Phorbol esters induce nitric oxide synthase activity in rat hepatocytes ...

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Dec 15, 2017 - The incubation of primary cultures of rat hepato- cytes with lipopolysaccharide (LPS) or biologically ac- tive phorbol esters promotes the release ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 35, Issue of December 15,pp. 24937-24940,1992 0 1992 hy The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication

endothelial enzyme is also constitutively expressed and exhibits the sameregulatory sites, but thesize of the protein is shorter in both theN and C termini (4). A form of constitutively expressed membrane-bound, Ca2+-and calmodulin-dependent NOS hasbeen reported, but the exact natureof this enzyme remains to be established (10). In addition to this ANTAGONISM WITH THE INDUCTION ELICITED “constitutive” enzyme, an “inducible” formof NOS exists, but BY LIPOPOLYSACCHARIDE* this NOS is independent of Ca2+ and calmodulin, being in(Received for publication, August 21, 1992) duced by bacterial lipopolysaccharide or interferon-y in different cell types (10-12). Sonsoles Hortelano, Ana M. Genaro, and Lisardo BoscaS One of the targets of NO in all tissues so far analyzed is the activation of soluble guanylate cyclase (13, 14). Although From the Znstituto de Bwquimica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain a role of cGMP as regulator of ionhomeostase has been proposed (E),itsfunctionremains elusive (4). However, The incubation of primary cultures of rat hepato- activation of guanylate cyclase has proved to be a useful tool cytes withlipopolysaccharide (LPS) or biologically ac- for the assessmentof NO release (8, 16, 17). tive phorbol esters promotes the releaseof nitric oxide In liver,Kupffer cells (hepatic macrophages) have some to the incubation medium. This process is the result of constitutive NOS activity, but it is immunologically undethe induction of the Ca2+-and calmodulin-independent tectable in hepatocytes(18-20). However, hepatocytes exhibit form of nitric oxide synthase. Both the releaseof nitric specific NO responses such as the inhibitionof mitochondrial oxide to the incubation medium and the expression of respiration(21),thecontrol of glycogenolysis (22), or the nitric oxide synthase activity exhibited a lag period of regulation of protein synthesis inco-cultures of Kupffer and about 45-60 min after cell stimulation. Exposure of hepatocyte cells (18,23). Moreover, treatment of animals with hepatocytes to both stimuli produced an antagonistic LPS promotes the expression of the inducible form of NOS effect on nitricoxiderelease,with a half-maximal in both types of hepatic cells (23, 24), and an important role inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of of liver for NO production in Gram-negative sepsis has been cells witha-phorbol 12,13-didecanoate failed tocoun- proposed (23). The absence of constitutive NOS in hepatoteract the effect of LPS or to induce nitric oxide syn- cytes and the ability of LPS or interferon-y to induce the thase, suggesting that activation of protein kinase C Ca2+-independentenzyme offer the possibility of the use of primary cultures of hepatocytes as a model system to study was involved in this process. the process of NOS induction by different extracellular factors. In this work, we show that phorbol estersalso promote the The identification of NO’ as the endothelium-derived re- expression of the inducible form of NOS in cultured hepatolaxingfactorhas openedanew view in the field of cell cytes, exhibiting a time course of NO production similar to communication, since this molecule due to its diffusible nature that elicited by LPS. In addition to this, a clear antagonism process of NO release may easily transpass the cell membrane and therefore, may between LPS and phorbol esters in the act both as an intracellular and intercellular messenger (1- and NOS induction hasbeen observed. 4). MATERIALS AND METHODS NO is produced from L-arginine by NOS, a n enzyme constitutively expressed in cells fromthe cardiovascular and Chemicals-[U-”C]Arginine was from Du Pont-New England Nuneural system and requires NADPH, Ca2+,and calmodulin to clear. Bovine hemoglobin was from Serva. Adenosine 2’,5‘-bisphosbeactive (3-6). Thebrain enzyme has beenpurified and phate-Sepharose was from Pharmacia. LPS was from Difco, and characterized and it shows a n absolute dependence for BH, Dowex AG50W-X8 was from Bio-Rad. Nitrate reductase from Asperand other enzymes were from Boehringer Mannheim. BH, was to be active (5, 7). This enzyme structurally resembles cyto- gillus from Dr. B. Schircks Laboratories (Jona, Switzerland). cGMP assay chrome P-450 reductase (4, 8) and is probably identical to kit was from Amersham Corp. Arginine derivatives and other chemneural NADPH diaphorase(9). In addition to brainNOS, the icals and biochemicals were from Sigma or Merck.

Phorbol Esters Induce Nitric Oxide Synthase Activity in Rat Hepatocytes

Cell Culture-Hepatocytes were prepared by the classical collagenase perfusion method (25), and the cells were preferentially sediMadrid andGrant PB91-134 from Comisi6n Interministerial de mented by controlled centrifugation at 40 X g for 4 min (Minifuge T, Ciencia y Tecnologia, Spain (CICYT). The costs of publication of Heraeus). Cell viability was assessed by the Trypan blue exclusion this article were defrayed in part by the payment of page charges. criterion and was always higher than 85%. The isolated cells were This article must therefore be hereby marked“advertisement” in resuspended(107/ml) in phenol red-free DMEM medium suppleaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. mented with antibiotics, 2% fetal calf serum, and 1 mM arginine (pH 4 To whom correspondence should be addressed. 7.4). To remove residual adherent cells (Kupffer), the suspension was The abbreviations used are: NO, nitric oxide; NOS, nitric oxide allowed to incubate in a plastic dish (9-cm diameter, 6 ml of cell synthase; BH,, (6R)-5,6,7,8-tetrahydrobiopterin; DMEM, Dulbecco’s suspension) for 30 min. After this incubation period, the suspension modified Eagle’s medium; D-NMA, p-methyl-D-arginine; L-NMA, was transferred (4 X lO‘/cm*) to rat-tail collagen precoated plastic P-methyl-L-arginine; L-NNA, Nu-nitro-L-arginine; LPS, lipopoly- culture dishes for 2 h, after which the incubation the medium was saccharide; PDBu, P-phorbol12,13-dibutyrate; a-PDD,a-phorbol aspirated toremove the unattached cells, and fresh medium contain12J3-didecanoate;PMA,P-phorbol12-myristate13-acetate; PBS, ing 5 mg/ml fatty acid-free bovine serum albumin instead of calf phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic serum was added. The cells were challenged with the appropriate acid. additions which were prepared in less than 10%of the final incubation

* This work was supported by Grant C183/91 from Comunidad de

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NOS Induction byPhorbol Esters volume, except for ligands with low solubility that were prepared in complete medium and filtered through a 0.2-pm pore membrane. Cell Homogenates-Hepatocyte homogenates were prepared from cultures (9-cm diameter dishes, Costar) after aspiration of the incubation medium and two washes of the cell layer with 10 ml of icecold PBS. The cells were scraped off the dishes and homogenized with an Ultraturrax in 2 ml of a medium containing 20 mM Tris, pH 7.5 (4 "C), 0.5 mM EGTA, 0.5 mM EDTA, 1 mM dithioerythritol, 1 p~ BH4, 1p~ leupeptin, and 0.2 mM phenylmethanesulfonyl fluoride (homogenization buffer). Adenosine 2',5'-Bisphosphate-Sepharose Chromatography-The cell homogenate was centrifuged at 20,000 X g for 15 min, and the supernatant was partially purified by a 2',5'-ADP-Sepharose column (0.5 X 5 cm) equilibrated with homogenization buffer supplemented with 1 mMMgC12 and 100 mM NaC1. After washing the column with this medium containing 0.5 M NaCl until no more protein emerged, NOS activity was eluted in homogenization medium supplemented with 5 mM NADPH and 10%glycerol (v/v) (5). Fractions containing NOS activity were concentrated by ultrafiltration through a cellulosetriacetate membrane with a cut-off of 30 kDa (Sartorious). The presence of 1 p~ BH4 during the purification was critical in maintaining an active enzyme (12, 26). Enzyme assays were carried out immediately after purification. Determination of NO-NO release from the cell culture or in NOS assays was determined either by the accumulation of nitrite and nitrate (total NO) or by the formation of a NO. hemoglobin complex (released NO). For the determination of total NO, 200 p1 of culture medium were transferred to 1.5-ml Eppendorf tubes and the nitrate was reduced to nitritewith 0.5 units of nitrate reductase (Boehringer) in the presence of 50 p~ NADPH, 5 p~ FAD (10, 27). The excess of NADPH, which interferes in the chemical determination of nitrite, was oxidized in the presence of 0.2 mM pyruvate and 1 pg of lactate dehydrogenase. Nitrite was determined with Greiss reagent (27, 28) by adding 1mM sulfanilic acid and 100 mM HC1 (final concentration). After incubation for 5 min the tubes were centrifuged and 150 p1 of supernatant were transferred to a 96-well microtiter plate. After a first reading of the absorbance at 595 nm, 50 pl of naphthylenediamine (1 mM in the assay) were added. The reaction was completed after 15 min of incubation, andthe absorbance at 595 nm was compared with a standard of NaN02.Alternatively, NO was measured by the difference in absorbance at 401 and 411 nm (extinction coefficient 99,580 M" cm") using the oxidation of oxyhemoglobin to methemoglobin (29). When NO release from cell cultures was measured, the incubation medium contained 1.5 p~ oxyhemoglobin. Purified bovine hemoglobin was converted into oxyhemoglobin by reduction with dithionite as described (30) and was submitted to gel filtration (Sephadex G-25 fine) prior to use. Authentic NO was used asstandardto establish the correlation between the changes in absorbance a t 401 and 411 nm and the concentration of NO. All solutions used to process NO were bubbled with pure helium to release the soluble air. Determination of cGMP-Cultures intended for the measurement of cGMP (9-cm dishes) were incubated for 10 min prior to ligand addition with 0.5 mM isobutyl-1-methylxanthineto favor the accumulation of this messenger. The medium was aspirated and replaced by 1 ml of an ice-cold mixture of ethano1:water (21, v/v). After homogenization and centrifugation in an Eppendorf centrifuge, samples were speed vacuum-dried andcGMP was measured using a specific binding kit, following the recommendation of the supplier (Amersham). Assay of NOS Actiuity-NOS activity was measured in crude hepatocyte homogenates and after partialpurification by 2',5'-ADPSepharose chromatography (5). The enzyme was assayed by three independent criteria: (a) theproduction of [U-'4C]citrulline from [U14C]arginine, (b) the release of NO, and (c) the NADPH-dependent diaphorase activity. Briefly, citrulline release was measured as described (5) at pH 7.4 in a buffer that contained 20 mM Hepes, 10 p M [U-"Clarginine (0.3 pCi), and 0.5 mM NADPH (200 p1 of incubation volume). After 10 min of incubation, the reaction was stopped by adding 1ml of ice-cold 10 mM EGTA, 1 mM citrulline, 100 mM Pipes, pH 5.5. One ml of this mixture was applied to a 1-ml Dowex AG50WX8 column (Na+-form), and [U-'4C]citrulline was eluted in 3 ml of water. The radioactivity was measured by liquid scintillation counting. The release of NO was measured as described for the method of the NO. hemoglobin complex formation (29). The NADPH-dependent diaphorase activity was measured at pH8.0 according to Hope et al. (9) by the reduction of nitro blue tetrazolium (0.5 mM) in the presence or absence of 1 mM NADPH. Samples were filtered through

a Sephadex G-25(fine) column equilibrated with 20 mM Tris, pH8.0, 1mM dithioerythritol, and 1p~ BH4 toremove NADPH. The enzyme was incubated for 10 min with the substrates and the reaction was stoppedwithone volume (0.5 ml) of 100 mM sulfuric acid. The absorbance of the reaction product (nitro blue tetrazolium formazan) at 585 nm was recorded. All reactions were carried out at 30 "C, and the enzyme activities were expressed as the difference of product formation in theabsence or presence of 1 mM L-NMA in thereaction mixture (31, 32).Linearity was verified by the indicatedreaction times when using the partially purified enzyme. Protein Determination-Protein was measured according to the method of Bradford (33) using bovine serum albumin as standard. RESULTS

LPS and PMA Stimulate NO Production in Cultured Hepatocytes-Primary cultures of hepatocytes challenged with 50 pg/ml LPS or 100 nM PMA released NO to the culture medium in a time-dependent fashion as shown in Fig. 1A. The release of NO was assessed both by the chemical assay of NO, nitrites, and nitrates (after reduction to nitrites as described), andby the changes in the absorbance of hemoglobin at 401 and 411 nm, when 1.5 ~ L Moxyhemoglobin was added to the incubation medium. The amount of NO measured by both methods was similar, anda lag period of 45-60 min was observed prior to the start of NO release. This lag period was roughly similar in both cases. The NO released by LPS was 37% higher than those produced by PMA. LPS and PMA Antagonize NO Release-To analyze the pathway by which LPS and PMA elicited the production of NO in cultured hepatocytes, cells were simultaneously stimulated with a saturating concentration of LPS (50 pg/ml) and different amountsof PDBu, or the biologically inactive isomer a-phorbol 12,13-didecanoate (a-PDD). As shown in Fig. l B , PDBu specifically inhibited the stimulation produced by LPS, with a half-maximal inhibitionobserved a t 14 nM PDBu. The inactive a-phorbol ester did not affect the response to LPS or promote NO release when added to the culturemedium in the absenceof LPS. Similar resultswere obtained when PMA was used instead of PDBu. To assess that NO was effectively produced in cells incu-

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FIG. 1. NO release from primary cultures of hepatocytes. Cells were cultured in 24-well dishes in phenol red-free DMEM medium containing5 mg/ml fatty acid-free albumin and 1 mM arginine. Panel A, time course of NO release. The totalNO production was assayed with Greiss reagent (open symbols) or by the changes in the absorbance of hemoglobin (1.5 p ~ added ) to the culture medium at time zero (filled symbols). Hepatocytes were challenged with 50 pg/ml LPS (0,O ) , 200 nM PMA (A, A), or without additions (0). Panel B, phorbol ester effects on LPS-induced NO release. Cells were stimulated for 4 h in the absence (open symbols) or in the presence of 50 pg/ml LPS (filled symbols) and the indicated concentrations of a-PDD (0,0 ) or PDBu (A, A). NO was assayed by the method of Greiss. Results are means +. S.E. of four different cell preparations.

NOS Induction by Phorbol Esters

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bated either with LPS or PMA, a parallel experiment was Inhibitors of protein kinase C such as staurosporine or the effect of PMA. done in which the concentrationof cGMP was measured. As isoquinoline H-7 also inhibited the stimulatory Furthermore, lipid A, the proposed biologically active moiety shown in Fig. 2 , LPS and PMA produced anincreasein cGMP that was maximal 2 h after activation of the hepato- of LPS, was even more effective than the complete molecule cytes. Moreover, the antagonism between LPS and PMAwas in promoting NO release. When the arginine derivatives Lalso evidenced in the reduced concentration of cGMP meas- NMA,D-NMA, or L-NNA were addedtotheincubation release of NO was ured in hepatocytes incubated with LPS and 200 nM PMA medium prior to LPS stimulation, the (14% of the effect elicited by LPS). These changes in cGMP drastically decreased in thepresence of L-NMA (TableI) but were compared with those obtained afterexposure of cells to was unaffected by other arginine derivatives. NOS Activity Is Induced in Cells Incubated with P M A or culture medium containing 3 PM authentic NO. The maximal increase of cGMP concentration obtained in hepatocytes in- LPS-When NOS activity was measured in hepatocyte culcubated either with LPS or with PMA was below the effect tures incubated for 4 h with either LPS or PMA, aclear elicited by pure NO which suggests that thereleased NO was increase in the total activity was observed (Table 11). The antagonism between LPS and PMA onNO release correlated unable to saturate the guanylatecyclase activity. The observation that PMA stimulates NO production in well with a decreased NOS activity in cells incubated with cultured hepatocytessuggests that activationof protein kinase both factors. The activity was inhibited by L-NMA when be C is involved in the mechanism of NO production. In agree- present a t 300 FM (95% inhibition), and turned out to ment with this result, activatorsof protein kinase C such as completely independent of Ca2+and calmodulin (105% of the 1,2-dioctanoylglycerol and the Ca2+ ionophore A23187 en- basal activity when assayed in the presence of 100 p M Ca2+ hanced NO production when added simultaneously (Table I). and 20 nM calmodulin).Measurement of NOS activityin crude supernatants by the release of [U-’4C]citrulline from arginine was difficultdue to the presence of ahigh basal activity not inhibited by L-NMA or L-NNA. However, after partial purification by 2’,5’-ADP-Sepharose chromatography, more than 95% of the citrulline release was inhibited in the presence of 0.5 mM L-NMA. This preparation was also very useful for the measurement of NOS by the NO. hemoglobin method, and contained the NADPH-dependent diaphorase activity, as shown in Table 11. Agreement was observed between NOSandNADPH-dependentdiaphoraseactivities when the hepatocytes were stimulated with either LPS or PMA. 0

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FIG. 2. cGMP concentration in primary cultures of hepatocytes incubated with LPS or PMA. Cells (5 X lo6) incubated in the presence of 0.5 mM isobutyl-1-methylxanthinewere stimulated

with 200 nM a-PDD (U), 50 pg/ml LPS (a),50 pg/ml LPS plus 200 nM a-PDD (0),200 nM PMA (A), LPS PIUS 200 nM PMA (A),or 3 PM authentic NO (W), and the concentrationof cGMP was measured at the indicatedtimes. When cells were incubated with pure NO, the culture was pulsed with a fresh solution of NO 5 min prior to the sample collection. Results are means f S.E. of triplicates of one representative experiment.

TABLEI Effect of LPS analogues, protein kinaseC modulators, and arginine derivatives on NO release from primary culturesof hepatocytes Cells were incubated in 24-well dishes with 250 pl of phenol redfree DMEM medium containing 5 mg/ml fatty acid-free bovine serum albumin. Cells were incubated for 5 h withthe indicated ligands. NO production was assayed chemically using Greiss reagent. Results are means f S.D. of triplicates from one representative experiment. NO production

Ligand

nmollmg protein

None LPS (50 pglrnl) Lipid A (10 pg/ml) Dioctanoylglycerol (2 pg/ml) Dioctanoylglycerol (2 pg/ml) A23187 (1p M ) PMA (200 IIM) PMA (200 nM) H-7 (50 p M ) PMA (200 nM) staurosporine (0.5 p ~ ) LPS (50 pg/ml) + D-NMA (0.5 mM) LPS (50 pg/ml) L-NMA (0.5 mM) LPS (50 uelml) + L-NNA (0.5 mM)

+

+ + +

3+1 44 f 6 57 + 8 8+2 28 _t 4 29 f 5 9 f 1 14 f 2 42 f 5 11 f 2 44 f 6

DISCUSSION

The reported results clearly show that phorbol esters may induce the expression of the Ca2+ and calmodulin-independent isoform of NOS in primary cultures of hepatocytes. These cultures have been depleted from adherent cells, and, therefore, this inductionmay be consideredas a specific hepatocyte response. Moreover, the complete absence of NO release or NOS activity in controlcells, confirmed by several criteria (8, TABLEI1 NOS activity in primary culturesof hepatocytes Cells (9-cmdishes) were incubated for 4hwith the indicated ligands, and at the endof the incubation period the culture medium was aspirated and thecell layer was washed twice with ice-cold PBS. The homogenate was partially purified by 2’,5’-ADP-Sepharose chromatography, and the enzyme activity was measured as described under “Materials and Methods,” by the release of [U-14C]citrulline from arginine (10 p ~ )by , the changes in the absorbance of oxyhemoglobin after formation of the NO.hemoglobin complex, or by the NADPH-dependent diaphorase activity assayed with 0.5 mM nitroblue tetrazolium. Results are means f S.E. of three independent experiments. NOS activity (nmol X min” X mg protein”) Ligand

None LPS (50 pg/ml) PMA (200 nM) a-PDD (200 nM) LPS (50 pg/ml) + PMA (200 nM) LPS (50 pg/ml) + a-PDD (200 nM) Not determined.

Citrulline release

N O ’ hemoglobin complex

Nitro blue tetrazolium formazan

721 337 f 43 287 f 34 9 f l 31 2 8

5 f l 278 f 35 227 f 29 4 f 1 22 f 3

1.1 f 0.7 9.8 f 1.2 7.1 f 1.7 1.2 + 0.3 2.1 + 0.4

302 & 38

229 f 34

ND“

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34,35), allowed a cleardetermination of both parameters after cellular response. The involvement of protein kinase C in the stimulation with extracellularfactors. induction of NOS and in the modulation of the biological effects of other inductor cytokines may open a new aspect Incubation of various cell types with LPS promotes the expression of inducible NOS (10, 36). In primary cultures of which could unravel thecomplex regulationof inducible NOS. hepatocytes, LPS hasbeen described as a weak stimulator of REFERENCES NO production(%fold increase) when comparedwiththe 1. Palmer, R. M. J., Ferrige, A. G., and Moncada S. (1987) Nature 257,524synergistic action of other cytokines under identical condi526 2. Ignarro, L. J. (1990) Annu. Reu. Pharmacol. Toxicol. 30,535-560 tions (LPS tumor necrosis factor + interferon-y interleu3. Schmidt, H. H. H. W., Pollock, J. S., Nakane, M., Gorsky, L. D., Forsterkin-1; 21-fold increase). These datasuggest the existence of a mann, U., and Murad, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,365369 complex regulatory system in the controlof NOS expression 4. Lowenstein, C. J., and Snyder, S. H. (1992) Cell 7 0 , 705-707 (37). We have used LPS as a positive control to assess the 5. Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685 effectiveness of phorbol esters as inducers of NOS activity 6. Tayeh, M. A,, and Marletta, M. A. (1989)J. Biol. Chem. 264,19654-19658 and NO release. According to our results, a clear parallelism 7. Giovanelli, J., Campos, K. L., and Kaufman, S. (1991) Proc. Natl. Acad. Sci. (I. S. A. 8 8 , 7091-7095 was observed in the time course of NO release, and in the 8. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and degree of NOS induction by both factors.Moreover, this time Snyder, S. H. (1991) Nature 361,714-718 course was in the rangeof that observed in liver NOS activity 9. Hope, B. T., Michael, G. J., Knigge, K. M., and Vincent, S. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,2811-2814 from animals intraperitoneally injected with LPS (24, 34). In 10. Schmidt, H. H. H. W., Warner, T. D., Nakane, M., Forstermann, U., and addition to this parallelism, consistent results were observed 11. Murad, F. (1992) Mol. Pharmacol. 41,615-624 Marletta, M. A,, Yoon, P. S., Iyengar, R., Leaf, C. D., and Wishnok, J. S. in the measurementof NO release from cultured cells and in (1988) Biochemistry 27,8706-8711 the appearanceof NOS activity,regardless of the methodused 12. Kwon, N. S., Nathan, C. F., and Stuehr, D. J. (1989) J. Biol. Chem. 2 6 4 , 20496-20501 to assay NO or NOS. 13. Ignarro, L. J. (1989) FASEB J. 3,31-36 The intracellular concentration of cGMP increased in a 14. Southan, E., and Garthwaite,J. (1991) Neurosci. Lett. 130, 107-111 15. De Vente, J., and Steinhusch, H.W. M. (1992) Acta Histochem. 9 2 , 13-38 NO-dependent fashion. However, these changes were quan- 16. Ishii, K., Sheng, H., Warner, T. D., Forstermann, U., and Murad, F. (1991) Am. J. Physiol. 2 6 1 , H598-H603 titatively moderate when compared with the effect reported 17. Waldman, S. A,, and Murad, F. (1987) Pharmacol. Reu. 39,163-196 for other cell types (36, 38) and were below the maximal 18. Billiar, T. R., Curran, R. D., Stuehr, D. J., Stadler, J., Simmons, R. L., and Murray, S. A. (1990) Biochem. Biophys. Res. Commun. 168,1034-1040 activity of guanylate cyclase as deduced by the changes in 19. Billiar, T. R., Curran, R. D., Stuehr, D. J., West, M. A,, Bentz, B. G., and cGMP measured in cells stimulated with authenticNO (70% Simmons, R. L. (1989) J. Exp. Med. 169,1467-1472 H.,Oguchi, S., Adachi, H., Iida, S., Suzuki, H., Sugimura, T., and higher than in cells stimulated with LPS). Indeed, the pres- 20. Ohshima, Esumi, H. (1992) Biochem. Biophys. Res. Commun. 183,238-244 ence of soluble guanylate cyclase in liver has been reported 21. Stadler, J., Billiar, T. R., Curran,R. D., Stuehr, D. J., Ochoa, J. B., Simmons, R. L. (1991) Am. J. Physiol. 2 6 0 , C910-C916 and theenzyme has been purified (39), and an efflux of cGMP 22. Moy, J. A., Bates, J. N., and Fisher, R. A. (1991)J . Bid. Chem. 266,8092from the cell to the extracellularmedium has been described 8096 Billiar, T. R., Curran, R. D., Ferrari, F. K., Williams, D. L., and Simmons, 23. (37). R. L. (1990) J . Surg. Res. 48,349-353 The observationof a high stereospecificityin the hepatocyte 24. Knowles, R. G., Salter, M., Brooks, S. L., and Moncada, S. (1990) Biochem. Biophys. Res. Commun. 1 7 2 , 1042-1048 response to phorbol esters and in counteracting theeffect of 25. Hue, L., Feliu, J. E., and Hers, H. G. (1978) Biochem. J. 1 7 6 , 791-797 LPS was unexpected, and these resultssuggest the existence 26. Yui, Y., Hattori, R., Kosuga, K., Eizawa, H., Hiki, K., and Kawai, C.(1991) J. Biol. Chem. 6 , 12544-12547 of an antagonisticsignaling pathway between LPS and phor- 27. Tayeh, M. A,, and2 6Marletta, M. A. (1989)J. Biol. Chem. 264,19654-19658 bo1 esters. Although synergism between cytokines in promot- 28. Green, L. C., Wagner, D. A,, Glogowski, J,., Skipper, P. L., Wishnok, J. S., and Tannenhaum, S. R. (1982) Anal. Blochem. 126,131-138 ing NOS inductionseemsto beacommonprocess,only 29. Knowles, R. G., Merrett, M., Salter, M., and Moncada, S. (1990) Biochem. glucocorticoids and some growth factors have been described J. 270,833- 836 30. Feelisch, M., and Noak, E. A. (1987) Eur. J. Pharmacol. 1 3 9 , 19-30 as antagonists of NOS induction (24, 40). 31. Rees, D. D., Palmer, R. M. J., Schulz, R., Hodson, H. F., and Moncada, S. Phorbolestersare well known activators of all protein (1990) Br. J. Pharmacol. 101, 746-752 K., Chang, B., Kerwin, J. F., Huang, Z. J., and Murad, F. (1990) Eur. kinase C subspecies except forthe { isoform, and because this 32. Ishii, J . Pharmacol. 1 7 6 , 219-223 kinase is the most likely target of phorbol esters (41), it is 33. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 M., Knowles, R. G., and Moncada, S. (1991) FEBS Lett. 2 9 1 , 145tempting to speculate that activation of protein kinase C may 34. Salter, 149 be sufficient to promote NOS induction. In agreement with 35. Bredt, D. S., Hwang, P. M., and Snyder, S. H. (1990) Nature 3 4 7 , 768w. ,"n this suggestion, stimulation of hepatocytecultureswith a 36. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Reu. combination of a permeant 1,2-diacylglycerol and a Ca2+ 4 3 , 109-142 T. R., Curran, R. D., Harhrecht, B. G., Stadler, J., Williams, D. L., ionophore produced an important NOS activation. Inhibitors 37. Billiar, Ochoa, J. B., DiSilvio, M., Simmons, R. L., and Murray, S. A. (1992) of protein kinase C such as H-7 or staurosporine prevented Am. J. Physiol. 2 6 2 , C1077-C1082 J., Rob, P., Mdlsch, A., Fandrey, J., Vosbeck, K., and Busse, the release of NO (70-80% inhibition). Similar results have 38. Pfeilschifter, R. (1992) Eur. J. Biochem. 2 0 3 , 251-255 been observed in NO release in NOS induction by phorbol 39. Braughler, J. M., Mlttal, C. K., and Murad,F. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 219-222 esters in cultured peritonealmacrophages (work in progress), 40. Pfeilschifter, J. (1991) Eur. J. Pharmacol. 2 0 8 , 339-340 suggesting that this effect of phorbol esters could be a wide 41. Bell. R. M.. and Burns. D. S. (1991) J. Biol. Chem. 266,4661-4664

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