©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
S100 Stimulates Inducible Nitric Oxide Synthase Activity and mRNA Levels in Rat Cortical Astrocytes (*)

(Received for publication, July 24, 1995; and in revised form, November 13, 1995)

Jingru Hu (1) Francis Castets (1) José L. Guevara (1) Linda J. Van Eldik (1) (2)(§)

From the  (1)Department of Cell and Molecular Biology, Northwestern University Medical School and the (2)Northwestern University Institute for Neuroscience, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The glia-derived, neurotrophic protein S100beta has been implicated in development and maintenance of the nervous system. However, S100beta has also been postulated to play a role in mechanisms of neuropathology, because of its specific localization and selective overexpression in Alzheimer's disease. To begin to address the question of whether S100beta can induce potentially toxic signaling pathways, we examined the effects of the protein on nitric oxide synthase (NOS) activity in cultures of rat cortical astrocytes. S100beta treatment of astrocytes induced a time- and dose-dependent increase in accumulation of the NO metabolite, nitrite, in the conditioned medium. The S100beta- stimulated nitrite production was blocked by cycloheximide and by the NOS inhibitor N-nitro-L-arginine methylester, but not by the inactive D-isomer of the inhibitor. Direct measurement of NOS enzymatic activity in cell extracts and analysis of NOS mRNA levels showed that the NOS activated by S100beta addition is the calcium-independent, inducible isoform. Furthermore, the specificity of the effects of S100beta on activation of NOS was demonstrated by the inability of S100alpha and calmodulin to induce an increase in nitrite levels. Our data indicate that S100beta can induce a potent activation of inducible NOS in astrocytes, an observation that might have relevance to the role of S100beta in neuropathology.


INTRODUCTION

The normal development and maintenance of the brain involves the temporal and spatial coordination and proper functioning of a number of intracellular and cell-cell signaling events, and the contribution of glial cells to these signaling processes is becoming more widely appreciated. The classical concept of the role of glia in brain function is rapidly changing with newer evidence of the crucial nature of these cells in controlling neurotransmitter levels, maintaining calcium homeostasis, and synthesizing and releasing neurotrophic and growth factors (for review, see (1) ). One such glia-derived factor is S100beta, a protein that promotes neuritic outgrowth of specific neuronal populations (e.g. cortical(2, 3) , dorsal root ganglia(4) , serotonergic(5, 6) , and motoneurons(7) ) and enhances survival of neurons during development (7, 8) and after insult(9) . S100beta is also a glial mitogen, inducing phosphoinositide hydrolysis, increases in intracellular calcium, and protooncogene expression(10, 11) . These trophic functions require nanomolar concentrations of a disulfide-linked S100beta dimer (see (12) ). Thus, S100beta may be beneficial during development of the nervous system, and increased S100beta expression and secretion following acute glial activation in response to central nervous system injury may be one mechanism the brain uses in attempts to repair injured neurons.

However, S100beta may also reach concentrations that are deleterious, e.g. in neurodegenerative diseases like Alzheimer's disease and Down syndrome where chronic glial activation occurs(13) . It has been found that S100beta levels in severely affected brain regions of Alzheimer's disease patients are severalfold higher than in age-matched control samples(13, 14) , that S100beta is increased selectively in regions that exhibit the most neuropathological involvement(15) , and that S100beta overexpression is correlated with the prevalence of neuritic plaques(16) . These data raise the possibility that high concentrations of S100beta may be detrimental, an idea supported by the recent demonstration that an S100 isoform can induce apoptosis in PC12 cells through sustained increases in intracellular calcium(17, 18) .

As a first approach to addressing the question of whether S100beta can induce potentially toxic signaling pathways, we examined the effect of S100beta on nitric oxide (NO) generation. NO is synthesized by the enzyme nitric oxide synthase (NOS) (^1)through the conversion of L-arginine to L-citrulline. In the nervous system, NO has been implicated in the regulation of cerebral blood flow, synaptic plasticity, and cell growth (see (19) ). A large amount of data also supports the concept that NO production in the central nervous system may be involved in the neuropathology associated with ischemia, traumatic insults, and neurodegeneration (see (19, 20, 21) ). We report here that treatment of rat cortical astrocytes with S100beta results in a stimulation of NOS activity and generation of NO.


EXPERIMENTAL PROCEDURES

Purification of Proteins

Calmodulin and S100alpha were purified as described previously(22, 23) . Bovine S100beta was expressed from a synthetic gene in Escherichia coli(24) . Purification was achieved by DEAE anion exchange chromatography followed by calcium-dependent phenyl-Sepharose chromatography as described previously(11) . Elution of S100beta from phenyl-Sepharose was done with buffer containing 20 mM Tris-HCl, 500 mM NaCl, 1 mM EGTA, pH 7.4 (elution buffer). For experiments with tissue-isolated protein, S100 was isolated from bovine brain (Pel-Freez, Rogers, AR) by a similar procedure. In addition, E. coli lysates (lacking the S100 expression vector) were taken through the same purification protocol. The purity of the S100beta was analyzed by electrophoresis on 15% acrylamide-SDS minigels (Idea Scientific, Minneapolis MN) in the absence of reducing agents. S100beta was stored at -20 °C in storage buffer (elution buffer plus 4 mM CaCl(2)). Protein concentrations were determined by the method of Lowry et al.(25) using bovine serum albumin as standard or by amino acid analysis as described previously(26) .

Cell Culture

Primary astrocytes were prepared from neonatal (1-day-old) Sprague-Dawley rat pups as described by Levison and McCarthy(27) . Briefly, the cerebral cortex from one rat pup was dissected out and trypsinized, and cells were passed through a 136-µm and then a 40-µm nylon mesh. Cells were then seeded into two 100-mm tissue culture plates at a density of 2 times 10^6 cells/plate in alpha-MEM (Life Technologies, Inc.) containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), and grown at 37 °C in a humidified 5% CO(2)-containing atmosphere. After 11 days in culture, cells were trypsinized from the two plates and re-plated into 20 plates and grown until confluence.

Treatment of Cells for NO Assays

Confluent secondary astrocytes were trypsinized and re-plated into 24-well plates at 1 times 10^5 cells/well (for the nitrite assay) or into 100 mm plates at 1 times 10^6 cells/plate (for the citrulline assay and Northern blot analysis). Cells were allowed to attach for 4 h, at which time (time 0) S100beta (1-50 µg/ml) was added to the culture medium and cells were grown for various time periods. Some cultures were treated with lipopolysaccharide (LPS; 10 µg/ml) or storage buffer instead of S100beta, and these cultures served as positive and negative controls, respectively. When cycloheximide or the NOS inhibitor, N-nitro-L-arginine methylester (L-NAME) were used, these agents were added to the cultures at the same time as the S100beta.

Measurement of NO by Nitrite Assay

NO production was assessed by measurement of nitrite (a stable oxidation product of NO) in the conditioned medium, based on the Griess reaction(28) . Sodium nitrite dissolved in culture medium was used as the standard. Nitrite levels were determined by mixing 100-µl aliquots of conditioned medium with 50 µl of 1% sulfanilamide in water plus 50 µl of 0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric acid, and incubating for 10 min at room temperature. The absorbance at 540 nm was then measured by using a Titertek Multiskan MC plate reader (ICN/Flow Biochemicals, Huntsville AL).

Measurement of NOS by Citrulline Assay

NOS activity was determined by measurement of the conversion of [^3H]L-arginine (Amersham Corp.) to [^3H]L-citrulline according the the method of Bredt and Snyder(29) . Briefly, cells treated with various agents as described above were washed with ice-cold phosphate-buffered saline (PBS) and then scraped into homogenization buffer (20 mM HEPES, pH 7.4, 0.5 mM EGTA, 1 mM dithiothreitol, 0.32 M sucrose). Cells were homogenized by brief sonication, and then centrifuged at 12,000 times g for 10 min in a Sorvall MC-12V microcentrifuge. The resulting supernatant was passed through a Dowex AG50W-X8 (Na form) column (Bio-Rad) to remove endogenous arginine. Aliquots of the unbound material (300-µl reaction volumes containing 100 µg protein) were incubated at 37 °C for 45 min in homogenization buffer containing 200 µM NADPH, 50 µM tetrahydrobiopterin, 10 µML-arginine, and 1 µCi/ml [^3H]L-arginine in the absence and presence of 0.5 mM CaCl(2) (1 µM calculated free calcium) or 1 mML-NAME. Reactions were terminated by adding 2 ml of 20 mM HEPES, 2 mM EDTA, pH 5.5. One ml of each reaction sample was passed over a Dowex AG50W-X8 (Na form) column to separate [^3H]L-citrulline from [^3H]L-arginine. The level of [^3H]L-citrulline was determined by liquid scintillation counting and was expressed as the radioactivity (cpm/µg protein) obtained after correcting for nonspecific radioactivity in blank reactions containing all components except cell extract.

GFAP and OX-42 Immunohistochemistry

Secondary astrocytes were re-plated onto glass coverslips, fixed for 5 min in ice-cold methanol, and incubated for 1 h at room temperature with rabbit anti-GFAP (1:500 dilution; Dako, Carpinteria, CA) or mouse OX-42 antibody (1:500 dilution; gift of Dr. Sue Griffin, University of Arkansas for Medical Sciences). After washing with PBS, coverslips were incubated with fluorescein-conjugated goat anti-rabbit or anti-mouse IgG (1:20 dilution; Kirkegaard & Perry, Gaithersburg, MD), washed and mounted. The number of cells in 15 fields (magnification, times63) were counted under phase-contrast microscopy, and the percentage of GFAP- or OX-42-positive cells in those fields was determined by fluorescence microscopy on a Leitz Diaplan microscope.

RNA Isolation and Northern Blot Analysis

RNA was isolated from treated astrocyte cultures as described by Ausubel et al.(30) . Briefly, cells were washed twice with PBS and then lysed in a solution of 4 M guanidine thiocyanate, 25 mM sodium acetate, pH 6.0, 0.1 M 2-mercaptoethanol. The lysed cells were centrifuged through a 5.7 M cesium chloride step gradient at 140,000 times g for 16 h at 20 °C. The RNA in the pellet was dissolved, ethanol-precipitated, and quantitated spectrophotometrically at A.

Total RNA (10 µg/lane) was run under denaturing conditions on a 1% agarose-formaldehyde gel, transferred to Hybond-N membranes (Amersham), and cross-linked in a UV Stratalinker (Stratagene). Membranes were incubated for 3 h at 42 °C in pre-hybridization solution (5 times SSC (20 times SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7.0), 5 times Denhardt's solution (50 times Denhardt's = 100 g/liter Ficoll, 100 g/liter polyvinylpyrrolidone, 100 g/liter bovine serum albumin), 50 mM sodium phosphate, pH 7.0, 1% glycine, 0.3% sodium dodecyl sulfate (SDS), 50% formamide, 20 µg/ml denatured salmon sperm DNA). Pre-hybridization solution was removed and membranes were incubated for 18 h at 42 °C in hybridization solution (5 times SSC, 1 times Denhardt's solution, 20 mM sodium phosphate, pH 7.0, 0.3% SDS, 50% formamide, 20 µg/ml denatured salmon sperm DNA, 10% dextran sulfate) containing P-labeled iNOS cDNA probe. The iNOS cDNA clone was pASTNOS4, corresponding to the rat iNOS cDNA bases 3007-3943 ((31) ; gift of Dr. Elena Galea, Cornell University), and was labeled with [alpha-P]dCTP by using a Promega Prime-a-Gene random-primed labeling kit. After hybridization, membranes were washed once for 5 min at room temperature in 2 times SSC; twice for 30 min at 65 °C in 1 times SSC, 0.5% SDS; and once for 10 min at room temperature in 0.1 times SSC, 0.1% SDS. Membranes were exposed to film, and band intensity on the autoradiograms was quantitated on an imaging densitometer (Bio-Rad, model GS-670). To correct for potential differences in RNA loading, membranes were subsequently hybridized with a cyclophilin cDNA probe (pSP65.1B15; (32) ) and results were expressed as a ratio of density of iNOS mRNA versus cyclophilin mRNA.

Data Analysis

The significance of differences was determined with Student's t test. Statistical significance was established at a level of p < 0.05.


RESULTS

The ability of S100beta to stimulate NO production was determined initially by measuring the accumulation of nitrite (a stable NO metabolite) in the conditioned medium of astrocytes following exposure to S100beta. Tertiary cultures of neonatal rat cortical astrocytes were prepared as described under ``Experimental Procedures.'' These cultures contained approximately 98% astrocytes, as assessed by positive staining for the astrocyte intermediate filament protein, GFAP (data not shown). This result was consistent with the observation that only 2-3% of the cells showed positive staining for OX-42 (data not shown), a marker for microglia(33) . Fig. 1shows the time course of nitrite accumulation following exposure of these cells to S100beta (40 µg/ml S100beta for 6-72 h). S100beta-treated astrocytes showed increased nitrite accumulation in their conditioned medium compared to control cultures treated with storage buffer alone. The increased nitrite levels were evident after a 12 h exposure of cells to S100beta and reached a plateau at about 48 h. The maximal level of nitrite accumulation in response to S100beta was 3-4-fold higher than that generated by exposure of cells to buffer alone.


Figure 1: Time course of S100beta-stimulated nitrite accumulation in rat astrocyte cultures. Cells were incubated with 40 µg/ml S100beta (squares) or control storage buffer alone (circles) for the indicated times, and nitrite concentrations in the conditioned media were determined. Data shown are from one of three independent experiments, each done in triplicate. Values shown are the mean ± S.E. from the triplicate determinations. Error bars are shown only when larger than the symbol. S100beta-stimulated nitrite accumulation was significantly different from control values (p < 0.05) at 24-, 48-, and 72-h treatment times.



The response to S100beta was dose-dependent in the range of 1-40 µg/ml S100beta, assayed after 48 h treatment of cells (Fig. 2). The concentration of S100beta required to elicit 50% of the maximal nitrite increase was 20-25 µg/ml, and the maximal response was achieved by 35-40 µg/ml S100beta.


Figure 2: Dose-response curve of S100beta-stimulated nitrite accumulation in rat astrocyte cultures. Cells were incubated with increasing concentrations of S100beta for 48 h, and nitrite concentrations in the conditioned media determined. Data shown are from one of four independent experiments, each done in triplicate. Values shown are the mean ± S.E. from the triplicate determinations. Error bars are shown only when larger than the symbol. S100beta-stimulated nitrite accumulation was significantly different from control values (p < 0.01) at doses geq 5 µg/ml.



To test whether the S100beta -induced nitrite accumulation was dependent on NOS activity, we examined the effect of S100beta in the presence of the NOS inhibitor, L-NAME. Incubation of cells for 48 h with S100beta in the presence of L-NAME almost completely abolished the nitrite accumulation, whereas the inactive inhibitor isoform, D-NAME, was ineffective (Fig. 3). The basal level of nitrite accumulated during 48 h was also affected by L-NAME but not D-NAME.


Figure 3: Inhibition of S100beta-stimulated nitrite accumulation by NOS inhibitor. Cells were incubated for 48 h with S100beta (40 µg/ml) or control storage buffer in the presence or absence of the NOS inhibitor L-NAME (1 mM) or the inactive isomer, D-NAME (1 mM), and nitrite concentrations in the conditioned media determined. Data shown are the mean ± S.E. from three independent experiments done in triplicate. *, p < 0.05.



Incubation of cells with S100beta in the presence of the protein synthesis inhibitor cycloheximide prevented the S100beta stimulation of nitrite accumulation (Table 1). Moreover, the specificity of the response to S100beta was examined by comparing the ability of two other related calcium binding proteins, S100alpha and calmodulin, to enhance nitrite accumulation. Table 1shows that neither S100alpha nor calmodulin stimulated nitrite accumulation, under conditions where the LPS positive control induced nitrite accumulation to a similar extent as S100beta. To exclude the possibility that the S100beta stimulation of nitrite accumulation was a result of contaminating LPS in the recombinant S100beta preparation, we isolated S100beta from bovine brain tissue and we took an E. coli lysate lacking the S100 expression vector through the same purification protocol as S100. We found that bovine brain S100beta induced nitrite accumulation to a similar extent as recombinant S100beta, and that the E. coli lysate showed no induction above the buffer control (data not shown).



The ability of S100beta to enhance nitrite accumulation that was inhibited by NOS inhibitors suggested an activation of NOS enzyme activity. To test this directly, we examined NOS enzyme activity in cytosolic extracts of astrocytes treated with S100beta for 24 h, by measuring the conversion of L-arginine to L-citrulline. As shown in Fig. 4, S100beta treatment of astrocytes resulted in a 4-fold stimulation of NOS activity. The S100beta -evoked NOS activity was independent of the presence of calcium in the enzyme assay, suggesting that the NOS stimulated by S100beta is the inducible enzyme (iNOS). This was further confirmed by the demonstration that S100beta -treated astrocytes showed increased levels of iNOS mRNA, as measured by Northern blot analysis (Fig. 5).


Figure 4: S100beta stimulation of inducible iNOS activity in rat astrocyte cultures. Cells were incubated for 24 h with S100beta (40 µg/ml) or control storage buffer, and NOS activity in cytosolic extracts determined. Enzyme assays were done in the presence or absence of calcium as described under ``Experimental Procedures.'' Data shown are from one of four independent experiments, each done in triplicate. Values shown are the mean ± S.E. from the triplicate determinations. S100beta-stimulated NOS activity was significantly different from control (p < 0.05).




Figure 5: S100beta stimulation of iNOS mRNA. Cells were incubated for 24 h with S100beta (40 µg/ml) or control storage buffer, and iNOS mRNA levels were determined by Northern blot analysis of 10 µg/lane total RNA, as described under ``Experimental Procedures.'' The blot was re-probed with cyclophilin cDNA, and the iNOS mRNA levels were expressed as the ratio of density of iNOS mRNA versus cyclophilin mRNA. Inset shows the iNOS mRNA bands from control (C) versus S100beta-treated (S) astrocytes.




DISCUSSION

We have demonstrated that treatment of astrocytes with S100beta enhances NOS activity and release of NO, as demonstrated by the increase in accumulation of the stable NO metabolite nitrite in the conditioned medium. Measurement of NOS enzyme activity and mRNA levels confirms that the NOS stimulated by S100beta addition is the calcium-independent inducible iNOS isoform. The specific NOS inhibitor L-NAME, but not the inactive D-NAME, suppressed the S100beta-induced iNOS activity. Moreover, the specificity of the effects of S100beta on iNOS activation was demonstrated by the inability of S100alpha or calmodulin to induce an increase in NO. These data demonstrate that S100beta induces a stimulation of astrocytic iNOS activity and generation of NO.

The NOS enzyme responsible for synthesizing NO from arginine exists in three major forms: two constitutive cNOS isoforms (eNOS and nNOS) that are calcium-calmodulin dependent and present in a variety of cells, including endothelial cells, neurons, platelets, and astrocytes; and an inducible form (iNOS) that is calcium-independent and expressed after gene induction in a variety of cells, including macrophages, endothelial cells, and astrocytes (see (34) ). Induction of iNOS in astrocytes has been well documented previously by exposure of cells in vitro to bacterial endotoxin (LPS) or combinations of cytokines, such as interleukin-1beta, interferon- or tumor necrosis factor-alpha(1, 35) . Our data represent the first report that S100beta is another potent inducer of astrocytic iNOS activity. The S100beta-stimulated NO production exhibits similar characteristics to those reported for induction by LPS or other cytokines in terms of long (hour) time frame and narrow dose-response curve(31, 35) . The response occurs over several hours of continuous application of S100beta, and nitrite concentrations continue to increase up to 48 h after S100beta exposure. Similar to IL-1beta(35) , the effect of S100beta on iNOS activity was dose-dependent through a small concentration range. The magnitude of the nitrite accumulation in response to S100beta also varied somewhat among cultures and among S100beta preparations, suggesting that endogenous coordinators or specific S100beta conformational states might be required for maximal S100beta induction of iNOS.

We considered the possibility that our results might reflect an activation of NOS activity in microglia, rather than astrocytes, because microglia express high levels of iNOS activity(36) . However, this possibility seems unlikely because our tertiary cultures are 98% astrocytes, as determined by GFAP and OX-42 immunoreactivity, and because the intensity of the iNOS mRNA on Northern blots and the iNOS enzyme activity from cytosolic extracts are too high to reflect a signal from only 2% of the cells in the culture. Thus, our data support an S100beta-induced activation of iNOS activity in astrocytes.

The mechanisms involved in activation of iNOS by S100beta addition are unknown at present. Our observation that S100beta stimulates iNOS mRNA levels suggests that regulation may be at the level of transcription of the iNOS gene, as has been shown previously for iNOS stimulation by LPS and interferon-(34, 37, 38) . However, iNOS has also been found to be regulated at the level of mRNA and protein stability(39, 40) . Definition of the mechanisms by which S100beta induces iNOS activity, and whether S100beta interacts with other cytokines to modulate iNOS activity await further investigation.

The consequences of S100beta-induced NO release from astrocytes are not known. There is a wealth of sometimes conflicting evidence in the literature that NO can be beneficial or detrimental to nervous system function (see (19) and (21) ). For example, it has been found that NO can act as a diffusible messenger to mediate cell-cell signaling pathways involved in synaptic plasticity and regulation of cerebral blood flow. However, there is also a large amount of biochemical and pharmacological data to suggest that NO is involved in the neuropathology associated with traumatic or ischemic insults, autoimmune diseases, and neurodegenerative disorders. Relevant to our studies, it has been reported (41) that stimulation of astrocytic iNOS activity and release of NO leads to enhanced NMDA receptor-mediated neurotoxicity. We are currently pursuing studies with astrocytic/neuronal co-cultures to evaluate the effects of S100beta-stimulated NO release on the neuronal cell. In this regard, it is interesting to note that in some experiments, the S100beta-treated astrocytes exhibited morphological changes consistent with toxicity, such as release of lactate dehydrogenase and cell rounding and detachment from the substrate (data not shown). The reason for this observation has not been defined yet, but appeared to correlate with the levels of nitrite accumulated in response to S100beta. It is probable that the toxic potential of NO in vivo depends, at least in part, on the concentration released from cells. However, the biology of NO is complex and the susceptibility of a cell to NO-mediated toxicity likely depends on a number of variables, such as acute versus chronic exposure to NO, the array of redox forms in which NO can exist, availability of reactive oxygen species and anti-oxidant defenses, changes in ion homeostasis, presence of other cytoprotective or cytotoxic agents, and the immune status of the organism.

Our data demonstrate that treatment of astrocytes with S100beta results in a potent induction of iNOS activity and NO production. Stimulation of iNOS required micromolar concentrations of S100beta, unlike the nanomolar concentrations required for neurotrophic and mitogenic effects. The requirement of higher concentrations of S100beta for activation of iNOS suggests that S100beta might possess dual roles in regulation of cell function, being beneficial to cells at low doses and detrimental at higher doses. If S100beta stimulates astrocytic iNOS activity in vivo, this might be relevant to the role of S100beta in neurodegenerative disorders like Alzheimer's disease and Down syndrome, where S100beta levels are increased severalfold in temporal lobe samples compared to age-matched control samples(13) . It has been found that S100beta levels are elevated in specific brain regions from Alzheimer patients(15) , that the overexpression of S100beta correlates with the pattern of regional neuropathology and neuritic plaque involvement(15, 16) , and that S100beta is localized primarily in activated astrocytes surrounding neuritic plaques(15) . It should also be noted that the local concentration of S100beta in astrocytes surrounding neuritic plaques in Alzheimer's disease has not been determined, although the tissue levels are in the micromolar range (15) . The relationship between increased S100beta levels and neuropathology is not known. It may be that the up-regulation of S100beta after acute central nervous system injury or perhaps in neurodegenerative disease is part of a compensatory response the brain uses in attempts to repair injured neurons, through an action of S100beta in its neurotrophic and neuroprotective roles. However, the chronic overexpression of S100beta or expression of S100beta above some threshold level as in Alzheimer's disease may have deleterious consequences leading to neuronal dysfunction and eventual death, perhaps through altered calcium homeostasis or inappropriate neuritogenesis. Our data that S100beta can stimulate astrocytes to produce NO provide another possible mechanistic scenario by which the high levels of S100beta seen in Alzheimer's disease and other neurodegenerative disorders might contribute to neuropathology.


FOOTNOTES

*
These studies were supported in part by National Institutes of Health Grants AG10208 and AG11138. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-0697; Fax: 312-503-0007; :vaneldik{at}nwu.edu.

(^1)
The abbreviations used are: NOS, nitric oxide synthase; LPS, lipopolysaccharide; L- (and D-)NAME, N-nitro-L (and D)-arginine methylester; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. W. Sue T. Griffin for helpful discussions and Dr. Elena Galea for providing the rat iNOS cDNA clone.


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