Expression of Nitric-oxide Synthase in Rat Kupffer Cells Is Regulated by cAMP

Abstract

Treatment of cultured rat Kupffer cells with lipopolysaccharide (LPS) resulted in a time-dependent increase in the expression of the inducible isoform of nitric-oxide synthase (iNOS). Agents that elevated intracellular cAMP levels (e.g. forskolin, dibutyryl cAMP, cholera toxin, and isoproterenol) markedly decreased nitrite production and iNOS protein formation by LPS-stimulated Kupffer cells. Furthermore, inhibition of LPS-induced nitrite formation and iNOS protein levels by these agents was enhanced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Forskolin, the most potent inhibitor of LPS-induced nitrite formation by Kupffer cells, decreased iNOS mRNA levels in a time-dependent manner. Time course studies indicated that forskolin was most effective at inhibiting LPS-induced nitrite formation and iNOS mRNA levels by Kupffer cells when added before LPS. Message stability studies established that forskolin did not enhance the rate of decay of LPS-induced iNOS mRNA. Nuclear run-on assays revealed that forskolin decreased LPS-induced transcription of the iNOS gene. Treatment of Kupffer cells with LPS induced the translocation of the p65 subunit of nuclear factor κB (NF-κB) into the nucleus, and this process was abolished by forskolin. In addition, the LPS-dependent degradation of IκBα was not observed in forskolin-treated cells; the levels of the p65 subunit of NF-κB were minimal in the nucleus at the same time. Also, we observed that forskolin induced transcription of the IκBα gene in a time-dependent manner and in addition up-regulated LPS-induced IκBα mRNA levels. Taken together, this study indicates that the attenuation of LPS-induced iNOS formation in Kupffer cells by elevated intracellular cAMP levels occurs by preventing the degradation of IκBα which suppresses the activation of NF-κB and inhibits the onset of transcription of the iNOS gene. Treatment of cultured rat Kupffer cells with lipopolysaccharide (LPS) resulted in a time-dependent increase in the expression of the inducible isoform of nitric-oxide synthase (iNOS). Agents that elevated intracellular cAMP levels (e.g. forskolin, dibutyryl cAMP, cholera toxin, and isoproterenol) markedly decreased nitrite production and iNOS protein formation by LPS-stimulated Kupffer cells. Furthermore, inhibition of LPS-induced nitrite formation and iNOS protein levels by these agents was enhanced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Forskolin, the most potent inhibitor of LPS-induced nitrite formation by Kupffer cells, decreased iNOS mRNA levels in a time-dependent manner. Time course studies indicated that forskolin was most effective at inhibiting LPS-induced nitrite formation and iNOS mRNA levels by Kupffer cells when added before LPS. Message stability studies established that forskolin did not enhance the rate of decay of LPS-induced iNOS mRNA. Nuclear run-on assays revealed that forskolin decreased LPS-induced transcription of the iNOS gene. Treatment of Kupffer cells with LPS induced the translocation of the p65 subunit of nuclear factor κB (NF-κB) into the nucleus, and this process was abolished by forskolin. In addition, the LPS-dependent degradation of IκBα was not observed in forskolin-treated cells; the levels of the p65 subunit of NF-κB were minimal in the nucleus at the same time. Also, we observed that forskolin induced transcription of the IκBα gene in a time-dependent manner and in addition up-regulated LPS-induced IκBα mRNA levels. Taken together, this study indicates that the attenuation of LPS-induced iNOS formation in Kupffer cells by elevated intracellular cAMP levels occurs by preventing the degradation of IκBα which suppresses the activation of NF-κB and inhibits the onset of transcription of the iNOS gene. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, nNOS, and iNOS, endothelial, neuronal, and inducible NOS, respectively; LPS, lipopolysaccharide; PAGE, polyacrylamide gel electrophoresis; NF-κB, nuclear factor κB; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]]amino}ethanesulfonic acid; IBMX, 3- isobutyl-1-methylxanthine.1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, nNOS, and iNOS, endothelial, neuronal, and inducible NOS, respectively; LPS, lipopolysaccharide; PAGE, polyacrylamide gel electrophoresis; NF-κB, nuclear factor κB; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]]amino}ethanesulfonic acid; IBMX, 3- isobutyl-1-methylxanthine. has been identified as an important signaling molecule that is involved in regulating a wide array of biological activities in neural, vascular, and immune cellular systems (1Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar). NO is generated froml-arginine and molecular oxygen in the presence of the enzyme NO synthase (NOS) (2Nathan C. FASEB J. 1992; 6: 3051-3064Google Scholar). To date three distinct NOS isoforms have been identified from molecular cloning and sequencing analyses (3Lowenstein C.J. Glatt C.S. Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6711-6715Google Scholar). The endothelial (eNOS) and neuronal (nNOS) isoforms are expressed constitutively in endothelial and neuronal cells, respectively. The amount of NO generated by these cell types is dependent upon the cellular content of NOS (4Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Google Scholar). NO generated by endothelial cells plays an important role in the control of vascular tone, whereas in neuronal tissue NO acts to regulate cGMP-mediated neurotransmission (1Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar, 2Nathan C. FASEB J. 1992; 6: 3051-3064Google Scholar). The third isoform of NOS, termed inducible NOS (iNOS), was first identified in macrophages stimulated with interferon-γ and bacterial lipopolysaccharide (LPS) (3Lowenstein C.J. Glatt C.S. Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6711-6715Google Scholar). iNOS has been identified in a wide variety of cell types including macrophages, mesangial cells, vascular smooth muscle cells, keratinocytes, chondrocytes, osteoclasts, and hepatocytes (1Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar, 5Nathan C. Xie Q.W. J. Biol. Chem. 1994; 269: 13725-13728Google Scholar). NO generated within these cells mediates macrophage cytotoxicity during host defense reactions, alterations in the contractile responses of mesangial cells, and in instances where NO exceeds normal physiological levels, instigates the inhibition of vascular smooth muscle tone, hepatocyte metabolism, and protein synthesis (6Kunz D. Walker G. Pfeilschifter J. Biochem. J. 1994; 304: 337-340Google Scholar, 7Wang M.H. Cox G.W. Yoshimura T. Sheffler L.A. Skeel A. Leonard E.J. J. Biol. Chem. 1994; 269: 14027-14031Google Scholar, 8Curran R.D. Billiar T.R. Stuehr D.J. Ochoa J.B. Harbrecht B.G. Flint S.G. Simmons R.L. Ann. Surg. 1990; 212: 462-471Google Scholar). Recent evidence indicates that elevated levels of NO play a major role in the pathogenesis of several chronic disorders and inflammatory processes. In particular, studies have indicated that an overproduction of NO in response to LPS and cytokines contributes to the development and prolongation of severe hypotension and peripheral vasodilation observed during endotoxic shock (9Szabo C. Wu C.C. Mitchell J.A. Gross S.S. Thiemermann C. Vane J.R. Circ. Res. 1993; 73: 991-999Google Scholar). The activities of nNOS and eNOS are regulated by rapid, transient elevations of intracellular free calcium which enhance the binding of calmodulin to the NOS enzyme resulting in NO release over a time frame of seconds and minutes (10Bredt D.S. Ferris C.D. Snyder S.H. J. Biol. Chem. 1992; 267: 10976-10981Google Scholar). In contrast, the expression of iNOS is thought to be regulated primarily at the transcriptional level of the iNOS gene. Once induced, iNOS produces NO for periods of several hours or days. Thus, given the magnitude of the wide variety of inhibitory actions of NO, it is of considerable interest and even may be of some therapeutic utility to delineate the mechanism(s) by which the production and resultant activity of NOS can be controlled or attenuated. In the presence of LPS, Kupffer cells, the resident macrophage found in the sinusoids of the liver, produce large amounts of nitrite and nitrate, the stable end products of the NO pathway (11Billiar T.R. Curran R.D. Stuehr D.J. West M.A. Bentz B.G. Simmons R.L. J. Exp. Med. 1989; 169: 1467-1472Google Scholar). It has become apparent recently that overproduction of NO by hepatic cells plays a major role in hepatic injury/necrosis associated with endotoxic shock. Kupffer cells also synthesize and release several cytokines in response to LPS which in turn stimulate neighboring hepatocytes to generate NO (8Curran R.D. Billiar T.R. Stuehr D.J. Ochoa J.B. Harbrecht B.G. Flint S.G. Simmons R.L. Ann. Surg. 1990; 212: 462-471Google Scholar). The consequent overproduction of NO in the liver results in profound degenerative changes observed in hepatocytes (12Laskin D.L. Rodriguez del Valle M. Heck D.E. Hwang S.M. Ohnishi S.T. Durham S.K. Goller N.L. Laskin J.D. Hepatology. 1995; 22: 223-234Google Scholar). These changes include a decrease in total protein synthesis, cellular proliferation, and an increase in cGMP formation (13Nussler A.K. Di Silvio M. Liu Z.Z. Geller D.A. Freeswick P. Dorko K. Bartoli F. Billiar T.R. Hepatology. 1995; 21: 1552-1560Google Scholar). The induction of iNOS by LPS in Kupffer cells requires the initiation of gene expression and de novo protein synthesis over a period of several hours. It is unclear whether classical second messengers such as cAMP are involved in iNOS gene expression and NO formation. Recent studies have indicated that agents that elevate levels of cAMP improve circulatory function in animal models of endotoxic shock; in particular isoproterenol was found to inhibit the development of vascular hyporeactivity in the endotoxic rat (14Harbrecht B.G. Johnson B. Pitt B. Billiar T.R. Endothelium. 1995; 3: S95Google Scholar, 15Szabo C. Hasko G. Zingarelli B. Nemeth Z.H. Salzman A.L. Kvetan V. Pastores S.M. Vizi E.S. Immunology. 1997; 90: 95-100Google Scholar). In contrast, certainin vitro studies have shown that elevation of cAMP caused an induction of iNOS, whereas in other studies increased levels of cAMP caused a reduction in iNOS (6Kunz D. Walker G. Pfeilschifter J. Biochem. J. 1994; 304: 337-340Google Scholar, 7Wang M.H. Cox G.W. Yoshimura T. Sheffler L.A. Skeel A. Leonard E.J. J. Biol. Chem. 1994; 269: 14027-14031Google Scholar, 16Bulut V. Severn A. Liew F.Y. Biochem. Biophys. Res. Commun. 1993; 195: 1134-1138Google Scholar). The present study was designed to investigate whether iNOS gene expression activity is regulated by elevated levels of cAMP in cultured rat Kupffer cells. of the rat liver, Kupffer cells were by as D.L. Exp. Res. Scholar). The of the Kupffer cell was as by Kupffer cells were at in with and and in or tissue cells were in an of and the of the was Kupffer cells were used within of in of NO by iNOS was by the of nitrite in the the D.A. J. Biochem. Scholar). Kupffer cells were cultured in tissue at a of at in the cells were and was added to The cells were to several a the from was The in was to nitrite the by Johnson Scholar). were with an of the and at for The of was at nitrite as a formation was expressed as cells. the of from at Kupffer cells were at a of in the cells were with and stimulated with LPS or with the cells were with three of and was added to and the cells were The resulting cell were to three to the Kupffer cells of the cell was used for protein and the of the was at Nuclear and were to P. Res. 1989; Scholar). Kupffer cells were with and in of and The cells were to for which of was The was for and the was for in a The was and at The nuclear was in of nuclear and and the was for with The nuclear was for in a at and the was at for the cell were a were in and and to the of Scholar). The were to a and the were for in and for with in and The were with in the same for the were in and were by with the development and and nuclear were The were to The were in for and to the p65 subunit of NF-κB or IκBα at The were for at with were an enhanced Kupffer cells were at a of in and the total from cultured rat Kupffer cells was was by a gel and a a iNOS by S. Snyder of or by A. of was with a were in and at for The were in at for in at for and at in for was a were a to rat were from Kupffer cells to J.A. K. in and Scholar). cells were in in of and at at for was added by for to the cell The cells were for and by at at for The were in and in in vitro were in for at The was by the addition of and and for at were by with and with amounts were in of TES, and for at to the were in at for at in for and in at for was a iNOS was from p65 and IκBα were from and and were from LPS dibutyryl cAMP, cholera toxin, forskolin, IBMX, and were from Kupffer cells a level of nitrite whereas LPS caused an increase in nitrite formation during the period The addition of agents LPS-induced nitrite formation by Kupffer cells, in a manner not The of the of used in this study is in The forskolin resulting in an increase of intracellular cAMP forskolin LPS-induced nitrite formation The cAMP dibutyryl cAMP LPS-induced nitrite formation by isoproterenol and cholera stimulate the isoproterenol by binding to cell and cholera by of have used these agents to changes in factor binding in Kupffer cells, and we have that isoproterenol an increase in intracellular cAMP in these cells Liu D.J. J. Biol. Chem. 1990; Scholar). It is important to that of the agents the indicated caused in the rate of formation of nitrite the control when added to Kupffer cells in the of LPS and alterations or of Kupffer cells an period of In addition, LPS-induced nitrite formation by Kupffer cells in the presence of the agents and the phosphodiesterase inhibitor was to a in the of agents are of LPS-induced iNOS activity in Kupffer of agents LPS-stimulated nitrite production by Kupffer forskolin cholera cholera isoproterenol in a cAMP levels control iNOS or decrease the synthesis of iNOS To these the nitrite inhibition were and the levels of iNOS protein in Kupffer cells were by with in in Kupffer cells iNOS was whereas cells a The iNOS protein from Kupffer cells that been with LPS, and to be a decrease in the amount of iNOS protein forskolin was the most effective inhibitor of LPS-induced nitrite and iNOS protein formation by cultured Kupffer cells and it was used for Kupffer cells were with LPS or LPS and nitrite formation was at the time indicated in A. In with other LPS stimulated nitrite formation by Kupffer cells in a time-dependent The inhibitory of forskolin LPS-induced nitrite formation by Kupffer cells apparent of and to The time of LPS-induced iNOS mRNA in Kupffer cells is shown in and the iNOS mRNA level increased and and by The addition of forskolin caused a considerable decrease in the of iNOS and the levels of were at and was added to Kupffer cells at the same time or at the addition of LPS to the time for the inhibition of LPS-induced nitrite production and iNOS mRNA levels. in the was the addition of LPS. that of LPS-induced nitrite formation by Kupffer cells when forskolin was present before the addition of LPS. forskolin was added LPS, inhibitory decreased with time. a iNOS mRNA was and by Kupffer cells were with forskolin for before the addition of LPS, the level of iNOS mRNA was the of the iNOS mRNA increased when forskolin was added at the same time as LPS and and the addition of LPS. To whether forskolin LPS-induced levels of iNOS mRNA by we the of forskolin the of LPS-induced iNOS mRNA by of Kupffer cells with the transcriptional inhibitor D. Kupffer cells were with LPS in the presence and of forskolin for to iNOS mRNA was added to the cells at this to inhibit the addition of total was and by To for in the of to the iNOS was by that to the the decay of iNOS mRNA as time. these the of iNOS mRNA can be as (2Nathan C. FASEB J. 1992; 6: 3051-3064Google of The of iNOS mRNA in LPS-stimulated Kupffer cells in the and presence of forskolin were and respectively; the reduction of LPS-induced iNOS mRNA levels by forskolin in Kupffer cells was not caused by a decrease in iNOS gene transcription in Kupffer cells was a nuclear run-on to that forskolin caused inhibition of this Kupffer cells were or with LPS or LPS and forskolin for and cells were and The transcription of iNOS and by was by the to and that been a that iNOS gene transcription was in control cells was increased by to LPS and that with forskolin for the LPS In a a with LPS, a with forskolin caused apparent attenuation of iNOS gene transcription and in These results are in with time-dependent changes in iNOS mRNA. that a with LPS a of iNOS gene transcription has and the of added forskolin be the of the with and forskolin of LPS the of iNOS gene transcription is mRNA levels this time the of added forskolin be the of the with and forskolin as Taken together, the indicated that forskolin LPS-induced iNOS mRNA at the transcriptional Recent studies by Q.W. C. J. Exp. Med. 1993; have shown that activation of the transcription factor NF-κB and binding to the of the iNOS gene are in the induction of iNOS synthesis by LPS in In addition, K. Hepatology. 1995; 22: activity in Kupffer cells with LPS. In of these we to investigate the of forskolin NF-κB activation in the presence and of LPS. Kupffer cells were with LPS for with forskolin for and stimulated with LPS for or with forskolin for and stimulated with for and nuclear were is in and nuclear In cells the p65 subunit of NF-κB was in the amount of p65 was in the nuclear In contrast, LPS increased the amount of p65 in the nucleus of Kupffer cells. in forskolin-treated Kupffer cells, in the presence and of LPS, the amount of p65 in the nuclear was with cells of the p65 was in the and that forskolin with the translocation of p65 from the into the NF-κB in the of cells to a of inhibitory which IκBα and of NF-κB, which results in the translocation of the free protein into the nucleus, is by a and degradation of Biol. 1993; Scholar). we the levels of IκBα a major of in the of and Kupffer cells. is in In from Kupffer cells an an with a molecular of In contrast, of of Kupffer cells with LPS the amount of IκBα protein was with that in cells. in forskolin-treated Kupffer cells in the presence and of LPS, the levels of IκBα in the were to the levels of the protein in cells and indicated that in the presence of forskolin the IκBα protein in the of Kupffer cells and to the NF-κB The results that in the presence of forskolin, the of IκBα protein in the of Kupffer cells was whether forskolin IκBα gene expression in Kupffer cells. Kupffer cells were stimulated with forskolin or LPS or LPS and forskolin and at the indicated in was and in The mRNA for IκBα in Kupffer cells as a at It is important to that in Kupffer cells the level of IκBα mRNA was not In the presence of forskolin and IκBα mRNA levels of and to levels by In contrast, in the presence of LPS and IκBα mRNA levels increased of a by and elevated for to Kupffer cells were for with forskolin and stimulated with LPS over a period of and it was observed that forskolin up-regulated LPS-induced IκBα mRNA levels for to the addition of LPS These that forskolin has the to IκBα mRNA synthesis and in turn increase the levels of protein in the of Kupffer cells. In the of Kupffer cells with forskolin and by LPS the levels of IκBα are and the translocation of NF-κB into the The release of NO by Kupffer cells of endotoxic shock an important to the of the liver Hepatology. Scholar). it is of considerable interest to investigate the intracellular signaling that regulate the of iNOS gene In Kupffer cells, agents that elevate intracellular cAMP levels LPS-induced nitrite formation and LPS-induced iNOS protein Furthermore, in the presence of forskolin LPS-induced iNOS mRNA formation was and that elevated levels of cAMP may have the onset of iNOS gene decreased transcription of the iNOS or iNOS mRNA The of iNOS mRNA was the same in the presence or of forskolin, the of mRNA stability in which forskolin was added to cultured Kupffer cells or at LPS indicated that inhibition of iNOS mRNA the presence of forskolin before the addition of LPS of the initiation of of iNOS gene transcription that at LPS forskolin decreased the transcriptional process whereas by LPS forskolin The of the recently rat gene iNOS has been found to for the binding of transcription D. Pfeilschifter J. Biochem. Biophys. Res. Commun. Scholar). of these is in the induction of iNOS by LPS or transcription factor of during the induction of iNOS by LPS in macrophages is The of the rat gene iNOS of the NF-κB binding Q.W. C. J. Exp. Med. 1993; Scholar). NF-κB is an transcription factor present in the of cells. It is of a of and p65 In cells the NF-κB are in the by with a of inhibitory which IκBα and Biol. 1993; Scholar). of NF-κB can be by a variety of agents including such as and inflammatory cytokines such as LPS and factor cellular activation the IκBα and degradation the pathway Rev. Scholar). NF-κB protein into the nucleus and to Biol. 1993; regulating a variety of for and inflammatory S. J. Biol. Chem. 1995; Scholar). of Kupffer cells by forskolin resulted in attenuation of LPS-induced iNOS mRNA in Kupffer cells. that forskolin the LPS-stimulated nuclear translocation of NF-κB in Kupffer cells, resulting in decreased transcription of the iNOS gene and decreased levels of iNOS mRNA. of and nuclear that in Kupffer cells forskolin the amount of NF-κB which was to the nucleus by LPS was and nuclear from Kupffer cells that been with forskolin. These that forskolin inhibits LPS-induced iNOS mRNA formation in Kupffer cells by of NF-κB activity by forskolin by forskolin the degradation of IκBα and the translocation of NF-κB into the nucleus, or forskolin of NF-κB by some other preventing degradation of the IκBα LPS of Kupffer cells decreased levels of and this decrease with the of the p65 subunit of NF-κB in the In of Kupffer cells that been with forskolin to be decrease in IκBα upon with LPS and These results that forskolin IκBα by preventing and Nuclear NF-κB can the expression of the IκBα the IκBα protein in the of cells 1993; Scholar). The IκBα with NF-κB in the translocation into the in LPS-stimulated Kupffer cells, where IκBα mRNA levels elevated for it the most for LPS-induced iNOS mRNA levels in Kupffer cells are not expressed were observed to decrease by and induced transient IκBα mRNA formation in Kupffer cells, with the mRNA levels of and to control by and of Kupffer cells for with forskolin up-regulated LPS-induced IκBα mRNA levels and It is important to that of IκBα mRNA levels in Kupffer cells with forskolin for and stimulated with LPS in the Kupffer cells been in with forskolin for at which time forskolin amounts of IκBα mRNA in Kupffer cells It may be of to that the time at which inhibition of LPS-induced iNOS mRNA levels in Kupffer cells occurs is when the cells have been with forskolin for with the where forskolin was added LPS which with IκBα mRNA levels in the cells. In contrast, when forskolin was added to Kupffer cells several hours LPS, the transcriptional and of iNOS formation are and is for forskolin to inhibitory of iNOS Recent have shown that LPS of Kupffer cells NF-κB within in vitro E.S. Biochem. J. Scholar, E.S. J. and in the rat J. Scholar). NF-κB activation to nitric oxide production in Kupffer cells S. S. T. M. J. 1997; Scholar). To the present is the first of the NF-κB control during iNOS response to in the Kupffer Also, this is the first to the of cAMP this a study Kupffer cells of dibutyryl cAMP LPS-stimulated iNOS induction K. S. K. K. M. Chem. Med. Scholar). The of cAMP iNOS production are of interest the first M. T. M. J. Biol. Chem. 1993; that agents induced iNOS in cultured vascular smooth muscle cells and that this induction was with that by inflammatory cAMP elevation has been shown to have in mesangial cells (6Kunz D. Walker G. Pfeilschifter J. Biochem. J. 1994; 304: 337-340Google and in rat C. 1997; Scholar). cAMP not iNOS in it iNOS induction in cells Simmons R.L. B.G. J. 1995; 269: Scholar). is regulated by the cAMP to iNOS gene expression in response to LPS of the macrophage and inhibition of the activation of iNOS in these cells Pharmacol. Scholar). In signaling including elevation of cAMP to the induction of iNOS by NF-κB C. U. J. Biol. Chem. Scholar). In contrast, elevation of cellular cAMP has been shown to iNOS in of rat macrophages A. C. J. G. 1997; rat K. J. Biol. Chem. 1997; and cells macrophage J.R. Biochem. Pharmacol. 1997; present that of Kupffer cells with forskolin the degradation of IκBα and IκBα mRNA the of IκBα in the these same cells are stimulated with LPS, degradation of IκBα that are with NF-κB the NF-κB with the IκBα translocation into the translocation of NF-κB into the nucleus results in decreased iNOS gene transcription and levels of iNOS formation. The of cAMP to LPS-induced NO formation by Kupffer cells a in which to the intracellular signaling that regulate iNOS gene expression in the liver of endotoxic shock. and NF-κB, and cellular in shock and M.A. C. Scholar). Treatment of Kupffer vitro which models may shock to be from it be of interest to whether these NF-κB are by cAMP for the Kupffer cells and for this are to M. for and to A. K. for of the