Proc. Natl. Acad. Sci. USAVol. 90, pp. 522-526, January 1993Cell Biology

Cytokines, endotoxin, and glucocorticoids regulate the expressionof inducible nitric oxide synthase in hepatocytesDAVID A. GELLER*t, ANDREAS K. NUSSLER*, MAURICIo Di SILVIO*, CHARLES J. LOWENSTEINt,RICHARD A. SHAPIRO*, STEWART C. WANG*, RICHARD L. SIMMONS*, AND TIMOTHY R. BILLIAR**Department of Surgery, 497 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261; and tDepartment of Neuroscience, Johns Hopkins MedicalInstitutions, Baltimore, MD 21205

Communicated by Klaus Hofmann, September 18, 1992

ABSTRACT Nitric oxide (NO-) is a short-lived mediatorwhich can be induced in a variety of cell types and producesmany physiologic and metabolic changes in target cells. Theinducible or high-output NO' synthase (NOS) pathway was firstcharacterized in macrophages activated by lipopolysaccharide(LPS) and interferon v (IFN-y). Hepatocytes also express aninducible NOS following exposure to the combination of endo-toxin (LPS) and tumor necrosis factor (TNF), interleukin 1(IL-1), and IFN-y. In this study, to identify which of thesecytokines, if any, was acting to induce the gene expression forhepatocyte NOS, we measured the levels ofrat hepatocyte NOSmRNA by Northern blot analysis after stimulation by variouscombinations of endotoxin and cytokines in vitro. We found themRNA for hepatocyte NOS to be a single band at --4.5kilobases which was maximally up-regulated ('70-fold) by thecombination of TNF, IL-1, IFN-y, and LPS. Abundance ofNOS mRNA peaked 6-8 hr after stimulation and then declinedby 25% at 24 hr. Unstimulated hepatocytes in vitro showed onlya trace mRNA band after prolonged autoradiographic expo-sure. As single agents, TNF and IL-1 were the most effectiveinducers of hepatocyte NOS mRNA. Combinations of two orthree stimuli revealed strong synergy between TNF, IL-1, andIFN-y. The increased mRNA levels correlated with elevatednitrogen oxide release and cGMP levels in the culture super-natants. Dexamethasone and cycloheximide inhibited induc-tion ofmRNA for hepatocyte NOS in a dose-dependent fashion.The addition of NG-monomethyl-L-arginlne had no effect onmRNA levels but effectively blocked NO- formation. Theinducible hepatocyte NOS mRNA was also detected in rathepatocytes following chronic hepatic inflammation triggeredby Corynebacterium parvum injection in vivo. These data dem-onstrate that the inducible NOS is functional in rat hepatocytesboth in vitro and in vivo and that this pathway is under complexcontrol. Endotoxin and inflammatory cytpkines act synergis-tically to up-regulate gene expression for hepatocyte NOS,whereas glucocorticoids down-regulate the mRNA.

Following the discovery of the nitric oxide (NO-) pathwayand its identification as endothelium-derived relaxing factor,a variety of cell types such as macrophages (1, 2), endothelialcells (3, 4), smooth muscle cells (5), and neurons (6, 7) havebeen shown to produce NO' from L-arginine. Constitutiveand inducible isoforms of the NO' synthase (NOS) enzymeexist, and they differ in structure and regulation (8). Theneuronal constitutive NOS is a 150-kDa protein whose ac-tivity is dependent upon calcium and calmodulin (7); theinducible macrophage NOS is a 130-kDa protein which isthought to function independently of calcium/calmodulin (9,10). The constitutive NOS cDNA has been cloned from ratcerebellum and identifies an ==10-kilobase (kb) mRNA onNorthern blot analysis (11), while the inducible murine mac-

rophage NOS has been cloned from RAW264.7 cells by threegroups and identifies an -4.4-kb mRNA (12-14). The phys-iologic importance ofNON as a vasodilator, neurotransmitter,and antimicrobial/antitumor agent is rapidly becoming ap-parent.

Previous work showed that rat hepatocyte/Kupffer cellcocultures stimulated with lipopolysaccharide (LPS) producelarge amounts of nitrite (NO-) and nitrate (NO-), the stableend products of the NO- pathway (15). Further, it wasdemonstrated that hepatocytes also produce NO- in vivoduring chronic hepatic inflammation (16, 17) and in vitro inresponse to conditioned Kupffer cell supernatant (18) or to amixture of LPS and the cytokines tumor necrosis factor(TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (19).Human hepatocytes were also stimulated to produce NO' bythe same combination of endotoxin and cytokines as rathepatocytes (20). However, essentially nothing has beenreported about the direct signals required for inducible NOSgene activation. Therefore, the present study was undertakento characterize the molecular regulation of the inducible rathepatocyte NOS by endotoxin and cytokines known toup-regulate hepatocyte NO' synthesis.

MATERIALS AND METHODSIsolation of Hepatocytes. Hepatocytes were isolated from

male rats (200-250 g, Harlan-Sprague-Dawley) by a modi-fication of the in situ collagenase (Sigma) perfusion techniqueof Seglen (21). Hepatocytes were separated from nonparen-chymal cells by differential centrifugation at 50 x g and thenpassed over a 30%o Percoll gradient to obtain a highly purifiedcell population. Hepatocyte purity assessed by microscopywas >98% and viability consistently exceeded 95% by trypanblue exclusion. For the in vivo studies, hepatocytes wereharvested from rats 3 days after injection of Cornyebacte-rium parvum (Wellcome Biotechnology; 28 mg/kg, i.v.) andfrom normal rats as control.

Cell Culture. Hepatocytes (5 x 106 in 6 ml ofmedium) wereplated onto 100-mm gelatin-coated Petri dishes (Coming).Medium consisted of Williams' medium E (GIBCO) withL-arginine (0.50 mM), insulin (1 1LM), Hepes (15 mM),L-glutamine, penicillin, streptomycin, and 10% low-endotoxin calf serum (HyClone). After a 24-hr incubation,the medium was changed to include a cytokine mixture (CM)containing LPS (Escherichia coli 0111:B4, Sigma; 10 ,ug/ml),human recombinant IL-1p8 (Cistron, Pine Brook, NJ; 5 unit/ml), TNF (Genzyme; 500 units/ml), and IFN-y (Amgen; 100units/ml) or various combinations of LPS and cytokines.Other experimental conditions included addition ofcytokineswith dexamethasone, actinomycin D, or cycloheximide (all

Abbreviations: NO-, nitric oxide; NOS, NO' synthase; NMA, NG-monomethyl-L-arginine; CM, cytokine mixture; LPS, lipopolysac-charide; TNF, tumor necrosis factor; IL-1, interleukin 1; IFN-y,interferon fy.tTo whom reprint requests should be addressed.

522

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Sigma) or NG-monomethyl-L-arginine [NMA, prepared asdescribed (17)]. After cultures were incubated at 370C in 95%air/5% CO2 for 2-24 hr, supernatants were collected for NO-2+ NO3- and cGMP assays, and total RNA was extracted forNorthern blot analysis. After collection, supernatant samplesfor cGMP assay had the phosphodiesterase inhibitor 3-isobu-tyl-1-methylxanthine (0.5 mM) added and all samples werestored at -700C until used.NOj + NO- and cGMP Measurement. To determine the

amount of NO- produced by hepatocytes, the culture super-natants were assayed for the stable end products of NO-oxidation, NO- and NO3-, by an automated procedure basedon the Griess reaction (15). cGMP levels were measured witha commercially available radioimmunoassay according to themanufacturer's instructions (NEN).

Molecular Probes. The inducible-NOS probe used was amouse macrophage cDNA clone (14). Not I digestion ofpBluescript plasmid containing a fragment of the clonedmouse NOS cDNA yielded a 2.7-kb cDNA insert which wasfound to effectively identify the rat hepatocyte NOS mRNAon cross-species hybridization (see Results). After hybrid-ization with the NOS probe, Northern blot membranes werestripped with boiling 5 mM EDTA/0.1% SDS and rehybrid-ized with a probe specific for 18S rRNA to control forvariations in amount of RNA per lane.RNA Isolation and Northern Blot Analysis. Total RNA was

extracted from freshly isolated or cultured hepatocytes usingthe RNAzol B (Biotecx Laboratories, Houston) modifiedmethod of Chomczynski and Sacchi (22). All RNA sampleshad an A260/A280 ratio > 1.50. Aliquots containing 20 ug oftotal RNA were electrophoresed in 1% agarose gel containing3% formaldehyde. RNAs were blot-transferred to Gene-Screen membrane (NEN) and UV autocrosslinked. Mem-branes were hybridized with the probe overnight at 43°C in50% deionized formamide/0.25 M sodium phosphate, pH7.2/0.25 M NaCl/1 mM EDTA/7% SDS containing dena-tured salmon sperm DNA (100 ,ug/ml). DNA probes (2-4 x106 cpm/ml) were labeled with [a-32P]dCTP (specific activity,3000 Ci/mmol; NEN; 1 Ci = 37 GBq) by random priming.The hybridized filters were washed at 53°C in 2x standardsaline citrate (SSC)/0.1% SDS/25 mM sodium phosphate/imM EDTA/0.1% SDS and finally in 25 mM sodium phos-phate/1 mM EDTA/1% SDS. Autoradiography was per-formed by exposure (2 hr to 11 days) to Kodak X-Omat filmat -70°C in the presence of intensifying screens. RelativemRNA levels were quantitated by scanning densitometry.Each Northern blot shown is representative of at least threeseparate experiments performed at different times, unlessotherwise specified.

Statistical Analysis. Values for NOj + NO3- and cGMP aremeans ± SEM. The significance of differences was deter-mined with a two-tailed Student's t test. Statistical signifi-cance was established at a P value < 0.05.

RESULTSFig. 1 shows the time course ofNOS induction in hepatocytesin vitro following exposure to LPS, TNF, IL-1, and IFN-,y,a combination referred to here as CM. Cultured rat hepato-cytes were stimulated (S) with CM in vitro for 2-24 hr andwere compared with unstimulated (U) hepatocytes. Northernblot analysis revealed up-regulation of hepatocyte NOSmRNA levels by CM stimulation (Fig. 1A). The mRNA wasseen as a single band at -4.5 kb which was faintly present 2hr after stimulation, became maximal at 8 hr. and thendeclined slightly by 24 hr (Fig. 1B). In the unstimulatedhepatocytes, NOS mRNA was not detectable. To compareNOS mRNA levels with NOS activity, we determined hepa-tocyte NO- release by measuring culture supernatant NO- +NO3- levels (Fig. 1C). Increases in hepatocyte nitrogen oxide

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FIG. 1. Time course of hepatocyte NOS induction following LPSand cytokine exposure in vitro. (A) Northern blot analysis of NOSmRNA from cultured hepatocytes stimulated (S) for 2-24 hr or fromunstimulated cells (U). Membranes were hybridized with acDNA forinducible NOS (Upper) and then with a probe for 18S rRNA (Lower).(B) Autoradiographic mRNA signals were quantitated by scanningdensitometry and corrected relative to 18S rRNA; *, CM-stimulatedhepatocytes. (C) Hepatocyte NO release was determined by mea-suring culture supernatant NO- + NO- levels in CM-stimulatedhepatocytes (e) and in unstimulated control hepatocytes (A). *, P <0.05 vs. unstimulated hepatocytes at the same time point.

levels were first seen at 8 hr after CM stimulation, withsignificant further increases to 86.1 juM at 12 hr and to 284.6,uM at 24 hr.To determine whether the individual stimuli would induce

NOS mRNA expression, and if so, whether they would act ina similar time-dependent manner, experiments were per-formed using the single agents at concentrations previouslyfound to optimally induce hepatocyte NOS activity (19). Thepeak induction of NOS mRNA was seen at 4 hr afterstimulation with TNF or IFN-y, whereas IL-1 induced a peakat 8 hr (Fig. 2). No mRNA was detected with LPS stimula-tion, nor in the unstimulated hepatocytes. Unlike CM stim-ulation, where mRNA levels remained 75% of maximal at 24hr, with any single cytokine the NOS mRNA was decreasedby 12 hr following stimulation and was nearly gone at 24 hr.This suggests that the cytokine effects are additive or syn-ergize to produce a prolonged induction of NOS mRNA.

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FIG. 2. Time course of hepatocyte NOS induction followingsingle-cytokine exposure in vitro. Rat hepatocytes were stimulated(S) with TNF, IL-1,B, IFN-y, or LPS for 2-24 hr or left unstimulated(U). Northern blot analysis is shown with corresponding concentra-tion (IAM) of supernatant NO. release (NOr + NO-) below eachmRNA band. Each blot shown is representative of duplicate exper-iments.

Twenty-four hours after stimulation, only TNF (11.5 ,uM)and, to a lesser extent, IL-1 (7.6 ,uM) induced appreciablerises in NO- + NOj levels (Fig. 2). These levels arerelatively low (<5%) compared with the 284.6,uM NO- +NO- induced by the CM.Based on the time-course experiments, maximal levels of

NOS mRNA were seen between 4 and 8 hr; therefore, the6-hr time point was used in experiments designed to deter-mine which combinations of the CM were most potent intriggering NOS mRNA expression (Fig. 3). Based on anaverage of at least three different Northern blots per combi-nation, the stimuli can be ranked in potency for activating thehepatocyte NOS pathway. For single cytokines, TNF> IL-1> IFN-y> LPS. TNF was the most effective single cytokine,achieving 10%o of the maximal (CM-induced) hepatocyteNOS mRNA level. LPS alone was the weakest stimulator,with a trace mRNA signal after prolonged autoradiographicexposure (<1% of maximal). For double cytokines, TNF +IFN-y 2 TNF + IL-1 > IL-1 + IFN-y >> LPS + TNF >LPS + IL-1 > LPS + IFN-y. TNF + IFN-y or TNF + IL-1was most effective, achieving 35-45% of maximal induction.Clearly, the least effective double combinations were LPSwith any cytokine, producing only 7-15% of maximal levels.For triple cytokines, LPS + TNF + IFN-y - TNF + IL-1 +IFN-y > LPS + IL-1 + IFN-y > LPS + TNF + IL-1. Themost effective triple cytokine combinations were LPS + TNF+ IFN-y and TNF + IL-1 + IFN-y, achieving 65-75% ofmaximal induction.To correlate mRNA levels with NOS activity, NOj + NO3

levels were measured in the culture supernatants collected at

FIG. 3. Comparison of LPS and cytokine combinations in stim-ulating hepatocyte NOS mRNA expression. Cultured rat hepato-cytes were stimulated for 6 hr with various combinations ofLPS andcytokines. (A) Effect of single vs. double signal combinations inup-regulating hepatocyte NOS mRNA. (B) Effect ofdouble vs. triplesignal combinations in up-regulating hepatocyte NOS mRNA.

the time of RNA harvesting (Table 1). Culture supernatantsfrom unstimulated (control) hepatocytes had a basal concen-tration of4.1 ,uM NO- + NO-. This rose to 28.9 uM 6 hr afterCM addition and was reversed by the addition of NMA. In

Table 1. Rat hepatocyte nitrogen oxide and cGMP production 6hr after in vitro stimulation

NO- + NOT, cGMP, fmol perGroup ,uM 105 hepatocytes

Control 4.1 ± 0.63 103.1 ± 8.2CM 28.9 ± 5.40* 282.0 ± 37.6*CM + NMA 4.2 ± 0.92t 138.4 ± 34.8tLPS 2.5 ± 0.35 164.0 ± 52.5TNF 8.9 ± 3.52* 262.0 ± 145.2IL-1 4.5 ± 1.39 145.2 ± 36.7IFN-y 4.5 ± 0.35 158.0 ± 41.2LPS + TNF 9.6 ± 2.30* 202.2 ± 66.2*LPS + IL-1 4.1 ± 0.70 125.4 ± 38.1LPS + IFN-y 4.1 ± 0.50 119.2 ± 40.0TNF + IL-1 14.6 ± 3.70* 317.8 ± 80.3*TNF + IFN-y 15.7 ± 5.30* 319.2 ± 133.7*IL-1 + IFN-'y 10.4 ± 3.15* 266.4 ± 106.1*LPS + TNF + IL-1 13.9 ± 2.60* 266.2 ± 86.1*LPS + TNF + IFN-y 19.4 ± 3.00* 302.8 ± 75.5*LPS + IL-1 + IFN-y 10.3 ± 2.35* 251.2 ± 82.8*TNF + IL-1 + IFN-y 22.4 ± 5.75* 280.8 ± 65.3**P < 0.05 vs. control.t, P < 0.05 vs. CM.

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agreement with the mRNA levels, TNF as a single agent mosteffectively induced NOS activity. From the double signalcombinations, TNF with IFN-y or IL-1 stimulated the highestnitrogen oxide synthesis (NOr + NO3 concentrations of 15.7and 14.6 1LM, respectively), again correlating very well withmRNA levels. A similar association between mRNA levelsand NO- + NOj synthesis was seen with the triple cytokinecombinations.

Soluble guanylate cyclase activation is a sensitive indicatorof NO synthesis, and much of the cGMP produced bycultured hepatocytes is released into the medium (23). There-fore, cGMP levels were used as an additional marker ofincreased NO formation. Increased extracellular cGMP co-incided with increased NO- + NO- (Table 1). The additionof NMA reversed the rise in cGMP, and no combination ofsingle, double, or triple cytokines and LPS increased cGMPwithout also increasing nitrogen oxide release. Taken to-gether, the results are consistent with previous findingsassociating hepatocyte NO- with cGMP synthesis (23).

Glucocorticoids decrease NO- synthesis in vitro in endo-thelial cells (24) and macrophages (25). Therefore, we inves-tigated the effects of dexamethasone on CM-induced hepa-tocyte NOS mRNA levels (Fig. 4). Dexamethasone de-creased NOS mRNA levels by 36% at 10-8 M, and by 63%at 10-6 M. The addition of the protein synthesis inhibitorcycloheximide also decreased NOS mRNA levels in a dose-dependent manner, whereas the transcriptional inhibitor ac-tinomycin D blocked NOS mRNA induction. These agentsdid not decrease hepatocyte viability determined by trypanblue exclusion (data not shown). The addition of NMAeffectively inhibited NO- synthesis (Table 1) but had no effecton cytokine-induced mRNA levels for hepatocyte NOS.To detect basal levels of NOS mRNA in unstimulated

hepatocytes, longer autoradiographic exposures of Northernblots were required (11 days). Fig. 5 shows very low basallevels of NOS mRNA in unstimulated hepatocytes and per-mits a measurement of fold induction in the CM-stimulatedcells. Averaging the densitometry values of four different rathepatocyte cultures indicated that CM stimulation produced=70-fold induction of NOS message.To compare the in vitro induction of hepatocyte NOS by

CM with in vivo induction, rats were injected with heat-killedC. parvum, producing chronic hepatic inflammation andhepatocyte NO- biosynthesis (16). Fig. 6 shows both in vivoinduction of hepatocyte NOS mRNA 3 days after C. parvuminjection and in vitro induction by CM stimulation. After

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correction for RNA loading, mRNA levels are -7-foldgreater in vitro than in vivo.

DISCUSSIONIn this report, we describe the regulation of the induciblehepatocyte NOS by LPS, cytokines, and dexamethasone.Using a mouse macrophage NOS cDNA clone and cross-species Northern blot hybridization, we demonstrated thatLPS, TNF, IL-1, and IFN-y acted synergistically in vitro toinduce the highest rat hepatocyte NOS mRNA levels. ThemRNA levels correlated with NOS enzyme activity deter-mined by culture supernatant NO- + NO- levels as well ascGMP release. The great induction of NOS mRNA withinonly a few hours suggests that the regulation of hepatocyteNOS expression is primarily transcriptional, as seen withmacrophage NOS (13). Inhibition of NOS mRNA by actino-mycin D is also consistent with this notion. That cyclohex-imide inhibits NOS mRNA induction suggests that a keypolypeptide component of the cytokine signal transductionpathway, possibly including NOS transcriptional factors, islabile. It is unlikely that the inhibition by cycloheximide issimply due to cytotoxicity, as cycloheximide at 10 ,ug/mldoes not abolish mRNA induction by cAMP in cultured rathepatocytes (26). The increase in NOS mRNA from freshlyisolated hepatocytes following C. parvum injection verifiesthat hepatocyte NOS is also induced in vivo during certainconditions of hepatic inflammation.The time delay of 4-6 hr required for cytokine and LPS

induction of hepatocyte NOS activity is characteristic of theinducible macrophage NOS (27). However, the cytokinesignals which up-regulate hepatocyte NOS differ quantita-tively and qualitatively from those required to induce mac-rophage NOS. First, macrophage NOS activity has beenreported to be maximally induced by two stimuli (27, 28),whereas both rat (19) and human (20) hepatocytes require acombination of four stimuli for the greatest induction. Sec-ond, the relative potency of the stimuli are cell-specific.Maximal macrophage NOS activity is typically reportedfollowing stimulation with LPS and IFN-y (27, 28). In con-trast, in the current study in hepatocytes where NOS activityas well as mRNA levels were measured, we found that LPS+ IFN-'y was the least effective stimulus in activating hepa-

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FIG. 4. Effect of cycloheximide (Chx), actinomycin D (actin. D,1 jig/ml), dexamethasone (Dex, 10-8-10-6 M) and NMA (0.5 mM)on hepatocyte NOS mRNA levels in vitro as shown by Northern blotanalysis. Cultured rat hepatocytes were stimulated for 6 hr with CMin the presence of the indicated agents.

FIG. 6. In vivo vs. in vitro induction of hepatocyte NOS mRNA.(Left) Northern blot analysis of NOS mRNA from freshly isolatedhepatocytes ofnormal and C. parvum-injected rats. (Right) Northernblot analysis of hepatocytes exposed to CM for 6 hr in vitro.

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tocyte NOS, achieving only 6-9%o of maximal measurablemRNA levels. Instead, LPS + TNF + IL-1 + IFN-y pro-duced maximal hepatocyte NOS mRNA levels and nitrogenoxide synthesis. TNF + IFN-y and TNF + IL-1 were themost effective double signal combinations in hepatocytes,producing 35-45% of maximal mRNA levels. Numerousreports have documented synergistic actions between TNFand IFN-y (see ref. 29 for review) for activation of macro-phage cytotoxicity and between TNF and IL-1 for inductionof the hepatic acute-phase response. TNF has also beenshown to directly stimulate NO' formation in cultured hepa-tocytes isolated from rats exposed to LPS (30).Whereas strong induction of NOS activity and marked

increases in the mRNA were seen with cytokine and/or LPScombinations, increases in the mRNA were also seen whenTNF, IL-1, or IFN-y was used alone. However, only TNFand, to a lesser extent, IL-1 increased NO- formation asindicated by NOj + NO- or cGMP release. The lack ofNO-+ NO- increase with IFN-y may be due to the sensitivity ofour methods to detect NO- or the absence of NOS activitydespite the presence of mRNA. These data suggest that assingle agents, TNF and IL-1 are capable of stimulatinglow-level NOS activity in hepatocytes.The physiological significance of NO' biosynthesis in the

liver is only beginning to be elucidated. In vitro hepatocyteNO' production has been associated with a decrease inhepatocyte total protein synthesis (18, 31) and mitochondrialaconitase activity (32) and an increase in cGMP synthesis andrelease (23). The cytostatic effect of macrophage NO' pro-duction against intracellular parasites (see ref. 33 for review)has also been shown in hepatocytes in vitro for inhibition ofthe pre-erythrocytic liver stages of malaria (34). Followingthe in vivo administration of LPS, NOS activity in the liverincreased (35), and an endotoxin-induced NOS enzyme wasrecently purified (36). Chronic LPS infusion in vivo rendershepatocytes inducible for NO' production following single-cytokine stimulation (30). Taken together, these findingsindicate that hepatic NOS is expressed in endotoxemia andsepsis and suggest a critical role for NO- in the liver as partof the host immune response. Other studies from our labo-ratory have looked into the function ofNO- in the liver in vivoand have revealed that inhibition of NO' synthesis by NMAin a model of hepatic injury produced by C. parvum and LPSresults in micro infarcts and increased liver damage mediatedpartially by oxygen radicals (17). This indicates that NO- isprotective by preventing cell damage, organ ischemia, andinfarction. From the results presented in the current paper itis clear that this induction of NOS in the liver is regulated bymultiple signals often acting in synergy, and further studiesare needed to define the precise mechanisms by which thesesignals activate the NOS gene.

We thank Debra Williams for expert technical assistance andSidney M. Morris, Ph.D., and David J. Tweardy, M.D., of theUniversity of Pittsburgh for helpful discussions and suggestions. Thiswork was supported by National Institutes of Health GrantsGM44100 (T.R.B.) and GM37753 (R.L.S.).

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