Introduction

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Alcohol-induced brain damage has been consistently correlated with abnormalities in central nervous system cellular morphology, selected neuronal cell loss and astrogliosis (Hunt and Nixon, 1993), but it is still unclear how ethanol causes brain injury. An interesting hypothesis proposes that ethanol neurotoxicity is linked to NO formation and glutamate excitotoxicity (Lancaster, 1992, 1995). Studies show that NO synthesis or NO donors can either inhibit or mediate excitotoxicity induced by the glutamate receptor agonist N-methyl-D-aspartate (Dawson et al., 1991; Lei et al., 1992). NO seems to perform these opposing roles through different concentration-dependent mechanisms (Lipton et al., 1993; Kashii et al., 1996).

Formation of NO occurs through the conversion of L-arginine to L-citrulline by NOS (Griffith and Stuehr, 1995). Three isoforms of NOS have been identified by gene cloning. Two are constitutively expressed and one, the inducible NOS (NOS-2), is produced de novo in response to inflammatory cytokines, protein kinase C activators, LPS or viral infection (Nathan and Xie, 1994). Although requiring calmodulin binding for activity, NOS-2 is fully functional at resting intracellular calcium levels (Griffith and Stuehr, 1995) and is continually active for days, provided substrate is available. In the central nervous system, astrocytes and microglia are the major NOS-2-expressing cells after trauma, ischemia, viral infection or immunological challenge (Murphy and Grzybicki, 1996), although neurons can also express NOS-2 (Minc-Golomb et al., 1996). The consequences of prolonged continual NO generation by glial NOS-2 are not fully understood, but it seems clear that NOS-2 induction is an important cellular response to brain injury.

The proposed link between excitotoxicity, NO formation and alcohol brain damage may involve glial cells (Chandler et al., 1994). In a previous study, inhibition of glial NOS-2 activity by ethanol exposure was demonstrated using the C6 glioma cell line, an astrocyte-derived tumor line (Benda et al., 1968) that exhibits many astrocytic properties and can be induced to express NOS-2 (Feinstein et al., 1994a). Specifically, intact cells exposed to ethanol during NOS-2 induction by a 24-hr combined treatment with LPS and PMA accumulated significantly less nitrite in their culture medium, compared with cells not exposed to ethanol (Syapin, 1995). Nitrite is a stable oxidative product of NO (Stuehr and Marletta, 1985) and an indicator of NOS-2 activity. The inhibitory effect of ethanol exposure was both concentration and time dependent (Syapin, 1995) and was not a consequence of cytotoxicity (P.J. Syapin, A. Rendon, and J.D. Militante, submitted). In cells not previously exposed to ethanol, the inhibitory effect was relatively weak (IC₅₀  150 mM), whereas cells previously exposed to ethanol for 9 days became sensitized to the inhibitory effect (IC₅₀ 30 mM).

The aim of the present study was to characterize the mechanisms involved in the ethanol inhibition of NOS-2 activity in C6 glioma cells not previously exposed to ethanol. Experiments on intact cells already expressing NOS-2 and on in vitro NOS-2 activity measured in cytosolic extracts demonstrated no direct effect of ethanol on the catalytic activity of the enzyme. On the other hand, in vitro NOS-2 activity was significantly decreased in cytosolic extracts prepared from cells previously exposed to ethanol during induction with LPS plus PMA. Immunoblot and RNA analysis of cells previously exposed to ethanol during induction showed decreases in NOS-2 protein and mRNA levels, respectively. Additional experiments demonstrated that ethanol inhibition also occurred when NOS-2 expression was induced by cytokines. These data lead to the conclusion that ethanol inhibits NOS-2 activity in C6 glioma cells not previously exposed to ethanol by suppression of NOS-2 gene expression.

	Materials and Methods

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Materials. The following materials were obtained from the sources indicated: rat C6 glioma cells (American Type Culture Collection, Rockville, MD); Dulbecco's modified Eagle's medium with high glucose (MediaTech, Gaithersburg, MD); characterized fetal bovine serum (Hyclone Laboratories, Logan, UT); human recombinant TNF- (Calbiochem-Novabiochem, San Diego, CA); rat recombinant interferon- (GIBCO/BRL, Grand Island, NY); PMA, Salmonella typhimurium LPS, NADPH, FAD, protease inhibitors, N-(1-naphthyl)ethylenediamine, sulfanilamide, Tris, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, bovine serum albumin and sodium nitrite (Sigma Chemical Co., St. Louis, MO); 5,6,7,8-tetrahydro-L-biopterin dihydrochloride (ICN Biomedicals, Costa Mesa, CA); RNAzol-B (Biotecx Labs, Houston, TX); SuperSignal CL-HRP substrate system and bicinchoninic acid protein assay reagent A (Pierce, Rockford, IL); 95% (v/v) ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KY); horseradish peroxidase-conjugated anti-mouse polyclonal antibody and L-[³H]arginine (Amersham Life Science, Arlington Heights, IL); [³³P]dCTP (New England Nuclear, Beverly, MA); acrylamide, N,N,N,N-tetramethylethylenediamine, L-glycine and SDS (Bio-Rad, Richmond, CA); and clone 6 anti-NOS-2 monoclonal antibody (Transduction Laboratories, Lexington, KY).

Cell culture. C6 glioma cells were propagated and maintained according to methods previously described (Syapin, 1995). In brief, stock cultures were grown in high glucose-containing Dulbecco's modified Eagles's medium with 5% fetal bovine serum added. Experimental cultures were grown in medium containing 2.5% fetal bovine serum. All cultures were maintained at 37°C inside humidified incubators with 5% CO₂/95% air. Culture medium was replenished 2 to 3 days after seeding and every other day thereafter.

NOS-2 induction. NOS activity was induced in serum-free medium by simultaneous treatment with 500 ng/ml LPS and 400 ng/ml PMA, as previously described (Syapin, 1995). In some experiments, PMA was replaced by 60 ng/ml TNF- or 150 U/ml interferon-. TNF- and interferon- were also used alone at 60 ng/ml and 150 U/ml, respectively. Exposure to the inducing agents was for 24 hr unless stated otherwise. For postinduction experiments, cultures were washed once after induction, with the appropriate prewarmed medium, to remove LPS plus PMA, followed by addition of fresh prewarmed medium for measurement of subsequent 24-hr nitrite accumulations. For 12-hr ethanol exposure experiments, induction was initiated as described above, except that exposure to ethanol and LPS plus PMA was terminated at 12 hr by washing of the cultures once with prewarmed control medium, followed by addition of fresh prewarmed medium for measurement of subsequent 36-hr nitrite accumulations. Experimental controls included cultures that were incubated in the presence of LPS plus PMA for 48 hr before nitrite determinations and cultures for which inductions were interrupted at 12 hr by washing but that were subsequently re-exposed to LPS plus PMA in fresh prewarmed medium for the remaining 36 hr. These controls were designed to reveal whether the inhibition by ethanol persisted for 48 hr and to uncover possible untoward effects of having induction momentarily interrupted at 12 hr that might confound data interpretation.

Ethanol treatment. Cells were treated with ethanol as for previous studies (Syapin, 1995; P.J. Syapin, A. Rendon, and J.D. Militante, submitted). The cultures were placed in holding trays inside plastic bags containing a 400-ml reservoir of aqueous ethanol at the same concentration as the medium. The bags, located inside a water-jacketed, 37°C, CO₂ incubator, were gassed with compressed 5% CO₂/95% air and sealed. We previously determined that, using this procedure, ethanol concentrations in the medium decreased 30% after 48 hr. Therefore, ethanol concentrations refer to initial values. Control cultures and those no longer exposed to ethanol were maintained in the same manner, except that the reservoir contained only water.

Preparation of cell homogenates and cytosolic extracts. The method described by Galea et al. (1992) was followed in isolating cytosolic extracts from C6 cell cultures, with minor modifications. After induction with LPS plus PMA, culture medium was collected and assayed for nitrite accumulation. The cell layer was rinsed with cold Hanks' balanced salt solution, scraped off the dish in buffer A (320 mM sucrose, 50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM DL-dithiothreitol, 0.2 mM benzamidine, 10 µM pepstatin A, 10 µg/ml chymostatin, 10 µg/ml phenylmethylsulfonyl fluoride) and homogenized. The homogenate was centrifuged at 100,000 × g for 30 min at 4°C. The supernatant was collected and dialyzed for 2 hr against 1 liter of buffer B (50 mM Tris-HCl, pH 7.8, 1 mM DL-dithiothreitol, 0.2 mM benzamidine) to remove low-molecular weight solutes. The buffer B was replaced once during dialysis. The dialyzed cytosolic extracts were stored in aliquots at 80°C. Aliquots of homogenates and cytosolic extracts were assayed for protein content as described below.

Determination of NOS-2 activity. The NOS-2 activity of intact cells was usually assayed by measuring nitrite accumulation in the culture medium 24 hr after the start of induction. The accumulation of nitrite, a stable oxidative product of NO, in culture medium is a commonly used indicator of NOS-2 activity. For studies on preinduced cells (postinduction experiments), the culture medium was changed at the end of the 24-hr induction period and nitrite was allowed to accumulate for another 24 hr. For studies where cells were exposed to ethanol and LPS plus PMA for different times (12-hr ethanol exposure experiments), culture media were changed at 12 hr after the start of induction and nitrite was allowed to accumulate for another 36 hr, or the same medium was left on the cells for 48-hr accumulations. Nitrite levels were determined spectrophotometrically on duplicate samples. Briefly, 1.5 ml of culture medium was mixed with an equal volume of a 1:1 mixture of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine (Green et al., 1982). Absorbance was read at 546 nm. Sodium nitrite diluted into serum-free culture medium was used to prepare standards. Total cell protein/culture was also determined and nitrite accumulation was expressed as nanomoles per milligram of protein.

Immunoblotting. Homogenates and cytosolic extracts were used for immunoblotting. Samples were electrophoretically separated through 8% polyacrylamide gels containing 0.1% SDS in running buffer (25 mM Tris base, 190 mM L-glycine, 1% SDS). Proteins were electrotransferred to polyvinylidene difluoride membranes in transfer buffer (20 mM Tris base, 150 mM L-glycine, 10% methanol, 0.01% SDS) for 2 hr at 100 A. Membranes were blocked for 1 hr at room temperature in PBST containing 1% bovine serum albumin and were incubated overnight at 4°C with anti-NOS-2 monoclonal antibody (1:1000 dilution) in the same buffer. Membranes were then washed with PBST three times for 5 min, incubated for 30 to 60 min at room temperature with horseradish peroxidase-conjugated anti-mouse polyclonal antibody (1:3000 dilution) and then washed again with PBST twice for 30 min. Bands were visualized using chemiluminescence and autoradiography, and their densities were quantified with a Millipore Bio-Imager.

RNA isolation and mRNA quantitation. Total cellular RNA was extracted with RNAzol B from cultures induced in the absence or presence of ethanol and LPS plus PMA, following the manufacturer's instructions. The quality of the RNA was assessed by nondenaturing electrophoresis through 1% agarose gels for the presence of intact 18S and 28 S rRNA bands and by absorption at 260 and 280 nm. Levels of NOS-2 mRNA were determined by a competitive RT-PCR assay, as described (Galea et al., 1994). cDNA was prepared by RT of cytoplasmic RNA using random hexamers. The cDNAs (corresponding to 50 ng total RNA) were amplified in the presence of increasing amounts of an internal NOS-2 cDNA standard and 1.25 µCi of [³³P]dCTP (specific activity, 3000 Ci/mmol). PCR conditions were 35 cycles of denaturation at 93°C for 30 sec, annealing at 63°C for 45 sec and synthesis at 72°C for 45 sec, followed by a 10-min elongation at 72°C. PCR products were separated by electrophoresis through a 2% agarose gel and excised, and the incorporated radioactivity was measured by liquid scintillation counting. The primers used were NOS-1704F (5-CTGCATGGAACAGTATAAGGCAAAC-3), corresponding to bases 1704 to 1728, and NOS-1933R (5-CAGACAGTTTCTGGTCGATGTCATGA-3), complementary to bases 1908 to 1933 of the rat NOS-2 cDNA sequence (Galea et al., 1994). The internal standard was a 190-base pair NOS-2 cDNA fragment (bases 1704-1933) having an internal 40-base pair deletion. The identity of NOS-2 PCR products was confirmed by DNA sequence analysis of subcloned PCR products.

Protein assay. After the culture medium was removed, cells were dissolved in NaOH (0.5 M final concentration). Homogenates and cytosolic extract samples were dissolved in NaOH in the same way. The bicinchoninic acid protein assay was performed in duplicate according to the method of Smith et al. (1985), with crystalline bovine serum albumin standards.

Data analyses. Unless stated otherwise, results are from at least three independent experiments and data are presented as the mean ± S.E.M. of either actual or percentage of control activities for the number (n) of individual samples used for each data point. Bartlett's test for equal variances was used to determine whether statistical analysis should be parametric or nonparametric. One-way and two-way ANOVAs, post hoc multiple comparisons with Bonferrioni correction and paired or unpaired Student's t tests were used for parametric analyses. The Kruskal-Wallis test with Dunn's post hoc comparisons was used for nonparametric analyses.

	Results

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No direct effects of ethanol on enzymatic activity. The possibility that ethanol exposure reduced nitrite accumulation in the medium of C6 glioma cells induced with LPS plus PMA by a direct inhibition of NOS-2 catalytic activity was examined using two approaches. One approach was to add ethanol directly to the reaction mixture during determination of cytosolic NOS-2 catalytic activity from LPS/PMA-stimulated control cells. Ethanol at up to 200 mM final concentration had no effect on this activity (table 1). Reaction tubes were capped during incubation to eliminate evaporation of the ethanol.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Effect of exposure to 100 mM ethanol postinduction on NOS-2 activity. Notations under columns, ethanol concentration during induction  ethanol concentration after induction for 24 hr before nitrite determination. One-way ANOVA indicated statistically significant (F = 33.57, P < .001) differences between groups. Each column represents the mean ± S.E.M. of four independent experiments (100% control activity = 16.0 nmol/mg protein/24 h; n = 11 or 13/condition). ***P < .001, relative to unexposed cultures () and cultures exposed to ethanol after termination of induction ().

Reduced recovery of cytosolic NOS-2 activity, protein and mRNA. In contrast to a lack of direct effects of ethanol on cytosolic NOS-2 catalytic activity and on cultures already induced to express NOS-2, recovery of NOS-2 enzyme activity in cytosolic extracts was significantly reduced (P < .05) from cultures exposed to 100 mM ethanol during induction with LPS plus PMA (fig. 2). As expected, nitrite levels were similarly decreased (P < .05) in media from the same cultures (fig. 2). Cytosol from untreated C6 cells lacked detectable NOS catalytic activity (results not shown). Immunoblot (Western) analysis was used to determine the effect of ethanol exposure on induction of NOS-2 protein expression. A representative blot is shown in figure 3A. No detectable immunoreactive NOS-2 protein was present in cytosol from untreated C6 cells (results not shown), consistent with previous results (Feinstein et al., 1994a) and a lack of cytosolic NOS catalytic activity. The results (fig. 3) show that NOS-2 protein was decreased in the cytosolic extracts from cultures exposed to ethanol during induction with LPS plus PMA. Similar reductions were observed in immunoblots on total cell homogenates (results not shown). Computer-assisted quantitation of band intensities from three independent experiments with cytosol confirmed that the decreases were statistically significant (P < .05) (fig. 3B). An equally significant reduction (P < .05) in nitrite accumulation paralleled these reductions in cytosolic NOS-2 protein (fig. 3B). Ethanol-induced decreases in immunoreactive NOS-2 protein appeared concentration-dependent, because a greater percent reduction was observed at 200 mM ethanol (results not shown) (P.J. Syapin, A. Rendon, and J.D. Militante, submitted).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Reduction in cytosolic NOS-2 activity measured by in vitro enzyme assay from ethanol-exposed cells. Cytosolic extracts were prepared from control cells and cells exposed to 100 mM ethanol during induction with LPS plus PMA and were assayed for the conversion of l-arginine to L-citrulline. Nitrite levels were measured on culture media collected from the same cells used to prepare cytosolic extracts. Each column represents the mean ± S.E.M. of seven independent experiments assayed in quintuplicate (cytosolic NOS-2 activity) (left two columns) or duplicate (nitrite accumulation) (right two columns). *P < .05 vs. control activity by paired t test. C, control group; E, ethanol-exposed group.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Cytosolic NOS-2 protein and nitrite levels in control cells and cells exposed to 100 mM ethanol during induction with LPS plus PMA. A, immunoblot of NOS-2 protein in control (C1 and C2) and ethanol-exposed (E1 and E2) cytosol extracted from C6 glioma cells. Results are shown from two separate experiments with two independent extracts. Equivalent amounts of protein were analyzed within each experiment. Lane 9, control cell homogenate from C6 cells induced with LPS plus interferon- (positive control). B, quantification of NOS-2 protein content (left two columns) and in situ activity (right two columns) from three identical experiments, each with duplicate groups/condition (n = 6/column). The amount of immunoreactive protein was quantified by computer-assisted densitometry. *P < .05 vs. respective control value. C, control group; E, ethanol-exposed group.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   A, measurement of NOS-2 mRNA levels by RT-PCR. Total cellular RNA was extracted at 12 and 24 hr from control and 150 mM ethanol-exposed cultures and converted to cDNA with random hexamers, and equal aliquots corresponding to 50 ng of starting RNA were amplified in the presence of specific NOS-2 PCR primers and the indicated amount of competitive internal standard (CIS). The locations of the expected NOS-2 cDNA product (cDNA) and competitive internal standard product are indicated. The gel shown is representative of two independent experiments. B, calculation of NOS-2 mRNA levels. After PCR, the NOS-2 cDNA and standard bands were excised from the gel, incorporated radioactivity was determined and NOS-2 mRNA levels were calculated as described in "Materials and Methods." The results shown are mean ± S.D. from two independent samples measured at each time point and are presented as femtograms of the NOS-2 cDNA produced per microgram of cellular RNA. *P < .05 vs. respective control value. In situ NOS-2 activity determined in sister cultures induced with LPS plus PMA in the absence or presence of 150 mM ethanol were statistically different at P < .01 (2.63 ± 0.35 and 0.76 ± 0.13 nmol nitrite/mg protein/24 hr for control and ethanol, respectively; n = 4).

Reduced NOS-2 activity after 12-hr ethanol exposure. We examined whether 12-hr exposure to LPS plus PMA was sufficient to induce functional NOS-2 activity in C6 cells and, if so, whether the reduction in NOS-2 mRNA caused by coincident ethanol exposure (fig. 4) translated into reduced NOS-2 activity. The results are shown in table 2 and illustrate several important findings. The data indicate that a 12-hr exposure to LPS plus PMA is sufficient for induction of NOS-2 activity in C6 cells, determined by subsequent 36-hr nitrite accumulation, but re-exposure to LPS plus PMA during the 36-hr accumulation period leads to significantly greater NOS-2 activity, indicating additional gene induction (table 2; two-way ANOVA indicated a significant main effect of duration of LPS plus PMA exposure, P < .0001; nitrite values for 0 mM 12-hr vs. 0 mM 12- plus 36-hr re-exposed groups were significantly different at P < .005 by unpaired t test). Furthermore, 12-hr exposure to 100 or 150 mM ethanol during LPS plus PMA treatment significantly reduced NOS-2 activity (table 2), consistent with the mRNA data. Interestingly, 12-hr exposure to ethanol reduced LPS- plus PMA-induced NOS-2 activity by the same magnitude as did 48-hr exposure [table 2; one-way ANOVA indicated no significant differences between percentage of control values for 100 mM (P = .9642) or 150 mM (P = .5597) ethanol across 12-hr, 12- plus 36-hr re-exposed and 48-hr LPS- plus PMA-exposed groups].

Ethanol inhibition of cytokine-induced NOS-2 activity. The effect of ethanol exposure during induction was also tested on NOS-2 activity stimulated by agents other than LPS and PMA. Two-way ANOVA (induction condition × ethanol exposure) indicated significant interactions of both induction condition (P < .001) and ethanol exposure (P < .001) on nitrite accumulation, relative to control cells induced with LPS plus PMA (fig. 5). Ethanol at 100 mM was found to significantly inhibit nitrite accumulation induced by LPS plus TNF- (P < .01) and by TNF- alone (P < .01) (fig. 5A). Stimulation of C6 cells with LPS plus interferon- produced a much greater level of nitrite accumulation, compared with induction with LPS plus PMA (fig. 5B). Ethanol at a 100 mM concentration did not significantly inhibit (P > .05) nitrite accumulation induced by LPS plus interferon-, whereas significant inhibition (P < .01) occurred when cultures were exposed to 200 mM ethanol (fig. 5B). Exposure of C6 cells to interferon- by itself caused only minimal nitrite accumulation that was decreased also by ethanol exposure, although not significantly (P = .1078).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of exposure to ethanol on nitrite accumulation induced in C6 glioma cells by cytokines alone or in combination with LPS. Groups are control (), 100 mM ethanol () and 200 mM ethanol (). A, effect of 100 mM ethanol on induction with TNF- at 60 ng/ml. Two-way ANOVA indicated significant effects of both induction condition (F = 15.33, P < .0001) and ethanol treatment (F = 23.17, P < .0001). Post hoc comparisons indicated that ethanol exposure reduced nitrite accumulation significantly, regardless of induction condition. **P < .01; ***P < .001 vs. respective control group [100% control activity = 17.5 nmol nitrite/mg protein/24 hr; n = 16 and 16 (LPS plus PMA), 20 and 21 (LPS plus TNF-) and 7 and 6 (TNF-) for control and ethanol groups from five, five and two independent experiments, respectively]. B, effect of 100 and 200 mM ethanol on induction with 150 U/ml interferon- (IFN-). Two-way ANOVA indicated significant effects of both induction condition (F = 81.70, P < .0001) and ethanol treatment (F = 12.66, P < .0001). Post hoc comparisons indicated that 100 and 200 mM ethanol significantly reduced nitrite accumulation induced with LPS plus PMA, whereas only 200 mM ethanol reduced nitrite accumulation induced with LPS plus interferon-. Ethanol exposure had no significant effect on the slight amount of nitrite production that occurred with interferon- treatment alone. **P < .01, ***P < .001 vs. respective control group [100% control activity = 13.7 nmol nitrite/mg protein/24 hr; n = 26, 22 and 15 (LPS plus PMA); 23, 17 and 14 (LPS plus interferon-); and 15, 11 and 11 (interferon-) for control, 100 mM ethanol and 200 mM ethanol groups, respectively; data from four to seven independent experiments].

	Discussion

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The results of this investigation clearly indicate that exposure to ethanol at concentrations that significantly reduce functional NOS-2 activity, measured as nitrite production by intact C6 cells, also reduces the amount of NOS-2 protein expressed by the cells. This was evident whether the NOS-2 was measured as enzymatic activity in dialyzed cytosolic extracts or as immunoreactive protein by immunoblotting. Previous studies with C6 cells demonstrated that reduced nitrite production at 200 mM ethanol was not a consequence of cytotoxicity or reduced cell viability (Syapin et al., submitted). The observation that specific NOS-2 mRNA levels were also significantly decreased during exposure to ethanol suggests a pretranslational mechanism for the reduced NOS-2 protein content in C6 cells. This finding is consistent with reports that ethanol administered in vivo reduces LPSinduced NOS-2 mRNA expression in liver and lung cells (Spolarics et al., 1993; Xie et al., 1995).

Whether the reduction in NOS-2 protein content of C6 cells is sufficient to totally account for the reduction in nitrite production remains unclear. However, ethanol had no direct effect on NOS-2 catalytic activity when measured in cytosolic extracts. Similarly, Brien et al. (1995) reported no direct effect of ethanol on constitutive NOS catalytic activity from different regions of guinea pig brain. Furthermore, the postinduction studies indicated that nitrite production by intact C6 cells already expressing functional NOS-2 protein was not inhibited by ethanol exposure. This result demonstrates that ethanol inhibition of NOS-2 activity in C6 cells not previously exposed to ethanol is not due to direct effects on NOS-2 protein stability or substrate or cofactor availability but that the presence of ethanol during some phase of NOS-2 expression is necessary for inhibition.

The specific step where ethanol acts to suppress NOS-2 expression is not known. On one hand, the previous finding that ethanol exposure did not interfere with the PMA component of the synergistic induction of NOS-2 by LPS plus PMA (Syapin, 1995) and the present finding that ethanol exposure also inhibits NOS-2 activity when induced by LPS plus interferon-, LPS plus TNF- or TNF- alone are consistent with ethanol acting downstream of cytokine receptor activation of immediate second messengers. On the other hand, Western blot and RT-PCR data are consistent with an action upstream of protein translation. Thus, it seems that ethanol is acting on a common convergence pathway regulating NOS-2 expression.

It is known that NOS-2 can be regulated at all levels of expression, from transcriptional through translational to post-translational (Nathan and Xie, 1994). As mentioned above, the data do not support a translational or post-translational mechanism for ethanol inhibition of NOS-2 activity, because both mRNA and protein levels were significantly reduced and ethanol had no effect on NOS-2 activity if presented 24 hr after initiation of induction. Whether ethanol acts to reduce NOS-2 expression at the transcriptional level, post-transcriptional level or both levels cannot be distinguished from the available data and will require further investigation. However, the data suggest that ethanol likely has effects on NOS-2 gene transcription. It is known that transcription of NOS-2 mRNA in glial cells generally peaks between 4 and 8 hr after initial exposure to LPS and cytokines and then decreases rapidly (Galea et al., 1994; Park and Murphy, 1994). At the same time, active NOS-2 protein dimers accumulate for up to 24 hr. We reported above that both NOS-2 mRNA and functional activity can be significantly reduced if ethanol is present for only the first 12 hr of induction, coincident with the time of maximal gene transcription. In addition, the magnitude of the inhibition is the same whether ethanol is present for only 12 hr or much longer (48 hr), which is generally not expected of agents acting to destabilize mRNA transcripts. Furthermore, we observed previously that exposure of C6 glioma cells to 100 mM ethanol during the first 12 hr of a 24-hr treatment with LPS plus PMA did not result in inhibition of subsequent NOS-2 activity (Militante and Syapin, 1995; Militante, 1996). This result contrasts with the present findings and suggests that re-exposure to LPS plus PMA in the absence of ethanol, which we know can lead to additional NOS-2 expression, is able to negate the inhibitory effect of a previous 12-hr exposure to ethanol. This suggests that new transcripts replaced those reduced by previous ethanol exposure. It cannot be ruled out, however, that treatment with LPS plus PMA after ethanol elicited additional interactions between regulatory mechanisms of NOS-2 expression and ethanol in C6 cells (see below). Also, it is possible that other experimental differences (e.g., an additional wash step and measurement of nitrite accumulation from hours 24-40 after initial exposure to LPS plus PMA) may have contributed to the discrepancy.

Induction of NOS-2 is accompanied by coinduction of several other systems (Akarasereenont et al., 1994; Nussler et al., 1996), including GTP-cyclohydrolase-I, the rate-limiting enzyme in tetrahydrobiopterin biosynthesis (Sakai et al., 1995; D'Sa et al., 1996) and arginine transport (Schmidlin and Wiesinger, 1995). Thus, it is possible that the effect of ethanol exposure during NOS-2 induction may include effects on these ancillary induced proteins. Consistent with this suggestion is the observation that nonlinear regression analysis of concentration-response curves for ethanol inhibition of nitrite production provides the best data fit when slope factors (i.e., Hill coefficients) are approximately 2.0 (Syapin, 1995; Syapin et al., submitted). This implies that inhibition of nitrite production by ethanol exposure may involve more than one mechanism or site of action.

The proposed link between excitotoxicity, NO formation, glial cells and alcohol brain damage remains unsettled at this time. Our results do not support an ethanol-dependent increase in NOS-2 expression to account for the damage accompanying ethanol toxicity. On the other hand, NO has been reported to protect against certain forms of brain injury, such as that due to oxygen radical formation, whereas it can mediate other forms of damage (Choi, 1993). Therefore, the ultimate outcome of suppression of glial NOS-2 expression in the alcoholic brain remains to be determined.