Inhibitors of nitric oxide synthase attenuate ... - Wiley Online Library

Journal of Neurochemistry, 2002, 81, 624–635

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells Bettina E. Kalisch, Nicholas A. Bock, Wanda L. Davis and R. Jane Rylett Department of Physiology, University of Western Ontario, and The John P. Robarts Research Institute, London, Ontario, Canada

Abstract NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N x-nitroL-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO

production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells. Keywords: acetylcholine, choline acetyltransferase, cholinergic, nerve growth factor, nitric oxide, PC12 cells. J. Neurochem. (2002) 81, 624–635.

Cholinergic neurons control a wide range of physiological functions in central and peripheral nervous systems. Expression and activity of choline acetyltransferase (ChAT; EC 3.2.1.6), the enzyme that synthesizes acetylcholine (ACh), is down-regulated in basal forebrain cholinergic neurons during normal aging; this is paralleled by a decline in spatial learning ability in aged rats (Fischer et al. 1991; Dunbar et al. 1992). While processes underlying degeneration of basal forebrain cholinergic neurons are unknown, attempts to rescue these neurons have been made in Alzheimer patients (Olson et al. 1992; Nordberg 1993) and in aged rats or rats with experimental brain lesions (Fischer et al. 1987; Williams and Rylett 1990; Rylett et al. 1993) by administration of nerve growth factor (NGF). NGF, a neurotrophic factor known to enhance cholinergic function, is produced in target tissues of cholinergic neurons and interacts with the high-affinity tyrosine kinase receptor, TrkA. This interaction can directly influence synaptic responses (Knipper et al. 1994a,b; Thoenen 1995) or NGF can be taken up by a receptor-mediated process and transported to the cell body (Seiler and Schwab 1984; Lapchak et al. 1993) where it can alter cellular functions. Importantly, age-related deficits in cholinergic neurochemistry in rat brain respond to intracerebral administration of NGF with increases in cholinergic

neuronal markers (Williams and Rylett 1990), reversal of atrophy of cholinergic neuronal perikarya and improvement in memory function (Fischer et al. 1987). In neuronal cultures, NGF increases ChAT activity as well as ACh synthesis (Pongrac and Rylett 1996), content and release (Takei et al. 1989; Suzuki et al. 1994). Although mechanisms by which NGF enhances cholinergic function are not elucidated, several advances have been made in understanding the signal transduction pathways responsible for mediating some of the actions of NGF. The free radical nitric oxide (NO) may play a role in the neurotrophic actions of NGF. NO is produced during the

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Received December 20, 2001; revised manuscript received January 25, 2002; accepted January 29, 2002. Address correspondence and reprint requests to R. J. Rylett, Department of Physiology, University of Western Ontario, London, Ontario, Canada, N6A 5C1. E-mail: [email protected] Abbreviations used: ACh, acetylcholine; ChAT, choline acetyltransferase; DAF2-DA, 4,5-diaminofluorescein diacetate; DMEM, Dulbecco’s modified Eagle medium; NGF, nerve growth factor; NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; L-NAME, Nx-nitro-L-arginine methylester; MIU, S-methylisothiourea.

2002 International Society for Neurochemistry, Journal of Neurochemistry, 81, 624–635

Regulation of ChAT by NGF involves NO 625

conversion of arginine to citrulline by a class of enzymes referred to as nitric oxide synthases (NOSs; Mayer et al. 1989). Three NOS isoforms, neuronal NOS (nNOS; NOSI), endothelial NOS (eNOS; NOSIII) and inducible NOS (iNOS; NOSII) arising from three distinct genes have been described. Over the last 10 years, numerous conflicting reports regarding the role of NO in neurotoxicity versus neuroprotection have emerged. Although the expression of NOS mRNA in cholinergic neurons varies in different brain 1 regions (Sugaya and McKinney 1994), infusion of NGF into rodent brain increased constitutive nNOS mRNA in basal forebrain cholinergic neurons (Holtzman et al. 1994) in a manner parallel to the regulation of ChAT mRNA (Holtzman et al. 1996). In PC12 cells, NGF treatment increased expression of NOS isozymes and inhibition of NOS activity prevented NGF-mediated neurite outgrowth (Peunova and Enikolopov 1995). Furthermore, in NGF-differentiated PC12 cells, NO donors increased the number of cells bearing neurites (Hindley et al. 1997). This information suggests that NGF can regulate NOS expression and NO production, and that NO can modulate NGF-mediated neurotrophic responses. Both NGF and NO are involved in regulation of a number of neuronal processes including phenotypic and morphological differentiation, proliferation/cell cycle arrest and survival (Poluha et al. 1997). However, it is not known to what extent NO can act as a second messenger/mediator of responses initiated by NGF treatment. NGF regulates NOS expression and cholinergic neuronal phenotype, but the relationship between these two actions has not been investigated. Therefore, in the present study we tested the hypothesis that NO/NOS plays a role in NGF-mediated enhancement of cholinergic neuronal phenotype in PC12 cells.

Experimental procedures Materials Dulbecco’s modified Eagle medium (DMEM), horse serum, Trizol and reverse transcriptase (RT)-polymerase chain reaction (PCR) supplies were from Gibco Life Technologies (Rockville, MD, USA) and fetal bovine serum was from HyClone (Logan, UT, USA). nNOS antibody was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) and anti-iNOS was a generous gift from Dr D. Mercer (University of Texas). NGF 2.5 S was obtained from Harlan Bioproducts (Indianapolis, IN, USA), acetyl-coenzyme A [acetyl-3H], specific activity 7.2 Ci/mmol, was purchased from ICN (East Hill, NY, USA) and acetyl-coenzyme A trilithium salt was obtained from Boehringer Mannheim (Mannheim, Germany). 4,5Diaminofluorescein diacetate (DAF-2DA) was supplied by Calbiochem (San Diego, CA, USA), and eserine sulfate, 3-heptanone, tetraphenylboron sodium, NADPH, nitro blue tetrazolium, Nx-nitroL-arginine methyl ester (L-NAME) and s-methylisothiourea (MIU) were all purchased from Sigma Chemical Company (St Louis, MO,

USA). Liquid scintillation cocktail was from Amersham Pharmacia Biotech (Piscataway, NJ, USA). All other chemicals were at least reagent grade and were obtained from Sigma Chemical Company or VWR (Mississauga, ON, Canada). Tissue culture PC12 cells were maintained in DMEM, 5% fetal bovine serum, 5% horse serum and 50 lg/mL gentamycin at 37C and plated on collagen-coated plastic culture dishes for 24 h prior to experiments. To determine the extent of NADPH diaphorase-positive staining, PC12 cells were plated at a density of 5 · 104 cells/well and treated with 50 ng/mL NGF for 1–5 days and stained as described below. To examine the effect of NGF on NOS or ChAT steady-state mRNA levels, cultures were plated on 100-mm plates at a density of 5 · 106 cells/plate, treated with 50 ng/mL NGF for 12–72 h and assayed for NOS and ChAT mRNA by RT-PCR. For western blot analysis of NOS proteins, PC12 cells were plated on 60-mm plates at a density of 1.5 · 106 cells/plate, then treated with 50 ng/mL NGF for 2 or 5 days. For measurement of ChAT activity, cells were plated onto 12-well plates at a density of 5 · 104 cells/well, then treated with L-NAME (2–20 mM) or MIU (0.5–2.5 mM), alone or in combination with 50 ng/mL NGF every 48 h for 5 days and assayed as described below; in the case of measurement of NOS activity, cells were plated on 60-mm dishes at 2 · 106 cells/plate and treated as for ChAT activity measurements. To determine the extent of NOS inhibition by L-NAME and MIU, PC12 cells were loaded with the fluorescent probe DAF-2DA following treatment with NGF for 3 days in the presence or absence of NOS inhibitor and viewed by confocal microscopy. As visualization of fluorescence by laser scanning confocal microscopy requires that cells be on a glass surface, PC12 cells were plated onto collagen-coated 35-mm tissue culture plates 3 containing a glass bottom insert (MatTel, Ashland, MA, USA) at a density of 5 · 105 cells/plate. Histology and neurite outgrowth analysis PC12 cells were fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS) at pH 7.4 for 15 min. Staining of NADPH diaphorase activity was carried out using modifications of the procedure described by Vincent and Kimura (1992). Fixed cells were incubated in 1.2 mM NADPH, 0.12 mM nitro blue tetrazolium and 0.3% Triton X-100 for 30–60 min and subsequently rinsed with PBS. All cells were rinsed with water and dehydrated in a series of alcohol solutions, cover-slipped and viewed under a light microscope. To assess the effect of L-NAME on NGF-mediated neurite outgrowth, cells were grown in the presence of NGF (50 ng/mL) and L-NAME (20 mM) or D-NAME (20 mM) for 4 days, or were untreated (control). Living cells were viewed by phase-contrast microscopy with 20 random images captured per plate using ACT-1 software at 40· magnification. Images were printed for analysis, and cells bearing neurites with lengths greater than two cell body diameters were considered positive. All cells in each image (at least 20 cells per frame) were analyzed, with over 400 cells assessed per tissue culture plate. The experiment was repeated five times. RNA isolation and RT-PCR amplification Total RNA was isolated from PC12 cells using Trizol. Extracted RNA was treated with DNase I and reverse transcribed with


626 B. E. Kalisch et al.

Superscript II using oligo-dT as the primer for 75 min at 43C. The cDNA generated was used for PCR with Platinum Taq DNA polymerase. All PCR reactions were performed in a final volume of 50 lL with an initial 2 min strand separation at 94C and a final 2 min elongation at 72C after the last cycle. Primer pairs and cycling conditions were: b-actin: 5¢ primer 5¢-TCATGAAGTGTGACGG-TTGACATCCGT-3¢ and 3¢ primer 5¢-CCTAGAAGATTTGCGGTGCACGATG-3¢, 14–16 cycles of 94C: 20 s, 65C: 30 s, 72C, 45 s; ChAT coding region: 5¢ primer 5¢-GCCAATCGCTGGTATGACAAGTC-3¢ and 3¢ primer 5¢-AACTCCACAGACGAGGTCTC-TTTG-3¢, 24–28 cycles of 94C: 20 s, 65C: 30 s, 72C: 45 s; nNOS: 5¢ primer 5¢-GCCAGCAAAGACCAGTCATTAGCA-3¢ and 3¢ primer 5¢-ACCACGTCCGTCTCCCA-GTTCT-3¢, 30–34 cycles of 94C: 20 s, 60C: 30 s, 72C: 45 s; eNOS 5¢ primer 5¢-CACAGGCATCACCAGGAAGAAGAC-3¢ and 3¢ primer 5¢-GGAGCCCAGCCCAAACAC-A-3¢, 32–35 cycles of 94C: 20 s, 62C: 30 s, 72C: 45 s; iNOS 5¢ primer 5¢-CCG-CTTCGATGTGCTGCCTCTG-3¢ and 3¢ primer 5¢-CCCATCCTCCTGCCCACTTCCTC-3¢, 32– 34 cycles of 94C: 20 s, 62C: 30 s, 72C: 45 s. PCR products were separated on a 1.5% agarose gel and visualized with ethidium bromide under UV light. Images were captured digitally using the Pharmacia Biotech Image Master VDS, processed in Adobe Photoshop 5.0 and Corel Draw 8.0 with densitometric analysis performed using Bio-Rad Multi-Analyst/PC version 1.1. The specificity of RT-PCR products was verified by Southern blot analysis or DNA sequencing. PCR cycle numbers for individual PCR products were established so that product formation fell within the linear range for RNA samples obtained from both control and NGF-treated PC12 cells. Western blot analysis of NOS isoforms Control and NGF-treated PC12 cells were rinsed with ice-cold PBS, then harvested in 200 lL of RIPA buffer (final concentration: 50 mM Tris, 150 mm NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM each of EDTA, sodium fluoride, AEBSF and sodium orthovanadate, and 1 lg/mL each of leupeptin, aprotinin and pepstatin, pH 7.4). Samples were shaken on ice for 15 min, 4 centrifuged at 17 500 g for 15 min at 4C and protein content in the supernatant was measured by the method of Bradford (1976). Protein samples (100 lg) were boiled in sample buffer [final concentration: 62.5 mM Tris–HCl; pH 6.8, 2% sodium dodecly sulfate (SDS), 10% glycerol and 0.01% phenol red] and loaded onto 7.5% SDS/polyacrylamide separating gels. Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Trans-blot semidry transfer unit (Bio-Rad Laboratories, Hercules, CA, USA) with transfer buffer (final concentration: 48 mM Tris, 39 mM glycine and 20% methanol, pH 9.2), and the membranes were blocked in 5% non-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h. Membranes were then incubated in 1 : 200 rabbit polyclonal nNOS antibody (Santa Cruz Biotechnology) in 3% bovine serum albumin (BSA) in TBS-T overnight. Antibody detection was achieved using 1 : 2500 donkey anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Amersham) in TBS-T (1 h), followed by enhanced chemiluminescence (ECL; Amersham). Blots were stripped with 62.5 mM Tris, pH 6.7, containing 2% SDS and 100 mM 2-mercaptoethanol at 50C for 30 min. Membranes were rinsed in TBS-T, blocked in 5% milk in TBS-T for 1 h, then probed for iNOS using 1 : 2000 rabbit

polyclonal iNOS antibody (a gift from Dr David Mercer, University of Texas-Houston Medical School), as described above. Determination of choline acetyltransferase activity Cultures were washed with PBS and assayed immediately or frozen at ) 80C until time of assay (within 2 weeks). Cells in each well were solubilized in 100 lL of ice-cold 0.1 M sodium phosphate buffer, pH 7.4, containing 0.87 mM EDTA, 0.1% Triton X-100 and 0.15 mM eserine and left for 30 min at 4C. ChAT activity was measured by monitoring the conversion of [3H]acetyl-coenzyme A to [3H]ACh using a modification of the method of Fonnum (1968). Each supernatant sample (10 lL) was incubated with 10 lL of reaction buffer for 30 min at 37C; product formation was linear with time and tissue concentration under these conditions. The reaction buffer consisted of 0.285 M NaCl, 0.095% BSA, 0.19 mM eserine, 3.8 mM choline, 0.2 mM acetyl-coenzyme A and 37 lM [3H]acetyl-coenzyme A in 0.1 M sodium phosphate buffer, pH 7.4. The reaction was stopped by the addition of 100 lL of ice-cold water. Radiolabeled ACh was extracted by adding 300 lL of 3-heptanone containing 20 mg/mL tetraphenylboron. Each sample 5 was vortex mixed for 1 min and centrifuged for 5 min at 5700 g. Subsequently, 200 lL of the upper organic layer was added to 3.5 mL scintillation cocktail and the radioactivity counted. The amount of protein present in the solubilized PC12 cell samples was determined by the method of Bradford (1976). Determination of NO production and NOS activity To evaluate NO production in living cells, the fluorescent probe DAF-2DA was used. PC12 cells were treated with 50 ng/mL NGF in the presence or absence of 20 mM L-NAME or 2.5 mM MIU for 3 days, washed once with media and loaded with 10 lM DAF2-DA in 1 mL culture media. Following 1 h of incubation cells were washed 4 times with 2 mL media and DAF-2 fluorescence was visualized using a Zeiss 510 laser scanning confocal microscope (excitation at 488 nm emission at 520 nm). Digitally captured images were processed in Adobe Photoshop 5.0 and Corel Draw 8.0. NOS activity was measured radioenzymatically using the kit from Calbiochem as described by the supplier and [3H]arginine (New England Nuclear, Boston, MA, USA). Control (untreated) PC12 cells or NGF-treated cells grown in the presence or absence of L-NAME (2–20 mM) or MIU (0.5–2.5 mM) were rinsed with PBS, then scraped into PBS containing 1 mM EDTA and pelleted by centrifugation. Cell pellets were dispersed in homogenization solution to give protein concentrations of about 20–30 mg/mL; individual assay samples contained about 200–300 lg protein, with incubation reactions carried out for 90 min. Data analysis The ethidium bromide gels are representative of at least three independent experiments. The level of mRNA in individual samples was determined by densitometric analysis and expressed in arbitrary units. Semi-quantitative evaluation of the relative amounts of PCR product formed was calculated as a ratio of the value obtained for each sample compared with its corresponding b-actin value, with data represented as mean ± SEM. ChAT activity was expressed as nmol [3H]ACh formed/mg protein/h. NOS activity was expressed as dpm [3H]citrulline formed/mg protein/ h. The effect of NGF, in the



absence or presence of NOS inhibitors, on ChAT or NOS activity was expressed as a percentage of enzyme activity in untreated PC12 cells, with data presented as the mean ± SEM. All data were assessed for homogeneity of variance using Cochran’s test and parametric statistical analysis was performed using a randomized One-way ANOVA followed by Newman–Keul’s test to determine which groups were significantly different. Differences in mean values were considered significant if p < 0.05.

Results

Effects of NGF on NADPH diaphorase staining and NOS and ChAT mRNA To ensure that the PC12 cells used in the present study exhibit an increase in total levels of NOS in response to treatment with NGF, as demonstrated by others (Peunova and Enikolopov 1995), we examined NADPH diaphorase staining over a time course up to 72 h (data not shown). Briefly, low levels of NADPH diaphorase-positive staining were observed in the cell bodies of control (untreated) PC12 cells. In cultures treated with NGF for 12–24 h, short neuritic extensions were observed in some cells and the intensity of NADPH diaphorase staining appeared greater following exposure to NGF for 24 h than in untreated cultures. Exposure of PC12 cells to NGF for longer time periods (48 or 72 h) resulted in more extensive neurite outgrowth, with intense NADPH diaphorase staining being evident in the cell bodies and neuritic processes. Figure 1(a) shows representative ethidium bromidestained gels demonstrating the time course of NGF-mediated effects on steady-state levels of transcripts for ChAT and the different NOS isoforms following RT-PCR amplification of RNA extracted from control or NGF-treated (50 ng/mL) PC12 cells. The first two lanes in all panels are controls in which water was taken through the PCR and RT-PCR processes, respectively. The upper panel of Fig. 1(a) illustrates the temporal pattern of expression of ChAT mRNA in response to NGF treatment. Over this time period, an increase in steady-state ChAT transcript is observed beginning at the 24 h time point in this RNA set (see below), and ChAT mRNA levels remain above control for at least 72 h. Similarly, nNOS (second panel) and eNOS (middle panel) expression is increased in response to NGF in a manner that parallels ChAT mRNA expression. In the next panel, steadystate levels of iNOS are increased in PC12 cells exposed to NGF for 12 or more hours, with the magnitude of this increase becoming greater at later time points. The bottom panel in Fig. 1(a) demonstrates that the levels of b-actin mRNA were similar in samples obtained from control and NGF-treated PC12 cells, indicating that the amount of RNA and efficiency of cDNA production was approximately equal in all samples. Ratios obtained for densitometric analysis of ChAT or NOS isoform mRNA normalized to b-actin

expressed as a function of time are depicted in Fig. 1(b). Increases in steady-state levels of ChAT, nNOS and eNOS were not observed consistently following 24 h of NGF treatment, thus yielding densitometric ratios for these transcripts at this time point that were similar to those obtained for control (untreated) PC12 cells; longer exposure to NGF (36–72 h) revealed consistent increases in the densitometric ratios for ChAT, nNOS and eNOS mRNAs when compared to control cells. In comparison, steady-state levels of iNOS mRNA, normalized to b-actin, were increased consistently in PC12 cells exposed to NGF for 12 or 24 h, with this increase being greater at later time points. Expression of NOS protein isoforms was investigated in control and NGF-treated PC12 cells by immunoblot analysis. As shown in Fig. 1(c, upper panel), low levels of nNOS protein were detected in lysates of control or 2 day NGFtreated cells, with increased levels being observed by 5 days of NGF treatment. Figure 1(c, lower panel) illustrates iNOS protein levels in lysates of control and NGF-treated PC12 cells; samples obtained from untreated or 2-day NGF-treated cells exhibited little or no iNOS protein, with a substantial increase in cells harvested 5 days following NGF treatment. The effect of NGF on eNOS protein expression in PC12 cells is unclear as bands observed on blots incubated with eNOS antibody were weak, with positive identification of eNOS not possible in lysates of a number of cell samples. Effect of L-NAME and MIU on NGF-induced ChAT expression The role of NO in NGF-mediated enhancement of ChAT expression was investigated using the NOS inhibitors L-NAME and MIU. In preliminary experiments, we found that mRNA for ChAT was consistently elevated in PC12 cells following exposure to 50 ng/mL NGF for 36 h. In subsequent experiments, this time point was selected to study the action of NOS inhibitors on NGF-induced increases in ChAT mRNA. Representative ethidium bromide-stained gels demonstrating the effect of 20 mM L-NAME or 2.5 mM MIU on ChAT and b-actin transcripts from RNA extracted from control or NGF-treated PC12 cells are shown in Fig. 2(a). NGF induced an increase in steady-state levels of mRNA for ChAT when compared to control PC12 cells. The intensity of the ChAT band was decreased in samples obtained from PC12 cells treated with NGF in the presence of MIU or L-NAME. No change in b-actin mRNA levels was observed in any of the treatment groups, indicating that these changes in steadystate ChAT mRNA levels are due to an action of the NOS inhibitors. Ratios of densitometric analysis of ChAT or mRNA normalized to b-actin versus treatment are depicted in Fig. 2(b). Treatment of PC12 cells with NGF for 36 h resulted in a statistically significant, approximately twofold, increase in steady-state levels of ChAT mRNA (p < 0.05). This increase was attenuated by co-treatment with NGF and either



Fig. 1 Effect of NGF treatment on steadystate ChAT and NOS mRNA and protein expression in PC12 cells. (a) Representative ethidium bromide-stained gels demonstrating the time course of RT-PCR amplification of specific transcripts from RNA extracted from control or NGF-treated (50 ng/mL) PC12 cells. Control samples were obtained at 12 h (C12) or 72 h (C72) and NGF-treated samples were obtained at 12, 24, 36, 48, 60 and 72 h after the beginning of the experiment. Specific primer sets were used for ChAT, nNOS, eNOS, iNOS and b-actin. Lane 1 in all panels is a 100-bp DNA ladder with the brightest marker at 600 bp, and lanes 2 and 3 are controls in which water samples were taken through the PCR and RT-PCR processes, respectively. (b) Steady-state levels of mRNA for ChAT, nNOS, eNOS or iNOS were analysed densitometrically (arbitrary units), with values expressed as a ratio of b-actin mRNA from the same sample; data are presented as mean ± SEM and are representative of up to 6 independent experiments. (c) Immunoblots showing changes in levels of nNOS and iNOS protein in control cells and cells treated with NGF for 2 or 5 days.

of the NOS inhibitors; cells treated with NOS inhibitors plus NGF were statistically different from cells treated with NGF alone (p < 0.05), but not different from control samples. Treatment of PC12 cells with MIU or L-NAME alone reduced the densitometric value of basal steady-state ChAT mRNA levels below that of control, indicating these NOS inhibitors decreased basal steady-state levels of mRNA for ChAT. To

ensure that these doses of NOS inhibitors were not toxic, viability of PC12 cells incubated with MIU or L-NAME was assessed by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Following 36-h exposure of cells to 2.5 mM MIU or 20 mM L-NAME in the presence or absence of NGF, MTT activity in any treatment group was not less than 90% of control activity (data not shown).



Fig. 2 Effect of NOS inhibitors on NGF-mediated enhancement of steady-state ChAT mRNA expression in PC12 cells. (a) Representative ethidium bromide-stained gels demonstrating RT-PCR amplification of specific transcripts from RNA extracted from untreated PC12 cells grown in control medium, medium containing 50 ng/mL NGF, medium containing 20 mM L-NAME or 2.5 mM MIU and NGF (50 ng/mL), or medium containing 20 mM L-NAME or 2.5 mM MIU without NGF. The last two lanes in all panels are water samples taken through the PCR and RT-PCR processes, respectively. Specific primer sets were used for ChAT (upper panel) and b-actin (lower panel). (b) Steadystate mRNA levels for ChAT were analysed densitometrically (arbitrary units), with values expressed as a ratio of b-actin mRNA levels from the same sample. Results are representative of three independent experiments. Statistical analysis revealed that steadystate ChAT mRNA levels in NGF-treated samples were significantly greater than that found in control and inhibitor-treated samples (p < 0.05), as indicated by the asterisk, with the exception of the NGF plus MIU group where statistical significance was achieved at p < 0.1.

The effect of L-NAME or MIU on NGF-induced increases in ChAT activity was also tested. As illustrated in Fig. 3(a,b), growth of PC12 cells with 50 ng/mL NGF for 5 days resulted in a twofold increase in ChAT activity. Figure 3(a) shows the action of 2, 10 or 20 mM L-NAME alone or in combination with 50 ng/mL NGF on ChAT activity, expressed as a percentage of ChAT activity in control (untreated) PC12 cells. Treatment of cells with 2 or 10 mM L-NAME did not alter basal enzyme activity, while 20 mM L-NAME significantly reduced constitutive ChAT activity (p < 0.05). Co-treatment of PC12 cells with NGF and 2 mM L-NAME did not alter NGF-mediated increases in ChAT activity, whereas treatment of cells with NGF and 10 mM L-NAME consistently resulted

in ChAT activity values that were lower than those obtained with NGF alone; this latter effect, however, did not achieve statistical significance. Co-treatment of PC12 cells with NGF and 20 mM L-NAME prevented the NGF-mediated increase in ChAT activity (p < 0.05). To confirm that the effect of L-NAME was related to its NOS inhibitory potential, we treated cells with the inactive enantiomer D-NAME in the presence and absence of NGF. As illustrated in Fig. 3(a), 20 mM D-NAME had no effect on either basal or NGFenhanced ChAT activity in PC12 cells. Figure 3(b) shows the action of 0.5, 1 or 2.5 mM MIU alone or in combination with 50 ng/mL NGF on ChAT activity. Treatment of PC12 cells with 0.5 or 1 mM MIU had no effect on basal or NGF-induced increases in ChAT activity. Similar to the results obtained with 20 mM L-NAME, 2.5 mM MIU decreased both constitutive and NGF-induced ChAT activity (p < 0.05). Interpretation of data regarding the action of 1 mM MIU is complicated, however, by the fact that this dose of inhibitor prevents NGF-mediated neurite outgrowth (see below); this is reflected in reduced protein content measured in samples of PC12 cells treated with NGF plus 1 mM MIU compared to cells treated with NGF alone in which there is extensive neurite outgrowth and cellular hypertrophy. As the NGFmediated increase in ChAT activity is partially attenuated by 1 mM MIU and the protein content of these samples is close to that measured in control cells, normalization of ChATspecific activity to sample protein content, as represented in Fig. 3, leads to underestimation of the effects of MIU on ChAT activity. At this dose of MIU, expressing the data as dpm [3H]ACh formed/culture well indicates that the NGFmediated increases in ChAT activity are reduced by 56% in cells co-treated with 1 mM MIU. To ensure that the observed action of NOS inhibitors on ChAT activity was not due to a direct effect of the inhibitors on ChAT protein, lysates of PC12 cells were incubated with L-NAME or MIU in vitro and ChAT-specific activity measured in their presence. It was found that neither 20 mM L-NAME nor 2.5 mM MIU altered specific activity of the enzyme directly (data not shown). In addition, in culture media obtained from PC12 cells treated with 2.5 mM MIU or 20 mM L-NAME for 5 days, there was no change in lactate dehydrogenase (LDH) activity when compared with control indicating further that the NOS inhibitors at these concentrations were not toxic to the cells (data not shown). Effect of L-NAME and MIU on NGF-induced morphological changes Representative photomicrographs demonstrating the effects of L-NAME or MIU treatment on NGF-mediated morphological differentiation of PC12 cells are shown in Fig. 4. In general, control untreated PC12 cells appeared rounded with occasional, short neuritic extensions (Fig. 4a). Following



Fig. 3 Effect of NOS inhibitors on NGF-mediated enhancement of ChAT activity in PC12 cells. ChAT activity was measured in lysates of PC12 cells in: (1) untreated control cells, (2) cells treated with NGF (50 ng/mL), (3) cells treated with NGF (50 ng/mL) in the presence of L-NAME (2–20 mM) or MIU (0.5–2.5 mM), and (4) cells treated with L-NAME (2–20 mM) or MIU (0.5–2.5 mM) alone. Media was changed on the cells every 2 days, with fresh inhibitor or NGF additions, for a period of 5 days. ChAT activity is measured in nmol/mg protein/h and the effect of NGF and/or NOS inhibitor on ChAT activity is expressed as a percentage of the enzyme activity of untreated control PC12 cells; ChAT activity in control cells was 6.22 ± 0.99 nmol/mg protein/h. The black bars shown for each NOS inhibitor concentration represents the effect of NGF in the absence of NOS inhibitor on ChAT activity in PC12 cells in each individual experimental group. Each bar represents the mean ± SEM of 4–8 experiments. Statistical differences were found for cells treated with NGF alone compared to cells co-treated with NOS inhibitor, denoted by *p < 0.05; cells treated with NGF alone or cells treated with NGF and NOS inhibitor compared to their respective controls, denoted by #p < 0.05; and cells treated with NOS inhibitor alone compared to their respective control groups, denoted by + p < 0.05.

exposure to NGF for 5 days, PC12 cell bodies appeared larger and flatter with extensive neurite outgrowth, compared to untreated cells (Fig. 4b). Treatment of cells with 10 (not shown) or 20 mM (Fig. 4c) L-NAME alone did not alter PC12 cell appearance when compared to control. As well, the pattern of NGF-induced morphological differentiation of PC12 cells treated with either dose of L-NAME did not appear to be substantially different from that observed following treatment with NGF alone (20 mM L-NAME Fig. 4d). However, when quantitative comparisons were made of the percentage of NGF-treated cells bearing neurites

Fig. 4 Effect of NOS inhibitors on NGF-mediated neurite outgrowth in PC12 cells. Representative photomicrographs of PC12 cells grown for 5 days in control medium (a), or in medium containing 20 mM L-NAME (c), or 2.5 mM MIU (e), or with NGF (50 ng/mL) alone (b), or in combination with 20 mM L-NAME (d), or 2.5 mM MIU (f). The proportion of cells bearing neurites greater than two cell bodies in length was quantified to determine whether L-NAME affected this measure. As shown in (g), 20 mM L-NAME significantly decreased NGF-mediated neurite outgrowth compared to NGF alone or NGF and 20 mM D-NAME, as denoted by the asterisk (p < 0.05). These results are representative of five independent experiments, with data shown as mean ± SEM.

was made in the presence and absence of 20 mM L-NAME, it was found that this NOS inhibitor significantly decreased NGF-mediated morphological differentiation of PC12 cells (p < 0.05; Fig. 4g). The L-NAME effect on NGF-mediated neurite outgrowth was further confirmed by comparison with NGF-treated cells grown in the presence of the inactive analogue D-NAME at 20 mM (p < 0.05 compared to 20 mM L-NAME; Fig. 4g); there was no difference between NGFmediated differentiation of cells grown in the absence or presence of D-NAME. Exposure of PC12 cells to both 1 mM



(not shown) and 2.5 mM (Fig. 4f) MIU blocked NGFmediated neurite outgrowth, however, these cells assumed a more flattened appearance when compared with cells grown in the 2.5 mM MIU alone (Fig. 4e). Effect of L-NAME and MIU on NGF-induced increases in DAF-2 fluorescence To determine whether PC12 cells produce NO and whether this is enhanced by NGF and to address the degree of NOS inhibition by L-NAME or MIU, we monitored DAF-2 fluorescence in control and treated PC12 cells. As shown in Fig. 5(a), a moderate level of DAF-2 fluorescence is observed in control, untreated PC12 cells. Importantly, DAF2 fluorescence, indicative of NO production, was substantially increased in cells treated with NGF for 3 days as depicted in Fig. 5(b). Exposure of cells to NGF and 20 mM L-NAME (Fig. 5c) resulted in attenuation of DAF-2 fluorescence compared with NGF treatment alone, although fluorescence in these cells remained greater than that seen in untreated PC12 cells. In contrast, co-treatment of PC12 cells with NGF and 2.5 mM MIU virtually eliminated DAF-2 fluorescence (Fig. 5d). These data were confirmed by

measuring NOS activity directly and evaluating the effect of NOS inhibitors on NGF-mediated increases in NO/NOS. As illustrated in Fig. 5(e), NGF increased NOS activity about twofold in PC12 cells (p < 0.05), and this was reduced to (or below) control levels in NGF-treated cells grown in the presence of 20 mM L-NAME or 2.5 mM MIU (p < 0.05). Taken together, these data suggest that 2.5 mM MIU is likely inhibiting constitutive NOS activity in addition to iNOS. Discussion

NGF promotes neurite outgrowth and increases ChAT mRNA levels and specific activity in PC12 cells. In the present study, we show for the first time that: (i) these NGFmediated actions were attenuated by co-treatment of cells with the iNOS inhibitor MIU, and that under similar treatment conditions, the non-selective NOS inhibitor L-NAME attenuated NGF-activated expression of ChAT but had less effect on neurite outgrowth; (ii) both MIU and L-NAME caused a dose-related decrease in basal levels of ChAT mRNA and activity in PC12 cells; and (iii) using the fluorescent probe DAF-2 to visualize NO production in

Fig. 5 DAF-2 fluorescence in PC12 cells is enhanced by NGF and decreased in NGFtreated cells by NOS inhibitors. Representative confocal microscope images depicting DAF-2 fluorescence in untreated PC12 cells in control medium (a), cells treated with 50 ng/mL NGF for 3 days (b), and NGFtreated cells grown in the presence of 20 mM L-NAME (c) or 2.5 mM MIU (d). The left column depicts fluorescence, middle column is the Nomarski differential interference contrast image of the cells and right column is the overlay of these two images. Untreated PC12 cells exhibited basal DAF-2 fluorescence that increased in intensity with NGF treatment. L-NAME attenuated, while MIU prevented this NGF-mediated increase in DAF-2 fluorescence. These results are representative of four independent experiments. (e) Data for NOS assays for the control and NGF-treated cells and cells treated with NGF in the presence of various concentrations of NOS inhibitors. Data represent mean ± SEM for up to seven separate experiments with duplicate or triplicate determinations; NOS activity in control cells was 29 980 dpm/mg protein/h. Statistical significance was determined for cells treated with NGF compared to controls, denoted by *p < 0.05, and cells treated with NGF and NOS inhibitor compared to NGF alone, denoted by #p < 0.05.



living cells, PC12 cells produce low levels of NO under control conditions and substantially increased levels of NO when grown in the presence of NGF. Taken together, these data suggest that activation of NOS with consequent production of NO in NGF-treated PC12 cells plays a role in modulating signaling pathway(s) involved in cholinergic gene expression, and that the action of NOS inhibitors on NGF-induced neurite outgrowth show qualitative differences. The NGF-induced increases in NOS/NO, measured in these studies as changes in NOS mRNA, protein and activity, NADPH diaphorase staining and DAF-2 fluorescence, are in agreement with and extend previous findings that NGF increases NOS activity, NADPH diaphorase staining, NOS immunostaining (Peunova and Enikolopov 1995) and nNOS protein expression (Sheehy et al. 1997; Phung et al. 1999) in PC12 cells. We found that nNOS, eNOS and iNOS mRNA was expressed in the PC12 cells, with steady-state levels of all three transcripts increased by NGF treatment. In addition, we verified expression and NGF-mediated regulation of nNOS and iNOS by immunoblot analysis. These data support the findings of Peunova and Enikolopov (1995) that NGF increased activity of Ca2+-dependent and Ca2+-independent NOS isoforms, as well as immunohistochemical staining of PC12 cells using antibodies specific for the three types of NOS. In contrast, Sheehy and co-workers (Sheehy et al. 1997) detected only nNOS on immunoblots of control or growth factor-treated PC12 cells. Aside from using different methodological approaches to detect NOS isoforms, it has been suggested that different lines of PC12 cells express different NOS isoforms (Sheehy et al. 1997). Peunova and Enikolopov (1995) obtained PC12 cells from Dr L. Greene, whereas Sheehy et al. (1997) used PC12 cells from American Type Culture Collection (ATCC, Rockville, MD, USA). To address this, we compared control and NGFtreated PC12 cells used in the present study (obtained from Dr S. O. Meakin) with those from ATCC for the presence of nNOS, eNOS and iNOS mRNA using RT-PCR. We found that transcripts for all three NOS isoforms were expressed in control and NGF-treated cells obtained from ATCC as well as those used in the present study, and that they appeared modulated by NGF (Fig. 6). Interestingly, while eNOS transcript was regulated by NGF, expression of eNOS protein was not observed consistently in our cells. This may be due to low levels of translation of eNOS mRNA, even in NGF-treated PC12 cells, or related to low sensitivity of detection of the protein by immunoblot compared to that observed for mRNA with RT-PCR. Although it has been demonstrated in separate studies that NGF enhances expression of NOS (Peunova and Enikolopov 1995; Sheehy et al. 1997) and ChAT (Baskey et al. 2002) proteins in PC12 cells, the temporal relationship between these events has not been investigated. Therefore, to address the role of NO in NGF-mediated enhancement of cholinergic phenotype, we compared the time course of action of NGF

Fig. 6 Steady-state NOS mRNA expression in different strains of PC12 cells. Representative ethidium bromide stained gels demonstrating RT-PCR amplification products of specific NOS isoform transcripts from RNA extracted from control or NGF (50 ng/mL) treated PC12 cells. Primer sets were used for nNOS, eNOS, iNOS and b-actin. Lane 1 is the 100 bp DNA ladder, and lanes 2 and 3 in all panels are water samples taken through the PCR and RT-PCR processes, respectively. This data is representative of at least 2 different RNA preparations for the cells, with multiple RT-PCR amplifications.

on NOS and ChAT mRNA levels. While nNOS, eNOS and ChAT transcripts appear to be regulated similarly by NGF, iNOS mRNA may be increased earlier, indicating that NO production resulting from increased iNOS could modulate actions of NGF on ChAT expression. Furthermore, while this profile suggests that enhanced levels of constitutive NOS do not occur prior to increased steady-state ChAT mRNA levels, constitutive NOS isoforms may still play a role in the early actions of NGF. NGF can cause rapid elevation of intracellular Ca2+ levels (Bouron et al. 1999; Jiang et al. 1999), with this potentially activating constitutive NOS (Garthwaite 1991; Klatt et al. 1993) present in PC12 cells leading to NO release. In this way, NO could be involved in early NGFmediated signaling events through activation of constitutive NOS and/or up-regulation of iNOS, both of which would result in increased NO production. Up-regulation of constitutive NOS isoforms may also play a role in the longer-term maintenance of NGF-enhanced cholinergic phenotype through sustained activation of cell signaling constituents. NOS inhibitors L-NAME and MIU were used to block production of NO to evaluate its role in NGF-mediated



increases in ChAT expression. NOS inhibition was determined radiometrically by NOS activity assays, as well as visually in real-time by measuring NO production in living untreated or NGF-treated PC12 cells using DAF-2DA; NO production in cells is monitored as increased fluorescence intensity resulting from irreversible reaction of NO with DAF-2 in the presence of oxygen (Kojima et al. 1998). In the present study, DAF-2 fluorescence was observed in PC12 cells in their regular culture media, with or without addition of NOS inhibitors, to explore production of NO under our experimental conditions. Fluorescence was seen throughout the cytosol of a subset of untreated cells, indicating that these PC12 cells express functional NOS that can release NO into cytoplasm to react with DAF-2; it is not known whether this can be attributed to activity of constitutive or inducible NOS. After 3 days treatment with NGF, DAF-2 fluorescence was substantially increased in essentially all cells indicating enhanced NO production and suggesting that NGF is increasing activity and/or levels of NOS isoform(s). Inhibition of NOS by L-NAME (20 mM) during NGF-treatment largely attenuated DAF-2 fluorescence compared to NGF treatment alone suggesting that, under these experimental conditions, this concentration of L-NAME reduces but does not prevent NGF-induced NO formation in PC12 cells; it has been reported that this concentration of L-NAME did not affect cellular metabolism (Peunova and Enikolopov 1995). Little DAF-2 fluorescence was observed in PC12 cells treated with NGF and 2.5 mM MIU. If MIU is selectively inhibiting iNOS, this would imply that most NO produced in these cells is from iNOS. However, this dose of MIU may be inhibiting both constitutive and inducible isoforms of NOS, and raises the question of whether constitutive NOS is active in these cells. We did not detect DAF-2 fluorescence in neurites, even though NADPH diaphorase staining was present in these processes. This difference in NOS/NO detection could be due to inability of DAF-2 to monitor smaller changes in NO production occurring in neurites compared to the cell body or to lack of NO production in these processes. This latter possibility raises the issue of distribution of NOS isozymes in NGF-treated PC12 cells. While NADPH diaphorase staining detects the presence of NOS, DAF-2 fluorescence only occurs when NOS is activated. Thus, if neurites contain primarily inactive constitutive NOS, then DAF-2 fluorescence would not be observed. Co-treatment of PC12 cells with 1 or 2.5 mM MIU prevented NGF-mediated neurite outgrowth, while co-treatment with up to 20 mM L-NAME significantly attenuated, but did not block, this action of NGF. This effect of L-NAME in the present study is similar to findings of Peunova and Enikolopov (1995) who reported that 20 mM L-NAME decreased the number of PC12 cells bearing neurites in response to 50 ng/mL NGF; Phung et al. (1999) found that 1 mM L-NAME reduced neurite outgrowth resulting from

treatment of PC12 cells with 40 ng/mL NGF. The difference between effects of L-NAME and MIU may be due to differential action/location of NO from constitutive versus inducible NOS, the degree of NOS inhibition resulting from L-NAME versus MIU treatment, or a specific action of MIU on neurite outgrowth not shared by L-NAME. To investigate the role of NO in NGF-induced, TrkAmediated enhancement of expression of cholinergic phenotype, the effect of treatment of PC12 cells with NOS inhibitors on basal and NGF-stimulated ChAT mRNA levels and ChAT activity was determined. L-NAME or MIU reduced basal as well as NGF-stimulated steady-state levels of ChAT mRNA and ChAT activity suggesting that NO released in both untreated and NGF-treated cells regulates expression of ChAT. This attenuation occurred at a dose of L-NAME that did not completely block NO production (measured by DAF-2 fluorescence), suggesting that a threshold of NO is required for modulation of NGFenhanced cholinergic phenotype in PC12 cells. Several hypotheses have been suggested to describe the role of NO in cell signaling events. For example, NGF-mediated increases in NOS/NO could modulate ChAT gene expression by mechanisms involving regulation of transcription factor function. NGF increases AP-1 transcription factor levels and DNA binding in PC12 cells, and NO donors stimulate expression of immediate early genes such as c-Fos and Jun-B (Haby et al. 1994; Morris 1995; Pilz et al. 1995) and increase transcription from AP-1 responsive promoters (Haby et al. 1994; Pilz et al. 1995). The ChAT gene promoter contains a number of AP-1 consensus sequences that could bind Fos–Jun or Jun–Jun transcription factors (Toussaint et al. 1992), thus making it a target for modulation. Similarly, the transcription factor NGF1-A, which may be involved in transcriptional activation of ChAT (Quirin-Stricker et al. 1997), is induced by NGF (Kumahara et al. 1999) and NO donors (Morris 1995). Furthermore, NO could modulate NGF-mediated activation of mitogen-activated protein (MAP) kinase. Activation of p21ras by NGF propagates a signaling cascade leading to phosphorylation of p42 and p44 MAP kinase (Boulton et al. 1991; Robbins et al. 1993) and cell differentiation (Cowley et al. 1994; Kortenjann et al. 1994; Marshall 1995; Sano and Kitajima 1998). Like NGF, NO donors stimulate phosphorylation of MAP kinase in hippocampal neurons through activation of Ras (Kanterwicz et al. 1998), suggesting that NO could modulate NGF signaling through the Ras–MAP kinase pathway. The present study demonstrates that NOS inhibitors attenuate NGF-mediated increases in ChAT gene expression in PC12 cells, suggesting that NO is involved in aspects of NGF-TrkA signaling in cholinergic neurons. The NOS inhibitors used show differences with respect to the extent to which they block NGF-mediated neurite outgrowth; this difference requires further investigation.



Acknowledgements These studies were supported by operating grants from the Alzheimer Society of Canada and the Medical Research Council of Canada (now CIHR) to RJR, and a Postdoctoral Fellowship award to BEK from the Alzheimer Society of Canada. NAB was the recipient of the PMAC-MRC Summer Studentship. The authors thank Ms. Jacqui Baskey for technical assistance with aspects of the studies. The iNOS antibody was kindly supplied by Dr David Mercer, Trauma Research Center at University of Texas-Houston Medical School funded by NIGMS Grant GM 35829.

References Baskey J. C., Kalisch B. E., Davis W. L., Meakin S. O. and Rylett R. J. (2002) PC12nnr5 cells expressing TrkA receptors undergo morphological but not cholinergic phenotypic differentiation in 6 response to NGF. J. Neurochem. 80, 501–511. Boulton T. G., Gregory J. S. and Cobb M. H. (1991) Purification and properties of ERK1, an insulin stimulated MAP2 protein kinase. Biochemistry 30, 278–286. Bouron A., Becker C. and Porzig H. (1999) Functional expression of voltage-gated Na+ and Ca2+ channels during neuronal differentiation of PC12 cells with nerve growth factor or forskolin. Nauyn Schmeid. Arch. Pharmacol. 359, 370–337. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248. Cowley S., Paterson H., Kemp P. and Marshall C. J. (1994) Activation of MAP kinase kinase is necessary and sufficient for PC12 cell differentiation and for transformation of NIH 3t3 cells. Cell 64, 841–852. Dunbar G. L., Rylett R. J., Schmidt B. M., Sinclair R. C. and Williams L. R. (1992) Hippocampal ChAT activity correlates inversely with spatial learning in aged rats. Brain Res. 604, 266–272. Fischer W., Wictorin K., Bjorklund A., Williams L. R., Varon S. and Gage F. H. (1987) Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature 329, 65–68. Fischer W., Bjorklund A., Chen K. S. and Gage F. H. (1991) NGF improves spatial memory in aged rodents as a function of age. J. Neurosci. 11, 1889–1906. Fonnum F. (1968) Choline acetyltransferase binding to and release from membranes. Biochem. J. 109, 389–398. Garthwaite J. (1991) Glutamate, nitric oxide and cell–cell signalling in the nervous system. Trends Neurol. Sci. 14, 61–67. Haby C., Lisovoski F., Aunis D. and Zwiller J. (1994) Stimulation of the cyclic GMP pathway by NO induces expression of the immediate early genes c-fos and junB in PC12 cells. J. Neurochem. 62, 496– 501. Hindley S., Juurlink B. H. J., Gysbers J. W., Middlemiss P. J., Herman M. A. R. and Rathbone M. P. (1997) Nitric oxide donors enhance neurotrophin-induced neurite outgrowth through a cGMPdependent mechanism. J. Neurosci. Res. 47, 427–439. Holtzman D. M., Kilbridge J., Bredt D. S., Black S. M., Li Y., Clary D. O., Reichardt L. F. and Mobley W. C. (1994) NOS induction by NGF in basal forebrain cholinergic neurons: evidence for regulation of brain NOS by a neurotrophin. Neurobiol. Dis. 1, 51–60. Holtzman D. M., Lee S., Li Y., Chua-Couzens J., Xia H., Bredt D. S. and Mobley W. C. (1996) Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: regulation by NGF. Neurochem. Res. 21, 861–868.

Jiang H., Takeda K., Lazarovici P., Katagiri Y. YuZ. X., Dickens G., Chabuk A., Liu X. W., Ferrans V. and Guroff G. (1999) Nerve growth factor (NGF)-induced calcium influx and intracellular calcium mobilization in 3T3 cells expressing NGF receptors. J. Biol. Chem. 10, 26209–26216. Kanterwicz B. I., Knapp L. T. and Klann E. (1998) Stimulation of p42 and p44 mitogen activated protein kinase by reactive oxygen species and nitric oxide in hippocampus. J. Neurochem. 70, 1000– 1016. Klatt P., Schmidt K., Uray G. and Monyer B. (1993) Multiple catalytic functions of brain nitric oxide synthase. Biochemical characterization, cofactor requirement and role of NG-hydroxy-L-arginine as an intermediate. J. Biol. Chem. 268, 14781–14787. Knipper M., da Penha Berzaghi M., Blochl A., Breer H., Thoenen H. and Lindholm D. (1994a) Positive feedback between acetylcholine and the neurotrophins nerve growth factor and brain-derived neurotrophic factor in the rat hippocampus. Eur. J. Neurosci. 6, 668–671. Knipper M., Leung L. S., Zhao D. and Rylett R. J. (1994b) Short-term modulation of glutamatergic synapses in adult rat hippocampus by nerve growth factor. Neuroreport 5, 2433–2436. Kojima H., Nakatsubo N., Kikuchi K., Urano Y., Higuchi T., Tanaka J., Kudo Y. and Nagano T. (1998) Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF2 DA. Neuroreport 9, 3345–3348. Kortenjann M., Thomae O. and Shaw P. E. (1994) Inhibition of v-raf dependent c-fos expression and transformation by kinase-defective mutant of the mitogen-activated protein kinase ERK2. Mol. Biol. Cell 14, 4815–4824. Kumahara E., Ebihara T. and Saffen D. (1999) Nerve growth factor induces zif268 gene expression via MAPK-dependent and -independent pathways in PC12D cells. J. Biochem. Tokyo 125, 541–553. Lapchak P. A., Araujo D. M., Carswell S. and Hefti F. (1993) Distribution of [125I]nerve growth factor in the rat brain following a single intraventricular injection: correlation with the topographical distribution of trkA messenger-RNA-expressing cells. Neurosci. 54, 445–460. Marshall C. J. (1995) Specificity of receptor tyrosine kinase signalling: transient versus sustained extracellular signal regulated kinase activation. Cell 80, 179–185. Mayer B., Schmidt K., Humbert P. and Bohme E. (1989) Biosynthesis of the endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts 1-arginine into an activator of soluble guanylyl cyclase. Biochem. Biophys. Res. Commun. 164, 678–685. Morris B. J. (1995) Stimulation of immediate early gene expression in striatal neurons by nitric oxide. J. Biol. Chem. 270, 24740– 24744. Nordberg A. (1993) Clinical studies in Alzheimer patients with positron emission tomography. Behav. Brain Res. 57, 215–224. Olson L., Nordberg A., von Holst H. et al. (1992) Nerve growth factor affects 11C-nicotine binding, blood flow, EEG and verbal episodic memory in an Alzheimer patient (case study). J. Neural Transm. Park. Dement. Sect. 4, 79–95. Peunova N. and Enikolopov G. (1995) Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature 375, 68–73. Phung Y. T., Bekker J. M., Hallmark O. G. and Black S. M. (1999) Both neuronal NO synthase and nitric oxide are required for PC12 cell differentiation: a cGMP-independent pathway. Mol. Brain Res. 64, 165–178. Pilz R. B., Suhasini M., Idriss S., Meinkoth J. L. and Boss G. R. (1995) Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells. FASEB J. 9, 552–558.



Poluha W., Schonhoff C. M., Harrington K. S., Lachyankar M. B., Crosbie N. E., Bulesco D. A. and Ross A. H. (1997) A novel, nerve growth factor-activated pathway involving nitric oxide, p53, and p21WAF1 regulates neuronal differentiation of PC12 cells. J. Biol. Chem. 272, 24002–24007. Pongrac J. L. and Rylett R. J. (1996) Differential effects of nerve growth factor on expression of choline acetyltransferase and sodiumcoupled choline transport in basal forebrain cholinergic neurons in culture. J. Neurochem. 66, 804–811. Quirin-Stricker C., Mauvais C. and Schmitt M. (1997) Transcriptional activation of human choline acetyltransferase by AP2- and NGFinduced factors. Mol. Brain Res. 49, 165–174. Robbins D. J., Zhen E., Owaki H., Vanderbilt C. A., Ebert D. and Geppert T. D. (1993) Regulaton properties of extracellular signalregulated protein kinase 1 and 2 in vitro. J. Biol. Chem. 268, 5097–5106. Rylett R. J., Goddard S., Schmidt B. M. and Williams L. R. (1993) Acetylcholine release following chronic intracerebral administration of nerve growth factor in adult and aged Fisher 344 male rats. J. Neurosci. 13, 3956–3963. Sano M. and Kitajima S. (1998) Activation of mitogen-activated protein kinases is not required for the extension of neurites from PC12D cells triggered by nerve growth factor. Brain Res. 785, 299–308. Seiler M. and Schwab M. E. (1984) Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat. Brain Res. 300, 33–39.

Sheehy A. M., Phung Y. T., Riemer R. K. and Black S. M. (1997) Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells. Mol. Brain Res. 52, 71–77. Sugaya K. and McKinney M. (1994) Nitric oxide gene expression in cholinergic neurons in the rat brain examined by combined immunocytochemistry and in situ hybridization histochemistry. Mol. Brain Res. 23, 111–125. Suzuki T., Kanagawa M., Takada Y., Fujimoto K. and Kawashima K. (1994) Nerve growth factor treatment induces high-potassiumevoked calcium-dependent acetylcholine release in cultured embryonic septal cells. Brain Res. 665, 311–314. Takei N., Tsukui H. and Hatanaka H. (1989) Intracellular storage and evoked release of acetylcholine from postnatal rat basal forebrain cholinergic neurons in culture with nerve growth factor. J. Neurochem. 53, 1405–1410. Thoenen H. (1995) Neurotrophins and neuronal plasticity. Science 270, 593–598. Toussaint J. L., Geoffroy V., Schmitt A., Garnier J. M., Simoni P. and Kempf J. (1992) Human choline acetyltransferase (CHAT): partial gene sequence and potential control regions. Genomics 12, 412–416. Vincent S. R. and Kimura H. (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755–784. Williams L. R. and Rylett R. J. (1990) Exogenous nerve growth factor increases activity of high-affinity choline uptake and choline acetyltransferase in brain of Fisher-344 rats. J. Neurochem. 55, 1042– 1049.
