Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 991646510
Received June 10, 1999; accepted August 10, 1999
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ABSTRACT |
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Key Words: nitric oxide; hepatocytes; nitric oxide synthase; endotoxin; nitrite; iNOS; NOS II.
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INTRODUCTION |
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There is increasing evidence that NO can function as a cellular antioxidant and can, under certain conditions, protect against reactive oxygen species-induced toxicity (Wink et al., 1995). Sergent et al. (1997) reported that NO protected against iron-mediated oxidative stress in primary rat hepatocyte cultures and hypothesized that NO-iron binding prevented the iron catalyzed formation of reactive oxygen species. Although NO can function as an antioxidant, NO can also react with superoxide anion to form the reactive oxidant species peroxynitrite. The dual nature of NO actions is also seen in studies conducted with hepatotoxicants. Muriel (1998) demonstrated that NO formation was protective against carbon tetrachloride-induced liver damage while Gardner et al. (1998) and Hinson et al. (1998) have proposed that NO is an important mediator of acetaminophen hepatotoxicity. Therefore, since NO can modulate toxicity and affect cellular functions, it is important to recognize when NO production is occurring in in vitro cell models.
The enzyme nitric oxide synthase (NOS) converts L-arginine to NO. At least three isoforms of NOS are known to exist in tissues. The inducible form of the nitric oxide synthase or type II nitric oxide synthase (iNOS) is expressed in several cell types including parenchymal liver cells (hepatocytes), smooth muscle cells, and macrophages (Harbrecht and Billiar, 1995). Kupffer cells are generally recognized as a source of NO in the liver, but hepatocytes can also contribute to liver NO production. Once iNOS is expressed in hepatocytes, extremely high levels of NO can be produced. For example, Curran et al. (1989) measured extracellular nitrite/nitrate (stable end products of NO formation) levels of over 500 µM in cultured rat hepatocytes after 18 h. Several treatments have been shown to induce iNOS in rat hepatocytes. Endotoxin and cytokines (tumor necrosis factor, interferon
and interleukin 1) are potent inducers of iNOS and act synergistically to evoke maximal NO production (Nussler et al., 1993
). Oxidative stress has also been proposed to induce iNOS expression in rat hepatocytes (Duval et al., 1995
).
Previously we reported that isolated rat hepatocytes in suspension (free of Kupffer cells) produce high levels of NO shortly after isolation (Nicholls-Grzemski et al., 1999). Hepatocyte NO production occurred in a variety of incubation media, and was not affected by adding antibiotics to incubations suggesting that bacterial contamination was not involved in nitrite synthesis. However, the addition of the NOS inhibitors aminoguanidine or NG-nitro-L-arginine methyl ester (L-NAME) completely blocked NO production, implicating iNOS involvement. Since isolated hepatocyte suspensions are a commonly used in vitro model system in metabolism and toxicology studies, a better understanding of NO production in this cell system is needed. In the present manuscript, we further characterize the biochemical events involved in NO production by freshly isolated rat hepatocyte suspensions and explore possible mechanisms that could initiate NO production. We propose that hepatocyte suspensions offer a convenient model system to investigate the cellular signaling events involved in iNOS induction.
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MATERIALS AND METHODS |
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Preparation of isolated hepatocyte suspensions.
Adult male Sprague-Dawley rats (180220 g) were obtained from Simonsen Laboratories, Inc. (Gilroy, CA). They were housed in small groups and given food and water ab libutum in a 12 h light/dark cycle for at least 1 week prior to use. Rats pretreated with gadolinium chloride (10 mg/kg) dissolved in phosphate-buffered saline (10 mg/ml) were iv-injected into the tail vein 24 h prior to hepatocyte isolation. Rat hepatocytes were prepared by collagenase perfusion as previously described (Fariss et al., 1985). Briefly, the liver was cannulated via the portal vein, surgically removed, and perfused in a recirculating system at 30 ml/min for 3 min with Hanks'-bicarbonate buffer containing 0.6 mM EGTA. The liver was then perfused in a recirculating system with Hanks'-bicarbonate buffer containing 3 mM calcium chloride and 60100 mg standard collagenase (Sigma L blend) for up to 10 min. To obtain the same degree of separation, 2 mg of Liberase was perfused for 4 min. After dispersion and washing, cells were assessed for viability by Trypan blue exclusion. A yield of 4.5 to 5.5 x 108 was routinely obtained with over 93% viability. Hepatocyte suspensions were prepared as described in Fariss et al., (1997). Briefly, hepatocyte suspensions (2 x 106 cells/ml, 12 ml total) were prepared in serum-free modified Waymouth's medium as previously described (Fariss et al., 1997
) with the addition of 0.12 mM ornithine but without cystine. Cells were placed in 125-ml boiling flasks and rotated at 37°C under ambient air. After a 15-min equilibration time, an aliquot of cells was taken as the 0 time point. Following collection of the 0 time point, the chemical treatments (prepared in media or as indicated) were immediately added. At hourly intervals thereafter, cells were sampled and pelleted, and supernatants were collected for nitrite and lactate dehydrogenase (LDH) leakage analysis. Cell pellets were stored at 80°C for iNOS Western blot analysis and quantitative RT-PCR.
Enzyme assays.
Hepatocyte calcium-independent NOS activity was determined by measuring the formation of nitrite as previously described (Nicholls-Grzemski et al., 1999). LDH activity was determined by monitoring the enzymatic formation of NADH from NAD+ in the presence of L-lactic acid. Post-centrifugation supernatants were diluted 1:80 with phosphate buffered saline, pH 7.4. A 100 µl aliquot was mixed with 100 µl of reagent to give a final concentration of 3.75 mM NAD+ and 25 mM L-lactic acid in 125 mM TrisHCl buffer, pH 8.9 in a 96-well plate. The increase in fluorescence (360 nm ex, 460 nm em) due to the formation of NADH was immediately monitored at room temperature (gain 70) using a CytoFluor 4000, Perseptive Biosystems (Framingham, MA). The percent LDH leakage was calculated by comparing values to total LDH activity. Total LDH was measured from a sample of hepatocytes collected at zero time and lysed with a final concentration of 0.2% Triton X-100.
Nitrite determinations.
Nitrite levels in media were determined according to the procedures of Misko et al. (1993) with slight modifications as previously described (Nicholls-Grzemski et al., 1999). Nitrite standards were prepared fresh and standard curves were constructed in the same media as samples. Samples and standards were read at 360 nm ex and 460 nm em with a gain setting of 90 on a CytoFluor 4000 fluorescence plate reader, Perseptive Biosystems (Framingham, MA).
Quantitative RT-PCR.
Total cellular RNA was isolated from the hepatocytes using the RNeasy Mini, RNA isolation kit from Qiagen (Valencia, CA) per the manufacturer's protocol. Total RNA was eluted from the matix with 35 µl of RNase-free water. Residual genomic DNA was removed by incubating the RNA solution with 15 units of RNase-free DNase I in 2 mM MgCl2 for 10 min at 37°C, followed by 5 min at 90°C to inactivate the DNase. DNase-treated RNA solution (25 µl) was reverse-transcribed to cDNA as previously described (Horikoshi, et al., 1992).
The amount of iNOS mRNA relative to the ß actin endogenous control was determined using real-time, quantitative PCR. The sequence of the ß actin primers was 5' ACCAACTGGGACGATATGGAGAAGA 3' bases 15571581, forward primer and 5' TACGACCAGAGGCATACAGGGACAA 3' bases 22102234 reverse primer of the genomic ß actin sequence (Nudel et al., 1983). The sequence of the iNOS primers was 5' CACGACACCCTTCACCACAAG 3' bases 301321, forward primer and 5' TTGAGGCAGAAGCTCCTCCA 3' bases 419438, reverse primer of the iNOS cDNA sequence (Kosuga et al., 1994
). The PCR was performed in the Perkin-Elmer Applied Biosystems GeneAmp 5700 Sequence Detection System (Foster City, CA) using the SYBR green PCR kit as recommended by the manufacturer (Foster City, CA). Briefly 2.5 µl of the 10x SYBR green buffer, 1 mM dA, dG, dC and dUTP, 2 mM MgCl2, 0.25 units of uracil N-glycosylase, 0.625 units of Amplitaq Gold DNA polymerase, 250 nM of the forward and reverse primer, 5 µl of a 1:10 dilution of the cDNA and water to 25 µl. The reactions were performed in the MicroAmp 96-well plate capped with MicroAmp optical caps Perkin-Elmer Applied Biosystems (Foster City, CA). The reactions were incubated at 50°C for 2 min to activate the uracil N'-glycosylase and then for 10 min at 95°C to inactivate the uracil N'-glycosylase and activate the Amplitaq Gold polymerase. The reactions were performed for 40 cycles of 15 sec at 95°C, 30 sec at 55°C and 30 sec at 72°C.
The data generated by real-time PCR were plotted as the Rn fluorescence signal versus the cycle number. The Applied Biosystems, Inc. (ABI) 5700 sequence detection system software from Perkin Elmer calculates the
Rn using the equation
Rn = (Rn+) (Rn-), where Rn+ is the fluorescence signal of the product at any given time and Rn- is the fluorescence signal of the baseline emission during cycles 3 to15. An arbitrary threshold was set at the midpoint of the log
Rn versus cycle number plot. The Ct value is defined as the cycle number at which the
Rn crosses this arbitrary threshold. The amount of iNOS cDNA relative to the ß actin endogenous control was determined using a modification of the 2-
Ct method as described in the ABI user bulletin number 2. The amount of iNOS mRNA relative to ß actin was calculated equal to 2-
Ct where
Ct = CtiNOS CtActin.
Western blot analysis of iNOS.
Hepatocyte pellets were resuspended in phosphate-buffered saline, pH 7.4, and protein levels were determined by the BCA protein assay (Pierce Chemical Co., Rockford, IL). Aliquots of resuspended pellets were heat-denatured and equal amounts of total protein (20 µg) were electrophoresed under reducing conditions on a 7.5% polyacrylamide-SDS gel. Proteins were transferred to nitrocellulose blots and blocked overnight at 4°C in 5% dried milk powder dissolved in 150 mM sodium chloride, 0.1% Tween-20 in 50 mM TrisHCl, pH 7.4. The blots were washed and expression of iNOS protein was detected with rabbit anti-mouse antibody Transduction Laboratories (Lexington, KY). This polyclonal antibody has been shown by the manufacturer to recognize 2 protein bands in mouse macrophage lysates. Specific immunocomplexes were detected using peroxidase-labeled anti-rabbit antibody with the ECL Western Blot Analysis System Amersham Pharmacia (Piscataway, NJ). Developed blots were immediately exposed to Kodak X-Omat scientific imaging film and both bands were quantified together using a Hewlett-Packard ScanJet 4c (Palo Alto, CA) equipped with Bioscan Analysis software, Biosoft (Ferguson, MO).
Statistics.
Results are presented as mean ± SD. Both the Student's t-test and ANOVA, followed by Dunnett's post hoc test were performed with the InStat 2.03 GraphPad Software Inc. (San Diego, CA) statistical package.
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RESULTS |
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Polymyxin B binds and neutralizes endotoxin and should therefore diminish any endotoxin-mediated induction of iNOS. The addition of 100 U/ml Polymyxin B to both the perfusion and incubation media significantly decreased nitrite production by hepatocyte suspensions (Fig. 4). Polymyxin B decreased nitrite production by about 50% in hepatocytes prepared from standard collagenase perfusions and completely blocked nitrite production (less than 0.8 µM) in hepatocytes isolated by the Liberase perfusion. Polymyxin B had no effect on cell viability (data not shown). In addition to binding endotoxin, polymyxin B also inhibits protein kinase activity (Rando, 1988
). To assess whether the inhibitory effects of polymyxin B on nitrite production are due to protein kinase inhibition, another protein kinase inhibitor was tested. The protein kinase inhibitor staurosporine was tested for its effects on nitrite production. In the results reported in Table 1
, polymyxin B and staurosporine were not present in the perfusion buffer, in contrast to Figure 4
, but instead were added at zero time. Staurosporine added at the onset of the hepatocyte incubations was as effective as polymyxin B in inhibiting nitrite production (Table 1
). Polymyxin B and staurosporine did not induce hepatocyte cell death during the 6 h incubation (data not shown).
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DISCUSSION |
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In our studies, increases in extracellular nitrite levels first occurred at about 4 h after the beginning of the hepatocyte incubations (Fig. 1). Since the collagenase isolation procedures require about 80 min to complete, the total time we observed between increases in nitrite levels and the isolation of hepatocytes was about 5 to 6 h. This time period is consistent with the time required to induce nitrite production in cultured hepatocytes following the addition of cytokines/endotoxin (46 h) reported by Geller et al. (1993). The time frame for nitrite production with hepatocyte suspensions suggests that the stimulus for nitrite production occurs during the isolation of hepatocytes. In addition, a comparison of the time-dependent formation of iNOS mRNA and protein (Fig. 2
) indicates that there is approximately a 1-h delay between mRNA expression and the subsequent formation of detectable levels of the iNOS protein. Once the iNOS protein is expressed, nitrite rapidly increases in the extracellular medium.
Curran et al. (1989) demonstrated that activated Kupffer cells release products which can induce NO formation in hepatocytes. Activated Kupffer cells produce cytokines and reactive oxygen species, both of which can induce iNOS expression in hepatocytes (Duval et al., 1995; Geller et al., 1993
). Studies have shown that gentle manipulation of rat livers can lead to the activation of Kupffer cells (Schemmer et al., 1998
). Since collagenase isolation techniques involve liver manipulations, this mechanism may be relevant to our studies. Our results with the Kupffer cell inhibitor, gadolinium chloride, also support a role for Kupffer cells in the sequence of events leading to the production of NO by hepatocyte suspensions. Pretreatment of rats with gadolinium chloride attenuated nitrite formation by freshly isolated hepatocyte suspensions (Fig. 3
).
In addition, Kupffer cells may become activated by exposure to endotoxin. Geller et al. (1993) found endotoxin ineffective in inducing iNOS in cultured hepatocytes, but endotoxin did potentiate the induction of iNOS by cytokines. In our experiments, hepatocytes isolated by Liberase, which contains very low levels of endotoxin, still produced nitrite. The addition of the endotoxin neutralizing agent, polymyxin B, inhibited NO production by rat hepatocyte suspensions, but polymyxin B was much more effective in blocking NO production in hepatocytes isolated by Liberase than by the standard collagenase preparations. There may be two explanations for this observation. Either the addition of polymyxin B was sufficient to bind and neutralize any residual endotoxin present in the Liberase preparation and block Kupffer cell activation, or the addition of polymyxin B inhibits NO production by a mechanism unrelated to endotoxin neutralization. Polymyxin B is known to be a protein kinase inhibitor and has been shown to affect protein kinase C activity (Rando, 1988). Reports have implicated protein kinase C as being involved in the signaling events triggering iNOS expression in hepatocytes (Hortelano et al., 1992
). Interestingly, Hortelano and colleagues also demonstrated that protein kinase C activation antagonized iNOS induction by endotoxin in rat hepatocytes, indicating that there is considerable complexity in the regulation of iNOS in hepatocytes. In our experiments, adding polymyxin B at zero time (Table 1
) was almost as effective in inhibiting NO production as including polymyxin B in the collagenase perfusion buffers and the hepatocyte suspensions (Fig. 4
). If polymyxin B is acting by binding endotoxin from collagenase preparations, then it should be much more effective if it is added during the collagenase perfusion. However, we did not find this to be true with either Liberase or the standard collagenase. In addition, the effects of the protein kinase inhibitor, staurosporine, on nitrite production were almost identical to that of polymyxin B (Table 1
). These results suggest that in addition to neutralizing endotoxin, polymyxin B may be affecting NO production by inhibiting protein kinases.
Adding the antioxidants DFO/Trolox to perfusion buffers and hepatocyte suspensions significantly inhibited nitrite production by hepatocytes isolated from Liberase preparations, but not from hepatocytes isolated from standard collagenase preparations. Duval et al. (1995) concluded that reactive oxygen species induced iNOS in rat hepatocytes and that DFO and Trolox inhibited this induction. Our results agree with this conclusion and suggest that reactive oxygen species may contribute to the induction of iNOS in hepatocytes isolated by Liberase. The sources of the reactive oxygen species are unknown, but may involve Kupffer cell activation, ischemia, the presence of trace amounts of iron, alterations in extracellular calcium, or other events occurring during isolation. Trace amounts of iron may be present as a contaminant in perfusion and incubation buffers or may be released from damaged cells. Several groups have suggested that protein kinase C is activated by reactive oxygen species (Bagchi et al., 1997; Gopalakrishna et al., 1991; Kass et al., 1989
). A reasonable hypothesis would be that reactive oxygen species are generated during collagenase perfusions and isolation procedures and these species may induce iNOS by activating the protein kinase C signaling pathway in hepatocytes. However, further work is required to specifically identify the role of protein kinase C in inducing iNOS in hepatocyte suspensions.
As Table 1 indicates, there are clear differences in the ability of hepatocytes isolated from standard collagenase and Liberase preparations to generate NO in the presence of polymyxin B, staurosporine, and DFO/Trolox. Our findings suggest that endotoxin may be involved in inducing iNOS in standard collagenase preparations, but other signaling pathways such as protein kinase C and reactive oxygen species may take a more prominent role in the absence of endotoxin. Our work suggests that endotoxin is not solely responsible for iNOS induction in isolated hepatocyte suspensions, but its presence may modify existing signaling pathways. Further work is necessary to characterize the differences between hepatocytes isolated with standard collagenase and Liberase preparations.
Recently, Wang et al. (1998) reported that collagenase perfusions induced the expression of iNOS mRNA in cultured mouse hepatocytes and speculated that the procedures involved in hepatocyte isolation may be responsible for these events. Although these researchers detected iNOS mRNA expression, they did not detect increases in extracellular nitrite. In agreement with our results, Lopez-Garcia (1998) recently reported that cultured rat hepatocytes produce large amounts of nitrite shortly after isolation, and they concluded that isolation procedures are involved in initiating this process. Jones and Czaja (1998) suggested that NO is generated in liver in response to injury. Hepatotoxicants, ischemia-reperfusion (Jones and Czaja, 1998), and partial hepatectomy (Hortelano et al., 1995
) have been shown to increase NO production by the liver. Our data are consistent with this viewpoint, and suggest that hepatocytes as well as Kupffer cells may contribute to NO production in the liver. Therefore, the induction of iNOS in isolated hepatocytes may represent a cellular response to the stresses imposed on the liver during the isolation procedures.
In summary, freshly isolated hepatocyte suspensions produce large amounts of nitrite as a result of NO production. Our data suggests that iNOS induction occurs during the isolation of hepatocytes as a result of several factors, including Kupffer cell activation, reactive oxygen species production, and to a certain extent endotoxin exposure. The induction of iNOS may be triggered in response to the stress of hepatocyte isolations. In our system, the large production of NO had no effect on cell viability; however, other cellular functions were not examined. Further research is necessary to determine if NO is also produced in cultured hepatocytes and to examine the toxicological implications of NO production in these cells.
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ACKNOWLEDGMENTS |
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NOTES |
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