©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Loss and Degradation of Enzyme-bound Heme Induced by Cellular Nitric Oxide Synthesis (*)

(Received for publication, September 6, 1994; and in revised form, January 18, 1995 )

Young-Myeong Kim (1) Hector A. Bergonia (1) Claudia Müller (3) Bruce R. Pitt (2) W. David Watkins (3) Jack R. Lancaster Jr. (1) (3) (2)(§)

From the  (1)Departments of Surgery, (2)Pharmacology, and (3)Anesthesiology and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report here that, like nonheme iron, protein-bound intracellular heme iron is also a target for destruction by endogenously produced nitric oxide (NO). In isolated rat hepatocytes NO synthesis results in substantial (approximately 60%) and comparable loss of catalase and cytochrome P450 as well as total microsomal heme, and decreased heme synthetic (-aminolevulinate synthetase and ferrochelatase) and increased degradative (heme oxygenase) enzymatic activities. The effect is reversible, and intact cytochrome P450 apoproteins are still present, as judged by heme reconstitution of isolated microsomes. The effects on -aminolevulinate synthetase and heme oxygenase are likely to be secondary to heme liberation, while the effects on ferrochelatase appear to be a direct effect of NO, perhaps destruction of its nonheme iron-sulfur center.


INTRODUCTION

Nitric oxide (NO) is produced by specific mammalian enzymes either in small quantities as a neural or vascular messenger or in large quantities by certain cells of the immune system as an effector where it is cytostatic/cytotoxic to many pathogenic organisms and also to infected or transformed host cells(1, 2) . Intracellular iron is a major target of NO as an immune effector, and previous studies have described loss of nonheme iron-containing enzyme activities(1, 2) . Although heme iron is also a well established target for exogenous NO(3) , few studies have demonstrated deleterious effects of endogenous NO synthesis on intracellular heme-containing enzymes. Recently it has been found that rat hepatic NO synthesis results in a decrease in both activity and levels of cytochrome P450 (CYP)(^1)(4, 5) . Understanding the mechanism by which endogenous NO inhibits heme-containing proteins is likely to explain such important phenomena as decreased drug tolerance in septic patients(6) . We describe here one basis for this phenomenon, which involves NO-induced intracellular heme loss and increased heme degradation.


EXPERIMENTAL PROCEDURES

Reagents

Williams media E, trypan blue, insulin, penicillin, streptomycin, L-glutamine, and HEPES were purchased from Life Technologies, Inc. Calf serum was purchased from Hyclone Laboratories (Logan, UT). Collagenase was purchased from Boehringer Mannheim. S-Nitroso-N-acetylpenicillamine (SNAP) was synthesized as described previously(7) , stored frozen as a solid in the dark, and routinely checked for stoichiometric S-nitrosothiol content by the method of Saville(8) . Other chemicals were obtained from Sigma unless otherwise stated.

Isolation and Treatment of Hepatocytes

Purified hepatocytes were isolated and treated with a cytokine mixture (tumor necrosis factor-alpha (Genzyme), interferon- (Amgen), interleukin-1beta (Cistron)) plus endotoxin/lipopolysaccharide (Escherichia coli 0111:B4, Sigma) (CME) with or without N^G-monomethyl-L-arginine (NMMA) for 24 h as described previously(9) . Cells were also exposed for 9 h to 1 mM SNAP in medium. Cell death (measured by crystal violet staining) resulting from the various treatments was found to be minimal.

Catalase Assay

Hepatocytes (1 times 10^7 cells) were suspended in 750 µl of PBS and homogenized on ice for 2 min by using a glass tissue homogenizer with a Teflon pestle. The solution was centrifuged at maximum speed (14,000 rpm) for 10 min at 4 °C in an Eppendorf microcentrifuge. The supernatant was designated as enzyme solution for catalase assay. Catalase activity was measured using a Clark type oxygen electrode (Yellow Springs Instruments Co.) as described(10) . The reaction mixture in a final volume of 1.5 ml of degassed PBS contained 20 µl of the enzyme solution and 50 µl of 400 mM H(2)O(2) in PBS.

Determination of P450, Heme, Nonheme Iron, Enzymatic Activities, and mRNAs

For isolation of microsomes, crude cytosol (11) was spun at 100,000 times g for 90 min to pellet the microsomes. Spectra and quantitation of the reduced versus reduced plus CO complex of CYP were as described(12) . Total extractable heme was quantified according to a previously described method(13) . Microsomes (10 µM total heme) were exposed to dissolved NO (88 µM) anaerobically for varying times at room temperature after which the NO was removed by gassing with argon, and total CYP heme was assayed as described above. For heme reconstitution of P450, each microsomal fraction (control, CME (24 h), CME + NMMA (24 h), and SNAP (12 h)) was divided between two dialyzing tubes. One tube was dialyzed aerobically for 6 h at 4 °C against phosphate buffer (0.1 M, pH 6.8), while the other was dialyzed aerobically against the same buffer but contained 50 µM heme (hematin reduced to heme by an equivalent amount of sodium dithionite in a small volume). All samples were dialyzed further against phosphate buffer for 24 h to remove excess heme, and CYP was determined as described above. Total iron was determined using a colorimetric micromethod after acid-permanganate treatment(14) . Nonheme iron is expressed as the difference between the total and heme iron. -Aminolevulinate synthetase, heme oxygenase (HO), and ferrochelatase activities were assayed as described(15, 16, 17) . Total RNA was extracted from the cultured hepatocytes, electrophoresed, and blotted as described by Geller et al.(18) . The probe to inducible nitric oxide synthase used was a 2.7-kilobase cDNA obtained by NotI digestion from a mouse macrophage cDNA clone(18) . The HO probe used was a 1.4-kilobase cDNA obtained by EcoRI digestion from a human macrophage (U937 histiolytic lymphoma) cDNA clone(19) . Radioactive membranes were quantified with storage phosphor screens (PhosphorImager, Molecular Dynamics), and the relative amount of mRNA is presented as the ratio of mRNA to 18 S RNA. The appearance of total medium NOx (NO(2) + NO(3)) was measured as described previously(9) . Protein concentration was measured by Lowry protein assay kit (P5656, Sigma).

Statistical Analyses

Results represent means ± S.D. from a minimum of three experiments. Significance between groups was determined using the Student's unpaired t test.


RESULTS AND DISCUSSION

Catalase is a heme-containing enzyme that catalyzes the dismutation of H(2)O(2) to O(2) and H(2)O. As shown in Fig. 1A, treatment of hepatocytes with CME (which we have shown previously to induce substantial NO synthesis(9) ) results in a decrease followed by gradual recovery of catalase activity. The minimum activity (65% loss) occurred at approximately the same time as the maximum rate of NO synthesis, as we have shown previously(20) . Inhibition of NO synthesis by NMMA results in oblation of catalase activity loss, demonstrating that it is due to NO production. This result raises the possibility that inhibition of catalase may be a previously unrecognized mechanism of NO-induced potentiation of oxidative injury(21) .


Figure 1: Effects of endogenous and exogenous NO on catalase, CYP heme, and total microsomal heme levels. Hepatocytes were isolated and treated and catalase, microsomal protein, CYP heme, and total extractable heme were determined as described under ``Experimental Procedures.'' A, effects of endogenous NO on catalase activity. Hepatocytes were incubated in the absence (circle) or presence (box) of CME or of CME + NMMA (bullet), and at the indicated times catalase activity was determined as described under ``Experimental Procedures.'' B, effects of endogenous NO on CYP and total microsomal heme. Hepatocytes were incubated without or with CME, NMMA, or SNAP as indicated (as described under ``Experimental Procedures'') and assayed for total microsomal protein, CYP heme, and total extractable heme. C, effects of NO on CYP heme in isolated microsomes after various times of exposure, as described under ``Experimental Procedures'': I = 0; II = 20 min; III = 60 min. Spectrum IV is free heme (1 µM)-CO complex. D, heme reconstitution of NO-induced loss of CYP heme. Treatment of hepatocytes, preparation of microsomes, and dialysis without (a) or with (b) heme were as described under ``Experimental Procedures.''



The CYPs are heme-containing enzymes that catalyze the metabolism of a variety of endogenous and exogenous compounds. We chose this class of enzymes for further studies on the effects of NO because they represent the major heme pool in hepatocytes(22) , and inactivation can be monitored by the reduced versus reduced + CO difference spectrum of the microsomal fraction(23) . Fig. 1B shows the effects of endogenous and exogenous NO on microsomal CYP heme and total extractable microsomal heme (13) in cultured rat hepatocytes. Exogenous NO exposure was accomplished by using SNAP, an NO donor. Compared with control cells there is an approximately 60% decrease in basal (uninduced) CYP heme induced by either endogenous or exogenous NO, as reported previously for inducible CYP heme(4, 5) . CYP heme loss is prevented by inhibition of NO synthesis (NMMA). The parent compound of SNAP (N-acetylpenicillamine, not containing a nitrosyl group) had no effect (data not shown); that was also true for all other studies below with SNAP. In order to determine whether this effect is due to prevention by NO of CO binding to heme (thus masking the appearance of the 450 nm peak(4) ) or instead loss of CYP protein and/or heme, we assayed total extractable microsomal heme. Fig. 1B shows that both endogenous and exogenous NO cause a loss of total microsomal heme that is quantitatively indistinguishable from CYP heme loss.

Having established that the effect of NO is due to decreased holocytochrome P450 (and not prevention of CO binding), we next determined whether this effect is due to a decrease in CYP protein expression (as reported by Stadler et al.(5) for inducible CYP) or to loss of enzyme-bound heme. We first determined if exposure of microsomes from untreated hepatocytes to NO induces loss of heme. As shown in Fig. 1C, exposure of microsomes from untreated hepatocytes (I) to NO for 20 min (II) results in partial conversion of P450 into P420, indicative of sulfur to nitrogen exchange of proximal axial heme ligand (23) . From comparison with the spectrum of free heme (IV), after a further 40-min exposure (III) it can be concluded that a substantial portion of CYP heme is fully detached from its normal binding in the proteins, as judged by the appearance of the peak at 405 nm(24) . Therefore it appears that NO exposure induces P450 to P420 conversion followed by release of heme from CYP.

In order to determine whether a decrease in CYP is due to decreased CYP protein or loss of heme, we determined whether CYP apoprotein is present after loss of heme. We determined this by isolating microsomes from NO-synthesizing hepatocytes (or from hepatocytes treated with SNAP), dialyzing the preparations versus heme, and then measuring the reduced versus reduced plus CO spectrum of the preparations to determine whether heme had reinserted into apocytochrome P450 (Fig. 1D). This method has been used successfully for reconstitution of several cytochrome P450s subsequent to several methods of inducing heme loss/destruction, including P450(25) , P450(26) , as well as the system studied here, rat hepatic microsomal cytochrome P450(27, 28) . Numerous studies have documented that the characteristic peak at 450 nm (for which these enzymes are named) is due to proximal cysteine ligation to the heme and that conversion to nitrogenous proximal heme ligation results in a shift in the peak to 420 nm with consequent loss of activity(23) . As shown in spectra I, dialysis versus heme of microsomes from control hepatocytes did not result in appreciable effects on the total CYP heme and resulted in no appreciable increase in either P420 or P450 heme absorption, as was also true for hepatocytes treated with the cytokine mixture plus NMMA (III). As shown in II, however, the substantial loss of CYP in microsomes from NO-synthesizing hepatocytes was restored by dialysis versus heme. Spectra IV show an effect with SNAP-treated cells that is similar to NO-producing cells, demonstrating that either endogenous or exogenous NO induces comparable effects. These results demonstrate that, in contrast to CYP induced by xenobiotic exposure (5) , the virtually exclusive effect of NO on basal CYP is loss of the heme prosthetic group with no appreciable decrease in CYP protein. In addition, the CYP apoproteins are left in a relatively native conformation since P450 (not P420) is reconstituted by heme. We note that while observation of the feature at 450 nm demonstrates that heme has indeed reinserted into the apoproteins (thus showing that CYP apoproteins are present), we have not attempted to determine whether dialysis versus heme results in reconstitution of any specific P450 activity because different CYP isoforms may be differently affected by NO (as described below).

We next determined whether intracellular heme levels in NO-producing hepatocytes change as a result of liberation from CYP, which is known to be the major heme pool in these cells(22) . Previous studies have demonstrated NO-induced loss of total cellular iron from cells (including hepatocytes(29) ), although no differentiation of heme versus nonheme iron has yet been made(1, 2) . As shown in Fig. 2A, there is a selective loss of whole cell total extractable heme iron upon induction of NO synthesis with CME, which is prevented by NMMA. Similar loss occurs with SNAP-treated cells. No significant change in total cellular nonheme iron occurs (Fig. 2B), although the total amount of nonheme iron is much more than heme iron. Similar relative changes in heme also occur in the microsomal and cytosolic subcellular fractions. Interestingly, there is a relatively modest but detectable increase in nonheme iron in the cytosolic fraction, which may represent increased iron storage as ferritin(30) .


Figure 2: Heme and nonheme iron distribution in various cellular compartments in hepatocytes exposed to either endogenous or exogenous NO. Subcellular fractions were isolated from the hepatocytes cultured with CME for 24 h, CME plus NMMA for 24 h, and 1 mM SNAP for 9 h. The microsomes were resuspended in 0.3 ml of PBS prior to iron and protein analyses. Nonheme iron (B) is expressed as the difference between the total (14) and heme iron (A). The levels of significance were determined using the Student's t test: *, p < 0.005;**, p < 0.03;***, p > 0.1 versus control.



In hepatocytes, free heme is known to up-regulate its own degradation (by transcriptionally regulated increase of HO) and down-regulate endogenous synthesis (by decrease of mRNA stability and mitochondrial import of -aminolevulinate synthetase(22) ). As shown in Fig. 3A, induction of NO synthesis by hepatocytes results in a decrease in -aminolevulinate synthetase and an increase in heme oxygenase activities, consistent with intracellular heme liberation with consequent effects on these two enzymes. This result also indicates that the increase in heme oxygenase and decrease in -aminolevulinate synthetase activities in rats injected with LPS (31, 32) are due to NO-induced heme liberation. It is worth noting that even in the presence of NMMA there is still an approximately 2-fold increase in heme oxygenase activity, which may indicate up-regulation by cytokine treatment (33, 34, 35) that is NO-independent.


Figure 3: Effects of NO on heme metabolic enzymes. A, effects of endogenous NO. Enzymatic activities were determined after the indicated treatments for 24 h as described under ``Experimental Procedures.'' B, time course of SNAP-induced effects on heme metabolic enzymes. At zero time, hepatocytes were exposed to 1 mM SNAP and activities assayed at the indicated times as described under ``Experimental Procedures.'' C, time course of induction of nitrite + nitrate (NOx) and inducible nitric oxide synthase and HO mRNA by endogenous and exogenous NO. NOx, inducible nitric oxide synthase (iNOS), or HO mRNA were determined as described under ``Experimental Procedures'' at various times after induction of endogenous NO synthesis (CME) or SNAP or no further treatment (CTRL). FC, ferrochelatase; ALAS, -aminolevulinate synthetase.



Ferrochelatase, recently discovered to contain an Fe(2)S(2) nonheme iron-sulfur center(36) , catalyzes the insertion of iron into porphyrin. As shown in Fig. 3A, NO synthesis also decreases ferrochelatase activity, which represents a newly identified nonheme iron enzymatic target for NO. Fig. 3B presents a time course for the changes in the activities of these three enzymes after exposure to SNAP. While a decrease in -aminolevulinate synthetase and an increase in heme oxygenase activities require a time lag of 2-3 h (consistent with previously described heme-induced regulatory mechanisms(22) ), the decrease in ferrochelatase activity is virtually complete within 1 h. This indicates that NO inhibits activity of this enzyme by a direct effect, perhaps by destruction of its nonheme iron-sulfur cluster(36) . This inhibition will contribute to a decrease in intracellular heme levels in addition to a decrease in -aminolevulinate synthetase and an increase in heme oxygenase activities.

Finally, Fig. 3C demonstrates that NO synthesis results in increased heme oxygenase transcription, as determined by Northern blot analysis using a cDNA probe to human HO1(19) . The increase occurs slightly after induction of NO synthase and concomitant with NO synthesis (appearance of NO(2) + NO(3) in the medium, NOx). Heme oxygenase mRNA is induced also by SNAP, without the lag required for induction of NO synthase by the cytokine mixture, demonstrating that NO alone (without cytokines) can induce heme oxygenase. Although not shown, addition of 8-bromo-cyclic GMP (20 µM) did not induce an increase in heme oxygenase mRNA, indicating that the effect is not due to a cGMP signaling mechanism.

With regard to the possible mechanism(s) of NO-induced heme loss, previous studies in oxygen-free conditions have shown that NO binds to both the reduced and oxidized forms of CYP and that conversion to the inactive P420 form can take place(37) . In the presence of oxygen, Wink et al.(38) have shown that NO causes both a transient, reversible inhibition of enzymatic activity and a long lasting irreversible inhibition. The transient reversible inhibition may be due to formation of nitrosyl-heme complexes; the irreversible inhibition is substantially prevented by the addition of albumin, suggesting the formation of a reactive nitrogen oxide species from the reaction of NO with O(2) (to form nitrosonium ion equivalents [NO]) or with O(2) (to produce peroxynitrite, ONOO). Early work demonstrated the formation of nitrosonium ion equivalents ([NO]) in activated macrophages synthesizing NO, and similar chemical reactivity is exhibited by the reaction of NO with O(2) in aqueous solution(39, 40) . In addition, activated macrophages synthesize peroxynitrite(41) . Previous studies have shown that both heme and albumin are nucleophilic targets for nitrosation (42, 43) and that albumin is a target for peroxynitrite(44) .

It is likely that all hemoproteins do not respond identically to endogenous nitrogen oxides. For example, as shown in Fig. 1B there appears to be a subpopulation of CYP proteins that are resistant to 60-min exposure to NO, and mitochondrial cytochromes are also resistant to enzymatic inhibition(1, 2) . In addition, at least two enzymes (cyclooxygenase (45) and guanylyl cyclase(46) ) are stimulated by NO. In the latter case, the results presented here may suggest that nitrosylation of the heme in guanylate cyclase is a transient event, terminated by dissociation of the NO-heme from the enzyme. This may explain the transient nature of stimulation of cGMP synthesis by NO.

These results also may have relevance to other well known pathophysiological conditions. For example, formation of NO by the reticuloendothelial system under conditions of immune activation may result in decreased erythropoiesis, perhaps contributing to the well known anemia of inflammation and infection(47) .


FOOTNOTES

*
This study was supported by American Cancer Society Grant BE-128 (to J. R. L.), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK46935 (to J. R. L.), National Institutes of Health Grant HL32154 (to B. R. P.), and a grant from the Max Kade Foundation, New York (to W. D. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Depts. of Physiology and Medicine, LSU Medical Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-6171; Fax: 504-568-6158.

(^1)
The abbreviations used are: CYP, cytochrome P450; CME, a cytokine mixture (tumor necrosis factor-alpha, interferon-, interleukin-1beta) plus endotoxin/lipopolysaccharide; HO, heme oxygenase; NMMA, N^G-monomethyl-L-arginine; NOx, nitrite plus nitrate; PBS, phosphate-buffered saline; P420, cytochrome P420; P450, cytochrome P450; SNAP, S-nitroso-N-acetylpenicillamine.


ACKNOWLEDGEMENTS

We thank Karla Wasserloos and William M. Konitsky for excellent technical assistance and are grateful to Dr. Shigeki Shibahara, Tohuku University, Japan for the gift of plasmid, pHHO1, containing cDNA to the human heme oxygenase 1 gene.


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