells overexpressing tyrosine kinase NGF receptor (TrkA), cPA elicited transphosphorylation of TrkA. cPA-elicited ERK activation was blocked by K252a and PKI. These results suggest that cPA mimics the effects of, and activates signaling pathways similar to, the neurotrophin NGF in cultured embryonic hippocampal neurons and B5P cells.

BAPTA/AM, an intracellular calcium chelator, induces delayed necrosis by lipoxygenase-mediated free radicals in mouse cortical cultures

1. Disruption of calcium homeostasis during neurodegenerative diseases is known to trigger apoptotic or necrotic death in neuronal cells. Recently, the authors reported that intracellular calcium restriction by NMDA receptor antagonists induces apoptosis in cortical cultures. To evaluate whether further restriction of intracellular free calcium can induce apoptosis or necrosis, we examined the neurotoxic characterization of BAPTA/AM, a permeable free calcium chelator, in mouse cortical cultures. 2. Exposure of mixed (glia and neuron) cortical cultures (DIV 13–16) to 3–10 μM BAPTA/AM (non-toxic concentration for glial cells) for 24–48 hr resulted in delayed and necrotic neuronal death. the necrotic findings included swelling and loss of mitochondria and endoplasmic reticulum (ER) with neuronal membrane rupture 24 hr after treatment with BAPTA/AM. Simultaneously, we observed a few TUNEL-positive cells in the neuronal subpopulation of the same cultures. 3. The neurotoxicity evoked by BAPTA/AM (10 μM) was significantly attenuated by the addition of 0.5 μM cycloheximide (a protein synthesis inhibitor), 10 μM actinomycin D (an RNA transcription inhibitor), a high extracellular potassium concentration (total 15 mM KCl), 100 μM t-ACPD (a metabotrophic agonist), 100 μM α -tocopherol (a free radical scavenger), 100 μM deferoxamine (a ferric ion chelator), 100 μM L-NAME (a nitric oxide synthase (NOS) inhibitor), 50 μM DNQX (a non-NMDA receptor blocker), and 3–30 μM esculetin (a lipoxygenase inhibitor). However, 0.3–3 mM ASA (a cyclooxygenase inhibitor), 100 ng/ml nerve growth factor (NGF), 10 μM MK-801 (a NMDA receptor antagonist), 20 μM zVAD-fmk (caspase inhibitor) and 50 U/ml catalase failed to inhibit the injury. 4. However, NGF and catalase blocked the neurotoxicity induced by BAPTA/AM in young neuronal cells (DIV 6). BAPTA/AM (10 μM) did not alter the expression of inducible nitric oxide synthase (iNOS) on glial cells. 5. These results suggest that the feature of neuronal death induced by BAPTA/AM exhibits predominantly delayed necrosis mediated by lipoxygenase-dependent free radicals.

Serum deprivation and NGF induce and modulate voltage-gated Na(+) currents in human astrocytoma cell lines.

Glial tumor cells derived from primary tissue express large voltage-gated Na(+) currents, whereas glioma cell lines usually lack this feature. We studied the effect of serum deprivation on the expression of Na(+) currents in two astrocytoma cell lines (1321N1 and A172). Serum deprivation for more than 2 days sufficed to induce large Na(+) currents in both cell lines; 300 nM of the specific blocker of voltage-gated Na(+) channels, tetrodotoxin, blocked these currents by about 85%. During serum deprivation, the cells also underwent morphological changes that were characterized by cell rounding and outgrowth of processes. Treatment with 100 ng/ml nerve growth factor (NGF) promoted these morphological changes and also accelerated the development of Na(+) currents. In 1321N1 cells, NGF increased the Na(+) current density after short serum deprivation (3--6 d) and changed several gating properties after longer serum deprivation (9--13 d). In comparison with cells from the early culture stage (3--6 d), the steady-state inactivation of the Na(+) current was shifted by -24 mV in NGF-treated cells from the late (9--13 d) culture stage. In untreated cells, this shift was only -13 mV. NGF accelerated the kinetics of inactivation and shifted the current-voltage relationship in cells from the late culture stage by -14 mV. In A172 cells, most of these effects were present already after short serum deprivation either in presence or absence of NGF. It is concluded that in astrocytoma cells, Na(+) currents are induced by serum deprivation and are modulated by NGF. This result supports the idea that NFG controls Na(+) currents in these cells by autocrine stimulation.

Soluble factors from rat basophilic leukemia (RBL-2H3) cells stimulated cooperatively the neurite outgrowth of PC12 cells.

Culture media from rat basophilic leukemia cells (RBL-2H3) induced the neurite outgrowth of rat pheochromocytoma PC12 cells, a model system for neuronal differentiation. The extension of the neurite outgrowth was dependent on the culture time of RBL-2H3 cells in the DMEM medium. The DMEM medium conditioned by RBL-2H3 cells for 48 h induced neurite outgrowth of PC12 cells significantly. The neurite extension was much higher than that by medium containing 1 ng/ml nerve growth factor (NGF) but was rather lower than that by medium containing 10 or 50 ng/ml NGF. The neurite extension by 50 ng/ml NGF was completely suppressed by excess anti-NGF antibody (1-1.5 microg/ml), while the extension by culture medium conditioned by RBL-2H3 cells for 48 h was not completely suppressed in the presence of the same amount of anti-NGF antibody. The neurite extension by the culture medium of RBL-2H3 cells was also suppressed by anti-interleukin (IL)-6 antibody (1 microg/ml), although IL-6 itself (20 units) could scarcely induce the neurite outgrowth of PC12 cells. This suggests that IL-6 in the culture medium of RBL-2H3 cells could be effective in inducing the neurite extension in cooperation with NGF. In the presence of an excess of both anti-NGF and anti-IL-6 antibodies, the culture medium of RBL-2H3 cells induced the neurite extension of PC12 cells. This suggests that the action of the various factors from RBL-2H3 cells may be synergistic as far as the neurite outgrowth of PC12 cells is concerned.

Inhibition of axonal growth from sensory neurons by excess nerve growth factor.

Fifteen-day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NGF). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain-derived neurotrophic factor or neurotrophin-3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.

In vitro regulation of insulin release and biosynthesis of fetal rat pancreatic cells explanted on pregnancy day 16.

Although the morphological development of the fetal pancreatic B cell has been studied in considerable detail, knowledge about the functional maturation, particularly in early stages of development, is still poor. The present paper describes a method for monolayer culture of fetal rat islet cells which allows a study of the regulation of insulin biosynthesis, release and content during critical stages of embryonic and fetal development. Suspensions of pancreatic cells were prepared from rat fetuses on pregnancy day 16 and cultured for 3 days. During the initial 2 days cultures were performed in the presence of 5 or 15 mmol/l glucose. During this initial period, culture at 5 mmol/l glucose was carried out in the presence or absence of either 10 mmol/l nicotinamide (NA) or 5 or 100 ng/ml nerve growth factor (NGF). After changing the media the cells were further exposed for 24 h to either 5 or 15 mmol/l glucose or 15 mmol/l glucose plus 5 mmol/l theophylline before measuring the insulin concentration in the culture medium. Cells that had initially been cultured for 2 days in 5 mmol/l glucose showed an increased insulin release, when subsequently cultured in 15 mmol/l glucose for 24 h. Theophylline potentiated the response and caused a decrease in cellular insulin content. Cells initially cultured in the presence of 15 mmol/l glucose showed unchanged insulin release during the subsequent 24-hour exposure to 15 mmol/l glucose, irrespective of the presence or absence of theophylline. The presence of NGF (100 ng/ml) during the initial 2-day culture period increased the insulin release in the presence of 15 mmol/l glucose and theophylline during the subsequent 24-hour culture period as compared to cells cultured in the absence of NGF. When cells were first exposed to either NA or NGF followed by exposure to 5 mmol/l glucose alone in the last 24-hour culture period, there was an increased insulin content. Rates of insulin biosynthesis remained unchanged irrespective of the glucose concentration in the culture medium. It is concluded that, already in early fetal development, B cells show glucose stimulation of insulin release albeit less pronounced than in the postnatal state.

An in Vitro Model for the Effects of Androgen on Neurons Employing Androgen Receptor-Transfected PC12 Cells

Androgen alters neurite outgrowth, synaptic organization, and cell survival in various portions of the brain and spinal cord. However, examination of the specific effects of androgen on neurons in vivo has been difficult. Previously, an in vitro model for the effects of estrogen on neurons was developed and characterized, using an estrogen receptor (ER)-transfected PC12 rat pheochromocytoma cell line. This model demonstrated estrogenic regulation of neurite outgrowth, spine formation, and gap junction formation. Similarly, an in vitro model for the effects of androgen on neurons is now described. Wild-type cells (PC12-WT) were stably transfected with an expression vector coding for the full-length cDNA for the human androgen receptor (AR). Resultant clones were isolated, screened for incorporation of vector and expression of AR mRNA and protein, and analyzed for morphologic responses to androgen. PC12-WT, NE09 (ER-negative, AR-negative), SER8 (ER-positive, AR-negative), and AR8 (ER-negative, AR-positive) cells were exposed to 10 ng/ml nerve growth factor (NGF), along with 0-10-7M dihydrotestosterone (DHT) for 2 days. AR8 cells demonstrated an androgen dose-dependent increase in mean neurite length, branch order, and neurite field area, whereas neurite branch segment length and soma area were not affected by androgen. PC12-WT, NE09, and SER8 cells exhibited no alterations in cell morphology with DHT exposure. Because of the synergistic effects of DHT and NGF, the regulation of NGF receptor mRNA by DHT was evaluated; however, no significant induction of either trkA or p75 mRNA expression by androgen was documented. The results suggest that in AR-positive PC12 cells, androgen acts additively with NGF to increase neurite outgrowth; but androgen effects are mediated specifically through branching and arborization. These responses are similar to developmental studies of androgen effects in vivo. Thus, androgen appears to induce an inherent neural morphologic program in AR-containing cells, which increases the receptive field of these cells, increasing the likelihood for interneural communication, although not promoting communication itself. These cell lines will provide a unique in vitro system for studying mechanisms of androgen-neuron interactions.

Botulinum toxin a inhibits acetylcholine release from cultured neurons in vitro.

Clostridium botulinum type toxin A (BoTx) blocks stimulus-induced acetylcholine (ACh) release from presynaptic nerve terminals at peripheral neuromuscular junctions. However, the detailed mechanism of this effect remains elusive. One obstacle in solving this problem is the lack of a suitable in vitro homogenous cholinergic neuronal model system. We studied the clonal pheochromocytoma PC12 cell line to establish such a model. PC12 cells were differentiated in culture by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release properties. Stimulation of these cells with high K(+) (80 mM) in the perfusion medium markedly increased calcium-dependent [(3)H]ACh release compared to undifferentiated cells. Stimulated [(3)H]ACh release was totally inhibited by pretreatment of cells with 2 nM BoTx for 2 h. BoTx inhibition of [(3)H]ACh release was time- and concentration-dependent. A 50% inhibition was obtained after 2 h incubation with a low (0.02 nM) toxin concentration. The time required for 2 nM BoTx to cause a measurable inhibition (18%) of stimulated [(3)H]ACh release was 30 min. Botulinum toxin inhibition of stimulated ACh release was prevented by toxin antiserum and heat treatment, suggesting the specificity of the toxin effect. Our results show that by differentiation with NGF, PC12 cells can be shifted from an insensitive to a sensitive state with respect to BoTx inhibition of stimulated ACh release. This cell line, therefore, may serve as a valuable in vitro cholinergic model system to study the mechanism of action of BoTx.